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Research Report 2000Covers the Period 1998/99

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Content

Foreword

Introduction

Genetics, Bioinformatics and Structural Biology

Cell Growth and Differentiation

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• Molecular Biology and Genetics of Detlev Ganten 20Cardiovascular Diseases

• Molecular Biology of Peptide Hormones Michael Bader 22

• Genome Research, Gene Variation, and Margret Hoehe 24Complex Disease

• Etiology and Pathogenesis of Friedrich C. Luft 26Hypertension and Vascular Disease

• Gene Mapping and Identification André Reis 28in Monogenic and Complex Diseases

• Tumor Genetics Siegfried Scherneck 30

• Clinical and Molecular Genetics Herbert Schuster 33of Cardiovascular Diseases

• Mouse Genetics Carmen Birchmeier 36

• Developmental Genetics Andreas Schedl 38

• Lipids and Experimental Gene Therapy Thomas E. Willnow 40

• Bioinformatics Jens Reich, Peer Bork 42

• Interactions of Biopolymers in Solution Joachim Behlke 44

• Folding and Misfolding of Proteins Gregor Damaschun 46

• Protein Folding and Misfolding Heinz Fabian 48

• Structural Studies of Proteins and Udo Heinemann 50Nucleic Acids by X-ray Crystallography

• Role of Protein Dynamics Christiane Jung 53in Enzyme Function

• Modeling Nucleic Acid Structure Heinz Sklenar 55and Recognition by Proteins

• Conformation, Stability and Interaction Heinz Welfle 57of Biological Macromolecules

• Protein Structure Analysis and Proteome Brigitte Wittmann-Liebold 59Research

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• Growth Control and Gene Regulation Achim Leutz 64in the Hematopoietic System

• Regulation of Transcription Claus Scheidereit 67in Mammalian Cells

• Differentiation and Growth Control Martin Lipp 69in Lymphocyte Development and Function

• Mechanisms Controlling the Initiation Manfred Gossen 72of DNA Replication

• Cell Cycle Regulation Hans-Dieter Royer 73

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Molecular Therapy

Molecular and Developmental Neurosciences

Structure and Organization

• Epithelial Differentiation, Invasion, Walter Birchmeier 75and Metastasis

• Cell Adhesion and Signal Transduction Jürgen Behrens 78

• Glycoconjugates Uwe Karsten 80

• Intracellular Signaling in the Myocardium Ernst-Georg Krause 82

• Molecular Muscle Physiology Ingo L. Morano 84

• Cell Biology of Cardiovascular Heinrich Leonhardt, 86Diseases M. Cristina Cardoso

• Surgical Oncology Peter M. Schlag 89

• Ubiquitin System and Endoplasmic Thomas Sommer 91Reticulum

• P450 Cytochromes and the Endoplasmic Wolf-Hagen Schunck 93Reticulum

• Vascular Biology Hermann Haller 95

• Functions of Dynamin II and PKC in Peter Westermann 98Post-Golgi Vesicle Formation

• Electron Microscopy 99

102

• Hematology, Oncology and Bernd Dörken 103Tumor Immunology

• Molecular Immunotherapy Antonio Pezzutto 106

• Experimental Pharmacology Iduna Fichtner 108

• Drug Targeting Regina Reszka 110

• Molecular Basis of Congestive Heart Failure Rainer Dietz 113

• Immunology of Cardiovascular Diseases Gerd Wallukat 114

• Molecular Immunology and Thomas Blankenstein 116Gene Therapy

• Cellular Immunology Kirsten Falk, Olaf Rötzschke 118

• Molecular and Cell Biology Martin Zenke 119of Hematopoietic Cells

• Phospholipids Dietrich Arndt 121

• RNA Chemistry Eckart Matthes 122

• Regulation and Deregulation Research Group of the 123of Cell Proliferation and Gene Therapy Humboldt University of Berlin

at the MDC

• Evolution, Regulation and Genetic Zoltán Ivics 125Applications of Transposable Elements in Vertebrates

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• Cellular Neurosciences Helmut Kettenmann 129

• Growth Factor and Regeneration Gary R. Lewin 131

• Synapse Formation and Function Frank W. Pfrieger 133

• Developmental Neurobiology Fritz G. Rathjen 135

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Organs of the Foundation 140

Supporting Divisions 143

Press and Public Relations 145

Administration 146

Central Facilities 148

Meetings, Workshops and Symposia 150

Awards 151

Adresses of Scientific Journals 152at the Berlin-Buch Campus

Index 153

Structure of the MDC 159

“Molecular Medicine”: Progressby Interdisciplinary Research

At the beginning of the 20th century,physics was the dominant and fast-moving science. This has changedtowards the end of the 20th century,when important progress was made inthe biological and life sciences. Duringthe last decades, the molecular basis oflife was elucidated in its fundamentalaspects. The basic mechanisms thatmake and maintain a living cell, forinstance energy metabolism,replication of DNA, RNA and proteinsynthesis, are understood. Theinformation can now be found intextbooks on biology or medicine in acondensed form. The final goal ofmodern life sciences, the understandingof a complex organism in molecularterms, is in reach. This implies that themolecular causes of human diseasescan and will be elucidated.

Such knowledge must be applied tothe maintenance of health, thediagnosis and treatment of humandisease. I see our mission at the MDCin the establishment of a life sciencethat improves the human condition. Tofulfill the promises and expectationsthat arise from this, modern medicinemust take advantage of many differentdisciplines and their methods. Inaddition to conventional anatomy,physiology, biochemistry, genetics,microbiology, pathology, and theclinical disciplines, new research areassuch as genomics, proteomics,bioinformatics, and even “phenomics”have emerged. Other areas such asphysics, informatics, material sciences,nanotechnologies etc. becomeincreasingly important. Todaydisciplines are not as clearly defined asin the past: The application ofmolecular biology, genetics, andgenomics to classical disciplines hasblurred their traditional borders.

“Molecular Medicine” requires aninterdisciplinary approach: on the oneside the understanding ofphysiological and pathologicalprocesses on basic levels and, on theother side, the application of theknowledge to clinical challenges.Classical discipline-oriented researchand educational institutions do notfavor interdisciplinary interactions.Progress, however, can only be madethrough intelligent cooperations.Creative scientists have always lookedbeyond their own disciplines. We arecoming to a point where not onlyindividuals, but also entire researchorganizations and institutions need tothink along such interdisciplinarylines. In my view, an interdisciplinarylife science is the science of thefuture, a “living science” in thegenuine sense of the word. Whenapplied to the human condition, it isthe essence of Molecular Medicine. Atthe Max Delbrück Center forMolecular Medicine, the “MDC”, wefoster such cooperations betweenbasic research and clinical disciplines.

Interdisciplinary research is persuedby the MDC, but also by all the other15 members of the HelmholtzAssociation of National ResearchCentres. One of the aims of thesecenters is the establishment ofprograms that address complexproblems by interdisciplinary, cross-country cooperation. At the MDC, this aim is addressed by assemblingbiomedical and clinical disciplines atone single center, and by fosteringtheir cooperation. I am convinced thattherein lies the future of medicine.

Let me look back in history andconsider the beginnings of MolecularMedicine on a very selective bias,focusing on the example of MaxDelbrück and his impact oninterdisciplinary research in medicine.

The Rockefeller Foundation, thatfunded among others Max Delbrück,had a pivotal impact on medicine andthe biological sciences at thebeginning of the 20th century. Animportant medical textbook, the“Principles and Practice of Medicine”(Appleton and Co. New York, 1893)written by William Osler, provided theimpetus for the establishment of theRockefeller Foundation. It was thefirst medical textbook clearlydescribing diseases in a mannerunderstandable to the layman. Osler’sbook was very honest on the subjectof therapy, which was basically non-existent. After he read this book,Frederick T. Gates, a non-physician,convinced the wealthy John D.Rockefeller to create this philantropicfoundation. The researchers, whichwere funded in Rockefeller’s program,included scientists from manydifferent fields, for instance physicsand chemistry. Warren Weaver, thedirector of natural sciences division ofthe Rockefeller Foundation, namedthe broad, well-funded program in1938 with the new but now familiarterm “Molecular Biology”. Weaveraimed to support “the application oftheoretical and experimentalprocedures to the study of theorganization and reactions of livingmatter”. This was the first majorinterdisciplinary biomedical program.Those at the Rockefeller Foundationunderstood better than anyone elsethat too many scientific efforts wereconducted in isolation, but neededcoordination. Born out of these ideaswas the “Science of Man” program, agreat success.

Max Delbrück from Berlin was one ofmany researchers that contributed andbenefited from this program. He wasat the center of a well-funded andintellectually fertile group, thefounders of today's molecular biology.His career provides a lesson for theadvantages of interdisciplinaryresearch. Max Delbrück was educatedas a physicist. Another physicist, NielsBohr, challenged him to start arevolution in biology similar to theone that was occurring already inphysics.

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Foreword

The quantum theory, that provided anew basis for the understanding ofmatter, was put forward by Niels Bohrand others who studied the interactionbetween light and matter. Bohrspeculated that a similar revolutioncould occur in biological sciences byapplying quantum mechanics to livingorganisms. In Berlin, Max Delbrückcame to know an equally youngRussian geneticist who worked inBerlin-Buch, Nikolai WladimirovichTimoféeff-Ressovsky. The twocollaborated and developed ideas thenunheard of. As an experimentalsystem, they used Drosophila flies,which were irradiated to increasemutation rates. Their results gave theterm “gene locus” a material basis andthey concluded that genes werephysical entities of a defined size, anassembly of atoms, “Atomverband”,as Max Delbrück called it. This was amajor conceptual breakthroughpublished in the famous paper “On theNature of Gene Mutation and GeneStructure” in 1935.

At this time, genetics was thriving asa discipline in its own right. ThomasHunt Morgan was mapping genes inDrosophila by the analysis of theirallelic association. BarbaraMcClintock was watching colorchanges in corn plants caused by“jumping genes”. When MaxDelbrück received his Rockefeller

Foundation fellowship to study in theUnited States, he visited severallaboratories and decided to establishhimself at the California Institute ofTechnology in Pasadena. There, a lonemaverick, Emory Ellis, was studyingbacterial parasites called “phages”.Phages turned out to be the tool withwhich physicists like Max Delbrückrevolutionized biology.

Max Delbrück’s ideas and his visionof the atomic constitution of genesstimulated the Nobel Prize-winningAustrian physicist Erwin Schrödinger,who left Berlin for Dublin in 1933, towrite a monograph entitled “What isLife?” Other physicists, GeorgeGamow, Leo Szilard, and later,Francis Crick, also started to applytheir knowledge, soon makingimportant contributions to biology.According to Schrödinger, the maintrick of life rested in the capability toproduce order from order, while ordertends to decay towards disorder ininanimate matter, according to the lawof entropy. He concluded thatbiologists had to know more about thegene's structure to understand thesecrets of living organisms. Untiltoday, the elucidation of the structureand function of genes and proteins is acentral and dynamic field of research.

That the chemical nature of genes isdeoxyribonucleic acid (DNA) was

indicated by the experiments of O. T.Avery. Two persons established thephysical structure of genes, i.e. ofDNA, which immediately impliedhow genetic information can bepropagated from one generation to thenext. They were the youngornithologist James Watson andFrancis Crick, a physicist turnedmolecular biologist. Watson and Crickprovide another example that it oftentakes several disciplines and differentlines of thought from separateindividuals for a great discovery. Theybuilt upon important concepts aboutthe nature of the chemical bond fromLinus Pauling, and were aided by theX-ray defraction studies of RosalindFranklin and Maurice Wilkins. Justlike Delbrück and Schrödinger,Watson and Crick concentrated on aproblem, and not on a traditionaldiscipline.

The advent of molecular biology,which came about by this fruitfulinteraction between physicists andbiologists, has lead within less than 50years to the understanding of manyfundamental biological processes. Theeffects of this research wereextraordinarily profound, andstimulated many scientists. Molecularbiology, cell biology and genomeresearch are still strongly influencedby these historical developments. Wenow know in principle how acomplete organism, such as a worm orfly, can emerge from an egg, and thatmany of these mechanisms areemployed even in higher animals andman. We have learned how the cellcontrols growth, and, in parallel, whatare the genetic causes of cancer. Thisknowledge can hopefully be appliedto the treatment of this disease in the

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Figure 2: The famous publication “On theNature of Gene Mutation and Gene Structure”is considered a milestone in the history ofmodern genetics. It was published by the Berlinscientists Nikolai Wladimirovich Timoféeff-Ressovsky, Max Delbrück, and Karl G. Zimmerin 1935.

Figure 1: Max Delbrück (1906 Berlin – 1981Pasadena/USA) in his office at the CaliforniaInstitute of Technology in 1970, shortly afterhaving been awarded the Nobel Prize forPhysiology and Medicine in 1969. Collaboratingclosely with Nikolai Wladimirovich Timoféeff-Ressovsky and Karl G. Zimmer during his stay inBerlin between 1932 and 1937, he is consideredone of the pioneers in the field of moderngenetics and molecular biology, renowned for hiswork on bacterial viruses (phages).

future. Glimpses of the molecularcauses of other important humandiseases, for instance cardiovasculardisorders, are emerging. What is stilllacking is a complete understanding ofcomplex organism in molecular terms.

The complete genome sequences ofvarious viruses, numerous bacteria,yeast, and of the first multicellularorganism, C. elegans, havedramatically demonstrated what liesahead of us. Already, the firstcomplete sequence of a humanchromosome is published; roughdrafts of the sequence of allchromosomes will be availablebetween 2001 and 2003. Quicklythereafter, the complete sequence ofthe human genome will be availableto anyone with an Internet access.This information will create acompletely new biology andmedicine, provided we are able tomake practical use of it. It can lead tonew diagnostic and therapeuticavenues, vaccines, drugs, procedures,and genetic tests. This will also bringalong thorny new questions aboutethics, fairness, and privacy. TheMolecular Medicine and the lifescience have therefore to be providedwith responsibility, an open and publicdialogue and a human touch. Subjectmatter and the research organizationmust respond to this challenge andmust be in harmony.

The future knowledge can only besuccessfully employed in medicine ifwe achieve such a harmony.

We can consider the Human GenomeProject as today’s “Science of Man”program. Weaver’s “mathematicalbiology” anticipated the emergingtheoretical biology and bioinformatics,in which the computer is as importantas the experiment. The “Science ofMan” program was singular in itssuccess because it viewed the scienceof man as interdisciplinary. To use theinformation generated by the HumanGenome Project will again require aninterdisciplinary effort. I firmlybelieve that integrating genomic,genetic and medical research will leadus to a new science of man that wecall Molecular Medicine. MolecularMedicine has great potential and alsogreat responsibilities for bringing thedisciplines together. We are convincedand dedicated to the notion that thisgoal can be achieved. Medicine hasalways benefited from discoveriesmade in physics (X-rays, isotopes),chemistry (reagents andpharmaceuticals), and engineering(biomaterials and instrumentation).Now new bridges must be constructedto link physics, biology, medicine, andother disciplines even closer.

At the MDC, we are determined tocreate an interdisciplinary researchenvironment. These efforts are timelyand well conceived. I am convincedthat they will be of advantage for alland a disappointment for no one. Weare trying our best at the MDC and weare grateful for your support.

Detlev Ganten

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Figure 5: Nikolai Wladimirovich Timoféeff-Ressovsky (1900 Province Kaluga – 1981Obninsk/Russia). He was one of the pioneers inthe field of modern genetics and molecularbiology, working in Berlin-Buch from 1925 to1945.

Figure 4: Erwin Schrödinger’s book ”What islife”, published in 1944, addresses the question,whether life can be explained by the laws ofphysics. Schrödinger worked on a model whichdescribed genes as ”aperiodic crystals”,referring to the famous work ”On the Nature ofGene Mutation and Gene Structure” by NikolaiWladimirovich Timoféeff-Ressovsky, whoworked in Berlin-Buch, Max Delbrück, andKarl G. Zimmer.

Figure 3: Nobel Laureate Erwin Schrödinger(1887 Vienna – 1961 Vienna), renowned for hisstudies on quantum mechanics and inspired byMax Delbrück’s model of the genes as“Atomverband”, successor of Max Planck atthe University of Berlin from 1927 to 1933.

Clinical Research

The collaboration between the MDCand the two universitarian clinicsFranz Volhard Clinic forCardiovascular Diseases and RobertRössle Cancer Clinic (both CharitéMedical School of the HumboldtUniversity of Berlin) Berlin-BuchCampus, has developed in anexceedingly satisfactory manner inrecent years. Annually, the MDCmakes available about 14 million DMfrom its budget for joint researchprojects. The following procedureswere set up very soon after theestablishment of the MDC to allocateMDC research funds to clinics:

1. The four senior professors andphysicians of the Franz VolhardClinic and the Robert Rössle Clinicare simultaneously in charge of aresearch group at the MDC,provided with staff, materials andequipment, investment andlaboratory space to a levelcomparable with that of the MDCresearch groups. This means thatthe clinicians are part of the MDCresearch activities and memberswith equal rights on all committeesof the MDC, including themanagement working party. Inrecent years, two research groupshave also been set up consisting ofclinicians working part-time asguest researchers at the MDC. Thisensures that there is an unhinderedinstitutional link between clinicaland basic research.

2. The improvement in the researchinfrastructure, as well as the settingup of Clinical Research Units(CRU), form the basis ofcollaborative projects under whichparticular diagnostic andtherapeutic procedures can becarried out on patients and

volunteers. This provides a soundbasis for clinical research. TheseClinical Research Units are notinvolved in routine patient care butare exclusively intended fordiagnostic procedures and thescientific investigation of patientsrequired as part of the collaborativeprojects. In this respect, theyrepresent an unusual feature of theresearch structure of a universityclinic.

Examples of the MDC-fundedmeasures include the equipping ofa center for the identification ofgenetic diseases and forestablishing experimental protocolsin the field of circulatory diseasesat the Franz Volhard Clinic as wellas setting up a tumor bank at theRobert Rössle Clinic. This isavailable to all researchers for theirresearch projects and provides auseful service involving future highthroughput procedures based onDNA chip technology to helpidentify altered disease genes.

Another important activity is thesetting up of a GMP (GoodManufacturing Practice) laboratoryat the MDC that will operate incollaboration with Schering AG toproduce drugs to be used in genetherapy.

3. All the research funding which theMDC provides for ClinicalCollaborative Projects (KlinischeKooperationsprojekte, KKP) ismonitored by internal and externalexperts.

The financing of clinical researchfrom MDC funds is basedexclusively on research projects.These projects obtain financialcontributions for research-associated additional expenditure

provided that it involvescollaborative projects between theMDC and the clinics. These KKPare proposed following closeagreement with the coordinationsectors of the MDC and, as far asthe topics are concerned, they areclassified under the researchspecialities of molecular therapy,genetics, cell growth anddifferentiation as well asneurosciences. Currently, 13 suchprojects are underway. A further 6 projects are due to start shortly.In general, they run for 2 – 3 years.The maximum period of funding is5 years.

This allows an important goal to beachieved, namely the application ofresearch methods under quality-controlled conditions and thesupport of collaboration betweenbasic research and the clinics. Inaddition, the two clinics have theopportunity to gain access to third-party funding, independently of theMDC, to help support their clinicalresearch.

4. Clinical Training Program(Klinisches Ausbildungsprogramm,KAP) for young doctors/internal.The fundamental idea behind thisclinical training program is to usetargetted trainee sponsorship ofdoctors who have completed theirclinical training to facilitate theswitch to basic research at theMDC and to lay a foundation fortheir further scientificdevelopment. At present 16 youngdoctors are taking part in thisprogram.

These four sponsorship programsform a substantive unit and have beenset up by means of agreed proceduresand evaluated. This guarantees thatoutstanding clinical research projectscan be supported for specific periodsfrom MDC resources.

Gene Therapy Program

One of the most important scientificand applied areas of cooperationbetween the MDC and the clinicsinvolves the development of strategiesand procedures for gene therapy. Asfar as this topic is concerned, theMDC and the clinics have come to anagreement with other working groupsand institutions in conjunction withthe Union of Clinical PharmacologyBerlin/Brandenburg and obtained

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Introduction

substantial funding from the GermanFederal Ministry for Education andResearch (Bundesministerium fürBildung und Forschung, BMBF) for aperiod of 2 x 4 years.

The Good Manufacturing Practice(GMP) laboratory includes fourlaboratories for vector production andtwo for analysis and cell culture. TheGMP laboratory is operated inconjunction with Schering AG and isalso available for use in collaborativeprojects with other partners e.g.biotechnology companies located onthe Campus.

Patient-oriented Research:Highlights in the Clinics

The Franz Volhard Clinic houses twodepartments, the Department ofCardiology and the Department ofNephrology/Hypertension. RainerDietz and Friedrich C. Luft are therespective heads. The Robert RössleClinic encompasses two departments,the Department of Hematology/Oncology and the Department ofOncological Surgery. Bernd Dörkenand Peter M. Schlag are the respectiveheads. The four chiefs are fully awarethat the goal of the unique cooperationwith the MDC is to help cliniciansformulate better hypotheses to pose atthe bedside, as well as to expedite thetransfer of basic knowledge intoclinical practice.

In the Franz Volhard Clinic twocardiologists, Ludwig Thierfelder andMatthias Friedrich, have teamed up toinvestigate arrhythmogenic rightventricular cardiomyopathy (ARVC).Usually, the cause for the responsiblecardiac arrhythmia is never identified.ARVC, which is inherited both as adominant and recessive trait, isresponsible for sudden death in youngpeople. The condition may be morecommon than believed because ofnew diagnostic tools that can beapplied before symptoms (fatalventricular arrhythmias) develop.With the help of genetic fieldworking, entire families can bestudied to identify asymptomaticindividuals. Such persons can begiven medication prophylactically ortreated with implantable defibrillators.In the Franz Volhard Clinic, JensJordan and other clinicians arestudying mechanisms of high and lowblood pressure. The group is studyinga rare form of monogenichypertension with brachydactyly.

Affected persons also feature as anadditional phenotype, an aberrantlooping posterior-inferior cerebellarartery, that impinges on theventerolateral medulla. This so-calledPICA loop could interfere withbaroreceptor reflex signaling. SimilarPICA loops are commonly observedin patients with primary essentialhypertension, raising the hypothesisthat a new, hitherto unrecognized formof secondary hypertension exists. Thegroup has also identified a defect in anorepinephrine transporter thatinterferes with sympathetic function.

In the Robert Rössle Clinic, clinicalscientists are working to offer patientswith renal cell carcinoma, a commonbut untreatable malignancy, a betteroutlook. A cell line that expresses atumor antigen recognized by T cellsfrom most HLA-A2 positive patientswith renal cell carcinoma has beengenetically modified in cooperationwith Thomas Blankenstein at theMDC and Dolores Schendel from theGSF in Munich. The geneticallyengineered cancer cell line expressescostimulatory molecules andinterleukin 7 and can effectivelyactivate cytotoxic T lymphocytes. Itwill be possible to overcome what hasbeen a major limitation in cancervaccines thus far; namely, thedifficulty of monitoring the ongoingimmune response in order to designpowerful vaccine schedules.

A further approach that utilizes theopportunities offered by recombinanttechnology is the generation ofbispecific-single chain antibodies.Such constructs can target Tlymphocytes to cells that express atumor-associated or a tissue-specificantigen recognized by monoclonal

antibodies. In collaboration with GertRiethmüller (Dept of Immunology,Univ. of Munich), Robert Rösslehematologists are planning a phase Istudy for the treatment of lymphomapatients.

The surgical oncologists at the RobertRössle clinic are working onimproving the survival of cancerpatients by improving both diagnosisand treatment. Recently 3 dimensionalultrasonography has been developed topermit characterization of the tumorextension far better than can beappreciated by the surgeons visualskills. Numerous treatment strategiesare being employed to optimizeoutcomes. Combined pretreatmentapproaches including hyperthermia arecurrently under clinical investigationfor esophageal and rectal tumors aswell as for soft-tissue sarcomas. In thetreatment of sarcomas and melanomasof the extremities, a surgical limbisolation provides for highly efficienthyperthermic combination therapies asa sole curative procedure or withcombined neoadjuvant interventions.Cytokines, such as tumor necrosisfactor are also being tested as adjuvanttherapies. Finally, in the framework ofthe OP 2000 initiative, we are makingsweeping changes in the surgeon’swork place. Digital data processing,the fusion of information frommultiple simultaneous sources,comprehensive virtual presentation ofcomplex surgical data sets, robotics,and interactive consultations withcolleagues outside the operating roomwill become routine.

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Figure 6: Concept of the Berlin-Buch Campus:basic research (MDC), clinical application(RRK, FVK) and commercial (BBB) use are realized in close collaboration.

Berlin - Buch Campus

RRK

FVK

ROBERT RÖSSLECANCER CLINICBERLIN-BUCH

FRANZ VOLHARDCARDIOVASCULAR CLINICBERLIN-BUCH

MAX DELBRÜCK CENTERFOR MOLECULAR MEDICINEBERLIN-BUCH

BIOMEDICALRESEARCH CAMPUSBERLIN-BUCH GmbH

Charité, Humbold University Berlin

MDC BBB

Patient Care

Researchand Teaching

Researchand Teaching

TechnologyTransfer

Researchand Teaching

Genome Research in Berlin-Brandenburg

The Berlin-Brandenburg region isoutstanding as far as German genomeresearch is concerned. Berlin-Brandenburg is deeply involved inboth the Human Genome Project aswell as the Plant Genome Project andthe genome-related BMBF (GermanFederal Ministry of Education andResearch) Priority Projects. Of all thefunding made available for theGerman Human Genome Projectalmost a third was won by the Berlinregion. Most of the key units formingpart of the infrastructure of genomeresearch are to be found in the region:the Resource Centre in Berlin-Charlottenburg, which plays a keyrole in both fields, the Gene MappingCentre of the German HumanGenome Project (DeutschesHumangenomprojekt, DHGP) at theMDC as well as the Max PlanckInstitute of Molecular PlantPhysiology in Golm. The Berlin-Potsdam area has one of the highestconcentrations of biotechnologycompanies which are growing at afaster rate than any other sector ofindustry.

In order to safeguard and expand thelocal potential and expertise of theregion and to strengthen itscompetitive position with respect torival regions (particularly Munich,Heidelberg, and Cologne), it has beensuggested that there should be a hugeexpansion of genome research in theBerlin-Brandenburg region.

The establishment of a Berlin-Brandenburg Centre for GenomeResearch will offer the following: • coordinated activities will reinforce

existing activities and promote asynergistic effect

• Berlin-Brandenburg will become aleader in German genome research

• the establishment of innovativecompanies will be accelerated

• the development of products withcommercial potential will bespeeded up

• there will be a sound foundationwhich will encourage talentedyoung researchers to remain in theBerlin-Brandenburg region ratherthan lose them to regions with astronger industrial base.

In 1999 a proposal for expansion wasmade by senior researchers at theMDC and with its support. The planinvolves a laboratory for medical andfunctional genome research (whichwill have to be built) to complementthe following areas: genetics andbioinformatics, functional genetics,and genetics and therapy.

Biotechnology Park

In accordance with therecommendations of the ScienceCouncil, the MDC has set up aBiotechnology Park on the Berlin-Buch Campus. In 1995, the MDC alsoestablished an offspring facility, theBiomedical Research Campus Berlin-Buch (BBB) GmbH. Co-partnersinclude Schering AG and theForschungsinstitut für MolekularePharmakologie (FMP), both of whichhave a 20 per cent share. Its task is todevelop the Berlin-Campus and attractBiotech companies to settle in Buch.

So far, over 30 biotech companieswith more than 400 employees havecome to the Berlin-Buch Campus towork closely with scientists from theMDC and clinicians from the twouniversity affiliated clinics – RobertRössle Cancer Clinic and FranzVolhard Clinic for CardiovascularDiseases.

In September 1998, the BBB GmbHopened a new Biotechnology andBusiness Development Center on theBerlin-Buch Campus. It was set upwith approximately DM 30 millionfrom the Common Mission for theImprovement of Regional Economics(GA) and the European Fund forRegional Development (German abbr.EFRE). In December 1999, the BBBGmbH laid foundations for a secondnew laboratory building. A productionfacility will also be built. Bothbuildings have received 19 millionDM in support from the GA andEFRE. In addition, due to the greatdemand for space from additionalentrepreneurs, a third new building forthe Development Center is undernegotiation with the SenateAdministration for Business andEconomy.

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Figure 8: Inauguration of the BBB’sBiotechnology and Business DevelopmentCenter for start-up companies on the Berlin-Buch Campus on September 8, 1998. From leftto right: Jürgen Rüttgers, the former FederalResearch Secretary, Gudrun Erzgräber,Business Director of the BBB BiomedicalResearch Campus Berlin-Buch GmbH,Eberhard Diepgen, the Mayor of Berlin, andPeter Strieder, a member of the Berlin Senate,looking at the bust of Erwin Negelein, theBerlin-Buch scientist after whom the newbuilding has been named. The bronze bust wasthe work of the Berlin sculptress SabinaGrzimek.

Figure 7: Expansion planned for the Berlin-Buch Campus, showing (in blue) theCommunications Center and the GenomeResearch Center.

The Helmholtz Association

The MDC is one of 16 researchinstitutions belonging to the Hermannvon Helmholtz-Association ofNational Research Centres. Theypursue long-term governmentalresearch goals with completescientific autonomy. The concentrationof considerable financial and staffresources, a sophisticated scientificand technical infrastructure, andefficient management enable theResearch Centres to deal withcomplex scientific and technicalissues and interdisciplinary projects,to operate large-scale scientific andtechnical equipment and also todevelop system solutions.

The Helmholtz Centres performscientific and technical as well asbiological and medical research anddevelopment. In the next few years,the health research activities of theHelmholtz Association will becomemore important and will concentrateon the great scientific and therapeuticchallenges offered by medicine. Inspite of the great advances that havebeen made in medicine, in only abouta third of all known human diseasescan the cause actually be treated,while the remaining two thirds receiveonly symptomatic treatment or cannotbe treated at all. The key areas ofcompetence of members of theHelmholtz Association in basicbiomedical research, clinical research(in collaboration with external partners),medical technology and “public health”research is an absolutely crucialprecondition for establishing a majorscientific network which will functioneffectively in an interdisciplinarymanner in the long-term.

The centres of the HelmholtzAssociation which are activelyengaged in research into health set upa scientific network in 1998, “HealthResearch Group” in order to develop ascientific network aimed atcoordinating all the health-orientedresearch carried out by the HelmholtzAssociation. In addition, via itsrepresentatives on scientific andpolitico-research committees, thegroup will actively play a part indecisions involving all health mattersof scientific and political importance,even acting as an advisor whenrequired. The Health Research Groupis also an umbrella organization forthe Clinical-Biological ResearchGroup (Klinisch-BiomedizinischerVerbund, KBF), which covers in thecontext of the Helmholtz Associationclinics as well as university and non-university institutions such that thekey areas of interest of the KBFGroup are much more orientedtowards clinically important topics.

In the next few years all the followingkey areas will be investigated:• biomedical basic research into gene

regulation, cell biology,transcription control, cell-cellinteraction and virus-cellinteractions

• functional genome and proteomeanalysis, particularly by developingDNA-chip technology and protein-chip technology.

• bioinformatics as the basis for thedevelopment of a new form oftheoretical biology.

• tumor immunology, protectionfrom infection, gene therapy andnew vaccination strategies.

• development of preclinical animalmodels

The three Helmholtz Centres, theGerman Cancer Research Center(DKFZ, Heidelberg), GSF-NationalResearch Center for Environment andHealth (Neuherberg) and MDC, due totheir collective expertise play a keyrole as local sites of competence as faras the German Human GenomeProject is concerned. In allbiomedically oriented HelmholtzCentres, structures will also bedeveloped to improve collaboration inclinical research, since this is the onlyway of effectively testing the newapproaches to therapy that aredeveloped in the laboratory.

The Helmholtz Centres receive 90 percent of their basic funding from theFederal Government and 10 per centfrom the respective host state. Theyhave a total budget of about 4 billionDM and a staff of roughly 22,000.

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Figure 9: One of the techniques students learnin the ”Hands-on Laboratory” is how to isolatethe genetic material DNA from tomatoes. Thelaboratory was opened to the public on theBerlin-Buch Campus in April 1999.

External Evaluation

Over the period November 1996 toApril 1998 three external assessmentwere carried out at the MDC. In April1998, under the chairmanship of Prof.Pasko Rakic, New Haven, USA, anassessment was carried out of theStructural Biology, Genetics andNeuroscience research groups. Thesethree assessments meant that all thekey research areas of the MDC weresubject to external evaluation.

All three assessments were carried outin accordance with the same basicprinciples as used for the specialresearch areas of the DFG. Thecommission of assessors visited theMDC for a two-day period duringwhich they had the opportunity to getto know the researchers during aseries of presentations made by thelatter.

Following the “site visit”, thecommission of assessors prepared areport in which they evaluated indetail, among other things, thecooperation between the basicresearchers of the MDC and theircounterparts in the Robert RössleClinic and the Franz Volhard Clinic.

Based on the recommendations of theassessors, the MDC adopted andimplemented a series of measures. Forexample, there was a change in theway the budget was allocated and along-term allocation was made for thegroups assessed and the organizationalstructure was modified to target thelatest key areas of scientific interest. Afurther example of the changesimplemented was the establishment ofa service “Career Guidance forTrainee Scientists” at the MDC. Theseand other measures were discussed atlength by the Scientific Committee ofthe Board of Trustees and the resultsof these measures are regularlymonitored by means of a check list.

Graduate Student Education – Dean of Graduate Students

The support and structure of graduatestudent education at the MDC is ofextreme importance. We haveestablished a graduate program toaccommodate highly qualifiedcandidates and prepare them forcareers in scientific research. Theprogram provides training andresearch opportunities at the highestlevel within existing resources of theMDC. Ph.D./M.D. students participatein lectures and seminars held at theMDC and receive a broad training inthe biomedical sciences. A strongstudent-advisor relationship isessential for outstanding academicperformance and is the basis forturning students into independent andcreative researchers.

The approximately 120 MDCgraduate students elect representativeswho negotiate graduate student affairswith the institute and interact closelywith the elected dean of graduatestudents. In addition, the studentrepresentatives organize scientific andsocial meetings for their fellowgraduate students such as thetraditional annual StudentSymposium.

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Figure 10: The winners of the Max Delbrückscholarship 1997, Anja Plewinsky (2nd fromleft) and Judith Kreutzberg (3rd from left),being congratulated by Detlev Ganten,Scientific Director of the MDC (on the right),and Hans-Jürgen Delbrück from the DelbrückFamily Foundation (on the left) on the occasionof the MDC’s New Year’s Reception on January19, 1998.

Scientific Journals

The MDC is the site of severaleditorial offices.

Journal of Molecular Medicine(JMM)

The Journal of Molecular Medicine(JMM) is published monthly bySpringer Verlag Heidelberg since1995. The focus is in molecularmedicine, a field which applies themethods and knowledge of molecularbiology and gene technology tomedical research, therapy, and diseaseprevention. JMM’s goal is to bringtogether basic science and clinicalmedicine in the field of molecular andgene technology research which hasbecome particularly important for theprogess of medicine in all aspects.The journal’s editor-in-chief is DetlevGanten, Scientific Director of theMDC.

Neuroforum

Neuroforum is the Newsletter of theGerman Neuroscience Society(NeurowissenschaftlicheGesellschaft). The journal wasfounded in 1994. Neuroforum givesan overview of the activities in thefield of neuroscience research inGermany. The journal publishesreview articles covering all aspects ofneuroscience research. Besides that,Neuroforum publishes articles on keypeople involved in the history of theneurosciences, meeting reports,methodological aspects, book reviews,opinions, portraits of industrialresearch institutions, information oneducational and research programs, and news from the GermanNeuroscience Society. The journal ispublished by Spektrum AkademischerVerlag, Heidelberg, Germany. Theeditor-in-chief is Helmut Kettenmann.

Glia

Glia, founded in 1988, provides adedicated forum for a broad range ofexperimental topics in the field ofglial research and is an indispensiblemedium for scientific exchangesamong researchers interested inneuroglial research. Original articles,short communications, review articlesand Special Issues on the physiology,anatomy, pharmacology, chemistry,and pathology of glia are published.The publisher of this journal isWILEY-LISS, New York, USA,editors-in-chief are Bruce Ransom andHelmut Kettenmann.

International CollaborationsChinese-German MicrosatelliteCenter in Beijing

The Chinese Academy of MedicalSciences (CAMS) and the MDC willopen a joint gene mappingmicrosatellite center, which iscurrently being set up at the CAMSFu Wai Hospital. The aim is toidentify genes involved in the onset ofcardiovascular diseases. The settingup of this microsatellite center isbeing financed by the German FederalMinistry for Education and Research.Collaborators also include cliniciansfrom the Franz Volhard Clinic forCardiovascular Diseases, Charité,Berlin-Buch Campus, and researchersfrom Hoffmann-La Roche, Basel(Schweiz).

The “Verein der Freunde undFörderer” of the MDC supports thisinitiative with stipends for visitingscientists. For further information onthis association contact Michaela Hen-selmann (mhensel@mdc-berlin.de).

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Figure 11: Welcome to Zhou Guangzhao, Vice-President of the National People’s Congress ofthe People’s Republic of China (in the middle),and his wife, by Detlev Ganten, ScientificDirector of the MDC (on the left), on July 9,1999.

Congresses

In the years reported, two majorconferences took place in Berlinwhich were organized by scientistsfrom the MDC.

6th International Gene TherapySymposium in Berlin-Buch

Approximately 250 scientists from theUnited States, Canada, France, GreatBritain, Austria, Switzerland, Israeland Germany attended the 6th GeneTherapy Symposium of the MDC onMay 4-6, 1998, in Berlin-Buch. Thefocus that year, under the title“Towards Gene Therapeutics”, wasbasic research. New insights andknowledge arising from thedevelopment of so-called “genevectors” and their target organs werekey points of interest.

In addition to the MDC, the MedicalBiotechnology Research Group of the German Society for ChemicalInstruments, Technology andBiotechnology (DECHEMA) co-organized the meeting. TheSymposium was accompanied by anexhibition of biotech companies. Itwas the last gene therapy symposiumorganized by the late Michael Strausswho initiated this series ofconferences in 1993.

1998 Forum of EuropeanNeuroscience

The 1998 Forum of EuropeanNeuroscience was organised by theEuropean Neuroscience Association(ENA) in collaboration with theGerman Neuroscience Society inBerlin from June 27 - July 1, 1998.

4,000 attended the meeting. The goalof the meeting was to integrate allNational Neuroscience Societies andmany European MonodisciplinarySocieties and Groups. All majornational neuroscience societies agreednot to hold their annual nationalmeeting in 1998, and to support theForum meeting. On the occasion ofthe 1998 Forum Meeting, FENS(Federation of European NeuroscienceSocieties) was founded. HelmutKettenmann is the treasurer of FENS.

Academic Appointments

Twenty-seven group leaders havebeen appointed to the MDC since itsfoundation in 1992, five of whomwere appointed in the years 1998 and1999. The five group leaders that havejoined the MDC during the last twoyears reported are André Reis (1998),Zoltán Ivics, Manfred Gossen, KirstenFalk and Olaf Rötzschke (1999).

Manfred Gossen

Biologist Manfred Gossen wasappointed group leader at the MDCfor five years in 1999. He heads theresearch group “Control of DNA-Replication” within the researchprogram “Cell Growth andDifferentiation”. Manfred Gossen wasborn in Siegburg, Germany. From1982 – 1987 he studied Biology at theuniversities of Bonn and Heidelberg.He gained his doctorate at theZentrum für Molekularbiologie derUniversität Heidelberg (ZMBH) in1993. He joined the University ofCalifornia in Berkeley, USA, as apost-doc in 1994 for five years beforecoming to the MDC.

Zoltán Ivics

The biologist and geneticist, ZoltánIvics, was appointed group leader atthe MDC for a period of 5 years insummer 1999. His field of expertiseinvolves transposable DNA elements,transposons, that can be found in thegenomes of most living organisms,from bacteria to humans. Zoltán Ivicsis a native of Budapest (Hungary) andstarted his biological studies there in1988. He studied at the University ofMinnesota (USA) in 1991 and gainedhis doctorate at the University ofAgricultural Sciences, Gödöllô,Hungary, in 1994. He continued as apost-doc in Minnesota until 1997. Hethen joined the Netherlands CancerInstitute in Amsterdam for two yearsbefore coming to Berlin-Buch.

Kirsten Falk and Olaf Rötzschke

Kirsten Falk and Olaf Rötzschke havebeen appointed joint leaders of thegroup “Cellular Immunology” for aperiod of 5 years. Kirsten Falk, bornin Bremen, and Olaf Rötzschke, bornin Wiesbaden, studied Biochemistry atthe Universities of Hannover andTübingen. They first worked at the

Max-Planck-Institute for Biology,Department of Immunogenetics, inTübingen and gained their doctoratein 1993. Since then, they werepostdoctoral fellows at HavardUniversity, Department of Molecularand Cellular Biology, and started workat the MDC in 2000.

Hermann Haller, Franz-Volhard-Clinicfor Cardiovascular Diseases of theCharité Medical School of theHumboldt University of Berlin andMDC, has accepted a professorship“Nephrology” at the MedizinischeHochschule Hannover and startedwork there in August 1999. LudwigThierfelder (Franz Volhard Clinic andMDC), accepted a professorship for“Genetics in Cardiology” at theCharité in February 1999.

Ingo Morano accepted a professorshipat the Charité in “Molecular MusclePhysiology”. Thomas Sommer, whosefocus of research is the ubiquitinsystem and the endoplasmaticreticulum, accepted a position at theMDC. Teymuras Kurzchalia accepteda position at the Max Planck Instituteof Molecular Cell Biology andGenetics in Dresden. BurkhardMicheel has become professor forBiotechnology at the University ofPotsdam. Dieter Riethmacher becamegroup leader at the Center forMolecular Neurobiology (Zentrum fürMolekulare Neurobiologie derUniversität Hamburg ZMNH) at theUniversity of Hamburg.

Geneticist André Reis, head of MDC’sgene mapping microsatellite center,has been offered a professorship at theUniversity Erlangen-Nürnberg.Thomas Blankenstein (MDC) hasbeen offered a professorship in“Immunology and Gene Therapy” atthe University of Hamburg. JürgenBehrens has been offered aprofessorship in “ExperimentalMedicine” at the University ofErlangen-Nürnberg and ReinholdFörster, in “Experimental Surgery”also at the University of Erlangen-Nürnberg. Stefan Schumacher hasbeen offered a junior group position atthe University of Hamburg.

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Obituary Michael Strauss

The staff of the MDC mourn thetragic death of Michael Strauss. Thisinternationally renowned cell biologistdied on April 29th, 1999, aged 49,after a severe illness. MDC not onlylost a brilliant scientist, whocombined a deep commitment with abreadth of vision, but also a dear andtreasured colleague.

Michael Strauss was born in Berlin onJanuary 12th, 1950. He studiedbiology at the Humboldt University,Berlin, gaining his doctorate in 1977and his post-doctoral lecturingqualification (Habilitation) in 1987.From 1981 to 1989, he led a researchgroup at Berlin-Buch and spentseveral periods abroad carrying outresearch in Great Britain and theUSA. This included work at theImperial Cancer Research Fund andRoyal Postgraduate Medical School(both in London) as well as ColdSpring Harbor Laboratory, New York(USA).

In 1992, Michael Strauss started a fiveyear period as leader of a researchgroup belonging to the Max PlanckGesellschaft at the HumboldtUniversity, Berlin, which was based atthe MDC. In 1993, he became head ofan international research group at theDanish Cancer Society in Copenhagenand, in 1994, he was invited tobecome Professor of Molecular CellBiology at the Humboldt University.

His main research interests includedthe regulation of cell division, thefunction of tumor-suppressor genes,developing gene therapy for cancerand genetically regulated conditionsas well as developing viral vectors fordelivering gene therapy. MichaelStrauss and his collaborators inBerlin, Copenhagen and Londondiscovered a mechanism whichcontrols cell replication and whichmalfunctions in virtually all cancercells. Using this finding, he and hiscolleagues developed a new approachto combat malignant diseases usinggene technology. He quickly

transferred this knowledge from thelaboratory bench to goodmanufacturing practice: he held about25 patents and, in 1996, he set up agene therapy company (HepaVec).

Michael Strauss received a number ofhonours including the Fichte Prizefrom the Humboldt University in 1971and the Virchow Prize (from the GDRMinistry of Health) in 1984. Inaddition, Michael Strauss receivedfellowships from the EuropeanMolecular Biology Organization(EMBO), the Union InternationaleContre le Cancer (UICC) and theImperial Cancer Research Fund(ICRF).

Michael Strauss was a member ofmany professional societies, both athome and abroad, including theAmerican Society for Gene Therapyand the European Working Group onGene Transfer (EWGT). In addition,he was a member of the ScientificAdvisory Council of the FederalMedical Council for Somatic GeneTherapy and chairman of the“Medical Biotechnology” workinggroup of the German Society forChemical Instrumentation, ChemicalTechniques and Biotechnology(DECHEMA).

Michael Strauss published over 80research papers as well as about 30reviews and book chapters. In 1993,he set up the international genetherapy symposia at Berlin-Buch andhe was involved running them eversince; the last one took place in 1998.

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Figure 12: The late Michael Strauss, researchgroup leader at the MDC, in his laboratory.

Awards

A number of prestigious prizes havebeen awarded to scientists of theMDC and clinicians of thecollaborating university affiliatedRobert Rössle Cancer and FranzVolhard Clinic for CardiovascularDiseases in 1998 and 1999.

Heinrich-Wieland Prize awardedto Thomas Willnow

Thomas Willnow, a Heisenbergscholar and research group leader atthe MDC, has discovered one of thecauses which lies behind themetabolic defect of a severe renaldisease called Fanconi’s Syndrome.He has been investigating a bindingsite (receptor) on the kidneys thefunction of which had remainedunknown until recently. He was ableto show that this receptor acted as asort of collecting point for the vitaminD filtered from the kidneys. Before itis removed, the receptor, known asmegalin (gr. mega = large), interceptsthe vitamin D and returns it back tocirculation via the cells which coat therenal tubules (epithelial cells). Now,scientists know why patients withrenal disease have bone defects. Forhis research Thomas Willnow wasawarded the Heinrich Wieland Prizeworth 50.000 DM in Munich inOctober 1998.

Deutscher Krebspreis awardedto Walter Birchmeier and PeterM. Schlag

Monika-Kutzner-Preis derBerlin-BrandenburgischenAkademie der Wissenschaftenawarded to Jürgen Behrens

For their research on the developmentof cancer and metastases, WalterBirchmeier and Jürgen Behrens havebeen awarded the DeutscheKrebspreis 1999, and the Monika-Kutzner-Preis der Berlin-Brandenburgischen Akademie derWissenschaften, respectivly. WalterBirchmeier shares the Krebspreisworth 30,000 DM with Peter M.Schlag, surgeon at the Robert-Rössle-Cancer Clinic (Charité, Berlin-BuchCampus). Peter Schlag has beenhonoured for the development of newtherapies against intestinal cancer andsoft tissue sarcomas. They receivedtheir award from the DeutscheKrebsgesellschaft in Heidelberg in

March 1999, Jürgens Behrensreceived his prize worth 20,000 DMin June 1999 in Berlin.

Galenus-von-Pergamon-Preis1999 awarded to Volhardclinicians and an MDC scientist

For their research on hypertension inpregnancy, Friedrich Luft, HermannHaller, Volker Homuth (Franz VolhardClinic, Charité, Berlin-Buch Campusand MDC) and Gerd Wallukat (MDC)received the Galenus-von-Pergamon-Prize 1999 (worth 25,000 DM) inNovember 1999 in Düsseldorf.Hypertension is one of the mostcommon complications of pregancy. Itcan interfere with the development ofthe fetus and can lead to prematuretermination of the pregnancy.Hypertension due to pregnancy isaccompanied with the excretion ofalbumin in the urine after the 20thweek. The reasons for this condition,known as pre-eclampsia or gestosis(lat. gestare – to carry), are unknown.During their research the cliniciansfound clues as to the cause of thisserious condition. In a collaborativeeffort with Gerd Wallukat (MDC),they discovered autoantibodies in theblood of pregnant women sufferingfrom pre-eclampsia. Theseautoantibodies bind to the receptorsfor Angiotensin II, a hormone thatplays a role in regulating bloodpressure. This activates the receptorsand leads to an increase in bloodpressure. It is not yet known whatcauses the formation of these

autoantibodies. The clinicians suspectthat the trigger for this comes fromthe fetus since after delivery there isno longer any sign of theseautoantibodies in the blood of womenwho had pre-eclampsia.

InnovationspreisBerlin/Brandenburg awarded toDr. Regina Reszka

Regina Reszka from G.O.T. GmbH &Co. KG and the MDC was awarded theInnovationspreis Berlin/Brandenburg1998 worth 100,000 DM shared withthree other companies in Novemberthat year. She received the prize forthe development of a “universally”applicable non-viral gene transfersystem (a cationic derivative ofcholesterol, DAE-Chol liposomes).This system is intended to be used forthe gene therapy of cancer, especiallybrain – and liver tumors, and againststenoses. It has been developed byDetlef Groth, Jana Richter and IngridBerger (all MDC) of Regina Reszka`sgroup.

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Figure 13: Lothar Weißbach, President of theGerman Cancer Society (on the right), handingover the ”Deutsche Krebspreis 1999” to WalterBirchmeier (MDC) and Peter M. Schlag(Robert Rössle Cancer Clinic, Charité, Berlin-Buch Campus, and MDC) in Heidelberg onMarch 24, 1999.

InnoRegio-Competition: Berlin-Buch reached the final round

In the competition announced by theFederal Ministry for Education andResearch (Bundesministerium fürBildung und Forschung, BMBF) forstructurally less developed regions inthe new German states, theInnoRegio, the GesundheitsregionBerlin-Buch e.V. reached the finalround. In November 1999, anindependent jury in Berlin chose fromthe 444 regions that had entered atotal of 25 for this round and theBerlin-Buch health region was one ofthese. The winners have eight monthsto develop their ideas into workableprojects and each of these regions willget up to 300 000 DM from theBMBF. The final decision will bemade in Summer 2000 and theprojects will be implemented beforethe end of 2005. The BMBF willprovide 500 million DM in funding for this. The “GesundheitsregionBerlin-Buch e.V.” hopes that thisInnoRegio programme will providetheir region with additional fundingfor the areas of Berlin-Buch, Karow,Blankenburg, and Heinersdorf as wellas the Panketal Office inBrandenburg. In the next few yearsthere will be a “future-oriented” totalinvestment of almost a billion DM tohelp allow the region to become anattractive site for all aspects ofhealthcare on scientific, economic,artistic, and cultural grounds as wellas being a provider of health-associated services. The aim is to

attract jobs to the region and generatenew employment opportunities. Agroup was set up to pursue this goal inJuly 1999 consisting of clinicians*,researchers from the MDC and theForschungsinstitut für MolekularePharmakologie (FMP), biotechcompanies on the Berlin-BuchCampus, teachers, self-help groupsand socio-cultural bodies as well asthe Academy of Arts in Berlin-Buchand the Barnim district. The group ischaired by Jens Reinwardt, managerand head of the School for HealthProfessions e.V.

*Representatives of the Berlin-Buch clinics,including the city, private sector, universitiesand other public bodies.

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Figure 14: Edelgard Bulmahn (3rd from right,front row), Secretary for Science andEducation, congratulates the Berlin-Buch“InnoRegio” team on its success in theInnoRegio Competition set up by the FederalMinistry to develop East German regions.

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Genetics, Bioinformatics and Structural Biology

Research in molecular genetics iscurrently undergoing a fundamentalchange triggered by the rapid progressof genome sequencing projects,notably the Human Genome Project.Landmark events such as thecompletion of the human chromosome22 sequence or the announcement of afirst working draft of the humangenome sequence by the Spring of2000 testify to the pace and vigor ofthis collaborative international project.At the same time, concepts for sciencein the post-genome era are gainingclearer contours. These concepts aredirected towards obtaining acomprehensive and general view ofprocesses including protein synthesisand processing, signal transductionfrom the cell surface to the nucleus,cell differentiation, or thedevelopment and function of entireorgans. These topics are oftensummarized under the term functionalgenomics. Proteomics, aiming atestablishing the complete cellularprotein inventory in a definedphysiological or pathophysiologicalstate, or structural genomics, an effortto map the entire protein “universe” atthe level of protein domain folds, areimportant components of functionalgenomics. MDC scientists are awareof the relevance of genome researchand functional genomics to molecularmedicine. We have already taken thefirst initial steps and are determined tomove into these fields in the nearfuture.

The research program Genetics,Bioinformatics, and StructuralBiology combines groups active invery different areas of research frompatient-oriented genetics, genetic fieldworking and genotyping projects toexperimental and theoretical studies ofmacromolecular folding and structuralcharacteristics. The connecting themeof these research endeavors is that

together they provide the coreexpertise for research into functionalgenomics. Crucial methodologicalapproaches in that context includeanimal models for studying genefunction and disease pathways, aswell as bioinformatics to evaluategenomic sequences and disease-related genetic variations. Part of theresearch in structural biology isdevoted to pilot projects in the areasof proteomics and structuralgenomics.

Important insights into thepathogenesis of human disorders canbe obtained by studying animal modelsystems. In hypertension and vascularresearch, multiple non-transgenic andtransgenic rodent strains offer theopportunity to identify causative ormodifying genetic components ofvarious disease phenotypes. DetlevGanten’s group is spearheading effortsat MDC to generate rat genome tools(genetic and physical maps, genomicand cDNA libraries etc.) necessary forthe identification of geneticabnormalities in the rat. The generationand molecular characterization ofcongenic rat strains should ultimatelyallow fine mapping and identificationof complex genetic traits.

Three groups in the program employmice as model organisms to study thefunction of genes, using targetedmutations, conditional mutations orother YAC transgenic technologies.

Thomas E. Willnow is analyzing thelow density lipoprotein (LDL)receptor gene family and the role ofthese gene products in the physiologyand pathophysiology of lipidmetabolism and other disorders. Bygenerating mice that lack the gene forthe LDL receptor, the LDL receptor-related protein (LRP) or both, hisgroup has shown that the clearance of

dietary lipids proceeds via a duallipoprotein receptor system, the LDLreceptor and LRP. Megalin, anothermember of the LDL receptor genefamily, has been found to act as anendocytic receptor for the uptake oflipophilic vitamins and regulatestransport and renal conversion ofvitamin D3 metabolites.

Andreas Schedl is analyzing theWilms’ tumor gene (WT1) that, whenmutated, causes Wilms’ tumor, acommon childhood malignancy, andthe Frasier and Denys-DrashSyndromes, which are characterizedby abnormal gonadal development.WT1 plays a crucial role in renaldevelopment. Following analysis ofYAC transgenic mice, WT1 has beenshown to be required continuouslyduring nephrogenesis, in particular,during the formation of matureglomeruli.

Carmen Birchmeier’s group has foundthat the EGF-like factor, neuregulinand its receptor, erbB2, play a dualrole during the expansion of theSchwann cell precursor pool andduring myelination. Moreover, thegroup has elucidated the function ofcryptic, another EGF-like factor, thathas been found to be essential forestablishing the left-right axis. Crypticmutant mice display laterality defects,such as malposition of the greatarteries, right isomerism of the lungand splenic hypoplasia. Thisphenotype is reminiscent of theasplenic syndrome in humans that istypically associated with lateralitydefects.

Further development of miniaturizedtechnology for a more detailedcharacterization of whole animals,isolated organs, and functional unitswill be required for a betterunderstanding of pathophysiologicaldisease pathways in rodent models.Friedrich Luft’s group has developedsophisticated tools to study rodentmodels in vivo. His group, as well asthe groups of Michael Bader andLudwig Thierfelder have applied thesetools to the characterization of rodentswith various genetic cardiovascularmodifications.

Although rodents and other animalscan be extremely useful in elucidatingdisease pathways, the ultimate modelsystem for human diseases is man.Great progress has been made inrecent years in the molecularcharacterization of single gene

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Genetics, Bioinformatics and Structural Biology

disorders. Various MDC groups havecontributed in the elucidation ofmonogenic diseases, including AndréReis and Herbert Schuster. Thecurrent scientific challenge is todelineate the genetic components ofcomplex traits. Extensive genetic fieldworking facilities established byHerbert Schuster, a gene mappingcenter with a capacity to generate2,000,000 genotypes per day led byAndré Reis, and the multiplexsequencing technology for theidentification and typing of singlenucleotide polymorphisms,established by Margret Hoehe, willprovide a framework for thecharacterization of complex geneticdiseases in humans.

The group of Siegfried Scherneck isstudying genetic susceptibility factorsfor breast cancer. His group hasanalysed the genes of Germanfamilies that have a high risk ofdeveloping breast cancer. Themajority of the families investigatedcarry mutations in known cancersusceptibility genes. However, everyforth family studied does not carrythese mutations and a novel breastcancer susceptibility gene has beenlocated on chromosome 8. Moreover,the group participates in a nationwideprogram initiated and supported bythe “Deutsche Krebshilfe”. Thisendeavor offers women options forrisk calculations, genetic counselingand provides clinical andpsychological support.

Important links between the geneticsand structural biology components ofthe program, as well as betweenMDC-based basic science and patient-oriented research at the clinics, areprovided by the Bioinformatics Unit.At present, the group operates at twosites with a genetics section in Berlin(Jens Reich) and a biocomputingsection at EMBL, Heidelberg, wherePeer Bork is currently a visitingscientist. Combining both approaches,the bioinformatics unit is evaluatingvariations in the human genome thatare relevant to the diseases treated andinvestigated at the Franz VolhardClinic (lipoprotein disorders,arteriosclerosis and hypertension, withFriedrich Luft) and at the RobertRössle Clinic (tumor and pertinentnormal tissue, with Peter M. Schlag).

The Structural Biology groups ofMDC use a wide range ofexperimental techniques to studyproblems of protein and nucleic-acid

structure, folding, dynamics andfunction. Together with complementarymethods offered at the Forschungs-institut für Molekulare Pharmakologie(FMP), they provide the Buch Campuswith expertise to address nearly allaspects of structural biology relevantto medicine and pharmacology. In the Berlin Brandenburg area, thesegroups are integrated in and provideleadership for the KoordinationszentrumStrukturforschung (KoSt), anorganization supported by the Senateof Berlin. The aim of KoSt is tocoordinate structural analyses of awide range of objects, frombiomolecules to shapes, surfaces andtextures important in the materialssciences. Responding to thechallenges posed by the internationalgenome programs, a Berlin-basedinitiative has begun to set up astructural genomics infrastructure forthe high-throughput structure analysisof proteins following the sequencingof their genes or cDNAs within theGerman human genome project. Thisinitiative, the “Proteinstrukturfabrik”,is coordinated at MDC and funded bythe BMBF.

Within the structural biology programof MDC, four main lines of researchare being followed. (1) The analysisof the three-dimensional structure ofproteins and nucleic acids by X-raydiffraction methods is the centraltheme of Udo Heinemann’s research.Recent projects of his group haveaddressed problems of specificprotein-RNA recognition, electrontransfer by [2Fe-2S] ferredoxins incytochrome P450 systems, in vivofolding of engineered glycosylhydrolases, sex steroid transport inplasma by the sex-hormone bindingglobulin, and blood coagulationmediated by tissue factor, a memberof the cytokine receptor superfamily.Computer simulations of nucleic acidstructure and ligand binding (HeinzSklenar) provide valuable informationwhere experimental data are notavailable or are inaccessible. Usingnew algorithms for the treatment ofsolvent electrostatics, molecularsimulations have been applied to thefunctional analysis of gene regulatoryDNA sequences, the characterizationof non-canonical structural motifs inRNA, and a binding study of singlet-oxygen generating dyes to DNA. (2) Protein misfolding events and theresulting aberrant protein conformationshave received considerable attentionrecently due to their relevance toamyloidoses, a family of diseases

characterized by deposits of β-strandedprotein aggregates in tissue. Applyinga variety of experimental techniques,the groups of Gregor Damaschun andHeinz Fabian are studying the foldingpathways and kinetics of a number ofmodel polypeptides to help shed lighton productive folding and pathologicalmisfolding of proteins. (3) To fulfilltheir diverse physiological functions,proteins interact with many ligands.These ligands vary widely in sizefrom small molecules to cellularstructures. The binding events arecharacterized by very different timescales and association constants.Time-resolved Fourier-transforminfrared spectroscopy is being used tostudy structural changes and reactionintermediates associated with electrontransfer in cytochrome P450(Christiane Jung). Antibody-peptideinteractions, the specific binding ofthe tetracyclin repressor to operatorDNA and initiator-tRNA binding bythe initiation factor IF2 are beinginvestigated by circular dichroism,fluorescence, infrared and Ramanspectroscopy as well as calorimetricmethods in Heinz Welfle’s laboratory.Finally, analytical ultracentrifugationis being employed by Joachim Behlketo study the interaction of proteindomains with unusually structuredDNA, protein oligomerization and thenucleation of protein crystal growth.(4) In a large number ofcollaborations with extramural andMDC groups, protein chemistry,peptide sequencing and massspectrometry (Brigitte Wittmann-Liebold) have proven to beindispensable tools for modernmolecular and cell biology research.In addition, highly sensitive protein2D-electrophoresis combined withMALDI mass spectrometry are keytechniques in proteome researchwhere the goal is to establish proteinpatterns characterizing cellular states,such as apoptosis, or processes suchas cell differentiation anddevelopment.

Carmen Birchmeier, Udo Heinemann,Friedrich C. Luft, Jens Reich, Ludwig Thierfelder

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Molecular Biologyand Genetics of Cardiovascular Diseases

Detlev Ganten

Analysis of complexcardiovascular diseases in the rat

The rat is one of the most importantmodel systems for complex, polygenicdiseases. Since all epidemiologicallyimportant human diseases belong tothis category, the potential for majoradvances through geneticinvestigation is substantial.

In recent years we have demonstratedthat multiple chromosomal loci in ratmodels contribute to blood pressureregulation and hypertension.Independent from elevated bloodpressure, additional genetic factorscontribute to end-organ damage andstroke in these animals.

Ongoing research in our laboratory isdirected towards the identification ofthe underlying predisposing genes andthe subsequent identification of theirmolecular variants responsible fordifferent cardiovascular diseasephenotypes.

To localize disease genes withinchromosomal regions linked toquantitative traits (e.g. bloodpressure), we are establishing multiplecongenic rat strains. These congenicstrains are being developed byintrogressing disease allelesencompassing the quantitative traitlocus (QTL) into a non-affectedreference strain by successivebackcrossing and molecular analysis.This strategy allows observation ofthe effect and genetic analysis of asingle QTL. We are currently applyingthis strategy to a number of QTLs forblood pressure regulation, stroke, andkidney disease in the stroke- prone,spontaneously hypertensive, rat. Asimilar strategy is currently beingadopted in collaboration with ourIsraeli partners to elucidate the genetic

basis of salt-sensitive hypertension inthe Sabra rat model.

Combination of congenicexperimentation with the developmentof subcongenic animals, having only afraction of the initial congenicsegment, will allow successive finemapping within a QTL.

Production and high throughputcharacterization of genomicresources for the rat genome

The ultimate identification of disease-relevant genes within QTLs bypositional cloning requires theavailability of a variety of genomictools, such as large insert genomiclibrary clones, cDNA libraries andmapping ressources. As a partner innational and international rat genomeprojects, our group has producedvarious genomic tools for the ratgenome, among them the first ratYAC library, a high resolutionmapping cross, and a hybridization-based Interspersed RepetitiveSequence (IRS-)PCR marker system.A set of about 800 IRS-markers hasbeen assigned to rat genetic andradiation hybrid (RH) maps. Apreliminary physical framework maphas been produced based onhybridization data from this set ofmarkers against high density griddedfilters representing about 90.000 YACclones (corresponding to 20-foldcoverage) of the rat genome.(for more details visit our webpage:http://www.mdc-berlin.de/ratgenom/)

The mapping efforts of complexcardiovascular traits by congenicexperimentation and positionalcloning will be used in ongoingprojects in combination with theestablishment of gene expressionsignatures in target organs of congenicanimals and their parental progenitors.High density arrays of cDNA clonesor gene-specific oligonucleotides areused for this approach. Acombinatorial approach of positionalcloning and expression profiling willprovide a powerful tool to identifypotential candidate genes withinchromosomal regions for geneticallydetermined cardiovascular diseases.

Transgenic rat technology

In order to study the functionalrelevance of genes linked tohypertension and stroke, transgenicrats are being produced withalterations in the expression of thesegenes. The power of this technologyhas been demonstrated in severaltransgenic rat models withmodifications in the renin-angiotensinsystem. Rats expressing the mouserenin-2 gene have helped inunderstanding the physiologicalfunctions of local renin-angiotensinsystems in tissues. Furthermore,transgenic rats carrying the humanrenin and angiotensinogen genes areexcellent models for studyinghypertension-induced end-organdamage, particularly in the kidney. Inaddition, numerous other transgenicrat models for the study ofcardiovascular physiology have beenproduced and analyzed incollaboration with other groups.Furthermore, transgenic technology inthe rat has been extended by thegeneration of transgenic animals withlarge genomic constructs and theestablishment of knockout technologyfor this species.

Selected Publications

Bohlender, J., Menard, J., Edling, O.,Ganten, D., and Luft, F.C. (1998)Mouse and rat plasma reninconcentration and gene expression in(mRen2)27 transgenic rats. Am. J.Physiol. 274, H1450-H1456.

Hübner, N., Lee, Y.A., Lindpaintner,K., Ganten, D., and Kreutz, R. (1999)Congenic substitution mappingexcludes Sa as a candidate gene locusfor a blood pressure quantitative traitlocus on rat chromosome 1.Hypertension 34, 643-648.

Yagil, C., Sapojnikov, M., Kreutz, R.,Zurcher, H., Ganten, D., and Yagil, Y.(1999) Role of chromosome X in theSabra rat model of salt-sensitivehypertension. Hypertension 33, 261-265.

Yagil, C., Sapojnikov, M., Kreutz, R.,Katni, G., Lindpaintner, K., Ganten, D.,and Yagil, Y. (1998) Salt susceptibilitymaps to chromosomes 1 and 17 withsex specificity in the Sabra rat modelof hypertension. Hypertension 31,119-124.

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Steen R.G., Kwitek-Black A.E., GlennC., Gullings-Handley J., Van Etten W.,Atkinson O.S., Appel D., Twigger S.,Muir M., Mull T., Granados M.,Kissebah M., Russo K., Crane R.,Popp M., Peden M., Matise T., BrownD., Lu J., Kingsmore S., TonellatoP.J., Rozen S., Slonim D., Young P.,Knoblauch M., Provoost A., GantenD., Colman S.D., Rothberg J., LanderE.S., and Jacob H.J. (1999) A highdensity integrated genetic linkage andradiation hybrid map of the laboratoryrat. Genome Res. 9, AP1-AP8.

Structure of the Group

Group leaderProf. Dr. Detlev Ganten

ScientistsDr. Jürgen Bohlender*Dr. Norbert HübnerDr. Margit KnoblauchDr. Kathrin MeißnerDr. Jan MontiDr. Xiao-Li Tian*Dr. Cui Zhaoqiang*

Graduate and undergraduate studentsClaudia Gösele Maolian GongLiu Hong*Liliana PantelicHeike Zimdahl

Technical assistantsSusanne BlachutAnja FeldnerHeide KistelAnita MüllerSabine ScheelBrigitte Hieke

*part of the period reported

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Figure 15: Integrated genetic and radiationhybrid map for rat chromsome 10. Informationof the high density genetic map based ongenotyping results derived from 48 animals of aBNxSHRSP intercross has been integrated withdata from two available radiation hybridframework maps that have been established bycollaborating groups from Milwaukee, USA(MCW) and Oxford, UK (OX), respectively.

These maps consist of genetic markersproduced in Berlin (MDC) and otherlaboratories and enables selection of markersfor further investigation of chromosomalregions of interest. Equivalent maps have beenestablished for all rat chromosomes and will bemade available via the WorldWideWeb.

D10Rat95D10Rat94D10Rat96D10Rat261D10Rat218

D10Rat184D10Rat65D10Rat259D10Rat110D10Rat91D10Mit17D10Rat103D10Rat185D10Rat49D10Rat260D10Rat51D10Rat117

D10Rat257D10Rat50D10Rat47D10Rat183D10Rat258

D10Rat121D10Rat64D10Rat256

D10Rat182D10Rat217D10Rat181D10Rat180D10Rat113D10Rat46D10Mit16D10Rat118

D10Rat45D10Rat216

D10Rat75D10Rat43D10Rat100D10Rat44D10Rat42D10Rat179D10Rat178D10Rat41D10Rat177D10Rat71D10Rat40D10Mit4

D10Rat72D10Rat254D10Rat253D10Rat39

D10Rat215D10Rat250D10Rat176D10Rat174D10Rat82D10Rat173D10Rat175

D10Mgh10D10Rat34D10Rat252D10Rat35D10Rat37D10Rat214D10Rat38D10Rat33D10Rat172D10Rat171D10Rat251D10Arb2

D10Rat104D10Rat36D10Rat167D10Rat168D10Rat213D10Rat79D10Rat130D10Rat249D10Rat169D10Mgh9D10Rat76D10Rat170

D10Rat166D10Rat247D10Rat164D10Rat246D10Rat165D10Rat248D10Rat126D10Rat73D10Rat163D10Rat85D10Rat63D10Rat81D10Rat212D10Rat245

D10Rat102D10Rat244D10Rat162D10Rat31D10Arb5D10Mgh8D10Rat32

D10Rat243D10Rat119D10Mgh6D10Rat116D10Rat77D10Rat161D10Rat29D10Rat158D10Rat160D10Rat30D10Rat239D10Rat240D10Mit8D10Rat241D10Rat157D10Rat133D10Rat211D10Mit2D10Rat69D10Rat242D10Rat156D10Arb8D10Arb7D10Rat80D10Rat159D10Rat238

D10Rat155D10Rat123D10Rat70D10Rat28D10Arb27D10Rat98D10Rat210

D10Rat131D10Rat25D10Rat26D10Rat27D10Rat153D10Rat154D10Rat24D10Rat92

D10Rat151D2Rat244D10Rat237

D10Rat93D10Rat150D10Rat148D10Rat86D10Rat236D10Rat106D10Rat147D10Rat128D10Rat22D10Rat21D10Rat146D10Rat145D10Rat107D10Rat114D10Rat149D10Rat124D10Rat20D10Rat209D10Rat144D10Rat19D10Rat208D10Rat127D10Rat99

D10Rat207D10Rat235D10Rat18D10Rat205D10Mit7D10Rat142D10Rat84D10Rat141D10Arb11D10Rat140D10Mit1D10Rat234

D10Rat17D10Rat204D10Rat202D10Rat203D10Rat233D10Rat14D10Rat15D10Rat138D10Rat13D10Rat16D10Rat139D10Mgh4

D10Rat12D10Rat137D10Rat10D10Mgh3D10Rat11D10Rat9

D10Rat267D10Rat132D10Rat232D10Rat231D10Rat136D10Rat201D10Rat101

D10Rat88D10Rat228D10Rat230D10Rat229D10Rat268

D10Rat227D10Rat8D10Rat7D10Mgh2D10Rat6D10Rat5D10Rat3

D10Rat226D10Rat134D10Rat108D10Rat109D10Rat1D10Rat2

D10Rat135D10Rat4

D10Rat67D10Wox28D10Rat96D10Got13D10Got14D10Rat94D10Got20D10Rat91D10Rat51D10Got24D10Got25D10Got27D10Rat48D10Got28D10Rat47D10Mdc31D10Rat64D10Got34D10Got35D10Rat45D10Mit5

D10Rat43D10Rat41D10Got43D10Mdc5D10Got46D10Rat39D10Got49D10Rat38D10Got54D10Rat37D10Got57D10Mdc10D10Rat36D10Wox25D10Got63D10Got64D10Got69D10Mdc4D10Mdc2D10Rat73D10Mdc11D10Rat83D10Rat63D10Rat85D10Wox11D10Mdc6

D10Mdc30D10Mdc3bD10Wox15D10Got80D10Mdc12D10Got85D10Rat58D10Got89D10Rat57D10Wox5

D10Mdc13D10Got102D10Mdc14D10Rat114D10Wox6

D10Rat107D10Got108D10Got11D10Mdc15D10Wox16D10Got120D10Wox19D10Got123D10Mdc16D10Rat12

D10Got128D10Mdc17D10Mdc18D10Rat120D10Got133D10Got134D10Mdc19D10Rat17D10Wox22

D10Mit1D10Rat15D10Rat16

D10Got148D10Got149

D10Rat9D10Mgh3D10Rat10D10Mit11Rr1023

D10Rat101D10Mdc20D10Wox7D10Rat8

D10Mdc21D10Mdc22D10Mdc23

D10Rat96D10Rat66D10Rat103D10Got25D10Rat48D10Rat47D10Mdc9

D10Rat121D10Rat64D10Rat182D10Rat113D10Rat43D10Rat41D10Got43D10Mdc5D10Mgh11D10Mdc24D10Rat40D10Mit9

D10Rat37D10Mgh10D10Rat33D10Mdc10D10Mgh9D10Rat79D10Rat130D10Rat73D10Mdc11D10Rat83D10Rat63D10Mdc4D10Rat85D10Rat32D10Rat69D10Mdc6

D10Mdc3bD10Mdc25D10Rat133D10Mdc12D10Mdc30D10Rat29D10Got85D10Got87D10Rat90D10Rat57D10Rat154D10Mdc13D10Rat25D10Rat27

D10Got101D10Rat24D10Rat150D10Rat93D10Rat107D10Mdc14D10Got115D10Mdc15D10Rat145D10Got120D10Rat144D10Rat127D10Mdc16D10Rat55D10Rat120D10Mdc18D10Mdc17D10Mgh12D10Rat84

D10Got140D10Mdc19D10Mit12

D10Got144D10Rat14D10Rat53D10Rat12D10Rat9D10Mit11

D10Rat101D10Mdc20D10Rat8D10Rat6

D10Mdc21D10Mdc22D10Mdc23D10Rat7

D10Mdc26D10Mdc27D10Mdc28D10Mdc29D10Rat135

Chromosome 10

SHRSP x BN MCW OX

Molecular Biology ofPeptide Hormones

Michael Bader

The group is interested in themolecular biology and function ofhormone systems involved incardiovascular regulation. Besidescloning and characterization of genesfor the components, the physiologicalfunctions of the systems are beinganalyzed by the production andanalysis of transgenic and gene-targeted animal models.

Renin-angiotensin system

The renin-angiotensin system (RAS)plays a key role in blood pressureregulation and, therefore, has beenstudied in detail employing transgenictechniques.

A major focus of our research is thetissue RAS in the brain. Transgenicrats expressing an antisense-RNAagainst angiotensinogen exclusively inastrocytes of the brain have beenproduced and show a decreased localconcentration of this protein andreduced blood pressure and plasmavasopressin levels. These animals aresuitable models for studying thefunction of local angiotensinproduction in the brain.

In order to investigate the function ofthe mas-protooncogene, a receptorexpressed in the brain and thought tobe involved in the behavioural effectsof angiotensins, we produced micelacking this protein by homologousrecombination in embryonic stemcells. Mas-deficient animals developnormally and exhibit normal bloodpressure and fertility. However, long-term potentiation in the hippocampusas well as anxiety behaviour issignificantly altered. Furthermore, theanimals show modified rhythms ofblood pressure and heart rate.The role of the RAS in hypertension-induced end-organ damage is of majorclinical importance. In a noveltransgenic mouse model we studiedthe function of locally producedangiotensin in the development ofcardiac hypertrophy andnephrosclerosis. These mice have atargeted disruption of theangiotensinogen gene compensated bya rat transgene exclusively expressedin liver and brain, but not in kidneyand heart, where the angiotensinogengene is expressed in normal mice.Because of elevated plasmaangiotensinogen levels, the animalsare hypertensive but suffer lessdamage to the target organs as a resultof a lack of local angiotensinsynthesis.

Kallikrein-kinin system

The kallikrein-kinin system (KKS) isan important hormone system forcardiovascular regulation mostlycounteracting the effects of the RAS.As a model for studying the functionsof the KKS in an intact animal,transgenic rats were produced

expressing the human tissue kallikreingene under the control of the heavy-metal responsive metallothioneinpromoter. The animals express thetransgene in all organs tested andexcrete human tissue kallikrein in theurine. In these rats, blood pressure andits diurnal rhythmicity, as measuredby telemetry, are significantly reducedcompared with control rats. The heartsof the animals are protected againstischemic and hypertrophic injury.

The functions of the kinin B1 receptorare unknown and so we producedmice lacking this subtype. Theresulting animals exhibited analgesiaand altered inflammatory reactionsdemonstrating an important role ofthe B1 receptor in pain transmissionand inflammation.

Embryonic stem cell technology

Using embryonic stem celltechnology, the gene for the cardiacfatty acid binding protein has beendeleted by homologousrecombination. The knockout miceexhibit a severe defect in long-chainfatty acid utilization causing exerciseintolerance and cardiac hypertrophy.Recently, we have started a series ofprojects to analyze the serotoninsystem by transgenic technology.Firstly, we have been able to showthat embryonic stem cells as well asmouse blastocysts express the keyenzyme in serotonin synthesis,tryptophan hydroxylase, implying animportant role for this hormone inearly embryogenesis. Mice lackingtryptophan hydroxylase have beenproduced and their genotype is underinvestigation.

We are also establishing embryonicstem cells from rats to allow gene-targeting experiments in this specieswhich is more suitable for research oncardiovascular diseases than themouse.

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Selected Publications

Binas, B., Danneberg, H., McWhir, J.,Mullins, L., and Clark, A.J. (1999)Requirement for the heart-type fattyacid binding protein in cardiac fattyacid utilization. FASEB J. 13, 805-12.

Schinke, M., Baltatu, O., Böhm, M.,Peters, J., Rascher, W., Bricca, G.,Lippoldt, A., Ganten, D., and Bader,M. (1999) Blood pressure reductionand diabetes insipidus in transgenicrats deficient in brainangiotensinogen. Proc. Natl. Acad.Sci. USA. 96, 3975-3980.

Voigtländer, T., Ganten, D., andBader, M. (1999) Transcriptionalregulation of the rat renin gene byregulatory elements in intron I.Hypertension 33, 303-311.

Walther, D.J., and Bader, M. (1999)Serotonin synthesis in murineembryonic stem cells. Mol. Brain Res.68, 55-63.

Walther, T., Balschun, D., Voigt, J.-P.,Fink, H., Zuschratter, W., Birchmeier,C., Ganten, D., and Bader, M. (1998)Sustained long-term potentiation andanxiety in mice lacking the Masproto-oncogene. J. Biol. Chem. 273, 11867-11873.

Structure of the Group

Group leaderDr. Michael Bader

ScientistsDr. Ovidiu BaltatuDr. Bert BinasDr. Cécile CaylaDr. Vassili GalatDr. Thomas LangenickelDr. Keiichi Sugimura*Dr. Thomas Walther

Graduate and undergraduate studentsNatalia AleninaDmitri AndreevGuido AxmannRonaldo de Carvalho AraujoHeike Danneberg*Guixuan Chai *Silvia Heringer-Walther*Ningling Kang*Jens-Uwe PeterJosé-Antonio Silva JuniorDiego Walther

Technical assistantsChristin Becker*Adelheid BöttgerMonika NitzSusan Radtke*Liselotte Winkler

SecretariatDana Hess

*part of the period reported

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Figure 16: Role of locally producedangiotensin II in cardiac fibrosis Mice carrying a rat angiotensinogen transgenedevelop hypertension, cardiac hypertrophy andfibrosis as detected by immunostaining forcollagen (c). Breeding these mice with animalslacking the endogenous angiotensinogen generesults in animals which are equallyhypertensive but lack local angiotensinproduction in kidney and heart. In theseanimals (b) perivascular collagen deposition isreduced to levels in normal mice (d) orangiotensinogen-deficient mice (a). Theseresults show that hypertension-induced cardiacfibrosis depends on local angiotensin synthesis.

Genome Research,Gene Variation, andComplex Disease Margret Hoehe

The systematic analysis of DNAsequence variation in biomedicallyrelevant genes is the key to a) theidentification of genetic risk factors incommon, complex diseases (‘MedicalGenomics’), and b) the identificationof genetic variation involved inindividually different drug responses(‘Pharmacogenomics’). To this end,the Genome Research Group has a)developed highly efficienttechnologies, which allow sequencecomparisons of candidate genes inlarge numbers of individuals in themegabase range (e.g. ‘MultiplexSequence Comparison’); b) appliedthese technologies to variationanalyses of candidate genes definedby biology and/or genetic mapping; c)identified numerous variants in genespotentially involved in hypertension,substance dependence, and othercommon, complex diseases; d)predicted numerous individuallydifferent forms of the genes by meansof haplotype analyses; e) developedbioinformatic approaches to haplotypeclassification into functionally relatedgroups; f) identified variants, orcombinations of variants (pattern),associated with complex disease.Thus, combined approaches have beendeveloped to establish complexgenotype-phenotype-relationshipsagainst a background of high naturalgenome sequence variability. Thisline of research, development andproduction has been supported by theGerman Human Genome Project. Ithas prepared the background forfunctionally characterizing geneticvariations conferring risk of disease; a‘Functional Genomics’ researchproject is presently being establishedin this group.

Gene sequence diversity,haplotypes, and genotype-phenotype-relationships

We have applied the approachesdescribed above to test the potentialinvolvement of the human mu opioidreceptor gene (OPRM1) in substancedependence. All functionally relevantregions of this candidate gene,including 6.7 kb regulatory, exonicand critical intronic sequences, wereanalysed by ‘Multiplex SequenceComparison’ in 250 subjects andcontrols. A total of 43 variants wereidentified, and 52 different haplotypespredicted in the subgroup of 172African-Americans. These haplotypeswere classified by hierarchical clusteranalysis into two functionally relatedcategories, one of which wassignificantly more frequent insubstance-dependent individuals.Common to this category was acharacteristic pattern of sequencevariants, which was associated withseveral forms of substancedependence (opioid and cocainedependence). This study provides thefirst example of the possibility ofestablishing genotype-phenotype-relationships in a situation ofabundant gene sequence variation.Moreover, to our knowledge, thiswork represents the largest body ofsequence data so far on multipleindividuals for the same gene(manuscripts in review). A largesample including 250 Israelisubstance-dependent individuals andcontrols has also been analysed, and aglobal survey has been performed.

Systematic comparative sequenceanalysis of the human beta2adrenergic receptor gene, including itsknown regulatory and coding regionsin more than 400 individuals, resultedin a total of 15 identified variants,several of which were functionallysignificant. An additional 700individuals were genotyped and theseincluded hypertensive patients,individuals characterized by salt-sensitivity/resistance, beta2 receptorbinding, vasodilator response, and aseries of other cardiovascularparameters including responsivenessto various forms of experimentallyinduced mental and physical stress, aswell as obese patients. Three majorhaplotypes of the beta2 adrenergicreceptor gene were identified, andobserved in 80-95% of all subjectsfrom several independent studies.Evidence of a genetic risk profile foressential hypertension has been

obtained. Generally, evidence of theinvolvement of beta2 variation inincreased blood pressure, in vivovasodilator response to beta2 agonists,catecholamines, and heart size wasobtained. Beta adrenergic receptorgene haplotypes are being expressedand functionally characterized. Anadditional technological developmenthas led to the first application ofMALDI-TOF mass spec for beta2genotyping.

Systematic analysis of geneticvariation in three chemokine receptorgenes (BLR1, BLR2, and the Fusingene) in more than 200 patientssuffering from tumors such as acuteleukemia, Hodgkin- and non-Hodgkinlymphomas, and controls, againresulted in numerous variations .Variants that cause an exchange ofconserved amino acids have beenidentified, and are now beingexpressed and functionallycharacterized. Additional genesstudied include the cannabinoidreceptor gene, the TRHR gene, thebeta1 adrenergic receptor gene, andthe beta myosine heavy chain gene.

These projects have been carried outin close collaboration with the MaxPlanck Institute for MolecularGenetics (Berlin), Department ofGenetics, Harvard Medical School(Boston), Department of Genetics,Yale University (New Haven), FranzVolhard Clinic and Robert RössleClinic at the MDC, Free University(Berlin), University of Graz, INSERM(Paris and Strasbourg), KarolinskaInstitute (Stockholm), andPennsylvania State University(Philadelphia).

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Technology transfer

Based on the fundamental researchcomponent in the ‘Genome ResearchGroup’, and as a direct spinoff fromthe German Human Genome Project,a genome research company,GenProfile AG, was founded inSeptember 1998 with M. Hoehe andR. Zettl as the executive board. Thecompany is based at the BiomedicalResearch Campus Berlin-Buch,Germany, with research facilitiesoccupying approximately 900 squaremeters. The company’s main aim isthe systematic identification of themolecular diversity within the humangenome. Special emphasis is placedon the functional significance of thisvariation for the pathogenesis ofhuman complex diseases (‘MedicalGenomics’) and the efficacy of drugs(‘Pharmacogenomics’). The companyhas established a powerful technologyplatform, in particular proprietaryhigh-throughput technologies forcomparative genome analysis as wellas appropriate bioinformatic strategiesfor data interpretation. With a total ofabout DM 12 Mio. from its first roundof financing, GenProfile AG has beenthe largest direct spinoff from theGerman Human Genome Project(funded by the Federal Ministry ofEducation and Research, BMBF).GenProfile AG has recruited 3i(Investors in Industry) Group plc,London, Europe’s leading venturecapital company, as lead investor.GenProfile AG has also recently beenawarded about DM 4 Mio. of fundingfrom the BMBF BioChance Project.More than thirty posts have beencreated.

Selected Publications

Delbrück, S.J.W., Wendel, B., Sander,T., Morris-Rosendahl, D., Crocq, M.-A., Berrettini, W.H., and Hoehe, M.R.(1997) A novel allelic variant of thehuman serotonin transporter generegulatory polymorphism. Cytogenet.Cell Genet. 79, 214-220.

Timmermann, B., Mo, R., Luft, F.C.,Gerdts, E., Busjahn, A., Omvik, P., Li,G.-H., Schuster, H., Wienker, T.F.,Hoehe, M.R., and Lund-Johansen, P.(1998) β-2 adrenoceptor geneticvariation is associated with geneticpredisposition to essentialhypertension: the bergen bloodpressure study. Kidney Int. 53, 1455-1460.

Gratze, G., Fortin, J., Labugger, R.,Binder, A., Kotanko, P., Timmermann,B., Luft, F.C., Hoehe, M.R., andSkrabal, F. (1999) β-2 adrenergicreceptor variants affect resting bloodpressure and agonist-inducedvasodilation in normotensivecaucasians. Hypertension 33, 1425-1430.

Smolka, M., Sander, T., Schmidt,L.G., Samochowiec, J.,Rommelspacher, H., Gscheidel, N.,Wendel, B., and Hoehe, M.R. (1999)Mu-opioid receptor variants anddopaminergic sensitivity in alcoholicwithdrawal. Psychoneuroendocrinology24, 629-638.

Busjahn, A., Li, G.-H., Faulhaber, H.-D., Rosenthal, M., Jeschke, E.,Schuster, H., Timmermann, B.,Hoehe, M.R., and Luft, F.C. (1999) β-2 adrenergic receptor gene variations,blood pressure, and heart size innormal twins. Hypertension, in press.

Structure of the Group

Group leaderDr. Margret Hoehe

ScientistsDr. Karla KöpkeDr. Katrin WenzelDr. Songjie Liu

Graduate StudentsGuo-Hua LiLars Ohl

Technical assistantsChristina FlachmeierPetra Heere

Guest ScientistsDr. Sebastian DelbrückBernd TimmermannKlaus NeffStefanie RechmannDr. Klaus-Ulrich Lenter

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Etiology andPathogenesis ofHypertension andVascular Disease

Friedrich C. Luft

This group is primarily interested inhypertension and the effect ofelevated blood pressure on thevascular wall. The focus has been onanimal physiology and vascular injury.Dominik Müller leads a teamconcentrating on unique transgenic ratmodels of hypertension. The focushere is to elucidate pathways resultingin end-organ damage. Volkmar Grosshas focused on establishingsophisticated physiological mousemodels because of the potential theyoffer in terms of studying gene-targeted conditions. In addition toelaborate renal function tests, he hassuccessfully conducted telemetrymeasurements of blood pressure andheart rate in conscious mice. Thegroup has a broad interest in patient-oriented research. Jens Jordan hasestablished a laboratory for studyinghuman vascular regulation. Withmicrodialysis, microneurography, andsophisticated autonomic pharmacology,he is identifying disease mechanismsas well as making clinical diagnoses.

Pathophysiology ofhypertension and vasculardisease in animal models

Dominik Müller is interested in theputative “tissue” renin-angiotensinsystem. He has focused on ratsharboring both the human renin andangiotensinogen strains. This modelwas established at the MDC by DetlevGanten’s group. The rats developsevere hypertension and die fromrenal and cardiac failure, beginning atthe seventh week of age. Focalnecrosis, increased matrix production,fibrinoid necrosis, leukocyteinfiltrates, and microthromboses in thekidney and the heart are hallmarkfeatures of this model. Together withEero Mervaala, Joon Keun Park, RalfDechend, and Anette Fiebeler, theteam has traced a pathway of eventsinvolving reactive oxygen species,MAP kinase activation, NFκB andAP1 activation, adhesion moleculeand MCP-1 expression, and tissuefactor production. With a novel set ofpharmacological studies (see figure),the team has shown that endothelin isinvolved, that the Rho pathway seemsto play a role, and that IκB kinase βmust also be activated. Their findingscould lead to a series of novelpharmacological interventions, aboveand beyond blockade of the renin-angiotensin system.

Ning Ling Kang, a doctoral student ofHermann Haller and Friedrich C. Luft,has studied streptozotocin-induceddiabetes in a rat model and found thatprotein kinase C isoforms weredifferentially regulated in the kidneyand heart in diabetes. High glucoseincreased PKC alpha expression,whereas PKC zeta was down-regulated.The finding that PKC alpha is mostlyincreased in endothelial cells supportsa role for PKC alpha in functionalendothelial disturbances observed indiabetes.

Volkmar Gross has continued hissuccessful development ofphysiological techniques in themouse. He has studied pressure-natriuresis in DOCA-salt-inducedhypertension and, more recently,observed that lovastatin lowers bloodpressure and restores normal pressure-natriuresis by influencing medullaryblood flow in the mouse. He andAnna Francka Milia have perfected asystem for 24 h telemetric monitoringin the mouse and have conducted aseries of studies defining the reasonsfor high blood pressure in AT2

receptor knockout mice. VolkmarGross has also established aproductive collaboration with Wolf-Hagen Schunck and they are nowexploring P450 enzyme-relatedchanges in the kidney in response tobezafibrate which stimulates 20-HETE and 11,12 ETE production.Finally, collaboration with Thomas E.Willnow’s group involves thecharacterization of renin bindingprotein knockout mice

Subject and patient-orientedresearch (POR)

Jens Jordan has rejoined the groupafter completing a fellowship in clinical pharmacology at VanderbiltUniversity. He is interested in the autonomic regulation of bloodpressure and cardiovascular reflexes. Recently, he studied subjects withmonogenic hypertension andbrachydactyly. These subjects allexhibit an aberrant loop of theposterior inferior cerebellar artery,which impinges on the ventrolateralmedulla. Such loops have beenimplicated in hypertension byputatively interfering withbaroreceptor reflex function. JensJordan, Jens Tank, and others havestudied these subjects systematicallyand observed that they exhibitorthostatic hypertension and do notbuffer increases in blood pressure byreducing sympathetic tone.Microneurography, microdialysis, anda battery of autonomic tests, includingganglion blockade, are the techniquesmost favored by this team.

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Selected Publications

Müller, D.N., Fischli, W., Clozel, J.P.,Hilgers, K.F., Bohlender, J., Menard,J., Ganten, D., and Luft, F.C. (1998)Angiotensin II formation pathwaysand angiotensin II-related functionaleffects in the rat heart. Circ. Res. 82,13-20.

Gross, V., Lippoldt, A., Bohlender, J.,and Luft, F.C. (1998) Cortical and medullary hemodynamics in DOCA-salt hypertensive mice. J. Am. Soc. Nephrol. 9, 346-354.

Kang, N., Alexander, G., Park, J.K.,Maasch, C., Buchwalow, I., Luft, F.C., and Haller, H. (1999)Differential expression of proteinkinase C, isoforms in streptozotocin-induced diabetic rats. Kidney Int. 56, 1737-1750.

Jordan, J., Beneke, R., Hütler, M.,Veith, A., Luft, F.C., and Haller, H.(1999) Regulation of CD11Bexpression on circulating granulocytesin endurance runners. Med. Sci.Sports Exerc. 31, 362-367.

Mervaala, E.M.A., Dehmel, B., Gross,V., Lippoldt, A., Bohlender, J., Ganten, D., and Luft, F.C. (1999)ACE inhibition and AT1 receptorblockade modify pressure-natriuresisby different mechanisms in rats withhuman renin and angiotensinogengenes. J. Am. Soc. Nephrol. 10, 1669-1680.

Gross, V., Schneider, W., Schunk, W-H., Mervaala, E., and Luft, F.C.(1999) Chronic effects of lovastatinand bezafibrate on cortical andmedullary hemodynamics in DOCA-salt hypertensive mice. J. Am. Soc.Nephrol. 10, 1430-1439.

Structure of the Group

Group leaderProf. Dr. Friedrich C. Luft

ScientistsDr. Volkmar GrossDr. Jens JordanDr. Volker HomuthDr. Dominik MüllerDr. Jens Tank

Graduate and undergraduate studentsMarkus BieringerBastian DehmelChimge ErdenechimegFranziska HampigNing Ling KangAnna Franca MiliaFolke SchmidtMathilde Schmidt

TechniciansSabine GrügerMandy Stoffels

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Figure 17: Electrophoretic mobility shift assayfor the transcription factor NFκB in kidney from double transgenic rats, doubletransgenic rats treated with PDTC an NFκBinhibitor, and control rats. The lower panelshows proteinuria, which is almost completelyblocked when NFκB activation is inhibited.

Gene Mapping andIdentification inMonogenic andComplex Diseases

André Reis

Positional cloning is now widely usedfor the identification of gene defectsthat underlie inherited diseases. Anecessary first step for positionalcloning is the mapping of the genelocus that co-segregates withinfamilies with a particular disease ortrait, which allows allocation of aspecific chromosomal position to theresponsible gene. Although mappingwas initially developed for monogenictraits, it is now possible to locategenetic factors involved in theaetiology of complex diseases. Themost powerful technique currentlyavailable is linkage analysis withhighly polymorphic microsatellitemarkers, which involves anexamination of the entire genome witha set of evenly spaced markers. Thistype of study is usually referred to asa whole genome scan.

The Gene Mapping Centre is aspecialised laboratory carrying outsuch high throughput genotyping forgene mapping in monogenic as wellas complex diseases. We havedeveloped various sets of wellestablished markers from theGénéthon reference genetic map withdifferent marker densities toaccommodate the requirements ofspecial study designs. The laboratoryis mainly funded through grants fromthe German Ministry of Science,Research and Technology (BMBF)and, since January 1997, we haveparticipated in the German HumanGenome Project. Additional funding isprovided through a strategy-fundproject, “genetics of complexdiseases”, from the Helmholtz Societyof National Research Centres. Thelaboratory is also available formapping projects by other groups.

Mapping of complex diseases

The main focus of the Gene MappingCentre is mapping genetic factors incomplex diseases. This type of studyinvolves the analysis of large numbersof phenotypically well characterisedfamilies. Hundreds of markers areused for genotyping and sophisticatedbiostatistical analyses are subsequentlyrequired to identify the genetic locithat contribute to a complex disease.For this purpose we have establishedsuitable techniques with an emphasison automation of the experimentalprocedures. In 2000 we expect toreach an annual capacity of 2.000.000genotypes. Currently, mapping isbased on highly-informativemicrosatellite markers but in thefuture analysis will shift more towardssingle nucleotide polymorphisms(SNPs). Two scientists are involved inproject management, genotyping andtechnology development. Onescientist concentrates on laboratoryinformation management (LIM) whichinvolves the integration of genotypeand phenotype data and thebiostatistical analysis of these data.This is done in close collaborationwith the bioinformatics group (Dr. K.Rohde) and the University of Bonn(Prof. T. Wienker) where data analysisis carried out.

A total of four genome scans forcomplex diseases have beencompleted and two further studies areunder way. In a German collaborativestudy on the genetics of asthma (Wjstet al. 1999) 100 families with twoaffected siblings each, totalling 400subjects were investigated.Furthermore, in a study to identifygenetic factors for susceptibility topsoriasis, 32 extended families withthree or more psoriasis patients within all, 500 subjects were investigated(Lee et al., in preparation). Each studyrequired a total of approx. 200.000genotypes. Besides confirmingalready known loci in both studies, wehave identified novel susceptibilityloci, which are currently beinginvestigated further with refinedmapping and testing of positionalcandidate genes.

Data analysis of two studies, forwhich we recently completedgenotyping, is well advanced. In astudy of the genetics of a subtype ofschizophrenia we were able to identifynew susceptibility loci and determinethe mode of inheritance, at least forthis subtype. The largest study in our

laboratory to date is a Europeancollaborative study of the genetics ofjuvenile myoclonic epilepsy. In total,we have investigated 130 familiescomprising 700 subjects. This is thefirst comprehensive genome-widestudy of this type of epilepsy and weexpect to gain important insights intothe aetiology of both disease groups.

Ongoing studies include two affectedsib-pair studies, one on atopicdermatitis from a Europeanconsortium headed by the CharitéHospital (Prof. Wahn) and a secondon juvenile obesity in collaborationwith the University of Marburg (Prof.Hebebrandt). Finally, genotyping of astudy of the genetic factors involvedin hypertension, in collaboration withthe Franz-Volhard-Clinic on thecampus (Prof. Luft), is scheduled forthe year 2000. The study design isbased on isolated populations andtakes advantage of the restrictedgenetic heterogeneity in thesepopulations. Running costs for all thestudies are funded through additionalexternal grants.

Mapping of monogenic diseases

In contrast to complex diseases,mapping of monogenic traits requiresmuch less genotyping and, usually, itis sufficient to analyse 30-50 subjects.The statistical evaluation is differentand often requires skilledinterpretation e.g. haplotyping. In thefour years since the lab opened, a totalof 31 monogenic traits have beenmapped in humans. For several ofthese the underlying gene defect hasalready been identified, completingthe process of positional cloning. Forinstance, the gene for an autosomalrecessive condition, NijmegenBreakage syndrome, was found to becaused by mutations in a proteininvolved in DNA double-strand repair(Varon et al. 1998). This importantfinding has solved a long-standingpuzzle and pointed research in thisfield in a new direction. In addition,we have also initiated mapping ofmonogenic traits in animal models,mainly the mouse. Severalspontaneous and ENU-inducedmutants have been mapped and, intwo cases, the underlying mutationshave also been identified. Themajority of these projects originatedin external laboratories in Germanybut also from England, TheNetherlands, Canada, United ArabEmirates and Australia.

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Selected Publications

Hennies, H.C., Wiebe, V., Krebsova,A., Hohl, D., Küster, W., and Reis, A.(1998) Genotype/phenotypecorrelation in autosomal recessiveLamellar ichthyosis. Am. J. Hum.Genet. 62, 1052-1061.

Varon, R., Vissinga, C., Platzer, M.,Cerosaletti, K.M., Chrzanowska,K.H., Saar, K., Beckmann, G.,Seemanová, E., Cooper, P.R., Nowak,N.J., Stumm, M., Weemaes, C.M.R.,Gatti, R.A., Wilson, R.K., Digweed,M., Rosenthal, A., Sperling, K.,Concannon, P., and Reis, A. (1998)Nibrin, a novel DNA double-strandbreak repair protein, is mutated inNijmegen Breakage syndrome. Cell93, 467-476.

Wallace, R.H., Singh, R., Scheffer,I.E., George, A.L.Jr., Phillips, H.A.,Saar, K., Reis, A., Sutherland, G.R.,Berkovic, S.F., and Mulley, J.C.(1998) Febrile seizures andgeneralised epilepsy associated withmutations in the sodium channelsubunit SCN1B. Nature Genet. 19,366-370.

Bittner, R.E., Anderson, L.V.B.,Burkhardt, E., Bashir, R., Vafiadaki,E., Ivanova, S., Maerk, I., Höger, H.,Jung, M., Storch, M., Lassmann, H.,Moss, J.A., Davison, K., Harrison, R.,Bushby, K.M.D., and Reis, A. (1999)Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limbgirdle muscular dystrophy 2B. NatureGenet. 23, 141-142.

Jung, M., Poepping, I., Perrot, A.,Ellmer, A.E., Wienker, T.F., Dietz, R.,Reis, A., and Osterziel, K.J. (1999) Afamily with autosomal dominantdilated cardiomyopathy defines anovel locus on chromosome 2q14-q22. Am. J. Hum. Genet. 65, 1068-1077.

Wjst, M., Fischer, G., Immervoll, T.,Jung, M., Saar, K., Rüschendorf, F.,Reis, A., Ulbrecht, M., Gomolka, M.,Weiss, E.H., Jäger, L., Nickel, R.,Richter, K., Kjellman, N.-I.M., Griese,M., von Berg, A., Gappa, M., Riedel,F., Boehle, M., von Koningsbruggen,S., Schoberth, P., Szczepanski, R.,Dorsch, W., Silbermann, M., Loesgen,S., Scholz, M., Bickeböller, H., andWichmann, H.-E. (1999) A genome-wide search for linkage to asthma.Genomics 58, 1-8.

Structure of the Group

Group leaderProf. Dr. André Reis

ScientistsDr. Johannes Becker-Follmann*Dr. Elke Burkhardt-Göttges *Dr. Wilfried Gunia*Dr. Hans Christian HenniesDr. Bories Jung*Dr. Martin Jung*Dr. Kathrin SaarDr. Raymonda Varon-Mateeva

Guest ScientistsDr. Katrin HoffmannDr. Young-Ae Lee*

Graduate and undergraduate studentsSilke AppelLisa Hauke*Britta Hinz*Jeanette Klautza*Julia Krause*Dirk Kuring*David Manasterski*Dietlind Pachale*Sascha Pommeranz*Dominik SeelowFabian Steinert*Freia Spillmann*

Technical assistantsFrançoise André*Alexandra Förster*Birgit Fädrich*Johanna Harder-d‘ Heureuse*Iska Liebner*Ulrike Mennier*Gudrun Nürnberg*Bianca SawitzkiMichaela Seeger*Madeleine Skorna*Christine Spingies*Inka Szangolies*Fabienne Trotier*Andrea Weller*

Guest assistantsSusanne SchmidtMonika Schwarz*

*part of the period reported

29

Tumor GeneticsSiegfried Scherneck

The research program of this group isaimed at obtaining a betterunderstanding of the genetic basis ofcancers, particularly human breastcancer (BC). BC has been shown tobe caused by a multi-step process inwhich a number of oncogenes andtumor suppressor genes contribute tothe cancer when their function isaltered. Most gene mutations have asomatic basis; germline mutations inthe BRCA1/2 genes make individualsmore susceptible to tumorigenesis andmainly occur in hereditary BC. Thereis strong evidence for the existence ofother BC (susceptibility) genes. Weare searching for BC families and areusing experimental models to identifysuch new genes. Molecular probeswill allow us to perform geneticscreening not only for those who areconsidered to be at higher risk butalso for the general population.Precise diagnosis of BC by geneticinvestigation may provide usefulinformation for choosing methods oftreatment and developing newtherapeutic strategies.

Detection and characterizationof germline mutations infamilies with a high incidence ofbreast cancer

W. Hofmann, E. Claßen, D. Horn, L. Estevéz-Schwarz, B. Jandrig, H. Zeidler, I. Sümnich in cooperationwith 11 centers for Familial Breastand Ovarian Cancers in Germany

Hereditary breast cancer accounts for5 – 10 % of all breast and ovariancancers that occur in the westernworld. Mutations in 2 genes, BRCA1and BRCA2, jointly explain the largemajority of families with breast-ovarian cancer syndrome. Using avariety of techniques, we haveidentified more than 30 differentBRCA1-, 15 BRCA2 germlinemutation and some 40 polymorphismsin about 200 German families with ahigh risk of BC. At present, we areparticipating in a nationwide,interdisciplinary approach(gynecological oncology, humangenetics, molecular biology,psychotherapy), initiated andsupported by the “DeutscheKrebshilfe”, to offer women optionsfor risk calculation, geneticcounseling and to provide clinical andpsychological support.

Genetic heterogeneity inhereditary breast cancer:Linkage analysis and the searchfor further breast cancersusceptibility genes

B. Jandrig, S. Seitz, A. Nothnagel,K. Poppe, K. Rücker, S. Hahn incooperation with K. Rohde, B. Hinzmann, A. Rosenthal, Centersfor Familial Breast and OvarianCancers in Germany and severalEuropean and American groupsorganized in the Breast CancerLinkage Consortium (BCLC)

Hereditary breast cancer has aheterogeneous genetic basis. Weassessed the contribution of BRCA1,BRCA2 and other genes to hereditarybreast cancer by linkage analysis inmore than 100 German families.Overall, an estimated 50 % of familiesshowed linkage of the disease toBRCA1, 25 % to BRCA2 and 25 % toother genes. Families with at least 4cases of breast cancer were chosen forBRCA1/2 mutation analysis andmutations were detected inapproximately 50 % of these families.At present, about 30 informative

families have tested negative forBRCA1/2. To investigate the role ofcandidate genes and/or candidategenome regions in hereditary breastcancer, BRCA1/2-negative familieswere used for mutation analysis aswell as linkage- and associationstudies. We have performed linkageanalysis in two BRCA1/2-negativefamilies using microsatellite markersfrom the chromosome region 8p12-p22. A maximum cumulative lodscore of 2.41 was obtained, whichconsiderably strengthens the evidencefor a third breast cancer susceptibilitygene within this genome region.

The respective chromosome 8p regioncould be narrowed down to 3cM usingmicrosatellite markers. A BAC contigof this region is under constructionand some anchor BACs have alreadybeen sequenced. Candidate genes orESTs are tested by mutation analysis.In addition, an electronic Northernblot analysis was performed to obtaindifferentially expressed genes andthese candidates are also included inthe mutation testing.

30

Somatic genetic alterations inbreast cancer: Association ofbreast cancer development andprognosis with geneticalterations

S. Seitz, A. Schwartz, K. Kölble, K. Poppe, S. Werner in cooperationwith P.M. Schlag, M. Dietel and theBCLC

Inter- and intratumoral molecularheterogeneity is one of thecharacteristics of breast cancer andgenetic mechanisms are likely tocontribute to it. We have studied lossof heterozygosity (LOH) at specificchromosomal regions in a large panelof breast tumors. The varyingincidence of different lesions that wedetected indicates intertumoralheterogeneity. We also observedheterogeneity within single tumors,since cases occur in which only somecells within a given tumor have aparticular LOH (intratumoralheterogeneity). At present, we areexamining the contribution andprognostic relevance of differentgenetic alterations to the complexprocess of breast cancer development.

Identification andcharacterization of genesrelevant to breast cancer: YACand BAC transfer studies andanalysis of differentiallyexpressed genes

H. Prokoph, E. Burghardt-Göttges,U.-H. Grasmo-Wendler, S. Seitz, B. Jandrig, R. Frege incooperation with B. Hinzmann, A. Rosenthal, B. Schlegelbergerand B.M. Jockusch

Two distinct chromosomal regionsinvolved in breast cancer wereidentified by chromosome transferstudies and microsatellite analyses.Our results strongly suggest theexistence of tumor suppressor gene(s)in a region distal to TP53 at 17p13.3. Differential display was used toidentify differential gene expressionbetween tumor cells andnontumorigenic hybrid cells obtainedafter transfer of chromosome 17p totumor cells. More than 150 sequenceswere cloned and sequenced. One ofthese sequences, the human profilin 1gene, a regulator of signal-dependentactin polymerization, has beencharacterized as a suppressor of thetumorigenic phenotype of breastcancer cells.

Another region for candidate gene(s)of about 10 cM was localized onchromosome 6q23-q25. To identifybreast cancer relevant genes, severalpositional and functional approachesare used in combination: identificationof differentially expressed ESTs byelectronic- and real Northern blottingand RT-PCR; fine mapping of LOHhotspots; construction of a BAC/PACcontig spanning 1-2 cM; mutationanalysis of candidate genes. Inaddition, functional complementationtests were carried out i.e. BAC/PACtransfer into breast cancer cell lines.

Molecular pathology of solidtumors

K. Kölble, B. Barthel, L. Estevèz-Schwarz, H. Pidde, O.M. Ullrich in cooperation with M. Dietel undP.M. Schlag

We have investigated the patterns ofchromosomal and microsatelliteinstability in human microdissectedtumors of the breast, gastro-intestinaland urogenital tract and havecorrelated these with the prevalence ofgerm line and somatic mutations inseveral genes known to be implicatedin tumorigensis (APC, PTEN,CTNNB1, PFN). Integrating thesegenetic approaches withimmunohistological expressionanalyses has led to the identificationof distinct genomic regions onchromosome 17 apparently involvedin producing pathomorphologicphenotypes common to a range ofdifferent solid tumors. Theconstruction of high resolution STS-and EST-maps has allowed theselection of candidate genes forfurther mutational screening.

Antibody engineering

B. Micheel, J. Schenk, G. Schartein cooperation with U. Heinemann

Antibody technology has been used toproduce and modify antibodies againsttumor antigens for the diagnosis andtherapy of cancer. Experiments usinghybridoma technology and phagedisplay are in progress to selectmonoclonal and recombinantantibodies against epitopes of theBRCA1 gene product. Theseexperiments will increase ourknowledge of the structure andfunction of the BRCA1 gene productand may also provide reagents for theimmunohistological diagnosis ofbreast cancer.

31

Selected Publications

An, H.X., Claas, A., Svelyeva, L.,Seitz, S., Schlag, P., Scherneck, S.,and Schwab, M. (1999) Two regionsof deletion in 9p23-24 in sporadicbreast cancer. Cancer Res. 59, 3941-3943.

Kölble, K., Ullrich, O.M., Pidde, H.,Barthel, B., Diermann, J., Rudolph,B., Dietel, M., Schlag, P.M., andScherneck, S. (1999) Microsatellitealterations in serum DNA of patientswith colorectal cancer. Lab. Invest.79, 1145-1150.

Kneissel, S., Queitsch, I., Petersen, G.Behrsing, O., Micheel, B., and Dubel,S. (1999) Epitope structuresrecognized by antibodies against themajor coat protein (g8p) offilamentous bacteriophage fd(Inoviridae). J. Mol. Biol. 288, 21-28.

Siebert, R., Gesk, S., Harder, S., Plotz,S., Matthiesen, P., Grote, W.,Schlegelberger, B., Jandrig, B.,Grasmo-Wendler, U.H., Scherneck, S.,Rosenwald, A., and Ott, G. (1998)Deletions in the long arm ofchromosome 10 in lymphomas with t(14; 18): A pathogenic role of thetumor suppressor genesPTEN/MMAC1 and MXI1? Blood 92,4487-4489.

Ford, D., Easton, D.F., Stratton, M.,Narod, S., Goldgar, D., Devilee, P.,Bishop, D.T., Weber, B., Lenoir, G.,Chang-Claude, J., Sobol, H., Teare,M.D., Struewing, J., Arason, A.,Scherneck, S., Peto, J., Rebbeck, T.R.,Tonin, P., Neuhausen, S., Barkardottir,R., Eyfjord, J., Lynch, H., Ponder,B.A.J., Gayther, S.A., Birch, J.M.Lindlblom, A., Stoppa-Lyonnet, D.,Bignon, Y., Borg, A., Hamann, U.,Haites, N., Scott, R.J., Maugard,C.M., Lindblom, A., Stoppa-Lyonnet,D., Bignon, Y., Borg, A., Hamann, U.,Haites, N., Scott, R.J., Maugard,C.M., and Vasen, H. (1998) Geneticheterogeneity and penetrance analysisof the BRCA1 and BRCA2 genes inbreast cancer families. Am. J. Hum.Gen. 62, 676-689.

Structure of the Group

Group leader Prof. Dr. Siegfried Scherneck

Scientist Dr. Burkhard Jandrig Dr. Helmuth Prokoph Dr. Arnfried Schwartz *Dr. Michael Theile *Dr. Susanne Seitz Dr. Elke Burghardt-Göttges *Dr. Denise Horn Elvira Claßen

Graduate and undergraduate students Unn-Hilde Grasmo-Wendler Jürgen Janke *Wera HofmannStefanie Hahn *

Technical assistants Renate Frege Konstanze Poppe Karin Rücker Ingeborg Sümnich Sabine Werner Helga Zeidler

Guest scientists Dr. Dr. Konrad Kölble Heiko Pidde *Oliver Ullrich *Beatrix Barthel *Dr. Lope Estevéz-Schwarz

Secretariat Renate Galle

Associated Research Group Group leader

Prof. Dr. Burkhard Micheel

Graduate studentJörg Schenk

Technical assistant Gudrun Scharte

* part of the period reported

32

Clinical andMolecular Genetics of CardiovascularDiseases

Herbert Schuster

This research group is conductingclinical genetic research into the basicmechanisms of cardiovascular disease.The primary emphasis has been onhypertension and lipid metabolism,although other topics are also beingpursued. The approaches haveincluded association studies, linkageanalyses, twin studies, and modifiedsib-pair analyses. Haplotype sharingstrategies are planned and will beconducted in the future. The groupconsists of a genetic field unit whichconcentrates on recruitment of indexpatients and their families, recruitmentof monozygotic and dizygotic twins,and a laboratory unit which isinvolved in DNA extraction,genotyping, mutation screening,mutation detection, sequencing and,most recently, functional andpositional gene cloning. The majorprojects are briefly outlined below:

Monogenic diseases

In the summer of 1994, we wereinformed of an extended family livingin northeastern Turkey on the coast ofthe Black Sea. This family featuressevere hypertension inherited in anautosomal-dominant fashion andbrachydactyly; the two traits exhibit100% cosegregation. Affected personshave severe hypertension and die ofstroke before the age of 50 ifuntreated. We visited this family andexamined over 60 members andmapped the gene to chromosome 12p.To narrow our critical segment, wehave identified additional familieswith this syndrome. A Canadian andan American family, neither ofTurkish origin, were located by DavidChitayat and Hakan Toka,respectively, and linkage studies inthese families have allowed us tonarrow our critical segment. We haveconducted additional clinical studiesand found that all affected individualshave vascular loops involving theposterior-inferior cerebellar artery,which impinges on the ventrolateralmedulla. Consistent with the notionthat such loops could interfere withbaroreceptor function and therebycontribute to hypertension, we haverecently shown that patients with thissyndrome exhibit orthostatichypertension and that their baroreflexdoes not buffer sympathetic toneadequately. In terms of positionalcloning, we have identified acandidate gene which we are currentlysequencing and are completing ourPAC contig.

We are continuing our studies offamilial hypercholesterolemia (FH). Incooperation with Eran Leitersdorf andhis team in Jerusalem, we havemapped a putative “lipid-lowering”gene in an Arab family living inIsrael. In this family, numerous FH-affected persons have normal LDLcholesterol concentrations. By meansof several linkage approaches, wehave been able to map this modifiergene to chromosome 13q. We havenow verified the relevance of thisfinding by performing a linkage studyin dizygotic twin subjects and theirparents. We have been able to showthat the gene locus on 13q is linked toLDL cholesterol and body mass indexin these subjects. The LOD scoresfrom these studies are shown in thefigure. We are now expanding thesestudies to other families in Israel andGermany and are starting positionalcloning studies.

Other monogenic projects of thegroup include the identification of anovel mutation in the elastin genecausing supravalvular aortic stenosisand a mapping study in a childexhibiting holoprosencephaly andrenal tubular dysfunction resemblingthe phenotype in mice with a megalingene disruption.

Genetic field working unit

A particular strength of the group isits genetic fieldworking and subjectrecruitment capability. This programwas developed by Herbert Schusterand details have been published. Inline with the MDC’s encouragementof commercial activities, HerbertSchuster has founded INFOGENGmbH. INFOGEN is a new companyspecializing in genetic field workingand cardiovascular risk assessment.INFOGEN is conducting studies on alarge scale throughout Germany. Oneproject concerns establishing thefrequency of APO-B mutations as acause for FH in Germany anddetermining which APO-B mutationsare most commonly involved. Thisstudy includes a prospective and aretrospective cohort, each containingover 3000 families. So far, thefrequency of APO-B mutations inGermany has been found to be higherthan expected. The study will becompleted in the year 2000. Anotherinvestigation by the group has verifiedlinkage between a chromosome 1qlocus and the syndrome of familial-combined hyperlipidemia (FCHL).This finding has been corroborated inChinese families by Weidong Pei. Aconcomitant twin investigation by thegroup has identified an exciting newcandidate gene for FCHL.

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Twin studies as a strategy toidentify quantitative trait loci

Andreas Busjahn and Hans-DieterFaulhaber have recruited over 200pairs of monozygotic (MZ) and 120dizygotic (DZ) normotensive youngtwins and the parents of the DZ twins.The subjects were carefully phenotypedin terms of blood pressure, bloodpressure in response to provocativemaneuvers, psychological testing, andserum lipid concentrations. Thestrategy is to use a standard twinanalysis to determine heritabilityestimates and to distinguish betweenhereditary and environmentalinfluences. This allows us to performa standard IBD linkage analysis in theDZ twins and their parents, as well asassociation studies in the entire twincohort.

With this approach, we recentlyidentified a series of quantitative traitloci (QTL) relevant to blood pressureregulation. The strongest linkage wasfound to the IGF-1 gene locus. Incollaboration with Margret Hoehe, wehave gained new insight into thecontribution of the β-2 adrenergicreceptor gene. Margret Hoehe’s teamsequenced the entire β-2 adrenergicreceptor gene in our twin cohort andfound 15 SNPs, including numerousnew mutations. Finally, we have anactive cooperation with Per Lund-Johansen’s group in Bergen, Norway.From the Bergen Hypertension Study,we have genotyped offspring fromtwo normotensive and hypertensiveparents and have been able toassociate the Arg16->Gly variant toblood pressure in this cohort.

We have used the QTL approach toshow that the loci for the long QTcgenes, which code for ion channelsand their regulators, are all linked toelectrocardiogram components. Thelong QTc syndromes are monogenicdiseases associated with suddencardiac death. Showing relevance ofthese genes to arrhythmias or risk ofarrhythmias in the general population,is the first step in identifying commonvariants indicating a risk to ventriculararrhythmia. The topic is also highlyrelevant to the tragic sudden infantdeath (SID) syndrome. Further studiesare in progress to investigate thisissue.

Finally, the twin studies have beenhelpful in identifying a new candidategene for FCHL. We first looked forlinkage between the loci for theperoxisome proliferator-activatingprotein receptor (PPAR) γ gene and itsbinding partner, the retinoid Xreceptor (RXR) gene. The formergene is strongly implicated in thedevelopment of obesity. We found thatthe PPAR γ gene locus is linked toHDL cholesterol and body massindex. Furthermore, the RXR genelocus was strongly linked totriglycerides. Since RXR is locatedprecisely at the chromosome 1q locuslinked to FCHL, RXR immediatelybecomes a very attractive candidategene for this condition.

New perspectives

Katrin Hoffmann is studying anisolated population in Germany,namely the Sorbs. She has collected60 families with hypertension and isin the process of performing a totalgenome scan in cooperation withAndré Reis. Tom Lindner, who hascollected 350 sibpairs with type 2diabetes from eastern Germany, joinsthe group after a fellowship withGraeme Bell at the University ofChicago. He is funded to conductfamily studies involving a cohort ofdialysis patients with type 2 diabetes.

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Figure 18: The results of linkage analysis usingMLB and MLBQTL in the FH pedigree areshown together with the linkage results for LDLin the DZ twins (p values transformed into LODscores). In the twins, the peak level ofsignificance was 0.0002, right on markerD13S1241 (Am J Hum Genet 66, 157-166,2000).

LOD MLB affected sib pairs

LOD MLB QTL

LOD twins

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Selected Publications

Toka, H.R., Bähring, S., Chitayat, D.,Melby, J.C., Whitehead, R., Jeschke,E., Wienker, T.F., Toka, O., Schuster,H., and Luft, F.C. (1998) Familieswith autosomal-dominantbrachydactyly type E, short stature,and severe hypertension. Ann. Intern.Med. 129, 204-208.

Schuster, H., Lamprecht, A.,Junghans, C., Dietz, R., Mueller-Myhsok, B., Baron, H., Nothnagel,M., and Luft, F.C. (1998) Approachesto the genetics of cardiovasculardisease through genetic field working.Kidney Int. 53, 1449-1454.

Nagy, Z., Busjahn, A., Bähring, S.,Faulhaber, H.-D., Gohlke, H.-R.,Knoblauch, H., Schuster, H., and Luft,F.C. (1999) Quantitative trait loci forblood pressure exist near the IGF-1,the Liddle syndrome, and theangiotensin II-receptor gene loci inman. J. Am. Soc. Nephrol. 10, 1709-1716.

Busjahn, A., Knoblauch, H.,Faulhaber, H-D., Uhlmann, R., Hoehe,M., Schuster, H., and Luft, F.C.(1999) The QT interval is linked totwo long-QT syndrome loci in normalsubjects. Circulation 99, 3161-3164.

Hanke, J., Brett, D., Zastrow, I.,Aydin, A., Delbrück, S., Lehmann, G., Luft, F.C., Reich, J., and Bork, P.(1999) Alternative splicing of humangenes: more the rule than theexception? Trends Genet. 15, 389-390.

Knoblauch, H., Busjahn, A., Müller-Myhsok, B., Faulhaber, H-D.,Schuster, H., Uhlmann, R., and Luft,F.C. (1999) Peroxisome proliferatoractivated protein γ gene locus, bodymass index and lipid values in normal subjects. Arterioscler. Thromb. Vasc.Biol. 19, 2940-2944.

Structure of the Group

Group leaderProf. Dr. Herbert Schuster

ScientistsDr. Sylvia BähringDr. Heike BaronDr. Andreas BusjahnProf. Dr. Hans-Dieter FaulhaberDr. Katrin HoffmannDr. Hans KnoblauchDr. Tom LindnerDr. Hakan TokaDr. Silke Zinke

Biotechnology engineerAtakan Aydin

Guest scientistsDr. Hussam Al KatebDr. Said Ali Al-YahyaeeDr. Thomas BöckelDr. Weidong PeiDr. Tamas Szelestei

Technical AssistantsChristine JunghansEirien KleinAstrid MühlYvette NeuhausRegina Uhlmann

35

Mouse GeneticsCarmen Birchmeier

We are using mice for the functionalanalysis of genes important fordevelopment and disease. Themolecular genetics of mice is welldeveloped, and homologousrecombination combined withembryonal stem cell technology canbe used to introduce deletions orinsertions into the genome. A furtherdevelopment of the technique, the cre-LoxP technology, now allows us tointroduce conditional mutations thatare restricted to a particular celllineage, or subtle alterations like pointmutations.

Peripheral nervous systemdefects in erbB2 mutants

S. Britsch, M. Woldeyesus, D. Riethmacher, E. Sonnenberg-Riethmacher

Neuregulins are EGF-like growth anddifferentiation factors, which signalvia the tyrosine kinase receptors of theErbB family. We have introducedtargeted null-mutations in the erbB2,erbB3 and neuregulin-1 genes. Thesethree mutations cause severehypoplasia of the primary sympatheticganglion chain. We have shown thatmigration of neural crest cells to themesenchyme lateral of the dorsalaorta, where they differentiate intosympathetic neurons, depends onneuregulin-1 and its receptors. A closeassociation between neuregulin-1expression and the migratory path andthe target site of sympathogenicneural crest cells has been observed.Moreover, these mice show severedefects in the development ofSchwann cell precursors and theircardiac system.

ErbB2-/- mice die at midgestation dueto heart malformation. We have beenable to gentically rescue their heartdevelopment by myocardial expressionof erbB2 cDNA. In rescued erbB2mutants, Schwann cells are lacking.Motoneurons form and can project tomuscle, but nerves are poorlyfasciculated and disorganized.Although neuromuscular junctionsform, there is a severe loss of cervicaland lumbar motoneurons, but not ofthoracic ones. These results define theroles of Schwann cells duringmotoneuron and synapse developmentand show that Schwann cells generateimportant survival factors for distinctmotoneuron populations. Our analysisprovides genetic evidence that themajor developmental role of ErbB2 isto provide a co-receptor function forthe neuregulin receptors ErbB4 andErbB3.

A role for erbB2 in myelination

A. Garratt

Neuregulin-1 provides an importantaxonally-derived signal for survivaland growth of developing Schwanncells, which is transmitted byErbB2/ErbB3 receptor tyrosinekinases. Null-mutations of theneuregulin-1, erbB2 and erbB3 mousegenes cause severe deficits in early

Schwann cell development, and themutant mice do not develop beyondbirth. We employed Cre-loxPtechnology to introduce erbB2mutations late in Schwann celldevelopment, using a Krox20-creallele. Cre-mediated erbB2 ablationoccurs perinatally in peripheralnerves. The mutant mice exhibit awidespread peripheral neuropathycharacterized by abnormally thinmyelin sheaths, containing fewermyelin wraps. Thus, the Neuregulinsignaling system functions duringmultiple stages of Schwann celldevelopment and is essential forproper myelination. The thickness ofthe myelin sheath is determined by theaxon diameter, and we suggest thattrophic signals provided by the nervedetermine the number of times aSchwann cell wraps an axon.

The cryptic gene is essential forcorrect establishment of theleft-right axis

U. Gaio, A. Garratt, T. Müller, C. Öczelik, W. Lankes, M. Strehle

During vertebrate embryogenesis, aleft-right axis is established. Theheart, associated vessels and innerorgans adopt asymmetric spatialarrangements and morphologies.Thus, the apex of the heart points tothe left side of the body, the liver islocated on the right side, stomach andspleen on the left, right and left lungdiffer in lobation, and the gut isasymmetrically curled. We havegenerated a mutant allele of cryptic,an EGF-CFC gene in the mouse.Homozygous cryptic mutants developto birth and die during the first weekdue to complex cardiac malformationsthat include malpositioning of thegreat arteries, and ventricular andatrial septal defects. A variety oflaterality defects are observed, such asrandomised heart looping, rightisomerism of the lung, and splenichypoplasia. This phenotype isreminiscent of the asplenic syndromein humans that is typically associatedwith laterality defects andmalpositioning of the great arteries.

36

Lbx1, c-met and the control ofcell migration of muscleprecursor cells

H. Brohmann

Muscle of the extremities is generatedby migrating myogenic precursorcells. These precursors delaminatefrom the lateral edge of thedermomyotome and form distinctstreams that migrate over largedistances, using characteristic paths.We are characterising the genetichierarchy that controls the migrationof this lineage. We have previouslyshown that the c-met tyrosine kinasereceptor and its ligand, SF/HGF, areessential for the delamination of cells.Moreover, SF/HGF is expressed alongthe entire migratory route of muscleprecursor cells, indicating that thissignaling system plays a role alsoduring the migration process. Indeed,we are currently analysing mice withreduced c-met signaling capacity,which show abnormal limb muscledevelopment and abnormal migrationof precursor cells. The homeoboxgene Lbx1 is expressed in migrating,but not in other types of muscleprecursor cells. We have used genetargeting to analyse the function ofLbx1 in the mouse. Myogenicprecursor cells delaminate from thedermomyotome in Lbx1 mutants, butmigrate in an aberrant manner, and donot reach the dorsal limb field. In theventral limb, precursors are presentbut distributed abnormally. As a

consequence, at birth some muscles inthe forelimbs are completely lacking(extensor muscles) or reduced in size(flexor muscles).

Selected Publications

Gaio, U. Schweickert, A., Fischer, A.Garratt, A. N., Müller, T., Özcelik, C.,Lankes, W., Strehle, M., Britsch, S.,Blum, M., and Birchmeier, C. (1999)A role of the cryptic gene in thecorrect establishment of the left-rightaxis. Current Biology 9, 1339-1342.

Woldeyesus, M.T., Britsch, S.,Riethmacher, D. Xu, L., Sonnenberg-Riethmacher, E., Harvey, R., Caroni,P., and Birchmeier, C. (1999) Geneticrescue of cardiac morphogenesis inerbB2 mutant mice reveals functionsof the ErbB2 receptor in developmentof the peripheral nervous system.Genes & Dev. 19, 2538-2548.

Dietrich, S., Abou-Rebyeh, F.,Brohmann, H., Bladt, F., Sonnenberg-Riethmacher, E., Yamaai, T.,Lumsden, A., Brand-Saberi, B., andBirchmeier C. (1999) The role ofSF/HGF and c-Met in thedevelopment of skeletal muscle.Development 126, 1621-1629.

Birchmeier, C., and Gherardi, E.(1998) Developmental functions ofscatter factor/hepatocyte growth factor(SF/HGF) and its receptor, the c-mettyrosine kinase. Trends Cell Biol. 8,404-410.

Britsch, S., Li, L., Kirchhoff, S.,Theuring, F., Brinkmann, V.,Birchmeier, C., and Riethmacher, D.(1998) The ErbB2 and ErbB3receptors and their ligand, neuregulin-1, are essential for development of thesympathetic nervous system. Genes &Dev. 12, 1825-1836.

Structure of the Group

Group leaderDr. Carmen Birchmeier

ScientistsDr. Stefan BritschDr. Alistair GarrattDr. Thomas MüllerDieter Riethmacher*Eva Sonnenberg-Riethmacher*Dr. Cemil Öczelik*Dr. Wolfgang Lankes*

Graduate and undergraduate studentsHenning BrohmannUrsula GaioLi LiMartin Sieber*Michael StrehleMas Woldeyesus

Technical assistantsSven BuchertKarin GottschlingCathrin Rudolph

SecretariatBrigitta Wedekind

*part of the period reported

37

Figure 19: Aberrant migration of myogenicprecursor cells in Lbx1 mutant embryos.Myogenic precursor cells in control (A), andLbx1-/- (B) embryos at E9.75 were visualizedby in situ hybridization using a Pax3 specificprobe. Myogenic precursor cells detach fromthe dermomyotome in Lbx1 mutants, but do notmigrate appropriately to the limb bud. Incontrol embryos, the precursor cells havereached the limb bud at this stage.

DevelopmentalGenetics

Andreas Schedl

Development and disease are twoclosely linked processes and manydisorders can be explained byabnormal cellular differentiation.Hence, it is essential to understand themolecular basis of development anddifferentiation, if we want to developtherapeutic interventions for humandiseases. Our group is particularlyinterested in developmental aspects ofthe urogenital and cardiovascularsystem. Using transgenic strategies inmice we are analysing the molecularfunction of several transcriptionfactors and cell surface receptors,developing model systems for humandiseases and trying to understand thecellular and pathophysiologicalprocesses leading to the diseasedphenotype in mouse and man.

WT1 in development anddisease

WT1 is a zinc finger protein, whichhas been shown to be mutated in apercentage of Wilms tumours, anembryonic kidney tumour arisingfrom undifferentiated mesenchymalcells. Dominant mutations have alsobeen found in patients suffering fromthe Frasier and Denys-DrashSyndromes, both of which arecharacterised by abnormal gonadaldevelopment and defects inglomerular function leading to end-stage renal failure early on in life.WT1 expression shows a very distinctexpression pattern throughout kidneydevelopment, with low levels in theundifferentiated blastema, slightlyhigher levels in the developingnephron and the highest levels withinthe podocyte layer, the filtrating celltype in the kidney. To investigate thefunction and regulation of the WT1gene at various stages ofdevelopment, we have generatedtransgenic mice carrying the humanWT1 locus. Using a lacZ reportergene inserted into a YAC construct,we have demonstrated that WT1 isexpressed in the early proepicardium,epicardium and subepicardialmesenchymal cells (SEMC). Lack ofWT1 leads to severe defects in theepicardial layer and a concomitantabsence of SEMCs, which explainsthe pericardial bleeding andsubsequent embryonic death observedin Wt1 null embryos. A human-derived WT1 YAC construct is able tocompletely rescue heart defects, butonly partially rescues defects in theurogenital system. Our analysis of theobserved dysplastic kidneysdemonstrates a continuousrequirement for WT1 duringnephrogenesis, in particular, in theformation of mature glomeruli.Furthermore, we have demonstratedthat the development of adrenalglands is also severely affected inpartially rescued embryos. Our datasupport a variety of new functions forWT1 and suggest a generalrequirement for this protein in theformation of organs derived from theintermediate mesoderm. Usingtransgenic mice, we are presentlymimicking several other diseasescaused by WT1, including the Frasierand Denys-Drash syndromes. Theseanalyses will allow us to gainadditional insight into the molecularfunction of WT1, the etiology ofWT1 diseases and, hopefully, allow usto develop therapeutic interventions.

WT1, SOX9 and thedetermination of sex

Sex determination is a fascinatingprocess in which an undifferentiatedgonad develops either into a testis orovary depending on the presence orabsence of a single gene, the SRYgene. Expression of SRY initiates amolecular cascade, which eventuallyresults in the expression of animportant male specific signallingmolecule, the Mullerian inhibitingsubstance (MIS). The precise factorsrequired for the activation of MIS arestill unclear, but a current modelsuggests that a combination oftranscription factors WT1, SOX9 andSF1 may synergistically activate theMIS promoter. We are presentlytesting this model by specificallyadding or removing some of thesefactors in an in vivo situation. Inaddition to being involved in gonadspecification, SOX9 is also importantfor normal differentiation of bonesand mutations have been found in thesyndrome campomelic dysplasia(CD). Patients present with eitherheterozygous mutations in the SOX9gene or chromosome rearrangementsmapping at least 50 kb upstream ofSOX9. Whereas mutations within thecoding region of SOX9 causehaploinsufficiency, the effects oftranslocations 5’ to SOX9 are unclear.To test whether these rearrangementsalso cause haploinsufficiency byaltering spatial and temporalexpression of SOX9, we havegenerated mice transgenic for humanSOX9-lacZ yeast artificialchromosomes containing variableamounts of DNA sequences upstreamof SOX9. We have shown thatelements necessary for SOX9expression during skeletaldevelopment are highly conservedbetween mouse and human and foundthat a rearrangement upstream ofSOX9, similar to that observed in CDpatients, leads to a substantialreduction in SOX9 expression,particularly in chondrogenic tissues.These data demonstrate that importantregulatory elements are scattered overa large region upstream of SOX9 andexplain how particular aspects of theCD phenotype are caused bychromosomal rearrangements 5’ toSOX9.

38

Selected Publications

Moore, A., McInnes, L., Kreidberg, J.,Hastie, N., and Schedl, A. (1999)YAC complementation shows arequirement for Wt1 in epicardium,adrenal gland and continuouslythroughout nephron development.Development 126, 1845-1857.

Moore, A.W., Schedl, A., McInnes,L., Doyle, M., Hecksher-Sørensen, J.,and Hastie, N.D. (1998) YACtransgenic analysis reveals Wilms’Tumour 1 gene activity in theproliferating coelomic epithelium,developing diaphragm and limb.Mech. Dev. 79, 169-184.

Wunderle, V.M., Critcher, R., Hastie,N.D., Goodfellow, P.N., and Schedl,A. (1998) Deletion of long rangeregulatory elements upstream ofSOX9 causes campomelic dysplasia.Proc. Natl. Acad. Sci. USA 95,10649-10654.

Menke, A., McInnes, L., Hastie, N.D.,and Schedl, A. (1998) The Wilms’tumor suppressor WT1: approaches togene function. Kidney Int. 53, 1512-1518.

Ericson, J., Rashbass, P., Schedl, A.,Morton, S., Kawakami, A., vanHeyningen, V., Jessell, T.M., andBriscoe, J. (1997) Pax6 controlsprogenitor cell identity and neuronalfate in the ventral spinal cord andhindbrain in response to graded Shhsignaling. Cell 90, 169-180.

Patent Application

Shuttle vectors for BACs and YACs

Structure of the Group

Group leaderDr. Andreas Schedl

ScientistsDr. Marie-Christine ChaboissierDr. Annette HammesDr. Norbert HübnerDr. Christian MrowkaDr. Valerie Vidal

Graduate and undergraduate studentsJiankan GuoJana Zenker

Technical assistantsDanilo LandrockSabine SchmidtUlrike Ziegler

SecretariatDana Hess

39

Lipids andExperimental GeneTherapyThomas E. Willnow

The low density lipoprotein (LDL)receptor is a 150 kDa endocyticreceptor that mediates cellular uptakeof lipoprotein particles and plays acentral role in the removal of lipidsfrom the systemic circulation. In patients with a genetic defect of the LDL receptor (FamilialHypercholesterolemia, FH), a massiveincrease in the concentration ofplasma lipoproteins results inhyperlipidemia and, as a consequence,in atherosclerosis and coronary arterydisease. In recent years, a number ofnew receptors have been identifiedthat are structurally related to the LDLreceptor and form members of theLDL receptor superfamily (Figure 18).The significance of these receptors forregulation of systemic and cellularlipid metabolism is unknown. We areusing gene targeting and somatic cellgene transfer approaches to generatemouse models with deficiencies inLDL receptor-related receptors and tostudy the consequence of suchreceptor defects in vivo.

The LDL receptor-related protein(LRP) is a 600 kDa cell surfacereceptor and a member of the LDLreceptor gene family. Because LRP ishighly expressed in hepatocytes, it hasbeen speculated that the receptor mayplay a role in the hepatic uptake ofdietary lipoproteins. Dietarylipoproteins are produced by theintestine and transport lipids and lipid-soluble vitamins absorbed from thediet. These particles are cleared fromthe circulation into the liver viahepatic lipoprotein receptors. TheLDL receptor mediates hepatic uptakeof dietary lipoproteins; however, it isnot the only receptor to do so, becausesystemic clearance of dietarylipoproteins is normal in patients withFH. To test the contribution of theLRP to the hepatic uptake of dietarylipoproteins, we analyzed micefunctionally deficient in the LDLreceptor, LRP or both receptors. Usingthese animal models, we have beenable confirm that the clearance ofdietary lipids proceeds via a duallipoprotein receptor system, consistingof the LDL receptor and the LRP.

Megalin is another member of theLDL receptor gene family. Thisreceptor is predominantly expressedon the epithelial cells of the proximaltubules in the kidney. Experimentalevidence suggests that the receptormay be involved in the uptake ofmacromolecules from the glomerularfiltrate. To test this hypothesis and toidentify its endogenous ligands, wegenerated mice genetically deficient inthe receptor and analyzed their tubularresorptive function. These studiesidentified megalin as a receptor forvitamin D binding protein (DBP), theplasma carrier for the steroid 25-(OH)vitamin D3, and demonstrated that thereceptor mediates the tubular retrievalof vitamin/ DBP complexes filteredthrough the glomerulus. This receptor-mediated uptake is required to preventthe loss of vitamin D3 metabolites byglomerular filtration.

Furthermore, it delivers 25-(OH)vitamin D3 to tubular epithelial cellsfor conversion into 1, 25-(OH)2vitamin D3, the active form of thevitamin and a potent regulator ofsystemic calcium and bonemetabolism. Urinary excretion of 25-(OH) vitamin D3 in megalin-/- miceresults in vitamin D deficiency andimpaired bone formation. Thus,megalin acts as an endocytic receptorfor uptake of lipophilic vitamins andregulates a crucial step in the transportand renal conversion of vitamin D3metabolites.

Ongoing research in our laboratory isdirected towards the generation ofnew mouse models with obligate andconditional gene defects of LDLreceptor-related receptors and theelucidation of their roles in thephysiology and pathophysiology oflipid metabolism.

40

Selected Publications

Herz, J., Willnow, T.E., and Farese, Jr.R.V. (1997) Cholesterol, hedgehogand embryogenesis. Nature Genet. 15,123-124.

Moestrup, S.K., Schousboe, I.,Jacobsen, C., Leheste, J., Christensen,E.I., and Willnow, T. E. (1998) β2-glycoprotein-I (apolipoprotein H) andβ2-glycoprotein-I-phospholipidcomplex harbor recognition site forthe endocytic receptor megalin. J.Clin. Invest. 102, 902-909.

Hilpert, J., Nykjaer, A., Jacobsen, C.,Wallukat, G., Nielsen, R., Moestrup,S., Haller, H., Luft, F.C., Christensen,E.I., and Willnow, T.E. (1999)Megalin antagonizes activation of theparathyroid hormone receptor. J. Biol.Chem. 274, 5620-5625.

Nykjaer, A., Dragun, D., Walther, D.,Vorum, H., Jacobsen, C., Herz, J.,Melsen, F., Christensen, E.I., andWillnow, T.E. (1999). An endocyticpathway essential for renal uptake andactivation of the steroid 25-(OH)vitamin D3. Cell 96, 507-515.

Willnow, T.E., Nykjaer, A., and J.Herz. (1999) Lipoprotein receptors:New roles for ancient proteins. NatureCell Biol. 1, E157-E162.

Structure of the Group

Group leaderDr. Thomas E. Willnow

ScientistsDr. Christian BönschDr. Jan HilpertDr. Christian Schmitz

Graduate and undergraduate studentsRegina Burmeister*Jörg-Robert Leheste

Technical assistantsDana Bischof*Charlotte Räder Hannelore Schulz

*part of the period reported

41

Figure 20: Structural organization ofmammalian receptors of the LDL receptor genefamily. Structural elements common to allmembers of the LDL receptor superfamily aredepicted. These include (i) ligand-binding type(filled circles) and epidermal growth factor(EGF) precursor type repeats (open circles), (ii)a single transmembrane domain (filled square)and (iii) internalization signals (asterisk) in thecytoplasmic tail that direct the receptors intocoated pits.

Bioinformatics Jens ReichPeer Bork

Analysis of genomic variationand its importance formultifactorial disease

The two sections of the group (geneticsection in Berlin, J. R., andbiocomputing section, P. B., at presentworking as a guest researcher atEMBL) have combined for a majorproject to evaluate variation in thehuman genome and its relevance fordiseases which are of key interest inthe Franz Volhard Clinic (FVK)(lipoprotein disorders, arteriosclerosisand hypertension, with F. Luft) and inthe Robert Rössle Clinic (RRK)(tumor and pertinent normal tissue,with P. Schlag). The goal is to test the“common variant – common trait”hypothesis (by Chakravarty andCollins) of the genetic causation ofpolygenic traits. A large-scale analysisof available EST databases has beencarried out on approximately 9000mRNAs revealing approximately 5500SNP candidates identified as variantsin alignments of all ESTs (Sunyaev etal.,1999). Possible sequencing errorsin the EST sources have been filteredout by applying sophisticatedalgorithms to the original EST traces(Phred program with a score above20) as well as the removal ofpseudogenes and paralogs. Usingthese EST-derived SNP, we have beenable to calculate the level of variancebetween non- coding and coding sites.As expected, the variation issomewhat higher in silent mutationsites than in non-synonymous sites (9per 10,000 vs. 4 per 10,000 bp).Surprisingly, the variation is less thanintuitively expected in non-codingregions (5 per 10,000). Combinationof EST-derived SNP data plus publicaccess data from sources such as theSNP data consortium should allow usto predict phenotypic effects bycomparative and statistical analysis ofhuman gene variants. Of particular

interest will be population-basedassociation studies to examinecholesterol and triglyceridemetabolism for the identification of“risk allelic variants”.

Alternative splicing (AS) allows onepre-mRNA to be processed into manydifferent mature forms within a cell,each of which can have a distinctfunction. Estimates of AS range from5 up to 30 % for specific tissue types.AS has also been shown to bespecifically associated with diseasephenotypes. The purpose of this studyis to create a bioinformatic method fordetecting possible AS forms bycomparison of the EST database witha large number of human genes.

We undertook two separate studies thefirst of 475 disease-associatedproteins, extracted from SWISSPROT, using TBLASTN to matchtranslations of ESTs to query proteins.We extracted some 204 candidatealternative splice sites and found that34 % of the proteins exhibitedalternative splicing (Hanke et al.,1999). Although this figure is higherthan in previous studies, it is quiteprobably an underestimate, as theratio of tissues per splice form foundis low and at least 18% of knownsplice forms already reported fromwithin this sample set were not foundby ESTs. In a second study on 3876mRNAs, a similar figure foralternative splicing was found (36 %).

Our department has developed in-house software to compare the tissueexpression profiles (normal v disease)of these newly discovered alternative splice forms. In collaboration withProf. Schlag’s group (Dr W. Kemmner,MDC) we are at present investigatinghow a number of these novelalternative splice forms relate to thedevelopment of colorectal cancer.

The effect of single gene loci onmultifactorial diseases, such asarteriosclerosis and hypertension, isweak. To understand their combinedgenetic effect on these commonphenotypes our department hasdeveloped a mathematical modeldescribing the metabolism andtransport of lipoproteins (see theattached metabolic scheme ). Inassociation with Prof Luft’s group, wehave collected and modeled bothepidemiological data together withgenetic analysis of specificlipoprotein-associated genes. Thisinformation has been used to simulatethe phenotypic effect of a number ofphysiological conditions and genedefects in the form of a system ofbalance equations describing thestationary state of a human being interms of its complex genotype(publication submitted).The next stepwill be to train a self-organizingneural network with information ongene variants derived from subjectdata collected in population studiespreviously initiated at FVK.

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Figure 21: Genotype-Phenotype Model of LipoproteinMetabolism, as a complex network of genetic,regulatory and metabolic reactions.

Lipoprotein Metabolism

Chyl_rem

HDL3

Chylo

discoid

VLDL IDL LDL

HDL2

LDLRec

HL

HL

HLLRP

LCAT

LPL

LPL

CETP/HL

CETP/HL

LCAT(LPL)

surface remnants

periph. cholesterol

LPL enhances

surface remnants

scavenger(on LDLRec defmodified LDL)

scavenger(on LPL def.)

downregul.of LDLRecand VLDL synthesis

blue: gene Productsboxes phenotype componentsarrow: intersectiondot arrow: weak reactions : feedback signal

(FFA activate synthesis)

(overflow)

(overflow)

SRB1

LDLRec

(LRP&HSPG)

HDLreccubulin

liver

nasc

insestine

We aim to study the geneticepidemiology of normal and aberrantlipid metabolism in man. Thecontribution of genotype, geneexpression type and conditioningenvironmental factors will be adressedin a systematic manner, making use ofmetabolic models of the lipoproteinsystem.

Selected Publications

Beckmann, G., Hanke, J., Bork, P.,and Reich, J. (1998) Mergingextracellular domains: Fold predictionfor laminin G-like and aminoterminalthrombospondin-like modules basedon homology to pentraxins. J. Mol.Biol. 275, 725-730.

Bork, P., and Koonin, E.V. (1998)Predicting function from proteinsequences: Where are the bottlenecks?Nature Genet. 18, 313-318.

Hanke, J., Brett, D., Zastrow, I.,Aydin, A., Delbrück, S., Lehmann, G.,Luft, F., Reich, J.G., and Bork, P.(1999) Alternative splicing of humangenes: More the rule than theexception? Trends Genet. 10, 389-390.

Huynen, M. A., and Bork, P. (1998)Measuring genome evolution. Proc.Natl. Acad. Sci. USA 95, 5849-5856.

Lucek, P., Hanke, J., Reich, J., Solla,S.A., and Ott, J. (1998) Multi-LocusNonparametric Linkage Analysis ofComplex Trait Loci Neural Networks.Human Heredity 48, 275-284.

Schultz, J., Milpetz, F., Bork, P., andPonting, C.P. (1998) SMART, asimple modular architecture researchtool: Identification of signallingdomains. Proc. Natl. Acad. Sci. USA95, 5857-5864.

Structure of the Group

Group leaderProf. Dr. Jens Reich

ScientistsDr. Concetta AmbrosinoDr. Miguel AndradeDr. Peer BorkDr. David BrettDr. Thomas DandekarTobias DoerksDr. Frank EisenhaberJens HankeMartijn HuijnenDr. Hans KnoblauchDr. Ina KochDr. Warren LatheGerrit LehmannHarald PankowDr. Klaus RohdeDr. Franz RüschendorfSteffen SchmidtDr. Willy SchmidtDr. Stefan SchusterDr. Chamil SiouniaevAlexej Tschapek Dr. Yanping Yuan

Graduate and undergraduate studentsBirgit EisenhaberRobert FürstFerdinand Moldenhauer Jörg SchultzInga Zastrow

Technical assistantsAnita NothnagelGudrun NürnbergBrunhilde PoppeVerena ThieleEdelgard Wolf

43

Interactions ofBiopolymers inSolutionJoachim Behlke

Our group is engaged in the analysisof the structure of proteins and nucleicacids in solution and their interactionsusing analytical ultracentrifugationmethods. Special programs have beendeveloped that allow us to determinethe gross conformation of polymers,self- and hetero-association as well asparameters of thermodynamicnonideality. The substancesinvestigated are of medical andbiotechnological relevance and thedata obtained may help us understandpossible regulatory mechanisms oftranscription or protein folding andmetabolic pathways within the cell.

Gross conformation of peptides

To obtain estimates of the possibleshape of angiotensin peptides whichbind to the AT1 receptor (seven-transmembrane-helix G-protein-coupled complex), we have analysedthe gross conformation of thesepeptides using measurements ofhydrodynamic mobility andtheoretical calculations. The mostprobable, extended structure ofangiotensin 2, about 3 nm in lengthwith a kink, seems to penetrateapproximately 2 nm into the AT1receptor where it binds to specificamino acids and induces the complexreaction.

Regulation of oligomeric proteinstructures and theirconsequences

Collaborations with E.-C. Müller, A. Otto, MDC, and T. Kriegel, TUDresden (hexokinase), P. Tavares,Inst. Pasteur, Paris (portal proteinSPP1) and S. Brantl and K. Steinmetzer, Univ. Jena (CopR)

Homodimeric hexokinase 2 fromSaccharomyces cerevisiae has onephosphorylation site at Ser 14. Thismodification is triggered in vivo byglucose exhaustion. We have shownthat, following site-directedmutagenesis (Ser 14 exchange by Glu)or phosphorylation, the dimericenzyme dissociates completely intomonomers. We assume that the in vivophosphorylation at Ser 14, astransiently occurs in low glucosestates, may be a mechanism toimprove glucose utilization at lowlevels and / or that nuclearlocalization of the monomer may beinvolved in signal transductionwhereby glucose causes cataboliterepression.

Bacteriophage SPP1 portal protein is alarge cyclic homo-oligomer composedof 13 subunits. It is stable in thepresence of 10-50 mM MgCl2.Decreasing electrolyte concentrationleads to a reversible dissociation intomonomers which are partiallyunfolded. The reassociation ofmonomers into the 13-mers requires achaperone-independent folding ofmonomers in the presence of Mg++.

CopR binds as a dimer with highaffinity to two consecutive majorgrooves (site I and site II) of the DNA(KD = 0.4 nM). The complex

formation is a coupled process and itsanalysis requires knowledge of thepreceding CopR dimerization whichhas a dissociation constant of 1.4 µM.Since the cellular concentration ofCopR is about 20-fold higher than thedimerization constant we can assumethat CopR binds in vivo as apreformed dimer.

Recognition of peptidesequences at the interface ofhomodimeric proteins

Collaboration with W. Höhne,Humboldt-Univ., Berlin

To map the putative dimerization sitein the capsid protein p24 (HIV-1) a setof overlapping peptides spanning thep24 sequence was synthesized andtested for the ability to modify themonomer-dimer equilibrium. Most ofthe candidates were inactive.However, one peptide was found tocompete with the monomers in thedimerization reaction. This sequence,therefore, may be part of the contactregion between two monomers.

Nucleic-acid protein interaction

Collaboration with A. Rich, MIT,Cambridge, MA, and H. Oschkinat,Inst. of Molecular Pharmacology,Berlin

The Z domain of the human RNAediting enzyme double-stranded RNAdeaminase I (ADAR1) binds to left-handed Z-DNA with high affinity(KD = 30 nM). Using sedimentationequilibrium techniques and CDspectroscopy, we found that two Zdomains bind to one d(CG)3T4(CG)3hairpin which contains a stem of sixbase pairs in the Z-DNA conformation.We suggest that short segments (6 bp)of the Z-DNA within a gene are ableto recruit two ADAR1 enzymes to thatparticular site.

44

Nucleation

Nucleation as a pre-requisite for thecrystallization of proteins can beconsidered as a special case of self-association. Using sedimentationvelocity experiments performed undercrystallization conditions, we wereable to detect oligomers of 15-20protein molecules. These complexesor nuclei can grow spontaneously tocrystals in supersaturated solution.Crystallization conditions are often farfrom the pI, where proteins are eitherpolyanions or polycations. The highnet charge, as reflected by nonidealitydata (second virial coefficient), canprevent oligomerization. By additionof neutral salts the charges arescreened resulting in a reduction inrepulsion between the proteinmolecules and the possibility offorming associates. The conditionsnecessary for protein crystallizationcan be derived from the value of thevirial coefficient or the ratio of theexcluded volume and the charge-dependent part of this parameter.

Selected Publications

Behlke, J., and Ristau, O. (1998) Animproved approximate solution of theLamm equation for the simultaneousestimation of sedimentation anddiffusion coefficients fromsedimentation velocity experiments.Biophys. Chem. 70, 133-146.

Behlke, J., Heidrich, K., Naumann,M., Müller, E.-C., Otto, A., Reuter, R.,and Kriegel, T. (1998) Hexokinase 2from Saccharomyces cerevisiae:Regulation of oligomeric structure byin-vivo phosphorylation at serine-14.Biochemistry 37, 11989-11995.

Behlke, J., and Ristau O. (1998) Grossconformation of dissolved angiotensinderived from sedimentation anddiffusion coefficients. Biochem. Soc.Trans. 26, 758-761.

Schade, M., Behlke, J., Löwenhaupt,K., Herbert, A. Rich, A., andOschkinat, H. (1999) A 6 basepair Z-DNA hairpin binds two Z domainsfrom the human RNA editing enzyme.ADAR1. FEBS Letters 458, 27-31.

Behlke, J., and Ristau O. (1999)Analysis of the thermodynamic non-ideality of proteins by sedimentationequilibrium experiments. Biophys.Chem. 76, 13-23.

Structure of the Group

Group leaderProf. Dr. Joachim Behlke

ScientistDr. Otto Ristau*

Technical assistantBärbel Bödner

*part of period reported

45

Folding andMisfolding of Proteins

Gregor Damaschun

The creation of proteins in living cellsconsists of two main processes:biosynthesis of the polypeptide chainand its folding into the native, three-dimensional structure with biologicalfunction. The first process has beenthoroughly studied, while the secondprocess is less well known. We havelearnt in recent years that the protein-folding process is not always flawlesswithin the cell and this can havepathological consequences. Thus, anumber of human diseases are relatedto the deposition of protein fibrilscausing tissue damage anddegeneration. Amyloid fibrils developfrom abnormal, misfoldedconformational states of differentnormally soluble proteins formingordered aggregates. The reasons formisfolding are unknown. Therefore,there are no causal treatments forthese diseases. The group “Physics ofBiopolymers” is engaged in studies ofthe folding pathways of proteins tounderstand the causes of misfolding.The main experimental methods usedin these studies include solution X-rayscattering (SOXS), dynamic lightscattering (DLS) and opticalspectroscopy, including kinetictechniques. Methods of statisticalphysics of chain molecules have beenapplied to modelling the experimentaldata.

Polymorphism of proteins

Textbooks state that the structure of aprotein is determined by its aminoacid sequence. However, we havebeen able to show experimentally thatthis so-called second genetic code isnot unambiguous. The three-dimensional structure of a protein isdetermined not only by the amino acidsequence but also by the environmentof the protein molecules and isinfluenced by interactions betweenstructural intermediates on the foldingpathway. Therefore, many proteinscan adopt differently folded three-dimensional structures and only oneof these structures is functionallyactive. For yeast phosphoglyceratekinase (PGK), we observed inaddition to the native structure twofurther, different conformations. Thestarting point for the formation ofthese misfolded conformations is theacid-unfolded state. At low pH values,PGK has the conformation of anexpanded random walk. If themolecule is transferred to ahydrophobic environment with a lowdielectric constant, the entire moleculeforms α-helix. On the other hand,anion-induced partial refolding of theacid-unfolded state leads to theformation of amyloid-like fibrils. Halfthe amino acids have the conformationof a cross-β-helix which is typical ofall amyloids.

Folding pathways and kinetics

The formation of amyloids starts fromnon-natively folded monomericintermediates. The monomersaggregate forming successivelydimers, tetramers and octamers. Moreand more cross-β-structure developsduring this aggregation process. Thekinetics of aggregation is stronglydependent on protein concentration.At room temperature, this processmay take several hours. Subsequently,the octamers grow in one directiononly and form fibrils. The growth ofthe fibrils, i.e. their time-dependentelongation, may take some months.Our results indicate that inhibitors ofcross-β-structure formation can beeffective only during the early phasesof amyloidosis. The slow kinetics aretypical of misfolding of proteins intoamyloids. In vivo, the progression ofthese processes is in some cases evenslower than in our in vitro experiments.By contrast, the folding of a proteininto its native structure is a fastprocess. Typical times for folding varyfrom milliseconds to minutes. Onecentral problem in protein folding isthe question, whether chain segmentswith a periodic secondary structuredevelop in a first step, then form in asecond step the compact globulethrough diffusion (framework model),or whether the chain initiallycollapses, driven by hydrophobicinteractions, with concurrent orsubsequent formation of segmentswith periodic secondary structure(hydrophobic collapse model). Wehave been able to show experimentallythat both models are not generalalternatives. There are proteins foldingmainly according to the mechanism ofthe framework model (e.g., bovineRNase A) as well as folding accordingto the hydrophobic collapse model(e.g., bovine α-lactalbumin). Furtherstudies are necessary to address theopen question: which of these foldingscenarios is more prone to themisfoldings that lead to amyloids? Up to now, a search for commonproperties of amyloid-formingproteins has been unsuccessful.

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Selected Publications

Damaschun, G., Damaschun, H., Gast,K., and Zirwer, D. (1998) Denaturedstates of yeast phosphoglyceratekinase. Biochemistry (Moscow) 63,259-275.

Gast, K., Zirwer, D., Müller-Frohne,M., and Damaschun, G. (1998)Compactness of the kinetic moltenglobule of bovine α-lactalbumin: Adynamic light scattering study. ProteinSci. 7, 2004-2011.

Nöppert, A., Gast, K., Zirwer, D., andDamaschun, G. (1998) Initialhydrophobic collapse is not necessaryfor folding RNase A. Fold. Des. 3,213-221.

Gast, K., Zirwer, D., Müller-Frohne,M., and Damaschun, G. (1999)Triflouroethanol-inducedconformational transition of proteins:insights gained from the differencesbetween α-lactalbumin and ribo-nuclease A. Protein Sci. 8, 625-634.

Damaschun, G., Damaschun, H., Gast,K., and Zirwer, D. (1999) Proteins canadopt totally different foldedconformations. J. Mol. Biol. 291, 715-725.

Structure of the Group

Group leaderProf. Dr. Gregor Damaschun

ScientistsHilde DamaschunDr. Klaus GastDr. Dietrich Zirwer

Graduate and undergraduate studentsAnsgar Siemer

Technical assistantReinhard Kröber

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Figure 22: Formation of amyloid fibrils bymisfolding of proteins. The blue bars representcross-β-structures of the polypeptide chain. U-unfolded state in an acidic environment, N-native state, I-folding intermediate.

U

I

N

Amyloid

Protein Folding andMisfolding

Heinz Fabian

Understanding the mechanism ofprotein folding is of considerableclinical importance since a number ofdiseases, such as Alzheimer’s diseaseand spongiform encephalopathies, arepathological consequences ofmisfolding. A characteristic feature ofvarious medical disorders is the self-assembly of β-sheet domains resultingin the formation of pathogenic proteinaggregates (amyloid fibrils). Agrowing body of data suggests thatpartially folded intermediates areprecursors of such aggregates. Fouriertransform infrared (FTIR) spectroscopyis particularly good at detecting thepresence of and changes in β-sheetstructures. Moreover, the FTIRapproach now allows the investigationof in vitro protein unfolding/foldingevents in the millisecond to minutetime scale and we have appliedinfrared spectroscopic techniques toinvestigate folding and misfoldingprocesses in peptides and proteins.

Peptides and proteins capableof forming amyloid fibrils in vitro

in collaboration with E.-G. Krause(Research Institute for MolecularPharmacology, Berlin)

The principles behind β-sheetformation are not well understood dueto difficulties in the development ofsimple model systems. The design ofβ-sheet peptides is complicated bytheir limited solubility in water anddue to the nature of their folding,which is dictated by long rangeinteractions. We have described denovo β-sheet peptides which self-assemble into fibrillar structures. Theinfluence of peptide length,concentration, and D-amino acidsubstitution on the ability to formamyloid fibrils has been analysed. Ourresults suggest that amyloid formationis not restricted to very few peptidesequences associated with diseasestates. Conformational studies ofsynthetic analogs of Alzheimer βA4peptides have revealed that the centralhydrophobic region plays a key role inthe conformational switch of thepeptide.

in collaboration with G. Damaschun(MDC)

Although unrelated to proteinsinvolved in known amyloid diseases,phosphoglycerate kinase is capable offorming amyloid fibrils under certainconditions. The ability to designconditions under which fibril formationcan be observed with otherwisesoluble proteins offers the opportunityto investigate the molecularmechanism of the underlying process.In the case of yeast phosphoglyceratekinase, a rapidly formed and partiallyfolded monomeric intermediateinvolved in the aggregation processhas been detected.

Folding of the enzymeribonuclease T1

in collaboration with D. Naumann(Robert Koch-Institute, Berlin)

Folding of the model proteinribonuclease T1 is known to becomplex, involving several fast andslow phases. Our time-resolvedinfrared studies have provided newinsights into the structural eventsaccompanying the folding ofribonuclease T1. In particular, anextremly slow folding process has

been observed, which was correlatedwith restricted structural changes dueto an isomerization of the proline-39bond in the protein.

Unfolding and folding of thelambda Cro repressor protein

in collaboration with V.V. Rogov(Institute of Protein Research,Russia), K. Gast (MDC) and H.H. Mantsch (Institute forBiodiagnostics, Canada)

The λ-Cro repressor is one of theproteins which can be used as a modelsystem to study the interplay betweenchanges in secondary structure and thestate of association upon unfoldingand refolding. In the active state of theCro repressor protein, two monomericunits form a dimer by aligning the C-termini of each monomer, allowingthe formation of an antiparallel β-ribbon across the dimer. The N-terminal parts form small globularsubdomains that consist of three α-helices and a short N-terminal β-strand connected to the β-ribbon.Conventional FTIR and dynamic lightscattering experiments have shownthat the first thermal transition of avariant of Cro, which contains adisulfide cross-link between theprotein subunits in the dimer, onlyinvolves unfolding of the three α-helices and the short N-terminal β-strand. The intermediate state has awell structured intermolecular β-sheetdomain still formed by the C-terminalparts of each polypeptide chain andassociates into a tetrameric structure.This stable intermediate unfoldsduring a second transition at highertemperatures, a process that is alsoaccompanied by dissociation of thetetramers. Our kinetic infrared studieshave revealed that oligomerization of the covalently cross-linked proteinstrongly decelerates its folding.Analysis of structural changesapplying 2D-IR correlationspectroscopy, a novel experimentalapproach, has provided fundamentalinsights into sequential events in theformation and also unfolding of thestable intermediate of the Cro protein.

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Selected Publications

Yuan, T., Walsh, M.P., Sutherland, C.,Fabian, H., and Vogel, H.J. (1999)Calcium dependent and -independentinteractions of the calmodulin-bindingdomain of cyclic nucleotidephosphodiesterase with calmodulin.Biochemistry 38, 1446-1455.

Janek, K., Behlke, J. Zipper, J.,Fabian, H., Georgalis, Y., Bienert, M.,and Krause, E.-G. (1999) Watersoluble β-sheet models which self-assemble into fibrillar structures.Biochemistry 38, 8246-8252.

Reinstädler, D., Fabian, H., andNaumann, D. (1999) New structuralinsights into the refolding ofribonuclease T1 as seen by time-resolved Fourier-transform infraredspectroscopy. Proteins: Struct. Funct.Genet. 34, 303-316.

Fabian, H., Fälber, K., Gast, K.,Reinstädler, D., Rogov, V.V.,Naumann, D., Zamyatkin, D.F., andFilimonov, V.V. (1999) Secondarystructure and oligomerization behaviorof equilibrium unfolding intermediatesof the λ-Cro repressor. Biochemistry38, 5633-5642.

Fabian, H., Mantsch, H.H., andSchultz, C.P. (1999) Two-dimensionalIR correlation spectroscopy:Sequential events in the unfoldingprocess of the lambda Cro-V55Crepressor protein. Proc. Natl. Acad.Sci. USA 96, 13153-13158.

Structure of the Group

Group leaderDr. Heinz Fabian

Graduate studentMartin Maetzig*

*part of the period reported

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Structural Studies ofProteins and NucleicAcids by X-rayCrystallography

Udo Heinemann

We rely on macromolecularcrystallography to study structuralaspects of proteins and nucleic acids.The crystal structures of thesemolecules serve to explain theirfunction in biological processes,conformational stability and folding.General areas of interest includenucleic acids and nucleic-acid bindingproteins, electron transport incytochrome P450 systems and thestructural determinants of the stabilityand folding of globular proteins. Y.A.Muller is engaged in studies ofhormone transport by the human sexhormone-binding globulin and oftissue factor. Many of these projectsinvolve collaborations with scientistsfrom Berlin and elsewhere. Inaddition, there is a growing number ofin-house collaborations focussing, forexample, on Wnt signal transductioninvolving β-catenin and conductin,inhibitors of the transcription factorNF-κB and G-protein coupledreceptors. In the newly developingfield of structural genomics, we havehelped create a Berlin-based researchproject, the Protein Structure Factory(PSF). Here, the aim is to set up alocal infrastructure for the semi-automated, low-cost, high throughputstructure analysis of proteins. The PSFcontributes to a world-wide effort todetermine the structures of arepresentative set of protein domainsthat will greatly facilitate futureprotein modelling and drug designstudies.

Nucleic acids and interactingproteins

H. Delbrück, A. Diehl, O. Gaiser, H. Lauble, U. Müller, Y. Roske, E. Werner

The sequence-specific recognition ofnucleic-acid molecules by proteinsand other ligands is thought to bemediated by local structural featuresof the nucleic acid. We havedetermined the crystal structures ofseveral synthetic RNA molecules inan effort to identify the determinantsof specific protein binding. A chimericDNA-RNA hybrid, that correspondsto the RNA-DNA junction formedduring minus-strand synthesis in thecourse of reverse transcription of theHIV-1 genome and carries specificcleavage sites of the reversetranscriptase-associated ribonucleaseH, has been shown to adopt thestandard A-type conformation. Thecleavage specificity of theribonuclease H has been suggested tobe associated with a structuralperturbation of the sugar-phosphatebackbone at the main cleavage site. Inanother study, the crystal structure ofthe acceptor stem helix of tRNAAla

was determined at atomic resolutionfrom pseudo-merohedrally twinnedcrystals. Here we have been able toshow that the G·U wobble base pairknown to be crucial for tRNArecognition by the cognate tRNAsynthetase is hydrated in acharacteristic way and embedded inthe unperturbed, standard A-formRNA. Significant progress has beenmade in the structure analysis ofseveral nucleic-acid binding proteins.The C-terminal domain of thetranscription factor KorB wasdetermined at high resolution andshown to adopt a SH3-like foldresponsible for KorB dimer formation.For the complex formed between theC-terminal domain of translationinitiation factor IF2 and initiatortRNA, crystallization and X-raydiffraction conditions will have beenoptimized to allow completion of thestructure analysis in the near future.

Electron transport incytochrome P450 systems

J.J. Müller

In vertebrates, enzymes of thecytochrome P450 family catalyse avariety of chemical reactions,including steroid hormone

biosynthesis. They receive electronsfrom a [2Fe-2S] ferredoxin which, inturn, accepts electrons from anNADPH reductase. We havedetermined the crystal structure ofadrenodoxin, the ferredoxin from thebovine adrenal gland mitochondrialmatrix, at 1.85 Å resolution (Figure21). In spite of the low-level sequencesimilarity, adrenodoxin bears closestructural similarity to the well knownclass of plant-type [2Fe-2S]ferredoxins and appears to share withthese proteins a common mode ofdocking to the cognate reductase andpredicted electron transfer pathway.Very recently, we have been able tosolve the crystal structure of thechemically cross-linked complex ofadrenodoxin with adrenodoxinreductase which will allow us tomodel electron transfer between theseproteins with some confidence. Thecrystal structures of adrenodoxin andits complex with the adrenodoxinreductase serve to explain a largebody of biochemical and mutationaldata.

Structural basis of proteinstability and folding

J. Aÿ, A.M. Babu, H. Delbrück, U. Müller

Selected aspects of protein foldingand thermodynamic stability can berelated to the native three-dimensionalprotein structure as determined by X-ray crystallography. Over the last twoyears, we have studied three differentmodel protein families in this respect.Biochemical and crystallographicanalyses of 1,3-1,4-β-glucanases haveshown that the jellyroll fold of theseproteins resists various circularpermutations of the protein sequenceand, in the case of the engineeredprotein GluXyn-1, even transplantationof the autonomous folding unit of axylanase into a surface loop of theprotein. These studies have beenexpanded using the proteinthiol/disulfide oxidoreductase DsbA,where we have demonstrated bycrystal structure analysis that movingthe polypeptide chain termini from thethioredoxin-like domain into the α-helical domain by circularpermutation of the sequence has littleeffect on the three-dimensionalprotein structure. Finally, we arecurrently investigating pairs ofbacterial cold-shock proteins ofclosely similar sequence butdrastically different conformational

50

stability. By determining the structureof the cold-shock protein, Bc-Csp, atatomic resolution we have shown thatits gain of more than 20 °C in thermalstability over a mesophilic homolog isentirely due to electrostaticinteractions of two exposed surfaceresidues. These findings open excitingnew possibilities for proteinengineering aimed at creating proteinsof predetermined stability.

Plasma sex steroid transport bySHBG

I. Grishkovskaya, G. Sklenar, Y.A. Muller

Human sex hormone-binding globulin(SHBG) is the major sex steroidcarrier in blood. In biological fluids,SHBG exists as a homodimer andeach monomer comprises two G-modules. These modules are about200 residues long and occur in avariety of proteins such asextracellular matrix proteins, proteinsinvolved in blood coagulation andligands of receptor tyrosine kinases.We recently solved the crystalstructure of the amino-terminal Gdomain of SHBG in a complex with5α-dihydrotestosterone andcharacterized both the architecture ofthe steroid binding site and thequaternary structure of the dimer. Wehave shown that G domains havejellyroll topology and are structurallyrelated to pentraxin. In each SHBGmonomer, the steroid intercalates intoa hydrophobic pocket within the β-sheet sandwich. The steroid and a 20Å distant calcium ion are not locatedat the dimer interface. Instead, twoseparate steroid binding pockets andcalcium binding sites exist per dimer.The structure shows why SHBG isable to bind a variety of syntheticsteroids used, for example, ascontraceptives. Future research willfocus on the crystallisation of SHBGwith various natural and syntheticsteroids.

Tissue factor, a member of thecytokine receptor superfamily

K. Fälber, Y.A. Muller

Tissue factor (TF), the obligatecofactor for coagulation factor VIIa(FVIIa) is a member of the cytokinereceptor family. Like growth hormonereceptor, TF is an integral membraneglycoprotein with a 219 residues longectodomain, a transmembranesegment (23 residues) and acytoplasmic domain (21 residues).Crystal structures of the ectodomainof TF and other members of thecytokine receptor family have beenreported. However, until now, nobodyhas succeeded in crystallizing anintegral receptor, thus leaving manyquestions related to the signaltransduction mechanism unanswered.Our crystallization trials onsolubilised intact TF, with or withoutmonoclonal antibodies, have yieldedcrystals but, so far, their quality hasnot led to a successful structureanalysis.

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Figure 23: Crystal structure of the truncatedbovine adrenodoxin Adx(4-108) (A. Müller etal., 1998). α-Helices and β-sheets are shown asblue spirals and grey arrows, respectively, andthe atoms belonging to the [2Fe-2S] cluster areshown explicitly. The core domain of the protein(right) is preserved in plant-type [2Fe-2S]ferredoxins, whereas the interaction domainresponsible for docking to adrenodoxinreductase or cytochrome P450 (left) is differentin the two subclasses of vertebrate and plant-type proteins.

Selected Publications

Müller, A., Müller, J.J., Muller, Y.A.,Uhlmann, H., Bernhardt, R., andHeinemann, U. (1998) New aspects ofelectron transfer revealed by thecrystal structure of a truncated bovineadrenodoxin, Adx(4-108). Structure 6,269-280.

Aÿ, J., Götz, F., Borriss, R., andHeinemann, U. (1998) Structure andfunction of the Bacillus hybridenzyme GluXyn-1: Native-likejellyroll fold preserved after insertionof autonomous globular domain. Proc.Natl. Acad. Sci. USA 95, 6613-6618.

Mueller, U., Maier, G., Mochi Onori,A., Cellai, L., Heumann, H., andHeinemann, U. (1998) Crystalstructure of an eight-base pair duplexcontaining the 3’-DNA-RNA-5’junction formed during initiation ofminus-strand synthesis of HIVreplication. Biochemistry 37, 12005-12011.

Mueller, U., Schübel, H., Sprinzl, M.,and Heinemann, U. (1999) Crystalstructure of acceptor stem of tRNAAla

from Escherichia coli shows uniqueG·U wobble base pair at 1.16 Åresolution. RNA 5, 670-677.

Müller, J.J., Müller, A., Rottmann, M.,Bernhardt, R., and Heinemann, U.(1999) Vertebrate-type and plant-typeferredoxins: Crystal structurecomparison and electron transferpathway modelling. J. Mol. Biol. 294,501-513.

Grishkovskaya, I., Avvakumov, G.V.,Sklenar, G., Dales, D., Hammond,G.L., and Muller, Y.A. (2000). Crystalstructure of human sex hormone-binding globulin: steroid transport bya laminin G-like domain. EMBO J.19, 504-512.

Structure of the Group

Group leaderProf. Dr. Udo Heinemann

ScientistsDr. Allampura M. Babu*Dr. Annette Diehl*Dr. Hanspeter LaubleDr. Jürgen J. MüllerDr. Yves A. Muller

Graduate studentsJacqueline Aÿ*Ralf BienertHeinrich DelbrückKatja FälberOlaf GaiserIrina GrishkovskayaUwe Müller*Yvette RoskeGisela TünnemannErik Werner

Technical assistantsAnette FeskeAndreas KnespelGisela Sklenar

SecretaryBirgit Cloos

*part of the period reported

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Role of ProteinDynamics in EnzymeFunctionChristiane Jung

The dynamic behaviour of proteinstructures and their relationship toprotein folding and function are themain focus of our research group. Thethiolate heme proteins cytochromeP450 and NO synthase are the mainsubjects studied. While cytochromesP450 are involved in severalmetabolic processes in animals andhumans, such as the biotransformationof drugs and the biosynthesis ofsteroid hormones, the NO synthasesplay a critical role in the production ofthe important signal molecule, nitricoxide. Both enzymes have a verysimilar heme iron coordination sphereand analogous intermediate steps intheir reaction cycles, although theirbiological function, secondarystructure and interaction with redoxpartners are very different.Uncovering the fundamental structuralrequirements for this differentbehaviour may contribute to a betterunderstanding of the reactionmechanisms of heme protein enzymesand to the development of newstrategies for the design of enzymeinhibitors which is of great medicalimportance.

In the last two years we have focussedon the structure analysis ofcytochromes P450 from varioussources and of inducible mouse NOsynthase in different states of thereaction cycle using Fourier transforminfrared (FTIR) spectroscopy.Bacterial cytochrome P450cam hasalso been studied in cooperation withother groups using NMR, EPR andMössbauer spectroscopy.

Structural changes implicated inelectron transfer in cytochromeP450

Within the last few years we haveestablished the laser flash-photolysistechnique coupled with time-resolvedFTIR spectroscopy. This techniqueallows study of the photoinducedprocesses like heme iron ligandbinding or photoreduction of hemeiron. We have found that reduction ofthe heme iron, as well as the bindingof iron ligands, induce changes in thesecondary structure of the protein andin intramolecular salt links betweenthe heme propionic acids and theprotein (see figure). It has beenestablished that formation or changesin salt links also play a significant rolein the intermolecular electron transferfrom redox partners (iron-sulfurproteins) to cytochrome P450 andinfrared spectroscopy is able to detectthese changes. In cooperation withF.W. Scheller and his group,University of Potsdam, we haveshown that bacterial cytochromeP450cam can also be reducedelectrochemically with a high electrontransfer rate, similar to that seenphysiologically, if the electrode ismodified by a negatively charged claywhich obviously mimics theelectrostatics of the natural redoxpartner, putidaredoxin. Electrontransfer rates between putidaredoxinand cytochrome P450cam have beendetermined in collaboration with G.Simonneaux and his group, UniversityRennes 1, France, using the protonNMR technique.

Unstable reaction intermediatesof cytochrome P450

The reaction cycle of thiolate hemeproteins proceeds via an unstableintermediate, called compound I, or[Fe-O]-species. The electronicstructure of this intermediate has notbeen characterized so far but it isthought to be similar to thecorresponding intermediates ofperoxidases. In cooperation with A.X.Trautwein and his group, MedicalUniversity of Lübeck, we havestabilized this intermediate in freeze-quench experiments and characterizedit by Mössbauer and EPRspectroscopy. It turns out that theheme iron is in the Fe(IV) state, butthe electronic structure of the wholespecies differs from that ofperoxidases.

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Figure 24: Fourier transform infraredabsorption difference spectrum induced by tris-bipyridyl-Ru(II)-complex-mediatedphotoreduction of 1R-camphor-boundcytochrome P450cam in the presence andabsence of carbon monoxide (100 mMdeuterated potassium phosphate buffer, pD 7, 1mM Ru(II)(bpy)3Cl2, 10 mM EDTA, 10 % (v/v)glycerol-d3, 13 mM 1R-camphor; 23 µmpathlength, 532 nm excitation by Nd-YAG laser)

Active site structure of iNOsynthase

In cooperation with D.K. Ghosh,Duke University Medical Center,Durham, USA, we have analysed theeffect of arginine andtetrahydrobiopterin on the active sitestructure of inducible NO synthase(oxygenase domain) from mouseusing the FTIR spectroscopy over thebroad temperature range from 20 K to298 K. The CO stretch mode of theheme iron CO ligand has been used asa spectroscopic probe. We have shownthat tetrahydrobiopterin has nosignificant effect on the active sitestructure. In contrast, arginine forms ahydrogen bond to the CO ligand andmakes the active site more rigid. Thissuggests that a hydrogen bond mayalso exist in the physiologicallyrelevant dioxygen complex whichwould have significant consequencesfor the reaction mechanism.

Selected Publications

Contzen, J., and Jung, C. (1999)Changes in secondary structure andsalt links of cytochrome P-450caminduced by photoreduction: A Fouriertransform infrared spectroscopicstudy. Biochemistry, 38, 16253-16260.

Mouro, C., Bondon, A., Jung, C., HuiBon Hoa, G., De Certaines, J.D.,Spencer, R.G.S., and Simonneaux, G.(1999) Proton nuclear magneticresonance study of the binary complexof cytochrome P450cam andputidaredoxin: Interaction andelectron transfer rate analysis. FEBSLetters 455, 302-306.

Behlke, J., and Jung, C. (1998).Dithionite reduced carbon monoxidecomplex of cytochrome P450cam is amonomer. Biochem. Biophys. Res.Commun. 253, 558-560.

Contzen, J., and Jung, C. (1998) Step-scan time-resolved FTIR spectroscopyof cytochrome P-450cam carbonmonoxide complex: A salt linkinvolved in the ligand rebindingprocess. Biochemistry 37, 4317-4324.

Schlichting, I., Jung, C., and Schulze,H. (1997) Crystal structure ofcytochrome P-450cam complexedwith the (1S)-camphor enantiomer.FEBS Letters 415, 253-257.

Structure of the Group

Group leader Dr. Christiane Jung

Graduate and undergraduate studentsJörg ContzenCorinne Mouro*Guillaume Jan*Elyzabeth Dehapiot*

* part of the period reported

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Modeling NucleicAcid Structure andRecognition byProteins

Heinz Sklenar

Understanding nucleic acid structuresis important due to the variety ofbiological functions fulfilled by DNAand different classes of RNAmolecules. Investigations usingcomputer simulations are based onphysical models that have beendeveloped in order to describe thedriving forces for the formation ofmolecular structures. Such studies arevery different from laboratoryexperiments, yet the capability forexploring the large diversity ofpossible structures, and of theirstability under given conditions, areoften complementary. The results leadto a better understanding ofbiomolecular structures in terms oftheir physical properties, help topredict what structures are formed andhow these structures interact in livingsystems. New algorithms using acontinuum treatment of solventelectrostatic effects in combinationwith molecular force field models haveenabled us to apply conformationalsearch techniques for structuralpredictions. The computationalapproach complements high-resolutionstructure determination using X-raycrystallography and NMRspectroscopy, with the advantage thatthe full sequence space can be readilyexplored.

Functional annotation ofregulatory regions in genomicsequences

Subtle sequence effects on the helicalgeometry of DNA have been found tobe critically important for theselective recognition of specific basesequences by regulatory proteins. Thestructural libraries, based on ourmodeling results and the analysis ofexperimental stuctures, permit fastconversion of base sequences intoprofiles of structural parameters.Thus, both systematic structuralanalysis of binding sites for specifictranscription factors, and the searchfor sites with characteristic andcommon features in long sequenceswith unknown function, have becomepossible. The applicability of thisapproach to the characterization ofindividual regulatory elements hasbeen confirmed. Recent data suggestthat there are specific examples oftranscription factor – DNAinteractions where consideration ofstructural features gives significantinsight into our understanding of therecognition of regulatory elementscompared with pure statisticalsequence analysis.

Conformation and stability ofnon-canonical structural motifsin DNA and RNA

The remarkable intrinsic stability ofcertain classes of structural motifs andtheir re-occurence in many RNAstructures indicate that they play animportant role in tertiary folding andin biological functions of RNAmolecules. The emerging “tool kit” ofRNA structural motifs will help usunderstand better the relationshipsbetween sequences, structures, andfunctions, and is also expected tosubstantially aid model building ofRNA 3D-structures. A computationalapproach to this task would be helpful,provided that any experimental data setcan be reliably extended. A force-fieldbased conformational analysis hasbeen applied to single-base bulges,GNRA tetraloops, and the asymmetricinternal E-loop. The structures arerepresented by an ensemble ofconformers that were selected purelyon the basis of calculated freeenergies from a large set ofconformations generated by asystematic combinatorial loop search.In contrast to the well-known failureof such predictions based on quasi-vacuum force fields, the inclusion ofreaction field contributions by thesolvent results in a selection of low-energy conformers in accordance withexperimental data. Besides thedetailed atomic resolution structure anunderstanding of the mobility andconformational deformability of RNAstructures is important for interpretingits function. Currently, only some

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Figure 25: Three binding modes of thephotoactive methylene blue molecule with DNA:intercalation (left, views perpendicular andparallel to the helical axis), minor groovebinding (right, upper panel), and major groovebinding (right, lower panel).

aspects of the dynamic behavior ofnucleic acids can be measuredexperimentally. The harmonic-modeanalysis method has been used tocharacterize the conformationaldeformability of regular Watson-Crickpaired, mismatch and bulgecontaining RNA fragments.

DNA – ligand interactions

Photoactive methylene blue is one ofthe most efficient singlet-oxygengenerating dyes. It binds to DNA andinduces photosensitized reactionswhich can be used for sequence-specific cleavage of the DNAbackbone. Photophysical dataobtained for methylene blue incomplexes with DNA indicatedifferent binding modes of the dyedepending on the base sequences.Based on a modeling study ofmethylene blue binding to a DNAdecamer with an alternating GC basesequence, we have derived sixstructural models. The conclusion ofthe detailed structural and energeticanalysis is that these structures can beconsidered as reprensentatives ofmethylene blue – DNA complexes indifferent binding modes (intercalation,minor and major groove binding). Thepredicted structures may be useful fora more detailed interpretation ofexperimental results and could serveas a starting point for studying basesequence effects in view of thephotochemical applications ofmethylene blue in gene therapy.

Classification of C2H2 zincfinger proteins in the C. elegansgenome

C2H2 zinc fingers, short repetitivesequence modules in zinc fingerproteins, are the most frequent nucleicacid binding motif in eukaryoticgenomes. Their high sequencevariability, combined with differentarrangements of the fingers, results inthe rather diverse functions of the zincfinger proteins, ranging fromsequence-specific binding to DNA orDNA/RNA hybrids, binding to RNAor heteroduplex DNA, to theirinvolvement in protein – proteininteractions. Using a substantiallyimproved sequence search pattern, thecomplete set of C2H2 zinc fingerproteins has been identified in the C.elegans genome. We have detectedzinc fingers in about 1 % of all ORFs.Our attempts at a functionalclassification are based on known dataof zinc finger – DNA recognition, onthe discovery of several C. eleganszinc finger proteins homologous tofunctionally characterized zinc fingerproteins in other species and onsequence pattern analysis using ourzinc finger protein databases. Inearlier studies, a similar approach wasapplied to the complete set of yeastzinc finger proteins.

Selected Publications

Ponomarenko, M.P., Ponomarenko,I.V., Kel, A.E., Kolchanov, N.A.,Karas, H., Wingender, E., and Sklenar,H. (1998) Computer analysis ofconformational featuresof theeukaryotic TATA-box DNApromotors. Mol. Biol. (Moscow) 31,733-744.

Zacharias, M., and Sklenar, H. (1998)Harmonic modes as variables toapproximately account for receptorflexibility in ligand-receptor dockingsimulations: applications to a DNAminor groove ligand complex. J.Comp. Chem. 20, 287-300.

Maier, A., Sklenar, H., Kratky, H.,Renner, A., and Schuster, P. (1999)Predicting RNA structural motifs byconformational search: GNRAtetraloops and their pyrimidinerelatives. Eur. Biophys. J. 28, 564-573.

Zacharias, M., and Sklenar, H. (1999)Conformational analysis of singlebase bulges in A-DNA and RNA usinga hierarchical approach and energeticevaluation with an continuum solventmodel. J. Mol. Biol. 289, 261-275.

Zacharias, M., and Sklenar, H. (2000)Conformational deformability ofRNA: A harmonic mode analysis.Biophys. J. in press.

Structure of the Group

Group leaderDr. Heinz Sklenar

ScientistsDr. Siegfried BöhmDr. Wolfgang SchulzDr. Martin Zacharias*

Graduate studentsAlexandra MaierAnnekathrin MüllerRemo Rohs

Technical assistentWerner Leistner

*part of the period reported

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Conformation,Stability andInteraction ofBiologicalMacromolecules

Heinz Welfle

Knowledge of the conformation,stability and interactions of biologicalmacromolecules is a basic prerequisitefor understanding the fundamentalproblems of molecular biology. Weare studying the physicochemicalproperties of selected targets, such asenzymes, transcriptional andtranslational factors and theircomplexes with nucleic acids, andantibodies and antibody-peptidecomplexes. Our main tools arecircular dichroism, fluorescence,infrared and Raman spectroscopy andcalorimetric methods.

Interaction of tetracyclin-repressor with operator DNA

in collaboration with WolframSaenger, Free University of Berlin

Tetracyclin repressor (TetR) isinvolved in the most commonmechanism of tetracycline resistanceof Gram negative bacteria. We haveanalysed the interaction of the class DTetR protein with anoligodeoxyribonucleotide with asequence corresponding to operatorsite O1. Infrared and Ramanspectroscopy were employed toinvestigate the solution structure ofTetR, and the TetR:operator complexwas studied by Raman spectroscopy.The following results were obtainedfor H2O and D2O samples: i) The B-DNA conformation of the operatorsite is conserved in the complex withonly small perturbations of thebackbone geometry; ii) TetR andoperator DNA interact at major-groove sites; iii) Minor changes inTetR secondary structure occur onoperator binding; iv) Localenvironments of aromatic amino acidsare altered. These data are consistentwith a model based on genetic andbiochemical experiments.

Antibody-peptide interaction

in collaboration with WolfgangHöhne and Jens Schneider-Mergener, Humboldt University,Berlin

High affinity antibodies are usuallythought to be monospecific,nevertheless, polyspecificity isfrequently observed. The murine anti-p24 (HIV-1) antibody, CB4-1, binds toa linear peptide epitope of the capsidprotein and also to several unrelatedpeptides. Using a synthetic positionalscanning combinatorial library, fiveunrelated peptides have beenidentified that compete with eachother for binding to the paratoperegion of the antibody (A. Kramer, T.Keitel, K. Winkler, W. Stöcklein, W.Höhne, J. Schneider-Mergener (1997),Cell 91, 799-809).

The crystal structures of the CB4-1Fab fragment alone and in complexwith epitope-homologous and non-homologous peptides have beensolved at 2.6 Å resolution (T. Keitel,A. Kramer, H. Wessner, C. Scholz, J.Schneider-Mergener, W. Höhne(1997) Cell 91, 811-820). CD spectraof the antibody and its Fab and Fcfragments exhibit the characteristicsexpected of β-proteins. Lowering thepH to 3.5 reduces the stability butdoes not change the conformationwhereas, between pH 3.5 and 2.0,conformational changes occured andnew, acid-induced and surprisinglythermostable forms are present. Tounderstand the main determinants ofthe complex formation between CB4-1 and peptides, we are studying thebinding reaction by isothermaltitration calorimetry. This methodprovides a complete thermodynamicdescription of the reaction. Bindingconstants, binding stoichiometry andbinding enthalpies have been obtainedexperimentally and allow calculationof binding free energies and bindingentropies. For the CB4-1-peptideinteraction, the enthalpy and entropycontributions to the free energy differsignificantly from peptide to peptidebut in each system studied so far thecomplex formation is enthalpicallydriven.

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Translational initiation factor IF2from Bacillus stearothermophilus

in collaboration with Claudio O.Gualerzi, University of Camerino,Italy, and Udo Heinemann, MDC

Initiation factor IF2 is involved in theinitiation step of eubacterialtranslation, and its main recognisedfunction is the correct positioning ofinitiator fMet-tRNAfMet in theribosomal P site. To accomplish itsfunction in translation, IF2 interactswith fMet-tRNAfMet via its C-terminaldomain (IF2 C) and with GTP/GDPand 50S ribosomal subunits via itscentral G-domain. Our efforts haveconcentrated on elucidating thestructure of IF2 C and the molecularnature of its interaction with fMet-tRNAfMet. Recently, we have shownin thermal and guanidinium chloride-induced unfolding studies that IF2 Cconsists of two subdomains. Isolatedsubdomain IF2 C-2 binds fMet-tRNAfMet with the same specificityand affinity as native IF2. IF2 C-2has been identified as a globularmolecule containing predominantlystructures (25% antiparallel and 8%parallel strands) and turns (19%)whose structural properties are notmarkedly affected by the presence orabsence of the N-terminal subdomainIF2 C-1. Functional and structuralcharacterisation of Cys mutants of IF2C have provided evidence that I) bothCys residues are buried within anhydrophobic core; II) neither Cys isfunctionally essential; III) both Cysresidues are located near the activesite, probably without participatingdirectly in fMet-tRNA binding.

Selected Publications

Krafft, C., Hinrichs, W., Orth, P.,Saenger, W., and Welfle, H. (1998)Interaction of Tet repressor withoperator DNA and with tetracyclinestudied by infrared and Ramanspectroscopy. Biophys. J. 74, 63-71.

Förster, C., Krafft, C., Welfle, H.,Gualerzi, C.O., and Heinemann U.(1999) Preliminary characterization byX-ray diffraction and Ramanspectroscopy of a crystalline complexof Bacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNAfMet. Acta Cryst. D55, 712-716.

Misselwitz, R., Welfle, K., Krafft, C.,Welfle, H., Brandi, L., Caserta, E.,and Gualerzi, C.O. (1999) The fMet-tRNA binding domain of translationalinitiation factor IF2: Role andenvironment of its two Cys residues.FEBS Letters 459, 332-336.

Reineke, U., Sabat, R., Misselwitz, R.,Welfle, H., Volk, H.-D., andSchneider-Mergener, J. (1999) Asynthetic mimic of a discontinuousbinding site on interleukin-10. NatureBiotechnol. 17, 271-275.

Welfle, K., Misselwitz, R., Hausdorf,G., Höhne, W., and Welfle, H. (1999).Conformation, pH-inducedconformational changes, and thermalunfolding of anti-p24 (HIV-1)monoclonal antibody CB4-1 and itsFab and Fc fragments. Biochim.Biophys. Acta 1431, 120-131

Structure of the Group

Group leaderProf. Dr. Heinz Welfle

ScientistsDr. Karin WelfleDr. Rolf Misselwitz

Graduate studentsAnnette Schaup*Christoph Krafft*Stefan Lättig

Technical assistantBrunhilde Kannen

*part of the period reported

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Protein StructureAnalysis andProteome ResearchBrigitte Wittmann-Liebold

The research of the group hasconcentrated on the structure andfunction of proteins and proteincomplexes and their protein-proteinand protein-RNA interactions at themolecular level. In addition to ownprojects, several long-termcollaborations with medical groups ofthe MDC and the Robert RössleClinic (the groups of Dr. KurtBommert and Dr. Peter Daniel /Department Prof. B. Dörken) havebeen started in order to studyapoptosis-induced processes and theprotein complexes involved intransport, signalling anddevelopmental processes (incollaboration with Dr. P.M. Kloetzel,HU Berlin and R.A. Kroczek, RobertKoch Institute, Berlin). Proteomeresearch has been successfully carriedout on several projects, and proteinsassociated to myocardial disease andapoptosis-associated processes havebeen identified. These studies have

yielded valuable data on the totalprotein expression in the cell indisease and under developmentalconditions. Our crosslinking data fromribosomes have established directcontact sites between the RNA andbinding proteins which have allowedinsertion of the 3D-structures of theseproteins into recent eubacterial RNAfine structure models (Brimacombe etal., in press).

The large increase in molecular dataobtained by proteome analysis hasbeen achieved by the application ofhighly sensitive protein 2D-electrophoresis isolation techniques incombination with MALDI-massfingerprinting and nanospray-ESI-TOF spectrometry. Mass fingerprintingprovides information on the masses ofthe peptides derived from theindividual proteins allowing us toperform searches in the databases forprotein identification. This yields 40-80% sequence coverage for most ofthe protein spots. In addition, usingnanospray-ESI-TOF massspectrometry, 4-8 partial peptidesequences can be derived from thepeptide mixture with minute sampleamounts (< 1pmol), and these datamake protein identification even morereliable. After separation of thecomplex total cell protein mixture byhigh-resolution 2D-electrophoresis in24x32 cm gels up to 5000 proteinscan be resolved (Klose and Kobalz,1995). Then, the proteins of interestare excised from the gel, cleaved insitu by trypsin, desalted and thepeptide mixture is subjected to mass

spectrometry. Often the identificationof the parent protein is possible fromthese masses, or alternatively, partialsequence information helps us assignthe correct protein. In this way, it ispossible to identify many proteinsoverexpressed or reduced after IgMapoptosis induction of Burkittlymphoma BL60 cells (Mülller et al.,1999). Among these, several new proteins have been detected which sofar are not connected with any of theapoptosis processes. Their genes arenow being cloned using appropriatepartial peptide sequences andcompletely sequenced. This will alsopermit recombinant protein isolationfor further functional assays.

In addition, using highly sensitiveprotein analytical methods incombination with truncationexperiments, sequences within theproteasomal prosequences have beendeduced which mediate efficientintegration of β-subunits into the 20S-proteasome complex (Schmidt et al.,1999). Employing human and yeastproteasomes, the function of theproteasome regulatory particle hasbeen studied. These exhibitchaperone-like activities as revealedby native citrate synthase recovery(Braun et al., 1999). Moreover,

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Figure 26: Silver stained 2-DE analytical gelsof the proteins derived from Burkitt lymphomaBl 60 cells: a) normal cells, b) cells after anti-lgM-induced apoptosis. Examples of proteinsaltered after treatment: 1-D4-GDI, fragmentedin b), 2-ribosomal protein Po, pl shift in b), 3-neutral calponin (not found in the apoptoticgel), 4-actin and actin fragments, 5-hnRNPC1/C2, pl shift in b).

besides the known T-cell specificsurface receptors CD28 and CTLA-4,a third member of this family, theinducible co-stimulator (ICOS) hasbeen identified (Hutloff et al., 1999)and results indicate that ICOS isanother major regulator of theadaptive immune system.

In recent years it became obvious thatgenome analysis alone cannotestablish structural-functionalcorrelations between biomolecules invarious cell processes. On the otherhand, the complete description andanalysis of all proteins within a cell,cell line or microorganism (proteomeanalysis) allows us to study dynamicstates within the cells, e.g. to get cluesabout cell development, proliferation,and regulation. We have been able todemonstrate the potential of the highlysensitive protein analysis toolsavailable now. These allow advancedstudies in the analysis of signaltransduction events, tumordevelopment, drug screening, andprotein marker assignment for earlydiagnosis. Great efforts have beenmade to make the group one of theworld leaders in proteome research.

Selected Publications

Braun, B.C., Glickman, M., Kraft, R.,Dahlmann, B., Kloetzel, P.-M., Finlay,D., and Schmidt, M. (1999) The baseof the proteasome regulatory particleexhibits chaperone-like activities.Nature Cell Biol. 1, 193-252.

Brockstedt, E., Otto, A., Rickers, A.,Bommert, K., and Wittmann-Liebold,B. (1999) Preparative high resolutiontwo-dimensional electrophoresisenables the identification of RNApolymerase B trancription factor 3 asan apoptosis-associated protein in thehuman BL-60-2 Burkitt lymphomacell line, J. Protein Chem. 18, 225-231.

Hutloff, A., Dittrich, A.M., Beier,K.C., Eljaschewitsch, B., Kraft, R.,Anagnostopoulos, I., and Kroczek,R.A. (1999) ICOS is an inducible T-cell co-stimulator structurally andfunctionally related to CD28. Nature397, 263-266.

Müller, E.-Ch., Schümann, M.,Rickers, A., Bommert, K., Wittmann-Liebold, B., and Otto, A. (1999) Studyof Burkitt lymophoma cell lineproteins by high resolution two-dimensional gel electrophoresis andnanoelectrospray mass spectrometry,Electrophoresis. 20, 320-330.

Schmidt, M., Zantopf, D., Kraft, R.,Kostka, S., Preissner, R., andKloetzel, P.-M. (1999) Sequenceinformation within proteasomalprosequences mediated efficientintegration of β-subunits into the 20Sproteasome complex. J. Mol. Biol.288, 117-128.

Structure of the Group

Group leaderDr. Brigitte Wittmann-Liebold

ScientistsDr. Joachim BoetzelDr. Dierk JorckeSusanne KostkaDr. Regine KraftDr. Eva-Christina MüllerDr. Albrecht OttoMargitta Schümann

Graduate studentsVolker BadockFrank EßmannOlaf Schäfer

Technical assistantsBettina FaassGerlinde GrelleHelga Neubauer

Visiting scientists and guestsDr. Peter Jungblut, BerlinDr. J. Klein, Louisville, KY, USAProf. Dr. Theodora Choli-Papadopoulou, Thessaloniki,GreeceProf. Dr. Tsezi Egorov, Moscowand coworkersDr. Anton Ivanov, Novosibirsk,Russia

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61

Cell Growth and Differentiation

The elucidation of mechanisms of cellgrowth and differentiation providesthe basis for understanding aberrantprocesses that occur in cancer andcardiovascular diseases. The geneticinformation contained in all cell typesof the human body, such ashepatocytes, blood cells andcardiomyocytes, is basically identical.Differences in the appearance andfunction of these cells are thengenerated during embryonicdevelopment through a range ofdifferentiation processes. Moreover,cell growth and cell death (apoptosis)are two basic principles ofdevelopment and homeostasis that areunder stringent regulation. Inpathophysiological situations, likecancer and cardiovascular diseases,these basic mechanisms becomederegulated. Cancer cells grow in anuncontrolled fashion. They fail toterminally differentiate, and theypenetrate surrounding tissue andmigrate to distant sites in the bodywhere they do not belong, i.e., theybecome invasive and metastatic.Disturbances of growth anddifferentiation also play crucial rolesin cardiovascular diseases: de-differentiation and proliferation ofsmooth muscle cells can cause asevere reduction in the vessel lumen.Moreover, the inability ofcardiomyocytes to proliferate preventstissue regeneration after injury to theheart.

The sequence of the entire humangenome will soon be available and afuture goal will be to understand theimplications of these gene sequenceswith respect to normal developmentalprocesses and to the development ofdisease. It will become possible tounderstand not only the function ofsingle genes, but also to identifyentire cascades of genes whoseproducts interact and regulate eachother. Research directed at obtaining acomprehensive and overall view ofthese subjects is termed FunctionalGenomics. Functional Genomics,which is the key interest of ourdepartment, complements the in-depth, molecule-by-molecule,pathway-by-pathway studies carriedout by many molecular and cellbiology laboratories around the world.

Today, we know that cancer is adisease of a distinct set ofmalfunctional genes. Malfunctionoccurs by mutations in these genes bytwo types of mechanisms: activationof genes that support cell growth(proto-oncogenes), and inactivation ofgenes that arrest cell growth (tumorsuppressor genes). Genes involved inthe control of growth anddifferentiation and in cancer havebeen found to function in I) signaltransduction (exemplified by the rasoncogene, which is part of thesignaling cascade of tyrosine kinasereceptors and plays a role in 20 - 30 % of human tumors), II) in generegulation (like the myc oncogene),III) in maintenance of genomicstability and control of apoptosis (likethe p53 tumor suppressor gene, whichis mutated in approximately 50 % ofall human tumors), or IV) inpreservation of normal tissue integrity(like the APC tumor suppressor gene,which is mutated in 80 % of coloncarcinomas).

In the Cell Growth and DifferentiationProgram of the MDC, several groupsare studying mechanisms of action ofproto-oncogenes and tumor suppressorgenes that function in the control ofsignal transduction and generegulation. Among the studiesperformed at the MDC, it has beenfound that the transcription factorC/EBPβ specifically interacts with theSWI/SNF chromatin remodellingcomplex, and that this interaction isrequired to activate a group ofmyeloid genes in collaboration withthe Myb proto-oncogen product.Grafting the N-terminus of C/EBPβonto Myb generates a chimerictranscription factor that recruitsSWI/SNF and activates chromosomalgenes, even in the absence of C/EBP.This shows that SWI/SNF recruitmentis an important feature of the Myb-C/EBP collaboration, and it is the firstdemonstration that in vertebrates theSWI/SNF complex may be recruitedby transcription factors to remodelchromatin at distinct sets of genes(Kowenz-Leutz, E. and Leutz, A.Molecular Cell 4, 735-743, 1999). Apotential new tumor suppressor gene,conductin, has been identified, and ithas been shown that conductininduces the degradation of β-cateninand blocks wnt signaling. Conductinis a scaffold protein which assemblesa multiprotein complex by binding toβ-catenin, the tumor suppressor geneproduct APC and the serine/threoninekinase GSK3 at separate domains.Complex formation is responsible forthe phosphorylation of β-catenin byGSK3β which leads to ubiquitinationof β-catenin and its subsequentdestruction by the proteasome(Behrens et al. Science 280, 596-599,1998). NF-κB is an important survivalfactor of tumor cells. It has beenfound that IκB kinases phosphorylateNF-κB p105 in the cytoplasm and thatthis results in rapid degradation ofp105 by the proteasome. NF-κB p50which is formed by processing ofp105, is liberated and transported tothe nucleus. Since p50 homodimersare specifically bound by theoncoprotein Bcl-3, TNFα or IL-1stimulation results in rapidaccumulation of transcriptionallyactive p50-Bcl-3 complexes in thenucleus (Heissmeyer et al. EMBO J.18, 4766-4778, 1999). To test thefunction of the chemokine receptorCCR7, mice were generated in whichthe CCR7 locus has been disrupted bygene targeting. Lymph nodes (LN) ofCCR-7 deficient mice were found tobe devoid of naive T cells and

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Cell Growth and Differentiation

dendritic cells (DC). Adoptive transferexperiments to wild-type recipientshas demonstrated that the migration ofCCR7-deficient T cells and B cellsinto LN, Peyers patches, and spleen isseverely hampered. The overalldisturbed microarchitecture ofsecondary lymphoid organs, causedby the impaired entry and retention oflymphocytes and antigen-presentingDC, may explain why CCR7-deficientmice fail to mount at rapid primary Bor T cell response (Förster et al., Cell99, 23-33, 1999).

Disturbances of growth anddifferentiation also play a crucial rolein cardiovascular diseases. Cardiacmyocytes build the contractileapparatus of the heart and respond toincreased work load by an increase incell numbers (proliferation) duringfetal development. However, soonafter birth cardiomyocytes lose theircapacity to proliferate and onlyrespond to changing physiologicalneeds by an increase in cell size(hypertrophy). Although adaptive bynature, this hypertrophic response canultimately lead to heart failure. Thepermanent withdrawal from the cellcycle (terminal differentiation)efficiently protects cardiac myocytesagainst malignant transformation, butalso prevents tissue regeneration afterinjury to the heart. In contrast,vascular smooth muscle cells (VSMC)maintain the ability to changebetween a proliferative and adifferentiated, non-proliferative, statethroughout their life. This allowsefficient damage repair after injury aswell as the formation of new or largervessels, but can also become a majorclinical problem as it contributes tothe reduction in the vessel lumen(stenosis). Interventional procedures(angioplasty) to restore normal vessellumen frequently fail as they excite aproliferative response of thesurrounding VSMC leading torepeated lumen loss (restenosis).

The ability of vascular smooth musclecells, as well as of endothelial cells toproliferate and form new vessels isalso crucial for tumor development,since the supply of nutrients andoxygen is required to sustain theuncontrolled growth of cancer cells.Several groups at the MDC arestudying the differentiation and cellcycle regulation of smooth musclecells and cardiac myocytes to developspecific approaches to control thegrowth and differentiation of thesecells in cardiovascular disease. Othergroups are investigating the regulationof the contractile apparatus of musclecells and the role of the calciumhomeostasis in healthy and diseasedhearts. Transgenic and experimentalanimal models have been establishedas disease models to develop novelstrategies to combat cardiovasculardysfunctions.

Walter Birchmeier, Achim Leutz,Heinrich Leonhardt, Claus Scheidereit

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Growth Control andGene Regulation inthe HematopoieticSystem

Achim Leutz

Red and white blood cells originatefrom hematopoietic stem cells locatedin the bone marrow. Stem cells giverise to progenitors that maydifferentiate into one of at least eighthematopoietic cell types, such aserythrocytes, neutrophils, ormacrophages. Hematopoieticproliferation, differentiation, andleukemogenesis are intimately linkedto a number of key transcriptionfactors that regulate expression oflineage-specific genes or entiredevelopmental programs.Dysregulation of the pathways thatcontrol the expression or activity ofcritical transcription factors, e.g., bymutation or viral interference, maycause various diseases such asimmune defects, anemia, or leukemia.Thus, hematopoiesis provides strikingopportunities to address bothfundamental biological questions andclinically relevant issues such as: Howare cell growth and differentiationregulated? How are proliferation anddifferentiation connected? How is cellidentity achieved during lineagecommitment? Obtaining answers tothese questions will improve ourunderstanding and treatment of manydiseases. We have set out to determinehow gene regulatory proteins controlcellular growth and differentiationprograms and how their dysregulationmay cause disease.

A bipartide gene switch

Proteins of the CCAAT/EnhancerBinding Protein family (C/EBP)induce expression of genes whichaccount for myelomonocyticcommitment, differentiation, andproliferation arrest. This becameevident when a conditional nuclearreceptor-C/EBP chimera wasexpressed and activated in progenitorcells that subsequently induced theirdifferentiation into eosinophils. Incollaboration with the cellular Mybproto-oncoprotein (c-Myb), C/EBPseven activate myeloid genes inheterologous cell types, e.g., infibroblasts. Such combinatorial geneswitches permit plasticity duringgrowth and differentiation and limitthe number of regulators andpathways required for cell typespecification. The concept ofconcerted action of transcriptionfactors has now been confirmed bymany research groups and has beenextended to other hematopoietictranscription factor interactions.

Chromatin remodeling and lineage-specific geneexpression

A prerequisite for ectopic activation ofsilent genes, such as myeloid genesinduced by Myb plus C/EBP infibroblasts, is to overcome therepressive effects of chromatin. This isaccomplished by large proteincomplexes that locally remodelchromatin. An assay that we haveestablished to monitor activation ofendogenous, chromatin embedded geneshas helped to unravel the mechanism ofthe collaboration between Myb andC/EBPβ. It became evident thatC/EBPβ specifically interacts with theSWI/SNF complex, and that thisinteraction is required to activate agroup of myeloid genes. An amino-terminal peptide which is containedonly in one particular isoform ofC/EBPβ (see below), is required forSWI/SNF recruitment. Grafting the N-terminus of C/EBPβ onto Mybgenerates a chimeric transcription factorthat recruits SWI/SNF and activateschromosomal genes, even in theabsence of C/EBPβ. This shows thatSWI/SNF recruitment is an importantfeature of the Myb- C/EBPβcollaboration. It is also the firstdemonstration in vertebrates that theSWI/SNF complex may be recruited bytranscription factors to remodelchromatin at distinct sets of genes.

Cell growth arrest anddifferentiation

In addition to inducing differentiation,C/EBPs arrest cells in the G1 phase ofthe cell cycle. To understand howC/EBPs mediate both proliferationarrest and differentiation, weinvestigated whether oncoproteinscould interfere with distinct C/EBPfunctions. Of various oncogenesexamined, only E7, from the high-riskpapillomavirus type 16 or 18 strains,abrogated C/EBPα-induced growtharrest. Remarkably, E7 did notinterfere with differentiation,suggesting that the two C/EBPfunctions can be separated (seefigure). Since C/EBPs are expressedin mammary epithelium, cervicalepithelium and skin, our results implythat elimination of C/EBP-mediatedproliferation arrest might contribute topapilloma pathology. Furthermore, theresults suggest that C/EBPs act astumor suppressor proteins and,therefore, are targets of tumorigenesis.

GBX2 is a homeobox targetgene of Myb

The product of c-Myb regulates genesinvolved in stem cell self-renewal andin progenitor differentiation. It is,therefore, important to identify criticalMyb target genes and determine theirfunction. Recently, we isolated thehomeobox gene GBX2 as a target ofMyb. Ectopic expression of GBX2 inprecursor cells changes theirphenotype and growth propertiessuggesting that GBX2 is involved inhematopoiesis and the establishmentof the transformed phenotype by theMyb onogene.

GBX2 gene expression is directlyinduced by a leukemogenic version ofMyb, whereas its activation by c-Mybdepends on a co-activated receptortyrosine kinase or ras pathway. Thus,leukemogenic Myb represents a gain-of-function derivative of its cellularcounterpart. Moreover, the resultssuggest that a signaling cascaderegulates c-Myb function.

Activation of GBX2 by c-Mybdepends on signaling from the cellsurface. This is of particular interestsince the Drosophila melanogasterhomologue of GBX2, the unpluggedgene, is downstream of the FGFreceptor during tracheal development.This implies that regulation of GBX2expression is part of a conserved

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developmental pathway that mayinvolve the Myb onco-protein. Insupport of such a speculation are ourobservations that murine GBX2 andFGF-2 knock-outs display epistatichematopoietic defects and GBX2 andFGF-2 are co-expressed inhematopoietic cell lines. We are,therefore, searching for a link betweenGBX2, FGF-2, its receptor, and Myb.

Interestingly, the same mutations inleukemogenic Myb that constitutivelyactivate GBX2 concomitantlyabrogate the collaboration betweenMyb and C/EBP. Accordingly, theyare loss-of-function mutations forC/EBP collaboration. Since C/EBPinduces cell differentiation andproliferation arrest, it appears that theoncoprotein abolishes the function ofa genetic switch that controls terminaldifferentiation of myeloid cells.

Translational regulation oftranscription factors

Several protein isoforms arise fromboth GBX2 and C/EBP mRNAs byalternative initiation of proteintranslation at different start codons.The isoforms give rise to DNAregulatory proteins with entirelydifferent functions. In the case ofC/EBPs, full-length proteins are trans-activators while an internally initiatedprotein is a repressor. The C/EBPtransactivator proteins mediateproliferation arrest and cellulardifferentiation, whereas the repressorpermits proliferation. Long and shortprotein isoforms are also generatedfrom the GBX2 mRNA. UnlikeC/EBPs, however, long GBX2isoforms are repressors whereas theshort form is an activator. Theactivator GBX2 supports expressionof at least one cytokine that promotesprecursor cell proliferation. Thus,internal start site usage will supportgrowth because short, growth-promoting isoforms replace the long,

differentiation-promoting isoforms ofC/EBP and GBX2. In contrast,preferential initiation from the firststart codons will supportdifferentiation by increasing the poolof long isoforms. Site-directedmutagenesis has revealed thattranslation initiation control relies ona highly conserved small upstreamopen reading frame (uORF). We havenow begun a detailed analysis of therelationship between GBX2 andC/EBP isoform expression, translationinitiation factor activity, regulation byuORF, and the biological functions ofprotein isoforms. From our results, wefound that two differentially initiatedC/EBPβ isoforms display strikingdifferences in recruitment of theSWI/SNF complex. It is anticipatedthat pathways and factors involved inthe control of translational initiationare important regulators ofhematopoiesis and may be noveltargets for innovative drug therapies.

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Figure 27: Proliferation in terminallydifferentiated adipocytes is induced by the E7oncogene. Cell division is evident by separatingmetaphase chromosomes and terminal fat celldifferentiation by storage of fat droplets in thecytoplasm. The model underneath indicates thatthe E7 oncoprotein uncouples C/EBP programsfor proliferationarrest and for differentiation.

C/EBPα

Differentiation Proliferationarrest

E7

Selected Publications

Kowenz-Leutz, E., and Leutz, A.(1999) A C/EBP-beta Isoform recruitsthe SWI/SNF complex to activatemyeloid genes. Mol. Cell 4, 735-743.

Müller, C., Alunni-Fabroni, M.,Kowenz-Leutz, E., Mo, X,Tommasino, M., and Leutz, A. (1999)Separation of C/EBPalpha mediatedgrowth arrest and differentiation. Proc. Natl. Acad. Sci. USA 96, 7276-7281.

Niss, K., and Leutz, A. (1998)Expression of the homeobox geneGBX2 during chicken development.Mech. Dev. 76, 151-155.

Kowenz-Leutz, E., Herr, P., Niss, P.,and Leutz, A. (1997) The homeoboxgene Gbx2 is a target of the Myboncogene that mediates autocrinegrowth and monocyte differentiation.Cell 91, 185-195.

Patent Application

198 59 780.0

Structure of the Group:

Group leaderDr. Achim Leutz

ScientistsDr. Stephane AnsieauDr. Valerie BegayDr. Cor CalkhovenDr. Elisabeth Kowenz-LeutzDr. Hubertus Pietsch

Graduate StudentsKnut NissSimone Joschko

TechnicansMarion BengsMaria HofstätterSabine Krause

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Regulation ofTranscription inMammalian CellsClaus Scheidereit

Cellular growth and differentiationlargely depends on the expression ofsets of genes which are controlledessentially at the level oftranscription. Differential geneexpression patterns are programmedby transcription factors, whoseactivity in turn is modulated bycomplex networks of signaltransduction cascades. The mainobjective of our laboratory is tounderstand how signal transductionprocesses are coupled to transcription.A model system with widephysiological and medical relevance isnuclear factor kappaB (NF-κB) and itsco-regulators and accessory proteins.A major goal of our research is tounderstand the structures andmechanisms underlying generegulation by this complex system.

Pathways and structures thatregulate NF-κB activity

The pleiotropic transcription regulatornuclear factor κB (NF-κB) plays animportant role in the inducibleexpression of a large number of geneswhich encode cytokines, surfacereceptors, adhesion molecules,transcription factors and othermolecules controlling various immunefunctions as well as cellular growth orprogrammed cell death. In its inactive,latent form NF-κB is kept in thecytoplasm by association with IκBmolecules, which inhibit nucleartranslocation and DNA binding of NF-κB. Stimulation of cells with a varietyof agents, such as bacteriallipopolysaccharides (LPS), phorbolesters (PMA), tumor necrosis factor α(TNFα), interleukin-1 (IL-1) or UVlight results in the proteolysis of theIκB molecules and liberation of activeNF-κB into the nucleus. Induced IκBproteolysis is triggered by IκBphosphorylation mediated by an IκBkinase (IKK) complex, which isactivated by the many NF-κB-stimulating pathways. Thecomposition and regulation of theIKK complex is under investigationand, of particular interest, is theidentification of molecules whichdirectly activate or inhibit thecomplex.

Differential regulation of NF-κBactivity by IκBα, IκBβ, p105 andBcl-3

The mammalian NF-κB familyconsists of five members, p50, p65,p52, the protooncogene product c-Reland RelB. These conserved proteinsform various hetero- and homodimersand are bound by IκB moleculesIκBα, β, and ε, the IκB-like precursorproteins for p50 and p52, p105 andp100, respectively, or by the nuclearIκB homologue Bcl-3. We have foundthat in human cells IκBβ is expressedas two distinct splicing variants,IκBβ1 and IκBβ2. While both formsequally well associate with NF-κB,they differ in their responsiveness tosignals and sub-cellular localization.Due to a lack of components of acarboxyterminal PEST sequence,IκBβ2 is only weakly degraded inresponse to inducing agents and so itsrelative abundance determines theresponsiveness of a given cell. In Blymphocytes, IκBβ1, but not IκBβ2,is found in the nucleus and maycontribute to the persistent NF-κB

activity in these cells. These findingsmay also indicate that the efficiencyof the IKK complex, whichphosphorylates IκBβ1 and 2 atinvariant aminoterminal residues, isaffected by the presence of the PESTdomain. The IKK complexphosphorylates IκBβ and IκBα at aconserved signal response domain andthis sequence, containing also lysinesfor phosphorylation-dependentubiquitin-conjugation, is sufficient toconfer inducible degradation. A short50 amino acid sequence of IκBα,when fused to other proteins, triggersdegradation of these proteins whencells are activated by TNFα or otheragents which activate IKKs. We havealso found that the NF-κB precursorproteins, p105 and p100, which onprocessing give rise to p50 and p52,sequester other NF-κB subunitsincluding their processing products inthe cytoplasm and so act like IκBmolecules. On stimulation with NF-κB activating agents, cellular p105 isphosphorylated by same kinetics asIκBα. We have now found that IKKsphosphorylate p105 and that the majorsites are three serines close to thecarboxyterminal end of p105.Phosphorylation at these sites byIKKs results in rapid, completedegradation of p105 by theproteasome. p105-associated NF-κBsubunits, such as p50, which isformed by processing of p105, areliberated and are transported to thenucleus. Thus, in parallel with therelease of NF-κB dimers by induceddegradation of IκBα or IκBβ1, otherNF-κB subunits, including p50homodimers, are released by p105degradation. Since p50 homodimersare specifically bound by the nuclearIκB homologue Bcl-3, TNFα or IL-1stimulation results in rapidaccumulation of p50-Bcl-3 complexesin the nucleus. The protooncogeneproduct Bcl-3 acts like atranscriptional co-activator for p50homodimers, which lack their owntransactivation domains. Severalnuclear cofactors and chromatin-modifying proteins have beenidentified which potentiate thetranscription activation potential ofBcl-3-p50 complexes. Theseaccessory proteins include the histoneacetylase, Tip60, whichsuperstimulates Bcl-3-mediatedtranscriptional activation and formsquarternary complexes with p50-Bcl-3bound to promoter DNA.

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Requirement of NF-κB forgrowth and survival oflymphoma and leukemia cells

In collaboration with the researchgroup of B. Dörken, we havediscovered the crucial role ofconstitutive nuclear NF-κB activity inthe viability of malignant cells inHodgkin’s disease (HD). NF-κBcounteracts programmed cell deathand, hence, may critically contributein the etiology of HD. Similarly,antiapoptotic effects of NF-κB havebeen demonstrated by other groups intransformed cell lines, primary murinecells or breast cancer cells.Constitutive NF-κB activity is furtherrequired for cell cycle progression ofHD cells. However, proliferation ofvirally transformed cell lines with aninactivated retinoblastoma protein(pRB) checkpoint do not require NF-κB activity. In collaboration with thegroup of M. Strauss, we can nowdemonstrate with primary non-transformed cells that NF-κB is, infact, also required for growth factorsignaling in normal primary cells andpromotes G1 to S phase transition byregulating the RB pathway. NF-κBactivates transcription of the cyclinD1 promoter in response to serumstimulation and, thereby, contributesto pRB phosphorylation. Furtherfunctional connections between NF-κB and cell cycle regulator proteinsare under investigation.

A characteristic feature of HD cells isthe constitutive presence of NF-κBp50-p65 in the nucleus. Our recentanalysis of Hodgkin cells has shownthat the NF-κB/IκB system isdysregulated in a cell-autonomousmanner, involving both mutations ofIκB genes and aberrant activation ofthe IKK complex. Similar constitutiveNF-κB activation has been found inacute lymphoblastic leukemia (C-ALL), again caused by IKKactivation. Further studies are beingperformed to elucidate the mechanismof constitutive NF-κB activation.

Selected Publications

Hirano, F., Chung, M., Tanaka, H.,Maruyama, N., Makino, I, Moore,D.D., and Scheidereit, C. (1998)Alternative splicing variants of IκBβestablish differential NF-κB signalresponsiveness in human cells. Mol.Cell. Biol. 18, 2596-2607.

Hirano, F., Hirano, Y., Tanaka, H.,Handa, H., Makino, I., andScheidereit. C. (1998) Functionalinterference of Sp1 and NF-κBthrough the same DNA binding site.Mol. Cell. Biol. 18, 1266-1274.

Krappmann, D., Emmerich, F.,Kordes, U., Scharschmidt, E., Dörken,B., and Scheidereit, C. (1999)Molecular mechanisms of constitutiveNF-κB/Rel activation inHodgkin/Reed Sternberg cells.Oncogene 18, 943-53.

Hinz, M., Krappmann, D., Eichten,A., Heder, A., Scheidereit, C., andStrauss, M. (1999) NF-κB function ingrowth control: Regulation of cyclinD1 expression and G0/G1 to S phasetransition. Mol. Cell. Biol. 19, 2690-2698.

Heissmeyer, V., Krappmann, D.,Wulczyn, F.G., and Scheidereit, C.(1999) NF-κB p105 is a target of IκBkinases and controls signal-inductionof Bcl-3-p50 complexes. EMBO J. 18,4766-4778.

Structure of the Group

Group leaderDr. Claus Scheidereit

ScientistsDr. Eunice Hatada*Dr. Michael Hinz*Dr. Fuminori Hirano*Dr. Uwe Kordes*Dr. Daniel KrappmannDr. Stefan Mathas*Dr. Felix Mehrhof*Dr. Benjamin Mordmüller*Dr. Ruth Schmidt-Ullrich

Graduate and undergraduate studentsAnnette Ahlers Vigo HeißmeyerSebastian Tegethoff*

Technical assistantsErika ScharschmidtSabine JungmannRudolf DettmerKarin Ganzel*

SecretariatDaniela Keyner

* part of the period reported

68

Differentiation andGrowth Control inLymphocyteDevelopment andFunction

Martin Lipp

The identification and functionalanalysis of differentiation and growthcontrol genes in lymphocytedevelopment will improve ourunderstanding of how these genes areinvolved in the multistep process oftumorigenesis and immuno-pathogenesis. Many of these genesmay also represent potential targetsfor novel therapeutical strategies. Inthis context, our investigations arefocussing on the following researchprojects:I) role of chemokines and

chemokine receptors inlymphocyte migration,organogenesis of lymphoid tissuesand immune responses.

II) immune modulatory and growth-inducing functions of chemokinereceptors encoded by humanherpesviruses

III) role of lysosphingophospholipidreceptors in the immune system.

IV) regulation and function ofCD155/polio virus receptor.

V) cell cycle-dependent control oftranscription.

Functional organization oflymphoid organs by thechemokine system

Chemokines are small basic proteinswhich exert their chemoattractiveactivities via binding to seven-transmembrane-domain receptorssignaling through heterotrimeric G proteins. Chemokines and theirreceptors can be broadly divided intotwo functionally distinct categories.On one hand, inflammatorychemokines, induced or upregulatedby inflammatory stimuli, areresponsible for recruiting cellsinvolved in acute inflammatoryreactions; on the other, constitutivechemokines, produced in bonemarrow, thymus and secondarylymphoid organs, are responsible forthe homeostatic control of leukocytetraffic and for mediating encountersbetween cells that need to interact togenerate an immune response. Ourrecent finding, that the chemokinereceptor BLR1/CXCR5 is needed forB cell migration into lymphoidfollicles, is the first experimentalevidence that the chemokine systemplays an essential role as a regulatorof migration of lymphocyte subsetsand is involved in the functionalcompartmentalization of lymphoidorgans.

Generation of monoclonal antibodiesspecific for human CCR7 revealedexpression of CCR7 on peripheral Tcell subsets, B lymphocytes andmonocytes. Furthermore, whereasCCR7 was not detected on monocyte-derived immature dendritic cells(DC), surface expression of CCR7was gradually up-regulated followingin vitro induced maturation of DC. Totest the function of CCR7, weproduced mice whose CCR7 locushad been disrupted by gene targeting.Lymph nodes (LN) of CCR7-deficientmice are devoid of naive T cells andDC and adoptive transfer experimentsto wild-type recipients demonstratedthat the migration of CCR7-deficientT cells and B cells into LN, Peyerspatches, and spleen was severelyhampered. Therefore, the overalldisturbed microarchitecture ofsecondary lymphoid organs, caused bythe impaired entry and retention oflymphocytes and antigen-presentingDC, may explain why CCR7-deficientmice fail to exhibit a rapid primary Bor T cell response. In collaborationwith A. Lanzavecchia, Basel, we haveshown that the memory response ismediated by two distinct T cell

subsets: tissue-seeking CCR7- effectormemory T cells (TEM) provideimmediate protection in inflamedtissue while lymph node-seekingCCR7+ central memory T cells (TCM)provide help for DC and B cells andgenerate a new wave of effector cells.Thus, by bringing togetherlymphocytes and DC to form thecharacteristic microarchitecture andfunctional microenvironments ofsecondary lymphoid organs, thehomeostatic chemokine system hasbeen shown to be an importantregulator of lymphocyte homing and,consequently, functions as acoordinator for initiating an antigen-specific immune response andcreating immunological memory.

Immune modulatory andgrowth-inducing functions ofviral chemokine receptors

We have previously shown thatEpstein-Barr-Virus (EBV) specificallytransactivates expression of thecellular chemokine receptor CCR7 byits regulatory nuclear factor EBNA2.In contrast to EBV, several otherhuman herpesviruses, likecytomegalovirus or the lymphotropichuman herpesviruses type 6 (HHV-6)and Kaposi’s sarcoma-associatedherpesvirus (KSHV), also termedHHV-8, encode viral chemokinereceptors and chemokines in theirgenome suggesting that herpesvirusesuse the chemokine system to interferewith the growth and differentiationprogram of the host and subvertspecific immune responses.Epidemiological and molecularevidence has linked infection withKSHV to the pathogenesis of allforms of Kaposi’s sarcoma, a non-Hodgkin’s B cell lymphoma, andmulticentric Castleman’s disease(MCD). This research project is aimedat establishing whether the KSHV-encoded chemokine receptor (KSCR),which is known to be constitutivelyactivated and able to induceproliferation, plays a role in thedevelopment of human herpesvirus 8-associated diseases and malignanciesas an essential oncogenic or paracrinefactor, or both. Murine tumor modelsand KSHV-specific vaccines based onrecombinant vaccinia viruses havebeen developed to prove whether theviral chemokine receptor induces aneffective immune response.

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Role of lysosphingophospholipidreceptors in the immune system

We have cloned human EDG6 from invitro differentiated dendritic cells inorder to identify novel G protein-coupled receptors (GPCR) that controlimmune functions. EDG6-specificRNA was detected almost exclusivelyin lymphoid and hematopoietic tissue,and in lung. Moreover the expressionpattern of EDG6 was found to beconserved in mouse and man.Homology of EDG-6 to the recentlyidentified sphingosine-1-phosphate(SPP) receptors EDG-1, -3 and -5 andlysophosphatidic acid (LPA) receptorsEDG-2 and -4. suggests that its ligandmay be a lysophospholipid orlysosphingolipid. In collaborationwith S. Spiegel, Washington, we haveshown that EDG-6 is a high affinityreceptor for SPP which couples to aGi/o protein resulting in the activationof growth-related signaling pathways.Although the biological significanceof SPP signaling via EDG-6 inlymphocytes and dendritic cells is

poorly understood, the wellcharacterized growth-related orcytoskeleton-associated activities ofSPP suggest that members of the EDGfamily may synergize with signalingpathways initiated by cytokines.Lysosphingophospholipids may play acritical role as potent autocrine andparacrine mediators in specific micro-environmental settings of normal andpathophysiological immune responses.

Differentiation-specificregulation and function ofCD155/poliovirus receptor

CD155, a transmembrane proteinpossessing an Ig-like architecture, wasdiscovered originally by its ability toserve as the cellular receptor forpoliovirus (PV). Since then, a lot ofeffort has been devoted to elucidatethe involvement of CD155 in PVinfection, a complicated series ofevents with the potential outcome ofthe poliomyelits syndrome in affectedindividuals. However, there is no

information about the natural functionof CD155 and, so, we initiated studiesI) to investigate the expression profileof CD155 (by means of analysing thepromoter of the CD155 gene) and II)to identify counter receptors/ligandsof CD155. Our findings suggest thatCD155 is one of the cell adhesionclass of molecules since it interactswith vitronectin and, to a lesserextent, with fibronectin. Currently, weare investigating these interactions inmore detail. Interestingly,immunohistochemical studies haveshown that expression of vitronectinand CD155 colocalizes to thegerminal centers of secondarylymphoid tissue.

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Figure 28: Lessons from chemokine receptorknock outs: Model of chemokine-directedtrafficking of lymphocytes and dendritic cells toand through secondary lymphoidorgans duringthe immune response.

B

T

T

B

B B

BB

TT

TT T

B zone

T

LTα1β2 LTβR

T

afferentlymphatic vessels

CXCR5

HEV

efferentlymphatic vessels

iDC

FDC

stromal cells

stromal cells

peripheraltissue

maturation& migration

DC

immature antigen-capturing LC

(skin)

CCR7

CCR7

CXCR5

CXCR5SLC

BLC

ELC

SLC

CCR7

CCR7

SpleenPPLN ?

CCR7

Cell cycle-dependenttranscriptional control via E2F

Cell proliferation is controlled by anetwork of extracellular andintracellular signalling pathwaysleading either to initiation andmaintenance, or arrest of cell cycleprogression. Transitions betweencertain cell cycle stages are regulatedat checkpoints monitored bycoordinately regulated kinase cascadesturning genes on and off. Recentevidence suggests that transcriptionfactors of the E2F-family and tumorsuppressor protein RB do not onlycontrol genes necessary for cell cycleprogression, but also induce growtharrest and apoptosis followingoncogenic and hyperproliferativesignals by activating p53, a tumorsuppressor protein known to becomephosphorylated and govern checkpointarrest in response to DNA- damagingagents. It is further supposed thatphosphorylation of p53 occursthrough a DNA-dependent kinase(DNA-PK) composed of a largecatalytic subunit and two DNA-targeting proteins, Ku70 and Ku80.DNA-PK is also involved in DNAdouble-strand break repair andrecombination of immunoglobulingenes. Based on our recent findingthat E2F factors physically interact viaa conserved domain with Ku70 andcan be phosphorylated by the DNA-PK holoenzyme, we have proposedthat functional interaction of E2F andDNA-PK abrogates E2F-dependenttranscription and, thereby, congregatesthe antiproliferative and apoptoticsignals induced by DNA-damagingagents.

Selected Publications

Förster, R., Schubel, A., Breitfeld, B.,Kremmer, E., Renner-Müller, I., Wolf,E., and Lipp, M. (1999) CCR7coordinates the primary immuneresponse by establishing functionalmicroenvironments in secondarylymphoid organs. Cell 99, 23-33.

Sallusto, F., Lenig, D., Förster, R.,Lipp, M., and Lanzavecchia, A.(1999) Two subsets of memory T cellswith distinct homing potential andeffector function. Nature 401, 708-712.

Wolf, I., Pevzner, V., Kaiser, E.,Bernhardt, G., Claudio, E., Siebenlist,U., Förster, R., and Lipp, M. (1998)Downstream activation of a TATA-less promoter by Oct-2, Bob1, andNF−κB directs expression of thehoming receptor BLR1 to mature Bcells. J. Biol. Chem. 273, 28831-28836.

Gräler, M.H., Bernhardt, G., and Lipp,M. (1998) EDG6, a novel G protein-coupled receptor related to receptorsfor bioactive lysophospholipids, isspecifically expressed in lymphoidtissue. Genomics 53, 164-169.

Solecki, D., Wimmer, E., Lipp, M.,and Bernhardt, G. (1999).Identification and characterization ofthe cis-acting elements of the humanCD155 gene core promoter, J. Biol.Chem, 274, 1791-1800.

Patent Application

Gräler, M., Bernhardt, G., Lipp, M.“G-Protein gekoppelter RezeptorEDG6 und seine Verwendung”.

Structure of the Group

Group leaderDr. Martin Lipp

ScientistsDr. Reinhold Förster Dr. Günter BernhardtDr. Felix CifireDr. Uta HöpkenDr. Angelika KuschDr. Christian Ried

Graduate and undergraduate studentsRalf Burgstahler* Markus GrälerFrank JeblonskiRudolf Gabor KabaRobert LangeThilo MokrosGerd MüllerLars Ohl Veniamin PevznerSteffen PosnerPhilipp Reiterer

TechniciansDagmar BreitfeldPeter Graßhoff Carmen Meese*Dagmar Meyer

SecretariatDaniela Keyner

Associated research projectDr. Gabriele Pecher, FU BerlinHeiko JohnenHagen KulbeThomas Schirrmann

* part of the period reported.

71

MechanismsControlling theInitiation of DNAReplication

Manfred Gossen

The research group is interested inthe mechanisms controlling theinitiation of DNA replication inmulticellular eukaryotes. Apart frombacteria, viral systems or yeast, boththe cis and the trans acting elementsin metazoans contributing to theinitation of replication are poorlycharacterized. This is, however, aprerequisite for a detailedunderstanding of those processescontrolling cellular proliferation. Itwould also allow new insights in theway the cell safeguards one aspect ofits its genomic integrity. To this end,we intend to investigate thearchitecture of chromosomalreplication origins as well as toanalyse the proteins binding to them.This work will be conducted withmammalian tissue cultures as well aswith Drosophila embryos or culturedcells.

Analyzing the functionalarchitecture of a Drosophilareplication origin

The chorion gene region of Drosophilamelanogaster encompasses an originof DNA replication, which by now isprobably the best characterized of allmetazoa. Its activity has beendemonstrated in specialized cells ofthe ovary (the follicle cells), where itdirects the developmentally controlledamplification of its neighbouringchromosomal regions. In recent yearswe have identified and analysed theorigin recognition complex (ORC) ofDrosophila. It is expected to interactwith origins in concert with otherreplication initiation factors like cdc6and MCM proteins (minichromosomemaintanance). Recent data show thisinteraction also occurs in chorionorigins. However, it remains to bedetermined if this origin is active incells other than follicle cells and, ifso, which of its sequences direct thetissue-specific amplification program.We plan to investigate the formerquestion in Drosophila embryos aswell as in insect tissue cultures. Suchstudies are needed to decide if thechorion replication origin can serve asa model system for origin architecturein metazoans.

Expression profiling of humanreplication genes

The proteins participating in theinitiation of DNA replication are onlypoorly characterized at thebiochemical level, which is largelydue to the lack of a suitable in vitroassay for replication. As an alternativeapproach to gain insight into theirfunction and regulation, we plan toundertake extensive quantitativeexpression profiling of the RNA aswell as at the protein level. Thisanalysis will be performed bycomparing cycling vs. resting and/ordifferentiated cells. We hope to learnwhich of the replication initiationfactors are downregulated innonproliferating cells and if this isactually one of the mechanisms bywhich unscheduled replication inthese cells is prevented.

Initiation proteins as diagnosticand prognostic markers forcancer cells

in collaboration with R. C. Bargou,K. Bommert and B. Dörken, Berlin

Unlike the downregulation of at leastsome of the replication initiationgenes in differentiated cells,reactivation of these genes has to takeplace in cells which reenter the cellcycle. Thus, one has to postulate thattumor cells, for example, express thecomplete set of DNA replicationinitiation genes described above. Wewant to analyze quantitative andqualitative parameters of thisreactivation and hope to learn, if theexpression of these genes can alsoserve as a diagnostic and prognosticmarker for various malignant diseases.This part of our work relies on theimmunohistochemical detection of thereplication proteins. We plan to testthe feasibilty of such an approach bycomparing expression levels inbiopsies from undiseased tissue withthose from tissue representing variousstages of multiple myeloma.

Selected Publications

Chesnokov I., Gossen M., Remus D.,and Botchan M.R. (1999) Assemblyof fuctionallytive Drosophila originrecognition complex from recombinantproteins. Genes Dev. 1999 13, 1289-1296.

Gossen M., Pak D.T.S., Hansen S.K.,Acharya J.K., and Botchan M.R.(1996) Drosophila homolog to theyeast origin recognition complex.Science 270, 1674-1677.

Structure of the Group

Group leaderDr. Manfred Gossen

ScientistDr. Katrin Engel

Graduate StudentPierre Debs

TechnicianMarion Papst

72

Cell Cycle RegulationHans-Dieter Royer

Recently, global expression studieshave been reported which revealedthat hundreds of different genes arespecifically expressed in variousphases of the cell cycle.Transcriptional control during the cellcycle, however, is poorly understood.Our current work is concentrating onseveral transcription factors whichoperate in the G1-, G1-/S- and S-phase. It is the aim of these studies toevaluate the functional role of thesefactors in cell cycle regulation andhuman diseases. In these studies, wehave discovered that the transcriptionfactor YB-1 is highly deregulated invarious malignant diseases, and wehave been able to establish agenotype-phenotype correlation forbreast cancer. We have shown thatYB-1 regulates the expression of thehuman mdr-1 gene which encodes P-glycoprotein, a multidrug transporter.Multidrug resistance is a phenotypewhich seriously hinderschemotherapy, and P-glycoproteinexpression is a major factor involvedin clinical multidrug resistance. Basedon these results, we have started novelprojects aimed at understanding thefunction of YB-1 in malignantdiseases, multidrug resistance and cellproliferation. It is the aim of our YB-1related research to identify signalpathways which lead to nuclear YB-1accumulation in multidrug resistanttumors and to develop selectiveinhibitors of this process. With thisapproach, we intend to develop noveltherapeutic strategies for the treatmentof clinical multidrug resistance.

Transgenic YB-1 mice

S. Bergmann in cooperation with F. Theuring (Berlin) and M. Dietel(Berlin)

The aim of this project is to analyzethe in vivo consequences of YB-1overexpression in breast epithelialcells. We have produced severaltransgenic mouse lines which expressan HA-tagged YB-1 cDNA under thecontrol of the beta-lactoglobulinpromoter. The expression levels ofYB-1 in these transgenic mice differconsiderably. These mice will bemonitored over time byhistopathological techniques. We arealso analyzing YB-1-regulated genesin mouse breast epithelial cellsoverexpressing YB-1.

Identification of YB-1-interacting proteins

K. Jürchott, Y. Shan, M. Janzin cooperation with R. Kraft (MDC)and J. Behrens (MDC)

The Y-box factor YB-1 is amultifunctional cellular protein which,besides being a transcription factor,participates in several aspects of RNAmetabolism. In order to obtain a betterunderstanding of YB-1 functions, weare identifying YB-1 interactingproteins. Cellular YB-1-interactingproteins have been identified byaffinity chromatography using acolumn with YB-1 peptide-antibodies.YB-1-protein complexes were elutedwith the immunizing peptide andseveral novel interacting proteins wereidentified by amino acid sequenceanalysis. In addition, we have used the yeast two-hybrid system to isolateseveral novel YB-1 interacting proteins.We are currently characterizing theseinteraction partners by GST-pulldownexperiments and in vivoimmunoprecipitation. These studiesare aimed at identifying proteinswhich are involved in regulating YB-1nuclear transport. The first candidatewhich could be involved in thisprocess has been identified.

Nuclear overexpression of YB-1as a prognostic marker formalignant diseases

M. Janz, A. Schmid in cooperationwith M. Schmitt, D. Dettmar(München) and M. Dietel (Berlin)

We were the first to discover thatnuclear overexpression of YB-1 inhuman breast cancers is associatedwith P-glycoprotein expression.Besides being responsible for amultidrug resistant phenotype, P-glycoprotein expression is also amarker for more aggressive tumorbehavior. We have initiated severalprojects which address this issue. Onemajor study has been completed andis currently being analyzed. We havedetermined the YB-1 expressionpatterns in over 100 breast cancerswhose clinical courses have beendetermined. This study will showwhether YB-1 is a prognostic markerin breast cancer. We are nowscreening systematically theexpression patterns of YB-1 in severalhuman malignancies. These studieswill show whether clinical multidrugresistance and deregulated YB-1expression are also correlated in othermalignancies. This work will establishthe biological relevance of YB-1 formultidrug resistance and provide abasis for the development of noveltherapeutic approaches to thetreatment of multidrug resistanttumors.

Development of gene transfervectors for the treatment ofmalignant tumors

Ch. Woischwill, M. Janz, K. Jägerin cooperation with G. Wolff and B. Dörken

Overexpression of YB-1 has beenobserved in breast cancer and certainother malignant diseases. It has beenreported that YB-1 is a prognosticmarker in osteosarcoma and ovariancancer. It is the aim of thiscooperative project to developadenoviral gene therapy vectors whichcontain the YB-1 promoter as acontrol element for the expression oftherapeutic genes. We have cloned theYB-1 promoter and critical promoterelements are being characterized byseveral approaches. A vector has beenconstructed containing the completeYB-1 promoter in order to examinetransgene expression levels in variousmouse tissues and human tumor

73

transplants. We are now generatingtherapeutic vectors expressingapoptosis-inducing genes under thecontrol of the YB-1 promoter.

Regulation of humanpapillomavirus 18 oncogenetranscription

E. Grinstein, I. Weinert, W. Jiain cooperation with R. Kraft (MDC),S. Hauptmann and M. Dietel (Berlin)

High risk human papillomaviruses e.g.HPV18 and HPV16 are causativeagents of squamous cell carcinomas ofthe cervix uteri and cervical cancer isthe leading cause of death in India andChina. Furthermore, the frequenciesof cervical cancers are markedlyincreased in HIV- infected women. Inthe last two years, we have studied thetranscription regulation of HPV18oncogene expression. The cell cycle-regulated HPV18 enhancer factor p92was purified and its amino acidsequence has been determined. Wehave used a p92-GST Fusion proteinand have shown that p92 binds in asequence-specific fashion to theHPV18 enhancer. Antisense knock-outexperiments have revealed that p92 isa key regulator of HPV18 oncogenetranscription. Using DNase I, we haveshown that p92 is involved inregulating chromatin accessibility ofthe HPV18 enhancer. In addition, wehave discovered that, in high gradesquamous intraepithelial lesions, p92expression is deregulated suggestingthat in vivo deregulated p92expression levels are a critical eventin cervical carcinogenesis. Theseresults have been submitted forpublication.

Selected Publications

Bargou, R.C., Jürchott, K., Metzner,S., Wagener, C., Dietel, M., Guski, H.,Dörken, B., and Royer, H.-D. (1997)Nuclear localization and increasedlevels of transcription factor YB-1 inprimary human breast cancers areassociated with intrinsic MDR1 geneexpression. Nat. Med. 3, 447-450.

Bargou, R.C., Emmerich, F.,Krappmann, D., Bommert, K.,Mapara, M.Y., Arnold, W., Royer,H.D., Grinstein, E., Scheidereit, C.,and Dörken, B. (1997) ConstitutiveNF-kappa B- RelA activation isrequired for proliferation and survivalof Hodgkin’s disease tumor cells. J.Clin. Invest. 100, 1-9.

Jundt, F., Leng, C., Krappmann, D.,Emmerich, F., Mapara, M.Y.,Bommert, K., Royer, H.-D.,Scheidereit, C., and Dörken, B. (1999)Hodgkin/Reed-Sternberg cells inducefibroblasts to secrete eotaxin, a potentchemoattractant for T cells andeosinophils. Blood 94, 2065-2071.

Royer, H.-D., Grinstein, E., and Janz,M. (1999) Regulation des Zellzyklusund therapeutische Implikationen. DerOnkologe 5, 884-890.

Patent Applications

Holm, P.S., Royer, H.-D., Dietel, M.,Bergmann, S., Lage, H., Jürchott, K.,Braandt, A., and Ladhoff, A. (1999)Mittel zur Behandlung malignerErkrankungen unter Verwendung desProteins YB-1. Aktenzeichen 199 29569.7.

Wolff, G., Royer, H.-D., Woischwill,C., Janz, M., Schumacher, A., andDörken, B. (1999) Gentransfervektorfür die Diagnostik und die Therapievon malignen Tumoren. Aktenzeichen198 60 602.8.

Bergmann, S., Jürchott, K., Brand, K.,Holm, P.S., and Royer, H.-D. (1999)Mittel zur Diagnose und Therapiemaligner Erkrankungen. Aktenzeichen199 29 747.9.

Weitz, J., Royer, H.-D., Stöhr, M.,Kopun, M., and Napierski, I. (1999)Nuclear Inhibitor I-92 and its use forthe production of a medicament. U. S.Patent Application No. 08/183,909.

Weitz, J., Royer, H.-D., Stöhr, M.,Kopun, M., and Napierski, I. (1999)Nuclear Inhibitor I-92 and its use forthe production of a medicament.Europäische Patentanmeldung Nr. 92115 730.1.

Structure of the Group

Group leaderDr. Hans-Dieter Royer

ScientistsDr. Martin JanzDr. Karsten JürchottDr. Edgar Grinstein*

Graduate studentsStephan BergmannYing ShanChristiane Woischwill (clinical cooperation, Dr. G. Wolff)Wentao JiaA. Schmid

Technical assistantsIngeborg WeinertKatrin Jäger (clinical cooperation, Dr. G. Wolff)Gerlinde Grelle

*part of the period reported

74

EpithelialDifferentiation,Invasion, andMetastasis

Walter Birchmeier

Our laboratory is concentrating on themolecular analysis of epithelialmorphogenesis and differentiation.Earlier, we defined the adhesion andsignaling capacities of the E-cadherin/catenin system. Moreover, we haveinvestigated the role of scatterfactor/hepatocyte growth factor(SF/HGF) and its receptor, c-mettyrosine kinase, in the morphogenesisof epithelial cells. Epithelial cells canlose expression of E-cadherin duringtumor progression, and this losscorrelates with the appearance ofhighly invasive carcinoma cells. Thefunction of cadherins depends directlyon cytoplasmic linkage molecules, β-catenin or plakoglobin, whichmediate interaction of cadherins withthe cytoskeleton. We have shown thatβ-catenin also binds to thetranscription factor LEF-1, and thatthis interaction translocates β-cateninto the cell nucleus and regulates geneexpression. This provides a molecularmechanism for transmission of signalsfrom cell adhesion components andthe wnt signalling pathway to the cellnucleus.

The scatter factor/c-met systemtransduces various signals in epithelialcells, such as scattering,differentiation and proliferation. Oneunique activity of SF/HGF and c-meton epithelial cells in culture is theability to induce branching or othermorphogenic events. We have recentlyidentified a new substrate of c-met,Gab1, which mediates the signalresponsible for branchingmorphogenesis. Gab1 is a member ofthe family of membrane-boundmultiadapter proteins which transmitsignaling of tyrosine kinase receptors.

Functional interactions of β-catenin with LEF-1, conductinand APC are specified throughdistinct hot spots in thearmadillo domain

Jens-Peter von Kries, GeorgiaWinbeck, Christian Asbrand, NataliaSochnikova and Andrea Dell’Oro. Incollaboration with Jürgen Behrens(MDC)

β-Catenin is a component of the wntsignalling pathway which plays animportant role in developmentalprocesses: wnt signals increase theamount of β-catenin in the cytosol byinactivation of the serine-threoninekinase GSK-3β. In the absence of wntsignals, GSK-3β phosphorylates β-catenin which inducesubiquitination and degradation of β-catenin by proteasomes. We haverecently found that β-catenin ispresent in the cytosol as a largemultiprotein complex with GSK-3βconductin or axin, and the tumoursuppressor gene product APC. Otherlabs have reported that mutations in β-catenin or mutations in APC inhuman tumors stabilise β-catenin andresult in constitutive LEF/TCFbinding and nuclear signalling. Thus,the regulation of β-catenin stability ispivotal for the transmission of wntsignals in embryonic development andtumor progression. β-Catenin containsa critical sequence, the armadillorepeats 3-8, that provides binding sitesfor the cytoplasmic fragment of E-cadherin, the 20 and 15 amino acidrepeats of APC, the N-terminal regionof LEF/TCF and a central domain ofconductin/axin.

We have now identified amino acidsof β-catenin that directly affect APC,conductin or LEF-1/TCF binding.These residues form separate clustersin the superhelix built by armadillorepeats 3-8. Point mutations in one ofthe APC or conductin binding sites donot stabilise β-catenin. Mutantsunable to interact with both APC andconductin are fully stabilised. So, fordegradation, it is sufficient ifconductin or APC is recruitedindirectly to β-catenin and, thus, tothe degradation complex. Thesemutants will now allow a functionalanalysis of the individual β-catenininteractions in development or tumorprogression.

Requirement for β-catenin inanterior-posterior axis formationin mice

Jörg Hülsken, Regina Vogel andVolker Brinkmann. In cooperationwith Carmen Birchmeier (MDC) andBettina Erdmann (MDC)

The anterior-posterior axis of themouse embryo becomes explicitmorphologically at E6.5, when thefirst mesoderm forms in the primitivestreak region at the posterior side.However, recent experiments showthat anterior-posterior polarity isestablished at least one day earlier: thefirst signs of anterior-posteriorpolarity are detectable by asymmetricexpression of Cerberus-like, Hex andother markers in the prospectiveanterior portion of the visceralendoderm. In Xenopus and Zebrafish,components of the wnt signalingpathway have been implicated in theinduction of embryonic body axis. InXenopus, accumulation of β-cateninon the dorso-anterior side of theembryo is the earliest sign of axisformation. Accordingly,overexpression of β-catenin inXenopus embryos induces formationof an additional embryonic axis.

We recently generated β-catenin-deficient mouse embryos andobserved a defect in anterior-posterioraxis formation at E5.5, as reflected inthe absence of Hex and Hesx1 and themislocation of Cerberus-like andLim1 expression. Subsequently, nomesoderm and head structures aregenerated. Intercellular adhesion ismaintained since plakoglobinsubstitutes for β-catenin. Our datashow that β-catenin function isessential for anterior-posterior axisformation in the mouse, andexperiments with chimeric embryosconfirm that this function is requiredin the embryonic ectoderm.

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Coupling of Gab1 to c-Met, Grb2and downstream effectorsmediate biological responses

Ute Schaeper, Martin Sachs, NielsH. Gehring, Renate Franke andIngrid Walther. In collaboration withBettina Kemkes (GSF Munich) andCarmen Birchmeier (MDC)

Gab1, like the insulin receptorsubstrates (IRS), the FGF receptorsubstrate FRS/SNT, and p62dokfamily members belongs to a newlyidentified group of docking proteinsthat function as specific substrates oftyrosine kinases. Gab1 contains an N-terminal PH domain and a novelphosphotyrosine recognition domainwhich mediate direct association withthe c-Met receptor. Gab1 binds to twosites of the cytoplasmic tail of c-Met,Y14 (Y1349) and to a lesser extentY15 (Y1356). Gab1 also forms aconstitutive complex with Grb2 andthis interaction is mediated via the C-terminal SH3 domain of Grb2.

We have now mapped the c-Met andGrb2 interaction sites using reverseyeast two-hybrid technology. The c-Met binding site is localized to a 13amino acid region unique to Gab1.Insertion of this site into the Gab1-related protein p97/Gab2 wassufficient to confer c-Met bindingactivity. Association with Grb2 wasmapped to two sites: a classical SH3binding site (PXXP) and a novel Grb2SH3 consensus binding motif(PP(V/I)(D/N)RXXKP). To detectphosphorylation-dependentinteractions of Gab1 with downstreamsubstrates, we have developed amodified yeast two-hybrid assay andidentified PI(3)K, Shc, Shp2 andCRKL as interaction partners of Gab1.In a trk-met specific branchingmorphogenesis assay, association ofGab1 with Shp2, but not PI(3)K,CRKL or Shc was essential to inducebranching morphogenesis in MDCKcells. A fundamental role of Gab1 forc-Met specific signaling is alsosupported by gene ablationexperiments in the mouse: Gab1 -/-embryos produced in our laboratoryare characterized by strongly reducedand delayed migration of myogenicprecursor cells into the limbs, aphenotype reminiscent of HGF/SF -/-and c-Met -/- mutant embryos.

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Figure 29: Ablation of the β-catenin gene inmice results in a defect of anterior-posterioraxis formation at embryonal day 6.0. Themarker Cerberus is mislocated in the -/- β-catenin embryos (arrow at the distal tip in d)whereas, in the wild-type embryo, Cerberusmarks the anterior side (arrow in c).Subsequent head development at the anteriorside is abrogated (as shown by the markerOtx2, compare e and f). Embryonal andextraembryonal tissues are properly developed(as shown by expression of BMP4, see a and b).Work by J. Hülsken, R. Vogel, V. Brinkmann, B. Erdmann, C. Birchmeier, W. Birchmeier. J. Cell Biol. 2000.

Selected Publications

Hartmann, G., Prospero, T.,Brinkmann, V., Ozcelik, Ö., Winter,G., Hepple, J., Batley, S., Bladt, F.,Sachs, M., Birchmeier, C.,Birchmeier, W., and Gherardi, E.(1997) Engineered mutants ofHGF/SF with reduced binding toheparan sulphate proteoglycans,decreased clearance and enhancedactivity in vivo. Curr. Biol. 8, 125-134.

Behrens, J., Jerchow, B.-A., Würtele,M., Grimm, J., Asbrand, C., Wirtz, R.,Kühl, M., Wedlich, D., andBirchmeier, W. (1998) Functionalinteraction of an axin homolog,conductin, with β-catenin, APC, andGSK3β. Science 280, 596-599.

Meiners, S., Brinkmann, V., Naundorf,H., and Birchmeier, W. (1998) Role ofmorphogenetic factors in metastasis ofmammary carcinoma cells. Oncogene16, 9-20.

Niemann, C., Brinkmann, V., Spitzer,E., Hartmann, G., Sachs, M.,Naundorf, H., and Birchmeier, W.(1998) Reconstitution of mammarygland development in vitro:Requirement of c-met and c-erbB2signalling for branching and alveolarmorphogenesis. J. Cell Biol. 143, 533-545.

Hülsken, J., Vogel, R., Brinkmann, V.,Erdmann, B., Birchmeier, C., andBirchmeier, W. (2000) Requirementfor β-catenin in anterior-posterior axisformation in mice. J. Cell Biol. 148,567-578.

Structure of the Group

Group leaderProf. Dr. Walter Birchmeier

ScientistsDr. Martin BehrendDr. Volker Brinkmann*Dr. Yasuyuki FujitaDr. Jens-Peter von KriesDr. Markus Morkel*Dr. Patricia Ruiz*Dr. Jörg HülskenDr. Martin SachsDr. Ute SchaeperDr. Martin SeidenstickerDr. Dietmar Zechner*

Graduate studentsChristian AsbrandSilvana Di CesareJan GrimmGunnar Schütz*Natalia Sochnikova*Michael TönjesGeorgia Winbeck

Diploma studentsNiels Gehring*

Technical assistantsAndrea Dell’OroRenate FrankeRegina VogelIngrid Walther

SecretariatIrmgard WiznerowiczGerhild Richter

*part of the period reported

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Cell Adhesion andSignal Transduction

Jürgen Behrens

Our group studies the dual role of thecytoplasmic component β-catenin incadherin-mediated cell adhesion andthe wnt signaling pathway. As part ofthe cell adhesion machinery β-cateninbinds to the cytoplasmic domain ofcadherins and provides a link to theactin cytoskeleton. In the wnt pathwayb-catenin transmitts signals to thenucleus by interacting with membersof the LEF/TCF family oftranscription factors. The wnt pathwaycontrols cellular interactions duringembryonal development, and theinappropriate activation of wnt signalsleads to cancer. In tumors, mutation ofthe tumor suppressor gene productAPC or of β-catenin lead to thestabilisation of β-catenin andactivation of oncogenic target genesby TCF/β-catenin complexes.

Regulation of b-catenin byconductin in the wnt signaltransduction pathway

Boris Jerchow, Martin Würtele,Thomas Schwarz-Romondin cooperation with W. Birchmeier(MDC)

In the past three years, we couldidentify new components that interactwith β-catenin and mediate essentialsteps in the wnt pathway. In 1996, weidentified the functional interaction ofβ-catenin with the HMG boxtranscription factor LEF-1. In 1998,we found that β-catenin interacts withthe novel protein conductin. We couldshow that conductin induces thedegradation of β-catenin and blockswnt signaling in Xenopus embryos.We could further demonstrate thatconductin is a scaffold protein whichassembles a multiprotein complex bybinding to β-catenin, APC and theserine/threonine kinase GSK3b atseparate domains. GSK3b isresponsible for the phosphorylation ofβ-catenin which leads toubiquitination of β-catenin and itssubsequent degradation inproteasomes. We propose thatconductin coordinates the interplay ofβ-catenin, APC and GSK3β in the b-catenin-degradation process byproviding a platform for the assemblyof these factors. Conductin negativelycontrols wnt signaling and might actas a tumor suppressor.

We are currently studying the role ofconductin in vivo by gene ablation inthe mouse. We will in particularanalyse whether the conductin-negative mice are prone to tumorformation and show lack of control ofβ-catenin. We also analyse theexpression of conductin in tumors andsearch for possible mutations in theconductin gene (collaboration withP.M. Schlag, Robert-Rössle-Klinik).Further studies aim at the analysis ofcellular consequences of conductin-mediated degradation of β-catenin intumor cells, and the identification ofregulators of conductin function byusing yeast two hybrid screeningmethods.

Interaction of β-catenin withSWI/SNF factors

Ralph Wirtz

Transcription of many genes iscontrolled by a multiprotein complexcomposed of SWI and SNF proteins,which regulate chromatin assembly byaltering nucleosome positioning. Wefound that β-catenin interacts with amouse homolog of the yeast SWI3protein named BAF155. Interestingly,BAF155 and TCF factors associatewith different parts of the central armrepeat domain of β-catenin, indicatingthat β-catenin may provide a linkbetween the two transcriptionalregulators. Indeed, inimmunoprecipitation experimentsLEF-1 co-precipitates with BAF155which is dependent on the b-cateninbinding domain of LEF-1. We proposethat β-catenin recruits the SWI/SNFcomplex to TCFs to promotetranscription of wnt target genes. Wecould also show that SNF5, which is acomponent of the SWI/SNF complex,competes with β-catenin for bindingto BAF155. SNF5 has been assigned arole as a tumor suppressor in earlychildhood rhabdoid tumors. Mutationsof the SNF5 gene lead to C-terminaltruncations of the protein. We foundthat SNF5 mutant proteins showreduced binding to BAF155 andcompete less efficiently with β-catenin than wild-type SNF5. We alsofound that TCF/β-catenin dependentgene transcription is active inrhabdoid tumor cells and can beblocked by re-introduction of wild-type but not mutant SNF5. We suggestthat SNF5 prevents the interaction ofthe TCF/β-catenin complexes with theSWI/SNF machinery. The loss offunctional SNF5 in rhabdoid tumorsmay allow recruitment of theSWI/SNF complex to oncogenic wnttarget genes.

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Selected Publications

Behrens, J., Jerchow, B.-A., Würtele,M., Asbrand, C., Wirtz, R., Grimm, J.,Wedlich, D., and Birchmeier, W.(1998) Functional interaction of anaxin homolog, conductin, with b-catenin, APC, and GSK3 β. Science280, 596-599.

Batsche, E., Muchardt, C., Behrens,J., Hurst, H.C., and Cremisi, C. (1998)RB and c-Myc activate expression ofthe E-cadherin gene in epithelial cellsthrough interaction with transcriptionfactor AP-2. Mol. Cell. Biol. 18,3647-3658.

Ghadimi, B.M., Behrens, J.,Hoffmann, I., Birchmeier, W., andSchlag, P.M. (1999)Immunohistochemical analysis of E-cadherin, α-, β-, and γ-cateninexpression in colorectal cancer:implications for cell adhesion andsignaling. Europ. J. Cancer 35, 60-65.

Behrens, J. (1999) Cadherin/catenin-mediated signal transduction. In:Guidebook to the ExtracellularMatrix, Anchor, and AdhesionProteins. Th. Kreis, R.Vale (eds.)Oxford.

Behrens, J. (1999) Cadherins andcatenins: role in signal transductionand tumor progression. CancerMetastasis Rev 18, 15-30.

Patent Application

“Mittel zur Diagnose und Therapievon Tumorerkrankungen”

Structure of the Group

Group leaderDr. Jürgen Behrens

ScientistDr. Lepu Zhou

Graduate and undergraduate studentsBoris JerchowThomas Schwarz-RomondRalph WirtzMartin Würtele*

Technical assistantKatharina Feller

*part of the time reported

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GlycoconjugatesUwe Karsten

Most cellular proteins requireposttranslational modifications inorder to function properly. The studyof these processes is a necessarycomplementary approach to researchon direct genetic causes of disease.

Glycosylations are among the mostcommon protein modifications.Essential properties of membraneproteins, such as conformation,charge, interaction with other proteins,or biological half-life are largelyinfluenced by glycosylation. Theglycans themselves are involved incell-cell, cell-matrix, and ligand-receptor interactions. Changes in theglycan composition of membraneproteins are regularly found during thecourse of normal development as wellas in cancerogenesis and tumorprogression. ‘Tumor antigens’ are, infact, often glycotopes and theycontribute to the invasive andmetastatic growth of cancer cells.

We are studying the structure,distribution, functional role andclinical relevance of cancer-associatedcarbohydrate antigens, their role inmetastasis, and possible interventionstrategies. In doing so, we closelycooperate with the Robert RössleClinic and others. Special emphasis isgiven to Thomsen-Friedenreich (TF)and related antigens and their mostprominent carrier molecule, epithelialmucin (MUC1). A major goal is thedevelopment of novel tumourvaccines against minimal residualcancer based on these antigens.

We are actively involved ininternational leukocyte and tumormarker workshops (TD-4, HLDA7).

Thomsen-Friedenreich-relatedantigens and tumor vaccines

U. Karsten, G. Butschak, S. GoletzCollaboration with H. Gollasch, A. Pezzutto, W. Kemmner, P.M. Schlag (Robert Rössle Clinic), S.E. Baldus, F.-G. Hanisch(Cologne), P. Kristensen (Aarhus), K. Lloyd (New York), S. v. Mensdorff-Pouilly (Amsterdam),H. Paulsen (Hamburg), J. Schreiber(Berlin), H. Shigeoka (Osaka), G. Winter (Cambridge)

In carcinomas the heavilyglycosylated, apically expressed high-molecular weight epithelial mucinMUC1 is underglycosylated. Thisleads to the exposure of otherwisemasked peptide epitopes, and to theappearance of new carbohydrateepitopes (TF). In comprehensivestudies we have shown that TF is anexceptionally specific tumor antigen,and that its expression in colorectalcarcinomas is an independentprognostic marker and risk factor forthe development of liver metastases.We have been able to demonstrate in amouse model that blocking TFepitopes on tumor cells with antibodyA78-G/A7 significantly reduces thenumber of liver metastases. We intendto follow up this finding as a newstrategy for the prevention of livermetastasis after resection of TF-positive colon tumors.

So far, tumor vaccines using syntheticMUC1 peptides have beenunsuccessful because of their lowimmunogenicity. We have observedthat most MUC1-specific antibodiesbind much better if the peptide isglycosylated at the immunodominantPTDR motif with GalNAc or TF.Based on this and other results, wehave devised a new MUC1 vaccinewith a glycosylated PDTR sequenceand a phase I clinical study with thisvaccine will start soon.

Carbohydrate antigens, albeit oftenhighly specific tumor antigens, aregenerally not well suited to vaccineformulations. Their synthesis isexpensive and, in most cases, theyevoke only incomplete immuneresponses consisting mainly of IgMantibodies and lacking cytotoxic Tcells. To circumvent these drawbacks,we are at present developingcarbohydrate vaccines based onmolecular mimicry. By employingadvanced phage display techniques,among them proteolytic selection, we

have been able to select a number ofhuman single-chain antibodyfragments (see figure) and peptideswhich mimic TF. This is not only thefirst successful mimicry of the TFdisaccharide, but also opens up thehitherto unavailable option ofdeveloping DNA vaccines for this andother carbohydrate antigens.

We are also examining thespecificities and titers of natural ‘anti-TF’ antibodies in human sera and theirchanges in cancer patients in order toexplore their potential application as aserum tumour assay.

Carbohydrate-mediated celladhesion to activatedendothelium

R. Stahn, C. GrittnerCollaboration with K. Wenzel(Humboldt University)

Cell adhesion to the vascularendothelium and subsequentextravasation into the surroundingtissue are important steps ininflammatory diseases and cancer.They are regulated by severaladhesion molecules and their ligands.E-selectin is crucial for cell ‘rolling’on vessel walls as a first step in theadhesion cascade. It is exclusivelyexpressed on activated endothelium,and specifically recognizescarbohydrates of the Lewis type (sLex,sLea).

We are pursuing two strategies to useE-selectin as a specific target tointerfere with the adhesion cascade: 1) blockade of E-selectin binding bymeans of glycoconjugates carryingsLex moieties, and 2) site-specific, E-selectin-mediated transport of agentswhich interfere with later stages of theadhesion cascade.

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Selected Publications

Karsten, U., Diotel, C., Klich, G.,Paulsen, H., Goletz, S., Müller, S.,and Hanisch, F.-G. (1998) Enhancedbinding of antibodies to the DTRmotif of MUC1 tandem repeat peptideis mediated by site-specificglycosylation. Cancer Res. 58, 2541-2549.

Cao, Y., Karsten, U., and Hilgers, J.(1998) TD-4 Workshop Group 9:Immunohistochemical characterizationof a panel of 56 antibodies withnormal human small intestine, colon,and breast tissues. Tumor Biol. 19(Suppl.1), 88-99.

Shigeoka, H., Karsten, U., Okuno, K.,and Yasutomi, M. (1999) Inhibition ofliver metastases from neuraminidase-treated Colon 26 cells by an anti-Thomsen-Friedenreich-specificmonoclonal antibody. Tumor Biol. 20,139-146.

Stahn, R., Schäfer, H., Kernchen, F.,and Schreiber, J. (1998) Multivalentsialyl Lewisx ligands of definitestructures as inhibitors of E-selectinmediated cell adhesion. Glycobiology8, 311-319.

Baldus, S.E., Zirbes, T.K., Hanisch,F.-G., Kunze, D., Shafizadeh, S.T.,Nolden, S., Mönig, S.P., Schneider,P.M., Karsten, U.R., Thiele, J.,Hölscher, A.H., and Dienes, H.P.(2000) Thomsen-Friedenreich (TF)antigen presents as a prognostic factorin colorectal carcinomas: a clinico-pathological study including 264patients. Cancer (in press).

Patent Applications

Karsten, U., Hanisch, F.-G., Paulsen,H.: Tumour vaccine against MUC1-positive carcinomas. PCT/DE 98/03819; 30.12.98.

Jeschke, U., Karsten, U., Haase, M.:Monoklonale Antikörper gegenGlykodelin A, Verfahren zu ihrerHerstellung und ihre Verwendung. 198 07 389.5; 21.2.98.

Goletz, S., Karsten, U.: Vakzine gegenkonformationsabhängige Antigene. 199 24 405.7; 27.5.99.

Structure of the Group

Group leaderDr. Uwe Karsten

ScientistsDr. Günter ButschakDr. Steffen GoletzDr. Renate Stahn

Graduate and undergraduate studentsChristine GrittnerAnnett KramerChristiane Kohrt

Technical assistantsAnnemarie KoberlingSabine RudolphHeidrun Wunderlich

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Intracellular Signalingin the Myocardium

Ernst-Georg Krause

The action of the heart as a pumpdepends on the coordination ofelectrical, mechanical and metabolicactivity of its cells. The primeregulator of these activities appears tobe the intracellular calciumconcentration which is subject tonerval and/or humoral mechanisms.These involve second-messengermediated phosphorylation of specificproteins which are known to directlyinfluence calcium transport throughthe sarcolemma and the vesicles of thesarcoplasmic reticulum (SR) incardiac as well as smooth musclecells.

Phospholamban, a pentameric proteincomplex composed of identicalsubunits, is thought to be the keyphosphoprotein in regulating calcium-pump (SERCA) activity of SRvesicles, thereby controlling therelaxation process. In thedephosphorylated state,phospholamban acts as an inhibitor ofSERCA, but this inhibition isabolished with increasingphosphorylation. Interestingly, bothcAMP-dependent protein kinase(PKA) and Ca2+/calmodulin-dependentprotein kinase (CaM Kinase)specifically phosphorylate eachphospholamban monomer at serine 16and threonine 17 residues,respectively. The functionalsignificance of this dual-sitephosphorylation is not yet known.

In the failing human heart, there isincreasing evidence that inotropicstimulation by cAMP-dependent, aswell as cAMP-independent,mechanisms results in inadequatediastolic relaxation with consequencesfor pumping efficiency. Thesealterations have been suggested to bedue to altered intracellular calciumhandling. Indeed, a reduced amount ofSERCA and an impairment in

phospholamban phosphorylation maylead to a slower diastolic calciumdecay. Thus, the function of the SRcalcium pump may be modulated attwo levels by I) changes in theexpression of SERCA, phospholambanand their respective protein kinasesand II) alterations of the regulation ofcalcium transport at the level of SRvesicles.

The research group “IntracellularSignalling in the Myocardium” isengaged in basic research that ismainly focused on molecularmechanisms related to proteinphosphorylation and dephosphorylationand to the short-term regulation ofcontraction and relaxation of thenormal and diseased myocardium, aswell as long-term processes withregard to Ca2+ homeostasis, growth,and differentiation of cardiac musclecells.

Intracellular signaling in themyocardium

S. Bartel and P. Karczewski incooperation with R. H. G.Schwinger, University of Köln; M. Kuschel, P. Xiao, E. Lakatta,NIH, Baltimore, USA; A. Kaumann,The Babraham Institute,Cambridge, U. K.; P. Molenaar,University of Melbourne, Australia

We have established that thephosphorylation of phospholamban byPKA at serine-16 prevails over thephosphorylation at threonine-17 andoccurs in parallel with the monitoredacceleration of relaxation after β-adrenergic stimulation. Interestinglythe detectable threonine-17phosphorylation was inhibited in thepresence of the L-type Ca2+ channelblocker, verapamil. These data favor aspatial Ca2+ signal for activation ofCaM kinase activity near the L-typeCa2+ channel. Indeed, L-type Ca2+

channel activators, like BayK8644 andBayY5959, induce phosphorylation ofthreonin-17 in the absence ofadrenergic stimulation. A dissociationof β2-adrenoreceptor signaling fromthe cAMP regulatory systems hasbeen evaluated for phosphorylation ofcytoplasmic proteins, like troponin Iand C protein, but not forphosphorylation of the L-type Ca2+

channel in the canine heart.

Our data on the short-term regulationby catecholamines in nonfailing andfailing human hearts clearly shows

that, in the post-adenylyl cyclasesignaling pathway of the failing heart,the phosphorylation of phospholambanis impaired, followed by disturbancesin Ca2+ sequestration, therebyprolonging the diastolic phase of eachcardiac cycle. Elucidating theselective activation of β2-adrenergicreceptors in the human heart allowedus to clearly show that β2-adrenoceptoractivation is mediated by activation ofPKA and phosphorylation ofphospholamban at serine-16 andthreonine-17, as well as of troponin Iand C protein. These data are relevantto strategies for therapeuticintervention(s) in patients with end-stage heart failure.

CaMKII in heart function andcardiac remodeling

P. Karczewski and B. Hoch incooperation with J. Bohlender,Franz Volhard Clinic, HumboldtUniversity Berlin; A. Remppis,University of Lübeck; R. Meyer,German Heart Institute Berlin; S. Hatem, INSERM Paris

The multigene family ofCa2+/calmodulin-dependent proteinkinases II (CaMKII), as a universalmediator in Ca2+ signaling, is involvedin many of the functions of eukaryoticcells. CaMKII is a multimerconsisting of 8 to 12 subunits encodedby four different genes (α,β,δ,γ). As amajor regulator of Ca2+ homeostasis,CaMKII is essential for heartfunction. Despite its importance, littleis known about the isoforms ofCaMKII expressed in the heart andtheir specific function in the normaland diseased myocardium. Inextension of our previous work, wehave characterized four isoforms ofthe δ-class, the dominant cardiacCaMKII, during rat heartdevelopment. Our data haveestablished that the isoform δ3 ischaracteristic for the adultmyocardium. Furthermore, δ4 so farcharacterized as the skeletal muscleisoform, is typically expressed in theembryonic and neonatal rat heart andbecomes down-regulated duringpostnatal development. Inhypertensive rat strains, whichdevelop a compensated cardiachypertrophy, δ4 is re-expressedwhereas the δ3 transcript levels fall.These alterations are accompanied bychanges in the δ-CaMKII proteincontent in membrane vesicles of theSR.

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Depressed contractility in human heartfailure has a variety of causes, such asimpaired Ca2+ cycling, electricalabnormalities and structuralremodeling of cardiac cells. We,therefore, characterized δ-isoforms ofCaMKII in the human ventricularmyocardium. Again, δ3 ischaracteristically expressed, whereasisoforms δ2 and δ9 are also found inhuman skeletal muscle. In explantedhuman hearts with dilatedcardiomyopathy, characterized by anincreased ANF transcript level andreduced amount of SERCA protein,there was a significant increase in δ3

transcripts and δ-CaMKII protein.This strongly suggests a role for δ-CaMKII in heart failure withisoform δ3 being a key determinant.

In the human atrium, the transientoutward K+ current is important forshaping the action potential and, thus,critical for the development ofelectrical abnormalities such asarrhythmias. We have obtainedevidence that CaMKII regulatesvoltage-gated K+ channels in humanatrial myocytes.Immunocytochemistry of atrial tissuesections shows an intense staining forδ-CaMKII in the intercalated disks,which contain most K+ channels.Using immunoblotting, we havedemonstrated increased δ-CaMKIIexpression in tissue specimens fromchronically fibrillating human atria.Thus, upregulation of δ-CaMKIIcould contribute to the electricalremodeling of the diseased atrialmyocardium.

Selected Publications

Hagemann, D., Hoch, B., Krause, E.-G., and Karczewski, P. (1999)Developmental changes in isoformexpression of Ca2+/calmodulin-dependent protein kinase II δ-subunitin rat heart. J. Cell. Biochem. 74, 202-210.

Hoch, B., Meyer, R., Hetzer, R.,Krause, E.-G., and Karczewski, P.(1999) Identification and expressionof δ-isoforms of the multifunctionalCa2+/calmodulin-dependent proteinkinase in failing and non-failinghuman myocardium. Circ. Res. 84,713-721.

Kaumann, A., Bartel, S., Molenaar, P.,Sanders, L., Burrell, K., Vetter, D.,Hempel, P., Karczewski, P., andKrause, E.-G. (1999) Activation of β2-adrenergic receptors hastensrelaxation and mediatesphosphorylation of phospholamban,troponin I and C protein in ventricularmyocardium from patients withterminal heart failure. Circulation 99,65-72.

Kuschel, M., Zhou, Y.-Y., Spurgeon,H. A., Bartel, S., Karczewski, P.,Zhang, S.-J., Krause, E.-G., Lakatta,E.G., and Xiao, R.-P. (1999) β2-adrenergic cAMP is uncoupled fromphosphorylation of cytoplasmicproteins in canine heart. Circulation99, 2458-2465.

Schwinger, R.H.G., Münch, G.,Bölck, B., Karczewski, P., Krause, E.-G., and Erdmann, E. (1999) ReducedCa2+-sensitivity of SERCA 2a infailing human myocardium due toreduced Serin-16 phospholambanphosphorylation. J. Mol. Cell. Cardiol.31, 479-491.

Tessier, S., Karczewski, P., Krause,E.-G., Pansard, Y., Acar, C., Lang-Lazdunski, M., Mercadier, J.-J., andHatem, S. N. (1999) Regulation of thetransient outward K+ current byCa2+/calmodulin-dependent proteinkinase II in human atrial myocytes.Circ. Res. 85, 810-819.

Structure of the Group

Group leaderProf. Dr. Ernst-Georg Krause

ScientistsDr. Sabine BartelDr. Brigitte HochDr. Peter Karczewski

Graduate and undergraduate studentsAmanda v. Epps*Ulrich Hildenbrand*Dirk Hagemann*Petra Hempel

Technical assistantsIngrid AmelnInge BeyerdörferDonathe VetterDorothea RiegeWolfgang Schlegel

SecretariatVerona Kuhle

*part of the period reported

83

Molecular MusclePhysiology

Ingo L. Morano

The aim of the group is to understandthe molecular mechanisms involved inthe regulation of cardiac and smoothmuscle contraction. Contractility ofboth cell types is regulated by calciumions (Ca2+) which penetrate the cellsthrough voltage-gated L-type Ca2+

channels and, subsequently, induce therelease of large amounts of Ca2+ fromthe sarcoplasmic reticulum into themyoplasm through Calcium ReleaseChannels (Ryanodin Receptors). Ca2+

activates the contractile apparatus bybinding to troponin C, allowing themolecular motor myosin to interactwith thin filaments to produce forceand shortening. Due to their key-rolesin muscle, we are studying theexpression regulation, post-translational modifications, andfunctional roles of the subunits of L-type Ca2+ channels, RyanodineReceptors, and Type II myosin incardiac and smooth muscle. Anystructural change in these keyproteins, by mutation, differentialgene expression, alternative splicingof the transcripts, or post-translationalmodification, modulates cardiac andsmooth muscle function.Understanding muscle contractionregulation at molecular and functionallevels provides the opportunity todevelop causal therapies for thetreatment of cardiac and smoothmuscle dysfunction. We are workingwith cardiac and smooth musclepreparations from transgenic/knock-out animals and humans as well ascultures of cardiomyocytes andsmooth muscle cells.

Understanding the molecularmotor

Essential myosin light chainisoforms regulate human heartcontractility

Type II myosin isoenzymes arehexamers of about 500 kDa consistingof two heavy chains (MHC) and 4light chains (MLC), designatedessential and regulatory MLC. Twogenes coding for cardiac MHC areexpressed, β-MHC and α-MHC,located as a tandem on chromosome14. The cardiac-specific MLCisoforms are designated as atrium- andventricle-specific essential (ALC-1and VLC-1, respectively) andregulatory (ALC-2 and VLC-2,respectively) MLC. Thecardiomyocytes of hypertrophiedventricles of patients with congenitalheart diseases and hypertrophiccardiomyopathy reexpress ALC-1,while MHC isoenzymes areunchanged. This is in sharp contrast tothe hypertrophied rodent ventriclewhich exhibits a change in MHCisoforms rather than MLC expression.The failing ventricles of patients withdilated cardiomypathy, however,hardly express ALC-1. Expression ofthe cardiac-specific basic-helix-loop-helix transcription factors, eHANDand dHAND which bind to E-boxelements in the ALC-1 promoter, isincreased in hypertrophied humanventricle.

Ventricular cross-bridges with ALC-1have revealed a higher shorteningvelocity and rate of force developmentthan normal cross-bridges withoutALC-1. Maximal isometric forceproduction per cross-sectional area aswell as the Ca2+ sensitivity of theforce-Ca2+ ratio are enhanced.

Besides MHC, essential cardiac MLCisoforms also bind via their N-terminus to actin. Inhibition of theactin-MLC interaction by peptidecompetition increases forceproduction and shortens the velocityof human heart fibers. We havesuggested that interaction betweenactin and MLC represents a“molecular load” for the cross-bridge,thus depressing its cycling kineticsand force production.

Regulation of smooth musclecontraction by recruitment of non-muscle myosin.

Prolonged smooth muscle activationproduces an initial transient state(phase 1) of high maximal shorteningvelocity (Vmax) and ATPconsumption which is followed by asustained state (phase 2) of forcegeneration with low Vmax and ATPconsumption. Three different genescoding for MHC are expressed insmooth muscle cells, namely, onesmooth-muscle-specific MHC (SM-MHC) and two genes coding for non-muscle MHC. We have mutated theSM-MHC gene by gene targetingtechnology and found that non-muscleMHC expression remained normal.

Smooth muscle from knock-out (KO)neonatal mice did not exhibit a phase1, although surprisingly, a phase 2was observed. Thus, the initialtransient phase 1 is generated by SM-MHC recruitment while the sustainedcontraction state can be generatedupon switching from SM- to non-muscle-MHC activation. Non-muscle-MHC-dependent sustained forcegeneration was sufficient for normalfetal development. However, phase 1i.e. high smooth muscle contractility,becomes indispensable for survivaland normal growth soon after birth,especially as far as homeostasis andcirculation functions are concerned.

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Understanding calcium handlingproteins

pp700/AHNAK is a cardiac PKAtarget and binds to the β-subunit ofCa2+ channels.

Ca2+ channels are multisubunitcomplexes composed of the pore-forming α1 subunit along withregulatory β and α2/δ subunits.Coordinated upregulation of Ca2+

channel subunit expression wasobserved in patients withhypertrophic, but not dilated,cardiomyopathy. Furthermore, wehave identified fetal isoforms of bothα2 - and β-subunits. A novel calciumchannel-associated protein of 700-kDawas detected in mammaliancardiomyocytes that undergoessubstantial protein kinase Aphosphorylation (pp700). Amino acidsequence analysis of pp700 revealedhomology to AHNAK andpp700/AHNAK is preferentiallylocalized in the plasma membrane ofcardiomyocytes. We believe that bothphosphorylation of pp700 and itscoupling to Ca2+-channels play aphysiological role in regulation ofcardiac contractility.

Selected Publications

Wetzel, U., Lutsch, G., Haase, H.,Ganten, U., and Morano, I.L. (1998)Alternative splicing of smooth musclemyosin jeavy chain in cardiac vesselsof normotensive and hypertensiverats. Circ. Res. 83, 204-20967.

Morano, I.L., Chai, G.-X., Walther, T.,Baltas, L.G., Lamounier-Zepter, V.,Kott, M., and Bader, M. (2000)Smooth muscle contraction withoutsmooth muscle myosin. Nat. CellBiol. in press.

Haase, H., Podzuweit, T., Lutsch, G.,Hohaus, G., Kostka, S., Lindschau,C., Kott, M., Kraft, R., and Morano,I.L. (1999) Signaling from β-adrenoceptor to L-type calciumchannel: identification of a novelcardiac protein kinase A targetpossessing similiarities to AHNAK.FASEB J. 13, 2161-2172.

Ritter, O., Luther, H., Haase, H.,Schulte, H.D., and Morano, I.L.(1999) Remodeling of thehypertrophied human myocardium bycardiac-specific bHLH transcriptionfactors. J. Cell Biochem. 74, 551-561.

Morano I.L. (1999) Tuning the humanheart molecular motors by myosinlight chains. J. Mol. Med. 77, 544-555.

Patent Applications

1998: “New substance to increasecardiac contractility”PCT/DE98/01240

1999: “A diagnostic marker for thehuman heart” DPA 199 19205.7

1999: “Cardiovascular activePeptides” DPA 199 33 090.5

1999: “Drug for the treatment ofcardiac insufficiency” DPA 19938 255.7

Structure of the Group

Group leaderProf. Dr. Ingo L. Morano

ScientistsDr. Hannelore HaaseDr. Monika KottDr. Leonidas BaltasDr. Udo ZacharzowskiDr. Ines PagelDr. Thomas Walther

Graduate and undergraduate studentsBirgit PfitzmaierUlrike WetzelCorina-Mihaela IsacKatarina WetzelValeria Lamounier-ZepterKatja Großmann

Technical assistantsDaniela BalzereitAnnette HohausHanna SydowUrsula KlammChristel Kemsies

SecretariatVerona Kuhle

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Figure 30: Localization of pp700/AHNAKin ratcardiac tissue (A: logitudinal and B: cross-section). Nuclei are stained with DAPI (inblue). Arrow heads: plasma membrane; largearrows: intercalated discs; small arrows:capillaries (micrograph taken by G. Lutsch,MDC)

Cell Biology ofCardiovascularDiseasesHeinrich Leonhardt M. Cristina Cardoso

Role and regulation of DNAmethylation during developmentand disease

DNA methylation is essential formammalian development and has far-reaching effects on gene expressionand genome structure. It has beenimplicated in a number of humanillnesses such as Angelman,Beckwith-Wiedeman, and Prader Willidisease and in cancer. In all thesecases, functional alleles are shut offby ectopic DNA methylation.Recently, mutations in one of theDNA methyltransferases (Dnmt3b;ICF syndrome) and in a methyl-cytosine binding protein (MeCP2;Rett syndrome) have been implicatedin human disease. Methylatedcytosine residues are also hot spots formutations resulting in C to Ttransitions, the most frequent type ofmutation found in human disease.

The longterm goal of this project is toelucidate the regulation of DNAmethylation in mammals, i.e. howDNA methylation patterns arechanged, how DNA sequences arechosen for methylation ordemethylation, how fatal errors in themethylation pattern occur and the roleplayed by genetic and/orenvironmental factors.

We are addressing these questions viathe identification and characterizationof functional domains of the knownDNA methyltransferases (Dnmt1, 2,3a and 3b) and searching forinteracting factors which mightcontrol and direct methylation activity.We have identified a targetingsequence in the regulatory domain ofDNA MTase that mediates theassociation with replication factoriesand, hence, might warrant the precisemaintenance of methylation patternsafter each round of DNA replication

(Cell, 71, 865-73). The most dramaticchanges in the overall DNAmethylation pattern occur duringpreimplantation development, whenmost methylation patterns are erased.We have now identified andcharacterized a regulatory elementthat is responsible for the cytoplasmiclocalization of Dnmt1 during earlydevelopment and, hence, might causedemethylation (JCB, 147, 25-32). Inaddition, we have been able toidentify and characterize differentisoforms of Dnmt1 (PNAS, 93,12920-5; JBC, 273, 32757-9). Incollaboration Dr. Jaenisch and hisgroup (MIT, Cambridge), we are nowstudying the role of these functionaldomains and isoforms in developmentand disease using transgenic mousetechnologies.

Functional organization of themammalian nucleus and cellcycle control

Several biological processes withinthe eukaryotic nucleus occur indiscrete subnuclear compartments (themost conspicuous being the nucleolus)which, in contrast to cytoplasmicorganelles, are not separated bymembranes. Different factors involvedin a particular process are foundconcentrated together at thesubnuclear sites where the respectiveprocesses take place, which isdesignated “functional organization ofthe nucleus”.

During our analysis of regulatorypathways leading from terminaldifferentiation to the S-phase, weobserved that cyclin A and cdk2(cyclin-dependent kinase 2) arespecifically localized at subnuclearsites of DNA replication and, hence,might function as a link between cellcycle regulation and the control ofDNA replication (Cell, 74, 979-992).We could also show that Dnmt1, aswell as replication proteins (RPA70,DNA ligase I), are specificallyredistributed to nuclear replicationfoci during the S-phase. Like Dnmt1,DNA ligase I contains a distincttargeting sequence that is necessaryand sufficient for association withreplication foci. This targetingsequence is dispensable for enzymeactivity in vitro but is most likelyrequired for the efficient ligation ofOkazaki fragments in vivo and, hence,may ensure genome integrity inmammalian cells (JCB, 139, 579-587).

Our longterm goal is to study thearchitecture, assembly and regulationof these replication factories duringthe cell cycle, including theirinteraction with cell cycle regulatorsand other nuclear components.

To study the dynamic regulation ofnuclear structures during the cell cyclein real time, we have developed aseries of fusion proteins and stablecell lines using a green fluorescentprotein (GFP) to visualize thesestructures in living cells. With thesecellular systems, we have been able toshow that replication foci patternschange throughout the S-phase in acharacteristic manner and that thechanging patterns of replication fociare not due to movements of fociwithin the nucleus. Individualreplication foci assemble at aparticular nuclear site, keep thisposition for a given period, anddisassemble after finishing DNAreplication at this site. Assembly anddisassembly of different foci occurasynchronously, suggesting thatreplication origins also fireasynchronously within thesemicroscopically visible clusters. Incollaboration with Dr. Zink and hergroup (LMU, Munich), we are nowlabelling subchromosomal domains invivo to study their nuclear localizationcompared with replication factoriesand other subnuclear compartmentsduring the cell cycle. In particular, weare investigating whether DNA isreeled through immobile replicationfactories rather than replicationmachines sliding down the DNA.

Differentiation and proliferationof smooth muscle cells

The uncontrolled proliferation ofvascular smooth muscle cells (VSMC)and the resulting formation of aneointima (called restenosis), aftercatheter-based therapy including theapplication of vascular endoprostheses(stents), is currently one of thegreatest unsolved problems ininterventional cardiology andangiology. This proliferation is mainlydue to the ability of SMCs to switchbetween contractile (differentiated)and synthetic (dedifferentiated) states,where the cells proliferate and migrateto form the neointima.

As far as basic science is concerned,we are investigating the signals andmechanisms controlling theproliferation and differentiation of

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VSMCs. We have cloned andcharacterized an isoform of acytoskeletal protein (smoothelin) thatis specifically expressed in VSMCs(JMM, 77, 294-301). We now intendto use smoothelin as a marker toidentify the signal transductionpathway controlling the differentiationof VSMCs.

As far as applications of our work areconcerned, we are collaborating withDrs. Dietz and Gross (InterventionalCardiology, FVK) and a stentmanufacturer to develop newtherapeutic options to prevent in-stentrestenosis. We are currently testingstents coated with a biodegradablepolymer for local drug administration.This approach offers the uniquepossibility of delivering activesubstances directly to the diseased andstented segment of the vessel and,thus, to directly influenceendothelialization, cell proliferationand migration and matrix deposition.

Terminal differentiation and cellcycle regulation in striatedmuscle cells

We are currently interested in themolecular mechanisms regulating theestablishment and maintenance ofterminal differentiation and indevising ways to transiently reversethis state to achieve tissueregeneration. During terminaldifferentiation in striated muscle, thelevel of retinoblastoma protein (pRb)is upregulated whereas cell cycleactivators are mostly downregulatedand differentiated myocytes arerefractile to mitogen stimulation. Wehave developed cellular systems inwhich skeletal myotubes can reenterthe cell cycle, after either transientexpression of viral oncogenes (SV40T antigen) or deletion of the Rb gene,indicating a central role of pRb in themaintenance of terminaldifferentiation (Cell, 74, 979-992). Incollaboration with Dr. Harsdorf andhis group (FVK, Berlin), we areinvestigating the moleculardifferences and similarities in terms ofcell cycle regulators among thecardiac and skeletal muscle types. Wehave tested the hypothesis that tumorsuppressors like pRb keep cardiacmyocytes out of the cell cycle byrepressing E2F transcriptional activity,thereby preventing expression ofproliferation-associated genes. Indeed,we have been able to induce S-phasereentry in cardiac myocytes usingrecombinant adenovirus

overexpressing E2F1 in the presenceof IGF-1 (Circ. Res., 85, 128-136).Using a cell-free S-phase assay inwhich isolated nuclei are incubatedwith extracts from different cell cyclestages, we have further establishedthat, although cardiac myocyte nucleican be induced to undergo S-phase inthe presence of S-phase extracts, anextract from adult cardiac myocytesinhibits S-phase entry (Circ. Res., 85,294-301). Concomitantly, we aredeveloping new approaches for directand transient delivery of proteins toaffect cellular functions in terminallydifferentiated cells. We have recentlyshown that proteins can be directlydelivered to differentiated musclecells by fusion to viral VP22 factor(JMM, 77, 609-613).

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Figure 31: Functional organization of thenucleus and subnuclear protein sorting. Upon translation by the ribosomes in thecytoplasm, a protein has several possible fates:it can stay in the cytosol or dependent onhaving specific signal peptides be imported intodifferent organelles including the nucleus.Within the nucleus, proteins (as well as othermolecules) can stay in the nucleoplasm or,although there are no separating membranes,be specifically targeted to one of an everincreasing number of subnuclear compartments.In this diagram we illustrate five of thesesubnuclear compartments visualized in livingmammalian cells by expression of differentfusion proteins tagged with the greenfluorescent protein.

Selected Publications

Gaudet, F., Talbot, D., Leonhardt, H.,and Jaenisch, R. (1998) A short DNAmethyltransferase isoform restoresmethylation in vivo. J. Biol. Chem.273, 32725-32729.

Krämer, J., Aguirre-Arteta, A. M.,Thiel, C., Gross, C. M., Dietz, R.,Cardoso, M.C., and Leonhardt, H.(1999) A novel isoform of the smoothmuscle cell differentiation markersmoothelin. J. Mol. Med. 77, 294-298.

von Harsdorf, R., Hauck, L., Mehrhof,F., Wegenka, U., Cardoso, M. C., andDietz, R. (1999) E2F1 overexpressionin cardiomyocytes inducesdownregulation of p21CIP1 andp27KIP1 and release of active cyclin-dependent kinases in the presence ofInsulin-like growth factor I. Circ. Res.85, 128-136.

Engel, F. B., Hauck, L., Cardoso, M.C., Leonhardt, H., Dietz, R., and vonHarsdorf, R. (1999) A mammalianmyocardial cell-free system to studycell cycle re-entry in terminallydifferentiated cardiomyocytes. Circ.Res. 85, 294-301.

Derer, W., Easwaran, H. P., Knopf, C.W., Leonhardt, H., and Cardoso, M.C. (1999) Direct protein transfer toterminally differentiated muscle cells.J. Mol. Med. 77, 609-613.

Cardoso, M.C., and Leonhardt, H.(1999) DNA methyltransferase isactively retained in the cytoplasmduring early development. J. CellBiol. 147, 25-32.

Cell Biology of CardiovascularDiseases

Structure of the Group

Group leaderDr. Heinrich Leonhardt

ScientistsDr. Jochen Krämer*Dr. Christina QuenselDr. Jean B. MargotDr. Jörg Meding

Graduate studentsFrançois Gaudet

Technical assistantIngrid Grunewald

* part of the period reported.

Differentiation and Cell CycleRegulation in Muscle Cells

Structure of the Group

Group leaderDr. M. Cristina Cardoso

ScientistsDr. Hans-Peter RahnDr. Anje SporbertDr. Wolfgang Derer

Graduate studentsAna M. Aguirre-ArtetaHariharan P. Easwaran

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Surgical OncologyPeter M. Schlag

Tumor progression, invasionand metastasis

W. Kemmner, U. Stein, W. Walther,W. Haensch, H. Schwabein cooperation with U. Karsten, J. Behrens, W. Birchmeier

Tumor-associated alterations of cellsurface glycosylation play animportant role in invasion andmetastasis of carcinoma cells.Accordingly, expression of theglycosyltransferases responsible forglycosylation is altered in tumor cellsand this influences their cell adhesionproperties. Therefore, sense- andantisense- transfectants of suchglycosyltransferases have beenestablished. Specific antisenseoligodeoxynucleotides, which inhibitenzyme activity have been studied inconnection with a possible clinicaltreatment of metastasis. Another ofour interests are Thomsen-Friedenreich (TF) related tumor-associated carbohydrates. We havefound that the balance between O-glycan chain termination throughsialylation and chain extensionthrough N-acetylglucosaminylation isdisturbed in colorectal carcinomas.Patients with high expression ofsialyltransferase ST6GalNAc-II are ata significantly higher risk of death andhave a shorter overall survival.

We have identified new genesinvolved in the organ-specificmetastases of colon carcinoma bydifferential display using surgicalspecimens. Three ESTs have beenidentified that are differentiallyexpressed in metastasizing versusnon-metastasizing primary tumors,distinct metastases (preferentially liverand lung) or normal epithelium.Expression of candidate genes wasconfirmed by quantitative real timeRT-PCR.

Expression of the transcription factorGLI has been analyzed in sarcomasand during metastasis and we foundsignificantly enhanced GLI expressionin tumors compared with normaltissues. Moreover, GLI expressioncorrelated with the grading of thesarcomas. Thus, enhanced GLIexpression might be indicative ofaggressiveness and dedifferentiationof mesenchymal tumors.

Multidrug resistance andhyperthermia

U. Stein, W. Walther, K. Jürchottin cooperation with B. Rau, P. Hohenberger, P.-U. Tunn

Multidrug resistance (MDR) stilllimits the successful chemotherapy ofcancer. The expression of MDR-associated genes is inducible byexternal stress factors such as heatshock. This is controlled by stressresponsive elements within the genepromoters and might be a molecularmechanism that hinderschemotherapy. Thus, the impact ofhyperthermia on the induction ofMDR-associated genes has beeninvestigated in colorectal carcinomas,before and after radiochemotherapy orradiochemothermotherapy,respectively. In the majority of thecases analyzed, the risk of inducingMDR gene expression byhyperthermia has been found to beminimal in a clinical setting.Investigations analyzing the influenceof hyperthermia on the expression ofthe MDR genes, 2 – 48h followingheat treatment, are currentlyunderway. The impact of isolatedhyperthermic limb perfusion on theexpression/induction of MDR-associated genes had been examinedin soft tissue sarcomas andmelanomas at several time pointsprior to, during and post treatment. Sofar, induction of certain MDR-associated genes, such as MDR1,MRP1, and LRP, has been observedwithin hours of hyperthermictreatment. Heat shock-inducedexpression of MDR genes should betaken into account when combininghyperthermia with MDR-associatedcytostatic drugs.

Detection and pathobiology ofsolid tumors with microsatelliteinstability

K. Kölble, B. Barthel, L. Estevéz-Schwarz, K. Krause, H. Pidde, O. M. Ullrich in cooperation with M. Dietel and S. Scherneck

Somatic alterations in simplerepetitive sequences that are presentas microsatellites throughout thehuman genome are characteristic of asubset of human tumors with defectsin DNA mismatch repair. Germlinemutations in various DNA mismatchrepair genes dramatically increase anindividual’s susceptibility to variousneoplasms and are the molecular basisfor the syndrome of hereditary non-polyposis colorectal carcinoma(HNPCC). HNPCC is characterizedby the early and familial occurrenceof intestinal and extraintestinalcancers. Collaborative efforts to betterdefine the epidemiological, clinicaland pathomorphologic features of thiscancer susceptibility syndrome arecritical for elucidating its pathogeneticpathways and improving the clinicalmanagement of patients and genecarriers. Potential cases of HNPCC,reported locally as well as regionally,have been registered and investigatedby a combination of detailed clinicaland pedigree studies, histopathology,immunohistology, microsatelliteinstability (MSI) analysis andgermline sequencing. However,identification of HNPCC carriers bybiochemical screening prior tocomplete genomic sequencing couldsignificantly improve these existingcancer preventive strategies. Cell-freeDNA in the blood of cancer patientshas been shown to harbormicrosatellite alterations frequentlymatching those of the primary tumors.We have investigated the patterns ofsuch microsatellite alterations in seraand microdissected tumors ofcolorectal cancer patients. Using anintegrative morphological,immunohistological and geneticapproach, high-grade serum MSI waspreferentially found in individualswith mismatch repair-defectivetumors. Although the molecularmechanisms of tumoral DNA-shedding remain to be elucidated, itsdetection by serum DNAmicrosatellite analysis appears to beuseful for the diagnosis andmonitoring of neoplasms caused bydefective DNA mismatch repair.

The subsequent development of a

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malignant phenotype in all solidtumors seems to be determined in partby actin-binding proteins. In earlierstudies, our group has shown aregression of the malignant phenotypein MCF7 cells after transfection of thegene of the actin-binding proteinprofilin. We are now analyzingimmunohistologically samples ofhighly malignant breast tumors inorder to select tumors which exhibitdefective expression of profilin and,subsequently, analyze the blockedexpression mechanism of the profilingene in these tumors using Western-blotting, quantitative RT-PCR andmethylation-specific PCR (MS-PCR).

Non-viral gene transfer for genetherapy of cancer

W. Walther, U. Stein, R. Cartier in cooperation with I. Fichtner andC. Engelmann

Non-viral gene transfer technologieshave developed into applicablealternatives to viral delivery systemsin gene therapy. A „High-speed Jet-Injection“ system has been tested forgene transfer of naked DNA intotumors. The in vivo experimentsshowed that naked DNA couldefficiently be delivered into tumortissue using the jet-injectiontechnology. The detection of reportergene expression in jet-injected tumorsrevealed strong LacZ- or GFP-expression. Therefore, high-speed jet-injection is feasible for an efficientgene transfer into tumors and isapplicable to the non-viral genetherapy of cancer. Since nucleartransport of transduced DNA limitsthe efficiency of non-viral genetransfer, peptides are employed fornuclear targeting of DNA. Peptidesharboring nuclear localizationsequences (NLS) are complexed withplasmid DNA for improved nucleartransport. Our initial studies indicatethe efficient gene transfer of thesepeptide-DNA complexes, asdetermined by reporter assays indifferent tumor cell lines in vitro.Confocal microscopy of transducedcells demonstrates the nucleartransport of these peptide-DNAcomplexes.

Selected Publications

Petretti, T., Schulze, B., Schlag, PM.,and Kemmner, W. (1999) AlteredmRNA expression ofglycosyltransferases in human gastriccarcinomas. Biochim. Biophys. Acta1428, 209-218.

Stein, U., Eder, C., Karsten, U.,Haensch, W., Walther, W., and Schlag,P.M. (1999) GLI gene expression inbone and soft tissue sarcomas of adultpatients correlates with tumor grade.Cancer Res. 59, 1890-1895.

Stein, U., Rau, B., Wust, P., Walther,W., and Schlag, P.M. (1999) Hyper-thermia for treatment of rectal cancer:evaluation for induction of multidrugresistance gene (mdr1) expression. Int.J. Cancer 80, 5-12.

Kölble, K., Ullrich, O.M., Pidde, H.,Barthel, B., Diermann, J., Rudolph,B., Dietel, M., Schlag, P.M., andScherneck, S. (1999) Microsatellitealterations in serum DNA of patientswith colorectal cancer. Lab. Invest.79, 1145-1150.

Walther, W., Stein, U., Fichtner, I.,Naundorf, H., Alexander, M,Shoemaker, R.H., and Schlag, P.M.(1998) In vivo evaluation of a drug-inducible vector system for thecombined gene and chemotherapy ofcancer. Adv. Exp. Med. Biol. 451,139-144.

Patent Applications

Stein, U., Walther, W., Schlag, P.M.The use of cytokines and cytostaticsin a new procedure for the treatmentof cancer. Patent filed: 96250206.8(1998): Europe, (1999) PCT.

Stein, U., Walther, W. Vector for theexpression of therapeutically relevantgenes. Patent issued: P 42 38 778(1995): Germany Patent issued:5,968,735 (1999): USA.

Structure of the Group

Group leaderProf. Dr. Peter M. Schlag

ScientistsDr. Lope Estevéz-SchwarzDr. Georgi GraschewDr. Wolfgang HaenschDr. Wolfgang KemmnerDr. Dr. Konrad KölbleDr. Karin SchumacherDr. Ulrike SteinDr. Wolfgang Walther

Graduate and undergraduate studentsYetunde Abdul*Martina Bosnar*Regis CartierVassilios Dakos*Sabine HaaseMike Hahn*Karsten JürchottChristian Lange*Nicolai KleinHan LiangShaoqian LinTobias PetrettiFrank SchneiderBettina SchulzeHolger SchwabeAlexandra Zuske

Technical assistantsLisa BauerGudrun FrankeSabine GrigullLieselotte MalcherekClaudia Roefzaad

*part of the period reported

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Ubiquitin System and EndoplasmicReticulumThomas Sommer

Proteolysis by the ubiquitin-proteasome pathway can be brieflysummarized as follows: componentsof the ubiquitin system recognizeproteins destined for proteolysis andcovalently conjugate multiple copiesof the polypeptide ubiquitin to thesubstrate. Proteins earmarked in sucha way are subsequently recognized bythe 26S proteasome which cleaves theproteins to peptides and releasesubiquitin. However, in some casesproteolysis is preceded by anintracellular transport process inwhich the substrate is presented to theproteolytic system. Currently, twoexamples of a link between transportand proteolysis are known: ubiquitin-dependent internalization of cellsurface proteins and endoplasmicreticulum (ER)-degradation, orERAD. Our latest results show theexistence of a third, and unexpected,linkage of this type.

Protein degradation at theendoplasmic reticulum

Proteolysis of ER-lumenal andmembrane proteins has beeninvestigated in detail by our group. Itis a process common to manyeukaryotic organisms and is ofsignificant medical importance. Inprinciple, ER-degradation can bedivided into three steps: firstly, an ER-lumenal detection system has to bepostulated which recognizesmisfolded proteins ; secondly, theproteolytic substrates are transportedback into the cytosol (retrogradetransport) via a channel formed by theSec61p-complex ; thirdly, theretrogradely transported substrates aremarked with the polypeptide ubiquitinand, subsequently, digested by thecytosolic 26S-proteasome complex .

The ubiquitin-conjugating enzymes,Ubc6p and Ubc7p, the latter of whichis anchored to the ER-membrane viaCue1p, are central to this proteolysis.In addition, we have now defined asecond pathway of ubiquitin-conjugation involved in ER-degradation (Friedlander et al.,manuscript in preparation). Ourprevious results show thatubiquitination and retrograde transportare tightly coupled, since proteolyticsubstrates accumulate in the lumen ofthe ER when ubiquitin-conjugation isabolished. This leads to the hypothesisthat ubiquitin-conjugation maycontribute to the driving force ofretrograde transport. In support of thismodel, we were able to demonstratethe accumulation of retrogradelytransported, ubiquitinatedintermediates in the cytosol when weabolished specific functions of theproteasome (Jarosch et al., manuscriptin preparation). Apparently, this ER-degradation pathway is conservedduring evolution since we recentlyidentified homologs of Ubc6p inhigher eukaryotic cells which areinvolved in ER-degradation of theδF508 CFTR protein which is foundin most cystic fibrosis patients (Lenket al., manuscript in preparation).

Degradation of nuclear proteins

Recently, we have investigated theturnover of a nuclear substrate, thetranscriptional repressor Matα2. Thisapproach was based on our previousobservation that degradation ofMatα2p was dependent onUbc6p/Ubc7p. In summary, our data

suggest that Matα2p is degraded viadistinct pathways in different cellularcompartments. We were able todistinguish a rapid and Cue1p-independent nuclear degradationpathway from a slow and Cue1p-dependent one taking place at the ER-membrane. Thus, a degradation signalonly functions in conjunction with itsrespective cellular localization signalwhich, in the case of Matα2p, is thenuclear localization sequence. In cellbiological terms this might represent amechanism for regulating the half-lifeof proteins. By transporting a proteininto a different cellular compartment,the turnover might be up- ordownregulated, probably because theubiquitination cascades are restrictedto certain areas within a eukaryoticcell. This mechanism may be animportant tool for regulation,especially for regulatory factors of thenucleus.

Furthermore, we have investigated theinfluence of nucleo-cytoplasmictransport functions on the proteolysisof the transcriptional repressor andobserved that the rapid turnover ofMatα2p completely relies on nuclearimport. Next, we determined theturnover of Matα2p when nuclearexport was blocked. Intriguingly, wefound that the breakdown of Matα2pis slowed down by mutations inCse1p, a karyopherin required forprotein export from the nucleus. Inaddition, we could demonstrate byimmuno electron microscopy that afusion protein containing thedegradation signal of Matα2p istransported from the nucleus back intothe cytosol. Next we asked whetherprotein export in general is required orwhether the Cse1p pathway isspecifically involved. To address thisquestion, we channeled Matα2p into adifferent export route from thenucleus. Such a hybrid protein wasrapidly transported out of the nucleus,but this did not result in rapidturnover, indicating that only theCse1p pathway channels Matα2p intorapid proteolysis. Thus, we concludethat Matα2p has to be shuttledthrough the cell nucleus for properdegradation and that rapid proteolysisis linked to Cse1p-dependent proteinexport from the nucleus.

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Our data are consistent with twoexport models: in one of these Matα2is recognized in the nucleus by aspecific ubiquitin ligase that carries anuclear export signal of the Cse1ppathway. Both Matα2p and E3 areexported together and, subsequently,Matα2p is ubiquitinated and becomesa target for the proteasome.Consequently, the E3 should alsocontain a nuclear localizationsequence to shuttle back into thenucleus for another round of exportand degradation. Alternatively,ubiquitination of Matα2p might occurin the nucleus and the exportmachinery would transport ubiquitin-conjugated Matα2p. This transportshould involve an adapter protein thatbinds ubiquitin and carries a nuclearexport signal recognized by the Cse1pathway. Since we do not observe anynuclear export of Matα2p from thenucleus in the absence of ubiquitin-conjugation via Ubc6p, we favor thesecond model. If ubiquitinatedproteins were linked to the Cse1pprotein export machinery via anadapter protein, all proteolyticsubstrates could be exported by suchan adapter protein. Thus, our modelwould be applicable to many short-lived regulators of the nucleus.Clearly, further experiments have tobe performed to distinguish betweenthese two possibilities.

Selected Publications

Biederer, T., Volkwein, C., andSommer, T., (1997) Role of Cue1p inubiquitination and degradation at theER surface. Science, 278, 1806-1809.

Sommer, T., and Wolf, D. H., (1997)ER-degradation: Reverse protein flowof no return. FASEB J., 11, 1227-1233.

Sommer, T. (1999) The ubiquitinsystem in yeast. In: Proteasomes, Edt.D. H. Wolf and W. Hilt. Landespublishing.

Sommer, T., Jarosch, E., and Lenk, U.(2000) Compartment-specificproteolysis by the ubiquitin-proteasome pathway. Rev. Physiol.Biochem. Pharmacol. in press.

Structure of the Group

Group leaderDr. Thomas Sommer

ScientistsDr. Ernst JaroschDr. Uwe LenkDr. Katrin Stade

Graduate and undergraduate studentsRuth FriedlanderBirgit MeusserJörg UrbanJan Walter

Technical assistantsAngelika WittstruckCorinna Volkwein

SecretariatSylvia Klahn

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P450 Cytochromesand the EndoplasmicReticulumWolf-Hagen Schunck

In the past two years, our laboratoryhas begun new projects aimed atelucidating the role of arachidonicacid metabolizing cytochrome P450enzymes in the regulation of vasculartone and renal function. The majortopics of the new projects are humanvascular endothelial cells and mousemodels of hypertension which arebeing studied in collaboration withgroups at the MDC (V. Gross, B.Erdmann) and Franz Volhard Clinic(H. Haller, F.C. Luft).

Expression of P450 isoforms inhuman vascular endothelialcells

Epoxy derivatives of arachidonic acidare important autocrine and paracrinemediators in the regulation of avariety of endothelial functions, suchas control of vascular tone andinflammation. However, little isknown about the molecular identityand regulation of the P450 isoformsactually expressed in endothelial cellswhich catalyze arachidonic acidepoxygenation and contribute to thedifferent signal transduction pathwaysinvolved. To identify potentialcandidates, we searched for theexpression of individual P450 genesbelonging to the P450 families 1, 2, 3,and 4. RT-PCR screening performedwith subfamily- and isoform-specificprimer pairs revealed the presence ofmRNAs for the P450 forms 1A1, 1B1,2C8, 2E1, 2J2, 3A7, 4A11, and 4F2.In addition, P450 1A2 was detectedafter induction with ß-naphthoflavonewhich also enhanced the expression ofP450s 1A1 and 1B1. Similar P450patterns were obtained analyzingprimary endothelial cells originatingfrom aorta, coronary arteries, dermalmicrovessels, and umbilical veins, aswell as an immortalized humanendothelial cell line (HMEC-1).HMEC-1 cells were found by gaschromatography/mass spectrometry(GC-MS) to contain a series ofregioisomeric epoxyeicosatrienoicacids and to actively produce thesemetabolites after extracellular additionof arachidonic acid. Among the P450isoforms detected, P450s 2C8 and 2J2are leading candidates for producing11,12-epoxyeicosatrienoic acid, ametabolite recently reported by otherresearchers to cause vasodilation andhave anti-inflammatory properties.Some of the other P450 formsdetected may be important undercertain pathophysiological conditions(P450s 1A1 and 2E1) or maycontribute to eicosanoid degradation(P450s 4A11 and 4F2).

P450-dependent renalarachidonic acid metabolism innormal and hypertensive mice

The starting point for these studieswas the physiological data obtainedby V. Gross and F.C. Luft showingthat hypertension in different mousemodels is associated with a reductionin total renal blood flow and a shift tothe right of pressure-natriuresis-diuresis curves. These features aretypical for desoxycorticosterone(DOCA)-salt hypertensive mice andfor angiotensin-type 2 (AT2) receptorknockout mice. Our laboratorybecame involved in these studies afterbezafibrate, an inducer of P450 formsmetabolizing fatty acids, was found toimprove renal hemodynamics. Thisfinding led to the hypothesis thatchanges in P450-dependent arachidonicacid metabolism may play animportant role in these models. Toaddress this question, we first studiedsome basic characteristics of mouserenal arachidonic acid metabolism.Using HPLC and GC-MS, theproducts formed were identified as20- and 19-hydroxyarachidonic acid(20- and 19-HETE), representingabout 80 % and 20 % of the totalhydroxylation products. Controlmicrosomes of untreated wild-typemice had arachidonic acidhydroxylase activities of about 200pmoles/min/mg. Antibody-inhibitionexperiments indicated the involvementof P450 forms belonging to the 4Asubfamily. Induction of hypertensionwith DOCA-salt resulted insignificantly reduced hydroxylaseactivities which were only about 40 %of the control values. Westernblotanalysis revealed that the specificcontent of P450 4A proteins wasmarkedly reduced. An even morepronounced reduction in the capacityto produce 20-HETE was observed inthe kidneys of AT2-receptor knockoutmice. Bezafibrate treatment partiallyrestored the low arachidonic acidhydroxylase activities in DOCA-saltmice and, in particular, induced theP450 isoform 4a-14. In situhybridization experiments performedin collaboration with B. Erdmanndemonstrated that this P450 isexpressed in the cortical-medullaryjunction where it is predominantlylocalized in the proximal tubules.Taken together, these results suggestthat hypertension in the mousemodels studied is associated with adeficiency in the production of 20-HETE by renal tubular structures.Since 20-HETE is known to inhibit

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ion channels responsible for saltreabsorption and, thus, stimulate saltexcretion, this deficiency provides areasonable explanation for theobserved changes in kidney function.Moreover, we speculate thatalterations in tubular 20-HETEproduction may also affect local renalblood flow assuming transcellulartransport and further metabolism of20-HETE to prostaglandin analogswith vasodilator effects.

Selected Publications

Ohkuma, M., Zimmer, T., Iida, T.,Schunck, W.-H., Ohta, A., and Takagi,M. (1998) Isozyme function of n-alkane-inducible cytochromes P450 inCandida maltosa revealed bysequential gene disruption. J. Biol.Chem. 273, 3948-3953.

Zimmer, T., Scheller, U., Takagi, M.,and Schunck, W.-H. (1998) Mutualconversion of fatty acid substratespecificity by a single amino acidexchange at position 527 in P450Cm2and P450ALK3A. Eur. J. Biochem.256, 398-403.

Scheller, U., Zimmer, T., Becher, D.,Schauer, F., and Schunck, W.-H.(1998) Oxygenation cascade inconversion of n-alkanes to α, Ω-dioicacids catalyzed by cytochrome P45052A3. J. Biol. Chem. 273, 32528-32534.

Sandig, G., Kärgel, E., Menzel, R.Vogel, F., Zimmer, T., and Schunck,W.-H. (1999) Regulation ofendoplasmic reticulum biogenesis inresponse to cytochrome P450overproduction. Drug Metabol. Rev.31, 393-410

Gross, V., Schneider, W., Schunck,W.-H., Mervaala, E., and Luft, F. C.(1999) Chronic effects of lovastatinand bezafibrate on cortical andmedullary hemodynamics in DOCA-salt hypertensive mice. J. Am. Soc.Nephrol. 10, 1430-1439.

Structure of the Group

Group leaderDr. Wolf-Hagen Schunck

ScientistsDr. Horst HoneckDr. Eva Kärgel

Graduate and undergraduate studentsEduardo Barbosa-SicardRalph Menzel*

Technical assistantsChristel Andreé*Ramona Zummach

Associated groupsDr. Solveigh Krusekopf / May-Britt KöhlerDr. Dieter Schwarz / Anne Sternke

*part of the period reported

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Figure 32: Localization of bezafibrate-inducedP450 4a-14 mRNA in the mouse kidney bymeans of in situ hybridization. Phosphor imagerpicture of a whole kidney section, black stainingshows that the P450 4a-14 mRNA isconcentrated in the cortical-medullary junction(arrow).

Vascular BiologyHermann Haller

This group is very interested in thecell biology of the vessel wall. Theapproaches being used are broad inscope and include patch clampelectrophysiology, signal transduction,cell differentiation anddedifferentiation, gene regulation, andapoptosis. In addition, the group isheavily involved in patient-orientedresearch directed at elucidatingmechanisms relevant to hypertension,including preeclampsia,atherosclerosis, and reperfusion injury.

Endothelial cell function

Endothelial cells and theirperturbations (endothelial celldysfunction) have recently become ofmajor interest in the pathophysiologyof chronic vascular disease. MarenWellner is leading a team that hasstudied the proliferative effect ofvascular endothelial growth factor(VEGF) on human endothelial cells.Two hypotheses have been tested: (1) VEGF affects intracellular calciumregulation and calcium-dependentmessenger systems and (2) thesemechanisms are important for theproliferative effects of VEGF. Theirdata show that VEGF induces initialand sustained calcium influx. VEGFleads to translocation of the calcium-sensitive PKC isoform alpha and theatypical PKC isoform zeta. Antisensemolecules for these PKC isoformsblock VEGF-induced proliferation.These findings suggest that PKCisoforms alpha and zeta are importantfor the angiogenic effects of VEGF.Maren Wellner has recently directedher attention to signals involved incausing endothelial cells to assume afenestrated phenotype. Phorbol esterstimulates endothelial cells in thisdirection and ESM-1 appears to be amarker protein.

Nuclear protein transportpathways

The study of the mechanisms involvedin nuclear transport is an exciting areaand Matthias Köhler’s team is leadingthis effort. Nuclear proteins, such astranscription factors and ribosomalproteins, are synthesized in thecytoplasm and must be transportedinto the nucleus to exert theirfunctions. The transport of proteins>20-60 kD through the nuclear porecomplex (NPC) into the nucleus is anactive, energy-requiring process.Transport substrates are recognized bytheir transport proteins via certainsignals. The best-characterized proteinimport pathway is the ‘classical’nuclear localization signal-dependentpathway with importin alpha and betacarrying the substrate to the NPC. Thetransport of the importin-substratecomplex into the nucleus is regulatedby the small GTPase Ran/TC4. Morethan ten proteins have beendiscovered which have already beenproven, or are very likely, to benuclear transport factors for distinctimport pathways. Members of theimportin alpha protein family are very

similar and transport, in a complexwith importin beta, nuclearlocalization signal-bearing proteinsinto the nucleus. Members of the Ran-binding protein family show a slightdegree of similarity to importin beta.Ran-binding proteins share a commondomain at the amino terminus whichenables them to bind RanGTP, aprerequisite for their function asnuclear import or export factors fordistinct proteins or RNAs. AlthoughKöhler has found that Ran/TC4 seemsto play a key regulatory role in allnuclear transport pathways, themolecular mechanism of thetranslocation step through the NPC isstill unclear.

Neutrophil apoptosis

Ralf Kettritz is interested in vasculitis,which invariably features neutrophilinfiltration and acute inflammation.Recently, he has branched out intoneutrophil apoptosis. Duringinflammation, neutrophils migrate intothe affected tissue interacting withextracellular matrix proteins. He hasrecently tested the hypothesis thatneutrophil-matrix interaction affectsneutrophil apoptosis. Kettritz hasfound that the extracellular matrix hasa significant effect due to processesregulated by tyrosine phosphorylation.Recently, he performed two-dimensional gel electrophoresis andWestern blotting to investigate this.He exposed neutrophils on fibronectinto TNFα and observed severaltyrosine phosphorylated proteins,which he subsequently sequenced.One of these proteins was LY-GDI.Ly-GDI cleavage was prevented bycaspase-3 inhibition, which alsodecreased apoptosis. Kettritz hasconcluded that tyrosinephosphorylation of Ly-GDI, followedby increased caspase-3-mediated Ly-GDI cleavage, is the signaling eventassociated with accelerated TNFα-mediated apoptosis on fibronectin.

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New modes of calcium signaling

Maik Gollasch, Matthias Löhn, andMichael Fürstenau are makingexciting advances in electrophysiology.During a Humboldt fellowship at theUniversity of Vermont, Gollaschworked with Mark Nelson and studiedlocal calcium transients termedcalcium sparks. These sparks areapparently caused by opening ofclustered ryanodine receptors in thesarcoplasmic reticulum. Gollasch’steam has investigated caveolae,cholesterol/sphingolipid-richinvaginations of the plasma membranewhich colocalize with both thesubsarcolemmal occurrence ofcalcium sparks and the junctionalsarcoplasmic reticulum. They havefound that a transient elevation incalcium at the inner mouth of a singleL-type calcium channel withincaveolae induces simultaneousactivation and opens severalryanodine receptors to generate a localcalcium spark. They are the first toshow that a caveolae-calciumsignaling pathway may regulatecellular functions via local ryanodinereceptors in the sarcoplasmicreticulum. The first illustration of acalcium spark in man (from acoronary artery vascular smoothmuscle cell) is shown in the figure.

Christian Maasch, Carsten Lindschau,Gabi Alexander and others are usinggreen fluorescent protein technologyto observe protein kinase Cα targetingin vascular smooth muscle cells. Theyrecently showed that local changes incalcium regulate PKCα translocation.The targeting mechanism is dependenton the C2 and P domains of theenzyme. Their observations are thefirst to show that localized calciumchanges determine the spatial andtemporal targeting of PKCα.

Gene therapy

The group has relied on antisensestrategy in a series of exciting studiesaimed at treating ischemia-reperfusioninjury occuring after organtransplantation and they have used arat renal transplant model. DuskaDragun has recently completed astudy in which she showed that theantisense treatment is very effective inthe prevention of ischemia-reperfusioninjury in transplantation. A chronicisograft transplantation study has alsobeen completed.

Patient-oriented research

Hermann Haller, Volker Homuth,Friedrich C. Luft, and Gerd Wallukathave collaborated with obstetricians inBerlin to elucidate new mechanismsresponsible for endothelial damage inpreeclampsia. In a cell culture systemof endothelial cell monolayers, thegroup showed that a factor from theserum of preeclamptic womenstimulates increased cell-layerpermeability. This process involvesPKC signaling, principally PKCα andPKCε. The permeability increase wasblocked with antisense against themRNA of these enzymes. Insubsequent studies, the team showedthat women with preeclampsiaproduce agonist autoantibodiesdirected at the AT1 receptor. Theantibodies are directed at the secondextracellular loop. Colocalizationstudies have demonstrated thenecessary specificity. The subsequentsignaling proved to be a PKC-mediated pathway. Very recently, thegroup has shown that the antibodiesare capable of making vascularsmooth muscle cells produce tissuefactor. The work of the fourinvestigators was awarded theGalenus von Pergamon prize for 1999.

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Figure 33: Calcium sparks are localizedintracellular events released through ryanodinereceptors. The sparks then control excitation-contraction coupling. The confocal line-scanimage of a fluo-3-loaded human coronaryartery vascular smooth muscle cell is shown.The fluoresence time course of the sparks wasdetermined over the time indicated by the twoarrows.

Selected Publications

Gollasch, M., Haase, H., Ried, C.,Lindschau, C,, Miethke. A., Morano,I., Luft, F.C., and Haller, H. (1998) L-type calcium channel expressiondepends on the differentiated state ofvascular smooth muscle cells. FASEBJ. 12, 593-601.

Kettritz, R., Xu, Y.-X., Kerren, T.,Quass, P., Klein, J., Luft, F.C., andHaller, H. (1999) Extracellular matrixregulates apoptosis in humanneutrophils. Kidney Int. 55, 562-571.

Wellner, M., Kupprion, C., Maasch,C., Lindschau, C., Luft, F.C., andHaller, H. (1999) The proliferativeeffect of vascular endothelial growthfactor (VEGF) requires protein kinaseC α and ζ. Arterioscler Thromb. Vasc.Biol. 19, 178-185.

Haller, H., Maasch, C., Dragun, D.,Wellner, M., von Janta-Lipinski, M.,and Luft, F.C. (1998) Antisenseoligodinucleotide strategies in renaland cardiovascular disease. KidneyInt. 53, 1550-1558.

Wallukat, G., Homuth, V., Fischer, T.,Horstkamp, B., Jüpner, A., Baur, E.,Nissen, E., Vetter, K., Dudenhausen,J.W., Haller, H., and Luft, F.C. (1999)Patients with preeclampsia developagonistic antibodies against theangiotensin AT1 receptor. J. Clin.Invest. 103, 945-952.

Köhler, M., Speck, C., Christiansen,M., Bischoff, F.R., Prehn, S., Haller,H., Görlich, D., and Hartmann, E.(1999) Evidence for distinct substratespecificities of importin alpha familymembers in nuclear protein import.Mol. Cell Biol. 19, 7782-7791.

Structure of the Group

Group leaderProf. Dr. Hermann Haller

ScientistsDr. Marek DrabDr. Ralf KettritzDr. Elke GenerschDr. Maik GollaschDr. Matthias KöhlerDr. Matthias LöhnDr. Maren Wellner

Doctoral studentsGabriele AlexanderMichael FürstenauChristian MaaschCarsten LindschauOlaf SchäferThorsten Kirsch

Technical assistantsJana CzychiPetra Quass

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Functions of DynaminII and PKC in Post-Golgi VesicleFormation

Peter Westermann

Cellular functions depend on propertransport and correct subcellularlocalization of proteins. To accomplishthis, secretory proteins, lysosomalproteins and membrane proteins haveto be sorted in the trans-Golginetwork (TGN) and packed intospecific transport vesicles. Vesicleformation is controlled by different Gprotein families. Therefore, functionsof inhibitory trimeric G proteins andof dynamin II have been studied. Inaddition, the PKC-dependentregulation of vesicle formation at theTGN is being investigated byanalyzing Golgi-bound PKCsubstrates.

New methods for investigatingmolecular interactions

K. Bulygin, A. Malygin, G. Karpova(Novosibirsk), J. Dong

Two methods have been developed forstudying molecular interactions.RNA-DNA interaction have beenstudied by cross-linking (K. Bulyginet al., 1998) while protein domaininteractions have been analyzed byaffinity binding of cytosolic ormembrane proteins to peptide-taggedprotein domains attached to agarosematrices.

Interactions between dynamin IIdomains and the Golgiapparatus

J. Dong, M. Knoblich incollaboration with A. Otto, E.-C.Müller, and C. Lindschau (FVK)

The functions of dynamins depend ontheir domain structure. To study thebinding of dynamin II to Golgimembranes, the pleckstrin-homologydomain (PHD), the proline-richdomain (PRD) and the C-terminal partof dynamin II, consisting of PHD,GTPase activator domain and PRD,were expressed and purified.Interactions between these domainsand cytosolic or membrane proteinswere studied by affinity binding andcross-linking. PHD binds with highaffinity to Golgi membranes, but doesnot interact with proteins suggestingbinding mainly to phospholipids. Theproteins that interact with PRD areSH3 domain-containing proteins,amphiphysin I , amphiphysin II andSH3GL2, while additional proteinsmay bind within larger complexes.The nature and composition of theseprotein complexes is presently beingstudied. In addition, membrane-bound, but not cytosolic profilin Ipromotes attachment of dynamin II tothe Golgi apparatus and supportstransport vesicle formation (J. Dong etal., in revision).

Identification of PKC substratesattached to the Golgi apparatus

B. Radau, M. Knoblich incollaboration with A. Otto, E.-C. Müller

Stimulation of vesicular transportbetween the TGN and plasmamembrane by activation of PKC(Westermann et al., 1996) may dependon the phosphorylation of Golgi-bound proteins. MARCKS,MacMARCKS, cytokeratin 8,cytokeratin 18 and synaptobrevin 2have been identified by in situphosphorylation, two-dimensionalprotein electrophoresis and peptidesequencing as Golgi-bound PKCsubstrates. The impact of individualphosphoproteins on vesicle formationis under investigation.

Selected Publications

K. Bulygin, K., Malygin, A., Karpova, G., and Westermann, P.(1998) Site-specific modification of4.5S RNA apical domain bycomplementary oligodeoxynucleotidescarrying an alkylating group. Eur. J.Biochem. 251, 175 - 180.

Westermann, P., Knoblich, M., Maier,O., Lindschau C., and Haller, H.(1996) Protein kinase C bound to theGolgi apparatus supports formation ofconstitutive transport vesicles.Biochem. J. 320, 561-568.

Structure of the Group

Group leaderDr. Peter Westermann

Guest scientistDr. Olaf Maier

Graduate studentsJiaxin DongBoris Radau

Technical assistantMaria Knoblich

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Electron Microscopy

Members of the electron microscopygroup have experience in variousmicroscopic techniques ranging fromlight microscopy to high resolutionelectron microscopy. Specialimportance is given to the applicationand improvement of immunohisto-and immunocytochemical methods.Recently, methods for correlativeimmunofluorescence andimmunoelectron microscopy havebeen introduced as well as markedimprovements in the preparation ofultrathin cryosections, the mostsensitive target for high resolutionimmunodetection of antigens

Molecular architecture of thenuclear pore complex

M. Vogel, F. Vogel in collaborationwith G. Schlenstedt (Homburg/Saar)

To understand the functional role of aparticular nucleoporin at the molecularlevel it is necessary to map itslocation within the three-dimensionalarchitecture of the nuclear porecomplex (NPC). Our new EM samplepreparation protocol applied to yeastcells expressing GFP-fused and myc-,Pk- and HA-tagged nucleoporins hasenabled the precise localization of anumber of transport factors andnucleoporins to distinct structuralcomponents of the yeast NPC.According to these results Nup1p,Nup2p, importin α, importin β andexportin (Cse1p) form a newstructural and functional complexinvolved in either nuclear proteinimport or RNA export.

Compartment-specificproteolysis

M. Vogel, F. Vogel in collaborationwith Th. Sommer

The group led by Thomas Sommerhas developed an assay to provideevidence for a new and unexpectedlink between protein export from thenucleus and degradation via theubiquitin-proteasome pathway (seeTh. Sommer´s report). We arecharacterizing this system by EMapproaches and are focussing, inparticular, on the subcellularlocalization of different GFP-taggedprotein fusions in wild type andexport-mutant cells.

Localization of plakoglobin in β-catenin-deficient mouseembryos

B. Erdmann in collaboration with W. Birchmeier, J. Hülsken

Using a combination ofimmunofluorescence and immunogoldlabeling methods, 6-7 days old wildtype and β-catenin-deficient mouseembryos could be distinguished andcharacterized. Followingimmunocytochemistry of selectedareas, an upregulation and redistributionof plakoglobin has been detectedalong membranes of the mutantembryos.

Structure of small heat shockproteins

G. Lutsch, M. Wieske, R. Wessel incollaboration with J. Behlke, MDC,Berlin, M. Gaestel, Halle, and F. Zemlin, Berlin

Mammalian small heat shock proteins(sHsps) are known to form oligomericcomplexes which can act as molecularchaperones. Using electron microscopy,it has been shown that phosphorylationof Hsp25 complexes in vitro results ina significant reduction in oligomericsize, accompanied by reducedchaperone activity of the protein. Thedata provide evidence for regulationof chaperone activity by phosphorylationand dissociation of Hsp25 complexes.Furthermore, cryoelectron microscopyand three-dimensional reconstructionhas revealed new details of the 3Dstructure of ice-embedded Hsp25complexes which might be ofimportance for the chaperone functionof the protein.

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Figure 34: Cryoelectron micrograph of Hsp25complexes. Inset: 3D structure of Hsp25complexes. Cropped view (left) and surface view(right).

Localization of smooth musclemyosin heavy chain B andpp700 in cardiac tissue

G. Lutsch, E. Kotitschke incollaboration with H. Haase, I.L. Morano

The 5´-spliced isoform of smoothmuscle myosin heavy chain(“intestinal” SM-MHC or SM-MHC-B) and the phosphoproteinpp700/AHNAK have been detectedfor the first time in cardiac tissue.SM-MHC-B was localized byimmunofluorescence microscopy inprecapillary arterioles of rat heart,with significantly reduced amounts inventricles of hypertensive rats,suggesting a role for SM-MHC-B inthe regulation of blood perfusion ofthe heart. pp700/AHNAK waslocalized to the plasma membrane ofcardiomyocytes in accordance with itsproposed function in β-adrenergicsignal transduction (see report of I.L. Morano).

Further collaborations

Electron microscopic investigationson • urokinase-induced mitogenesis in

human vascular smooth musclecells (F. Vogel with I. Dumler,D.C. Gulba),

• differentiation-induced changes inantigen uptake mechanisms ofavian dendritic cells (F. Vogel withM. Zenke),

• localization of the overexpressedhuman heart sodium channelprotein in EK293 cells (F. Vogelwith Th. Zimmer, Jena),

• cellular distribution of generatednon-infectious virus-like particlesfor gene transfer experiments (F.Vogel with R. Ullrich, Berlin),

• influence of different P450 geneson the regulation of ER biogenesisin transfected EK293 cells (F. Vogel with W.-H. Schunck),

• localization of digoxigenin-labeledforeign DNA in endothelial cellsfollowing non-viral gene transfer(B. Erdmann with M. Boettger),

• influence of Hsp25 peptides onactin polymerization in vitro(G. Lutsch, M. Wieske with R.Benndorf, Ann Arbor), and

• immunofluorescence microscopicinvestigations on localization ofHsp25 and αB-crystallin in normaland ischemic rat kidney (G. Lutschwith J. Stahl and R. Benndorf, W.Smoyer, Ann Arbor).

Selected Publications

Sandig, G., Kärgel, E., Menzel, R.,Vogel, F., Zimmer, T., and Schunck,W.-H. (1999) Regulation ofendoplasmic reticulum biogenesis inresponse to cytochrome P450overproduction. Drug Metabol. Rev.31, 393-410.

Dube, P., Wieske, M., Stark, H.,Schatz, M., Stahl, J., Zemlin, F.,Lutsch, G., and van Heel, M. (1998)The rat-liver 80S ribosome at 25Åresolution by electron cryomicroscopyand angular reconstitution. Structure6, 389-399.

Rogalla, T., Ehrnsperger, M., Preville,X., Kotlyarov, A., Lutsch, G., Wieske,M., Arrigo, A.P., Buchner, J., andGaestel, M. (1999) Regulation ofHsp27 oligomerisation, chaperonefunction and protective activityagainst oxidative stress/TNFα byphosphorylation. J. Biol. Chem. 274,18947-18956.

Wetzel, U., Lutsch, G., Haase, H.,Ganten, U., and Morano, I. (1998)Expression of smooth muscle heavychain B in cardiac vessels ofnormotensive and hypertensive rats.Circ. Res. 83, 204-209.

Haase, H., Podzuweit, T., Lutsch, G.,Hohaus, A., Kostka, S., Lindschau, C.,Kott, M., Kraft, R., and Morano, I.(1999) Signaling from β-adrenoceptorto L-type calcium channel:Identification of a novel cardiacprotein kinase A target possessingsimilarities to AHNAK. FASEB J. 13,2161-2172.

Structure of the Group

ScientistsDr. Gudrun LutschDr. Frank VogelDr. Ralf Wessel*

Graduate studentMartin Wieske*1

Technical assistantsDr. Bettina ErdmannErika Kotitschke Helga Rietzke Margit Vogel (W.-H. Schunck’sgroup)

* part of the period reported1 graduated in 9/98

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Molecular Therapy

The aim of this program is to developnew therapeutic strategies for thosediseases, such as cancer orcardiovascular diseases, which oftenremain resistant to treatment. Ourefforts are based on a wide variety ofexperimental strategies that exploit thelatest knowledge emerging from thefast-growing fields of cell biology,cancer biology, immunology andmammalian genetics. The molecularmechanisms that underlie malignanttransformation have been unravelledto a considerable degree, and a greatdeal is now understood about howtumors become resistant to standardtherapies and escape immunerecognition and destruction. Forinstance, it is recognized that tumorsoften express potentially immunogeneticantigens that, nevertheless, fail toelicit an effective immune responsefrom the host. It is also known that T cells must undergo an elaborateactivation process in order to rejectmalignant tissues. An arsenal ofcloned genes is now available whoseproducts are involved in cell-cyclearrest, apoptosis, selective killing oftumor cells, and the induction ofimmune responses. Combining thisknowledge and these reagents, anumber of gene transfer technologiesnow allow the development of veryprecise and, hopefully, more effectiveand less toxic therapeutic modalities.

The program “MolecularTherapy” consists of thefollowing groups:

Molecular Basis ofCongestive Heart Failure

Prof. Dr. Rainer Dietz

Immunology ofCardiovascular Diseases

Dr. Gerd Wallukat

Medical Oncology and Tumor ImmunologyHematology, Oncology and Tumor Immunology

Prof. Dr. Bernd Dörken

Molecular ImmunotherapyProf. Dr. Antonio Pezzutto

Molecular and Cell Biology ofHematopoietic Cells

Dr. Martin Zenke

PhospholipidsDr. Dietrich Arndt

Drug TargetingDr. Regina Reszka

Experimental PharmacologyDr. Iduna Fichtner

RNA ChemistryDr. Eckart Matthes

TranspositionDr. Zoltán Ivics(recently appointed)

Immunology and Gene TherapyProf. Dr. Thomas Blankenstein

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Molecular Therapy

Hematology,Oncology and TumorImmunologyBernd Dörken

Multimarker analysis of cellcycle and apoptosis regulators :definition of novel prognosticfactors

Peter T. Daniel, Isrid Sturm,Sandra Herrmann, Alicja Mrozek

Dysfunction of the apoptoticp53/Bax/caspase-3 signaling pathwayplays a role in tumorigenesis, tumorprogression and development of drugresistance. We are investigating genesand proteins known to be activated inthe p53-mediated response to genotoxicdamage: BAX, a pro-apoptoticmember of the bcl-2 family, and p21,a cyclin-dependent kinase inhibitor,known to mediate the p53-inducedG1-arrest. Both are transcriptionallyactivated by p53, and mutations in thep53 gene may prevent activation ofthese downstream effectors. Patientswith esophageal carcinoma orcolorectal cancer with high BAXexpression in their tumor lesions havea significantly better survival rate.Multivariate analysis showed that lowBAX expression was a highlysignificant independent negativeprognostic marker. In chroniclymphocytic leukemia, thederegulation of p53 or BAX impairsthe sensitivity of leukemic cells tocytotoxic drugs. Analysis of the wholesignalling pathway, rather thananalysis of single genes, such as p53,is crucial and could be useful inpredicting the response to cytotoxictherapy. Therapeutic approachesinvolving transfer of these genes tocancer cells to restore susceptibility toapoptosis are currently beinginvestigated.

Identification of molecularregulators during anti-IgMmediated apoptosis

Kurt Bommert, Anke Rickers, Volker Badock, Niels Peters, Claus Reimertz in cooperation withBrigitte Wittmann-Liebold (MDC)

In order to identify proteins involvedin anti-IgM induced apoptosis (whichis crucial for elimination ofautoreactive B cells) apoptotic andnon apoptotic cells of the BurkittLymphoma cell line BL 60-2 werecompared by high resolution two-dimensional gel electrophoresis anddifferentially appearing spots wereidentified by Edman microsequencingand/or peptide mass fingerprinting.The transcription factor SP1 iscleaved into two products of about 68KDa and 45KDa. Using massspectrometry, we identified a newCaspase-3 cleavage site at positionD584 leading to a 20kDa proteinfragment containing the DNA bindingmotif, which might act in a dominantnegative manner.

Inhibition of Caspase-3 by z-DEVD-fmk inhibits both the cleavage of SP1and apoptosis, indicating Caspase-3 asa central regulator in anti-IgMinduced apoptosis.

We have also identified an earlyresponse gene that is stronglyupregulated shortly after anti-IgMinduction of apoptosis in the BL60-2cell line but not in the apoptosisresistant subclone R37. The functionof these proteins is currently beinginvestigated.

Biology of Hodgkin’s disease

Franziska Jundt, Florian Emmerich,Stefan Mathas in cooperation withClaus Scheidereit (MDC)

We recently identified constitutiveactivation of NF-κB (p50/p65) as acommon feature of Hodgkin/Reed-Sternberg cells which prevents themfrom undergoing apoptosis andtriggers proliferation. To examinepossible alterations in the NF-κB/IκBsystem, which might be responsiblefor constitutive NF-κB activity, wehave analyzed the inhibitor IκBα inprimary and cultured Hodgkin/Reed-Sternberg cells. In the lymph nodebiopsy of 1 of 10 patients withHodgkin’s disease and in two celllines (L428 and KM-H2) we detected

mutations in the IκBα gene, resultingin C-terminally truncated proteins,which are presumably not able toinhibit NF-κB–DNA binding activity.Our data provide the first indicationthat constitutive NF-κB activity inHodgkin/Reed-Sternberg cells mightbe the consequence of mutations inthe inhibitor genes.

We have also reported that the CC-chemokine eotaxin is stronglyexpressed in fibroblasts of Hodgkin´sdisease tissues. Hodgkin/Reed-Sternberg cells induce eotaxinexpression in fibroblasts via TNF-α.Our data suggest that eotaxincontributes to eosinophil and T-lymphocyte recruitment in Hodgkin’sdisease.

Development of gene- andimmunotherapy strategies forthe treatment of multiplemyeloma

Dirk Hönemann, Patric Seibert,Freya Riechert, Kurt Bommert, Ralf Bargou in cooperation with Gert Riethmüller (Munich) and Axel Greiner (Würzburg)

Bone marrow stromal cells (BMSC)produce survival factors that supportthe growth of multiple myeloma(MM) cells. Interleukin-6 appears tobe essential for survival and growth ofMM cells. In the abscence of BMSCs,dexamethasone, all-trans retinoic acid(ATRA), or the IL-6 receptorantagonist Sant-7 inhibit MM cellgrowth. If MM cells are co-culturedwith primary human BMSCs, theybecome almost completely resistant tothe drugs, suggesting that the bonemarrow microenvironment contributesto drug resistance. If dexamethasoneand ATRA are given in combinationwith Sant-7, drug resistance isreversed resulting in almost completegrowth inhibition. We are nowplanning a gene therapy approach formultiple myeloma by stablyexpressing IL-6 receptor antagonistsin BMSCs and hematopoietic stemcells of plasmocytoma patients.

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Cytotoxic T cell targeting bybispecific antibodies andchimeric T cell receptors

Anja Löffler, Jan Schwenkenbecher,Ralf Bargou in cooperation with Bernd Groner (Frankfurt), Zelig Eshhar (Israel), and Gert Riethmüller (München)

Cytotoxic lysis by T cells requiresspecific binding of the T-cell receptorcomplex to antigenic peptidespresented by MHC molecules.Bispecific antibodies can bypass thisrequirement by targeting Tlymphocytes to cells that express anantigen recognized by a monoclonalantibody. Using a recombinantbispecific single-chain antibody(CD19 x CD3) we have been able toinduce rapid and highly effectivelymphoma cell cytotoxicity byunstimulated T cells. In collaborationwith G. Riethmüller we are planning aphase I study for the treatment oflymphoma patients. Currently, we aretrying to establish the same strategyfor the treatment of multiple myelomapatients using a novel plasma cell-specific surface antigen as a targetstructure. Tumor cell-targeting canalso be achieved by chimeric T-cellreceptors, whereby the cytoplasmicpart of the ζ-chain of the T-cellreceptor/CD3 complex is fused to arecombinant single chain antibody.Using retroviral vectors, we haveachieved high transfer rates and stablesurface expression of chimeric T-cellreceptors with different specificities inT cells. A clinical phase-I trial inpatients with metastatic breast canceris planned.

Cell-biologic features of acuteleukemias

Wolf-Dieter Ludwig, ChristianWuchter, Richard Ratei, Leonid Karawajew

Acute lymphoblastic (ALL) and acutemyeloid leukemias (AML) exhibit ahigh degree of genotypic diversity.Recently, significant associationsbetween immunophenotypic andgenotypic features have beendescribed that in the near future mightcontribute to the development ofindividually adjusted treatmentstrategies. In the last few years, wehave characterized the expression and function of molecules involved in apoptosis regulation andchemosensitivity modulation in

T-lineage ALL and AML subtypes.Spontaneous apoptosis, cytokineresponsiveness and expression ofapoptosis-regulating Bcl-2 and Baxproteins have been analyzed inleukemic blasts from T-ALL patients.IL-7, in contrast to IL-4 and IL-2, is ahighly efficient inhibitor of apoptosisand this correlates with the expressionlevels of the IL-7 receptor (α-chain aswell as upregulation of Bcl-2 proteinexpression. In a large series of T-ALLsamples (n=130), in vitro IL-7responsiveness is associated withcortical/mature immunophenotype andbetter in vivo early cytoreduction. Thissuggests that IL-7 responsivenessmight have potential prognosticrelevance as a surrogate markerreflecting differential survival factordependence, apoptosis regulation andtreatment response in T lineage ALL.

Resistance to chemotherapy-inducedapoptosis and multiple-drug-resistance(MDR) activity, mainly mediated by the efflux pump P-glycoprotein (P-gp), contribute to the failure ofchemotherapy in hematologicmalignancies. In a large series of adultde novo AML patients, the mostimmature AML cells exhibited asignificantly lower CD95 (Fas/APO-1) expression, CD95 sensitivity andextent of spontaneous apoptosis invitro as well as a significantly higherBcl-2 expression and P-gp function,compared with more mature AMLblasts. Several functional parameters,including high P-gp function, lowspontaneous apoptosis in vitro, highBcl-2 expression and low CD95sensitivity, have been found to bepredictive of a poor response toinduction chemotherapy in adult denovo AML. Prospective studiesmonitoring apoptosis-relatedparameters during chemotherapy incytogenetically defined risk groupsare in progress.

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Selected Publications

Sturm, I., Köhne, C.H., Wolff, G.,Petrowsky, H., Hillebrand, T.,Hauptmann S., Lorenz M., Dörken,B., and Daniel, P.T. (1999) Analysis ofthe p53/BAX pathway in colorectalcancer: low BAX is a negativeprognostic factor in patients withresected liver metastases. J. Clin.Oncol. 17, 1364-1374.

Rickers, A., Beyaert, R.,Vandenabeele, P., Dörken, B., andBommert, K. (1999). Cleavage oftranskription factor SP1 by caspasesduring anti-IgM-induced B Cellapoptosis. Eur. J. Biochem. 261, 269-274.

Löffler, A., Kufer, P., Lutterbüse, R.,Zettl, F., Daniel, P.T.,Schwenkenbecher, J.M., Riethmüller,G., Dörken, B., and Bargou, R. (2000)A recombinant bispecific single chainantibody CD19xCD3 induces rapidand high lymphoma directed T cellcytotoxicity. Blood, in press.

Jundt, F., Anagnostopoulos, I.,Bommert, K., Emmerich, F., Müller,G., Foss, H.-D., Royer, H.-D., Stein,H., and Dörken B. (1999)Hodgkin/Reed-Sternberg cells inducefibroblasts to secrete eotaxin, a potentchemoattractant for T cells andeosinophils. Blood 94, 2065-2071.

Emmerich, F., Meiser, M., Hummel,M., Demel, G., Foss, H.-D., Jundt, F.,Mathas, S., Krappmann, D.,Scheidereit, C., Stein, H., and Dörken,B. (1999) Overexpression of I kappaB alpha without inhibition of NF-κBacitivity and mutations in the I kappaB alpha gene in Reed-Sternberg cells.Blood 94, 3129-3134.

Ludwig, W.D., Rieder, H., Bartram,C.R., Heinze, B., Schwartz, S.,Gassmann, W., Löffler, H., Hossfeld,D., Heil, G., Handt, S., Heyll, A.,Diedrich, H., Fischer, K., Weiss, A.,Völkers, B., Aydemir, Ü., Fonatsch,C., Gökbuget, N., Thiel, E., andHoelzer, D. (1998) Immuno-phenotypic and genotypic features,clinical characteristics, and treatmentoutcome of adult Pro-B acutelymphoblastic leukemia: Results ofthe german multicenter trials GMALL03/87 and 04/89. Blood 92, 1898-1909.

Structure of the Group

Group leaderProf. Dr. Bernd Dörken

ScientistsProf. Dr. Wolf-Dieter LudwigDr. Ralf BargouDr. Kurt BommertDr. Sophie CayeuxDr. Peter DanielDr. Florian EmmerichDr. Franziska JundtDr. Leonid KarawajewDr. Ida-Johanna KörnerDr. Markus MaparaDr. Stefan MathasDr. Gerhard WolffDr. Christian WuchterDr. Isrid Sturm

Graduate and undergraduate studentsDirk HöhnemannFreya RiechertSilke RitschelSebastian TheuringAntje HaefkerNiels PetersPatric SeibertAnja LöfflerJan SchwenkenbecherUlrike Steinhusen

Technical assistantsKati HildebrandtAndrea KatzerKerstin KrügerIna KrukenbergSylvia MetznerUte Nitschke

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MolecularImmunotherapy

Antonio Pezzutto

Our group is focusing on thedevelopment of molecularly definedimmunotherapy strategies for thetreatment of renal cell carcinoma,colorectal carcinoma and chronicmyeloid leukemia. A gene-modifiedtumor cell vaccine, that expresses ashared renal carcinoma antigenrecognized by T cells in the context ofHLA-A*0201 (developed incooperation with Th. Blankenstein(MDC) and D. Schendel (GSF,Munich), is being developed in ourGMP laboratory in the clinic: the firstpatients will be recruited in Spring2000. Other approaches in renalcancer include cytokine administrationstudies and vaccination protocolsusing dendritic cells.

Induction of T-cell immunityagainst EpCam (Epithelial CellAdhesion Molecule)

Günther Richter, Frank Kaiser

We have found that some patientswith colorectal cancer develop anMHC-II restricted response againstpeptides of the epithelial adhesionmolecule EpCam, which isoverexpressed in several humanadenocarcinomas. The EpCam-specific monoclonal antibody CO17-1A (Panorex®) is used in the adjuvanttreatment of colon cancer, T-cellimmunity seems to contribute to thistherapeutic activity. A correlationbetween the presence of EpCam-specific T cells and the clinical courseis currently being investigated.Transgenic mice expressing humanEpCam and human HLA-A2 are beinggenerated for use in vaccinationexperiments in order to evaluate thesafety and toxicity of EpCam-directedimmunity. Dendritic cells (DC) pulsedwith recombinant EpCam protein orselected EpCam MMC-I and MMC IIpeptides, gene-modified EpCam-expressing DC or EpCam-Adenovirus,will be evaluated for their ability toinduce rejection of EpCam-positivetumors. The feasibility of a clinicalvaccination study in patients withEpCam-positive adenocarcinomas willbe evaluated.

Use of dendritic cells for theinduction of antileukemicimmune response

Monika Schwarz, Günther Richter,Jörg Westermann, Kang Hun Lee

Nonamer peptides derived from thebcr-abl fusion protein that is producedas a consequence of the t(9:22)chromosomal translocation in patientswith chronic myeloid leukemia(CML) can bind to HLA-A3, -A11, or-B8. Indeed, HLA-B8 and HLA-A3appear to protect against thedevelopment of CML as shown inrecent epidemiology studies. We havestarted a clinical trial using in vitro-generated, bcr-abl-positive DC inCML-patients with the aim ofinducing a CML-directed immuneresponse. Therapy appears to be safeand flexible. Evaluation of theimmune responses is ongoing. Incooperation with B. Wittman-Liebold,A. Otto, and B. Wittmann (MDC,protein biochemistry), we areanalyzing naturally processed peptides

from CML-cells in order to detectother potential candidate peptides forvaccination. We have established abcr-abl-specific Elispot assay fordetection of bcr-abl-specific T cells,and an assay based on the use ofHLA-Class I tetramers is beingdeveloped. We have already detectedbcr-abl-specific T cells in somepatients in clinical remissionfollowing interferon treatment. Theseassays will allow us to monitor anti-leukemic immunity in CML patients.

Gene modification of dendriticcells

Jörg Westermann, Tam Nguyen-Hoay, Andreas Molweide

Both human and murine DC can begene-modified using retroviral vectorsand receptor mediated endocytosis(targetting the mannose receptor).Complexes of DNA, polyethylenimine(PEI) and mannose are efficientlyinternalized, resulting in geneexpression. A particular advantage ofthis method is the possibility oftransferring several genes with thesame construct, allowing theexpression of both tumor-antigens andgenes that can modulate DC function,such as superantigens, chemokinereceptors, and adhesion molecules. Astronger stimulation of the immuneresponse or an altered immuneresponse with prevalence of TH1immunity can be achieved with thismethod. These studies are beingperformed in cooperation with M.Zenke (MDC). The use of cytokinesthat can modify the number andfunction of DC, such as Flt-3 Ligandand GM-CSF, is being investigated ingene transfer models in tumorvaccination experiments.

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Selected Publications

Westermann, J., Aicher, A., Qin, Z.,Cayeux, S., Daemen, K.,Blankenstein, T., Dörken, B., andPezzutto, A. (1998) Retroviralinterleukin-7 transduction into humandendritic cells: a strategy to increaseT-cell activation. Gene Therapy 5,264-271.

Daniel, P.T., Kroidl, A., Kopp, J.,Sturm, I., Köhne, C.H., Moldenhauer,G., Dörken, B., and Pezzutto, A.(1998) Immunotherapy of B celllymphoma with CD3xCD19 bispecificantibodies: costimulation with CD28prevents “veto” apoptosis of antibody-targeted cytotoxic T cells. Blood 92,4750-4757.

Cayeux, S., Richter, G., Becker, C.,Pezzutto, A., Dörken, T., andBlankenstein T. (1999) Direct andindirect T cell priming by dendriticcell vaccines. Eur. J. Immunol. 29,225-234.

Fan, W., Richter, G., Cereseto, A.,Beadling, C., and Smith, K.A. (1999)Cytokine response gene 6 induces p21and regulates both cell growth andarrest. Oncogene 18, 6573-6582.

Aicher, A., Shu, G., Magaletti, D.,Mulvania, T., Pezzutto, A., Craxton,A., and Clark, E.A. (1999)Differential role for p38 MAPK inregulating CD40-induced geneexpression in dendritic cells and Bcells. J. Immunol. 163, 5786-5795.

Structure of the Group

Group leader Prof. Dr. Antonio Pezzutto

Scientists Dr. Günther Richter Dr. Jörg WestermannDr. Kang Hun Lee

Graduate and undergraduate studentsFrank KaiserAndreas MolweideTam Nguyen-HoayMark SchnitzlerMonika Schwarz

Technical assistantsKerstin DaemenJessica Bigott

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Figure 35: Two gene-modified DC expressingthe β-Galactosidase reporter gene (a nuclearlocalisation signal determines the darkerstaining of the nucleus) close to a normal, nontransduced lymphocyte.

ExperimentalPharmacology

Iduna Fichtner

The group is continuing with thedevelopment of in vitro and in vivomodels of relevance to preclinicalinvestigations in cancer. With the helpof these models, we shall addressquestions concerning :

• the expression of tumor markers inrelation to biologicalcharacteristics, such as metastasis,drug- or hormone resistance,

• the pharmacological potential ofnovel therapeutic or diagnosticapproaches, or

• the possibility of mitigatingtherapy-induced side-effects.

Expression of tumor-relatedmarkers

In this research, the occurrence ofmarkers for metastasis (CD44),resistance (MDR), endocrinedependence (estrogen receptor) orimmune defence (RANTES) havebeen correlated with the properties oftumor growth.

The surface marker CD44 and severalof its splice variants are expressed in a very specific pattern in individualbreast cancer xenografts, as revealedby RT-PCR and immunohistochemistry.The detection of certain CD44-isoforms is not related to the hormonedependence or metastasis capacity ofthe tumors. Cytostatics andantihormones used clinically for thetreatment of breast cancer do notaffect the expression pattern of CD44in xenografts indicating that it is asuitable target molecule for gene- orimmunotherapeutic approaches.

In a clinically related study in 14human sarcomas, we have found aclose relationship between theexpression of the multidrug resistancegene (mdr1) and the response todoxorubicin, both in xenografts andpatient tumors, while for lungreistance protein (LRP) and MDR-associated protein (MRP) there wasonly a poor correlation. We haveconcluded that screening sarcomas forMDR-related markers clearly predictschemoresistance and helps avoidunnecessary and toxic treatment.

In cooperation with the University ofMannheim, the chemokine RANTEShas been found to be expressed by a subset of melanomas. It isresponsible for the recruitment ofmonocytes, T-cells and dendritic cellsbut, surprisingly, it favored tumorformation in nude mice.

Models for novel therapeutic ordiagnostic approaches

In cooperation with the Department ofPediatric Oncology/Hematology of theVirchow-Clinics leukemic blasts ofpatients have been transplanted toseverely immunodeficient NOD/SCIDmice. In all, 11/16 acute lymphaticleukemias were successfullyestablished in vivo and shown tomaintain their immuno- and genotypeduring passaging. The antileukemicactivity of allogeneic humanmononuclear cells as a graft versus

leukemia (GVL) reaction with anaccompanying graft versus hostdisease (GVHD) was simulated in themouse model. The chemo- andradiation sensitivity of the ALL linesresembled that in a clinical situation.We believe that xenotransplanted ALLcan be considered as clinicallyrelevant models mimicking humanconditions and are a useful preclinicaltool for the evaluation of novelimmuno- or gene therapeuticapproaches.

Another extended study deals with thedetection of minimal residual disease(MRD) in the bone marrow of patientswith solid tumors. At present, occultepithelial cells are determined byimmunohistochemical or PCRmethods in patient samples. However,nothing is known about the viabilityof these cells or their proliferating andmetastasizing potential. Therefore,bone marrow samples of 13 patientswith breast cancer, 30 from colorectalcancer (Robert-Rössle-Clinics) and 33from head and neck cancers (Mund-Kiefer-Gesichtschirurgie, VirchowClinics) were transplanted to NOD/SCID mice. Human and epithelialcell-specific detection methods havebeen developed for a sensitive proofof potential cancer cells in murineorgans. The results obtained show thatonly in rare instances can vital cancercells be found. These findingscorrelate with the poor prognosis forthe disease. Additionally, the resultssuggest that the evidence of epithelialcells in bone marrow samples resultsin too many false positives concerningthe survival potential of those cells.

Engraftment of non-hematopoietic progenitor cellsfrom human blood inimmundeficient mice

Human cord blood (CB) and humanmobilized peripheral blood (PB) areattractive cell sources for hematopoietictransplantation, but their potential toform non-hematopoietic cells is as yetpoorly characterized. Six to nineweeks after injection of separatedCD34+-cells from CB and PB intosublethally irradiated NOD/SCID-mice we found besides humanhematopoietic cells (up to 40 %) inchimeric bone marrow, cells stainingpositive with antibodies specific forhuman fibroblasts and humanendothelial cells. PB CD34+-cells wereflow-cytometrically sorted intoCD34+/CD38low and CD34+/CD38high-

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fractions. The hematopoietic potentialwas found predominantly in theCD34+/CD38low –fraction, whilehuman fibroblasts marker-positivecells and human endothelial cells weremuch more commonly detected aftertransplantation of the CD34+/ CD38high –fraction. These data showthat non-hematopoietic cellpopulations are present in humanblood cell transplants, engraft in vivoand probably support donorhematopoiesis. This techniqueprovides a preclinical model toevaluate clinical protocols involvingthe transplantation of hematopoiesis-supporting stromal populations intopatients with myelotoxic andmyelodysplastic disorders.

Selected Publications andPatents:

Dehmel, A., Becker, M., Lemm, M.,and Fichtner, I. (1999) Expression ofCD44 isoforms in human breastcarcinoma xenografts is notinfluenced by the treatment of micewith cytostatics or (anti-)hormones.Anticancer Res. 19, 1977-1988.

Hoffmann, J., Schmidt-Peter, P.,Hänsch, W., Naundorf, N., Bunge, A.,Becker, M., and Fichtner, I. (1999)Anticancer drug sensitivity andexpression of multidrug resistancemarkers in early passage humansarcomas. Clinical Cancer Res. 5,2198-2204.

Mrowietz, U., Schwenk, U., Maune,S., Bartels, J., Küpper, M., Fichtner, I.Schröder, JM., and Schadendorf, D.(1999) The chemokine RANTES issecreted by human melanoma cellsand is associated with enhanced tumorformation in nude mice. Br. J. Cancer79, 1025-1031.

Henschler, R., Möbest, D.,Spyridonidis, A., Goan, S.R.,Junghahn, I., Fichtner, I., Groner, B.,Wels, W., Bosse, R., Winkler, J.,Mertelsmann, R., and Schulz, G.(1999) Behavior of hematopoieticstem cells and solid tumor cells duringex vivo culture of transplants fromhuman blood. In: Autologous bloodand marrow transplantation. 550-560,ed by Dicke, K.A., and Keating, A. byJennings Publishing Co., Ltd. USA.

Fichtner, I., Goan, S.R., Becker, M.,Baldy, C., Borgmann, A., vonStackelberg, A., and Henze, H. (1999)Transplantation of humanHaematopoietic or leukaemic cellsinto SCID and NOD/SCID mice. Invivo models for hematopoiesis. FiebigHH, Burger AM (eds): Relevance oftumor models for anticancer drugdevelopment. Contrib Oncol. Basel,Karger, 54, 207-217.

Fichtner, I., and Nowak, C. (1996)Procedure for the detection ofmalignancy of occult tumor cells inbody fluids. Patent: Date ofApplication: 19.09.1996, Referencenumber: DE 196 36 219.9

Structure of the Group:

Group leader:Dr. Iduna Fichtner

Scientists:Dr. Michael BeckerDr. Silvia-Renate GoanDr. Ilse Junghahn

Graduate and undergraduate students:Anke DehmelChristina BaldyDiana BehrensBrigitte Jost-Reuhl

Technical assistants:Jutta AumannMonika BeckerClaudia NeumannMargit Lemm

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Figure 36: Detection of human endothelial cells(EN4-positive) in long term cultures of chimericbone marrow derived eight weeks aftertransplantation of separated cord blood CD34+cells in NOD/SCID mice.

Drug TargetingRegina Reszka

The major focus of our group is thedevelopment, characterisation, andtesting of new drug carrier systems forboth therapeutic and diagnosticapplications. Therapeutic approachesinclude the establishment andoptimisation of in vivo liposomal genetransfer of “suicide” genes, humanchemokine genes, as well as p53-independent apoptosis-inducing genesall for the treatment of primary braintumors and liver metastases. Withregard to immunological gene therapy,we are investigating the use of humanchemokine genes to attract leukocytesand to modulate the angiogenesis ofdifferent tumors.

Therapy of liver metastases

One strategy in anticancer genetherapy is the use of “suicide” genes.We have concentrated on the tumor-specific expression of Herpes simplexvirus thymidine kinase gene (HSV-tk)expression under the control of thecarcino-embryo-antigene promotor(CEA).

In contrast to viral-based strategies,our delivery approach uses in vivocationic and surface-modifiedliposomal gene transfer systemsadministered intrahepatically. For theeffective transfer of marker (figure)and therapeutic genes (includingsuicide genes) we have developed anew drug carrier embolisation system(DCES) which combines three novalprinciples to yield a hybrid techniquewith high transduction and therapeuticefficiency.

Therapy of glioblastomas

For the in vivo transfection of ratglioblastomas with the TK suicidegene, we are using three differentcationic liposomal formulations,including our own DAC-CHOL/DOPE, to deliver the pUT TK vector.The liposomal delivery of pUT TKhas been compared with adenoviraland retrovirus producing systemscarrying the HSVtk gene. Althoughnone of these systems is capable ofefficient gene delivery in vivofollowing a single application, a keyadvantage of the liposomal system isthat we can administer the liposome-DNA complex continuously over threedays via a mini-osmotic pump. Withthis technique, we can obtainsignificant tumor regression. Theassessment of the safety and toxicityof this gene delivery show no organpathology. We have demonstrated byimmunohistochemistry that onlyanimals with complete tumor rejectionexhibit macrophages as well as T- andB-lymphocyte infiltration in theformer tumor area. This suggests animmune host response followingtherapy and supports the hypothesisthat this effect is necessary fortherapeutic success.

Our present in vivo studies arefocused on optimising theadministration route and schedule ofganciclovir dosing. The non-viralsuicide gene delivery system, usingeither continuous i.v. infusion ordirect CNS application via pumps, is

now being introduced in a clinicaltrial involving two neurosurgerycenters, in Cologne and Düsseldorf.

An additional therapeutic approach isthe evaluation of the biological effectsof selected rat chemokines on ratbrain tumor development. F98 cellshave been stably transfected with thesub-cloned rat chemokines MIP-1α,MCP-1 and Mob-1 and inoculated inrat brains. Inoculated non-transfectedF98 cells serve as a control. Thebiological activities of MIP-1α, MCP-1 and Mob-1 have been evaluatedwith regard to tumor growth, immuneresponse and microglial activation.

We are currently investigating variousnewly synthezised biodegradablecationic lipids, with cholesterol orglycerol backbones, as improved genedelivery systems.

Another interest is the characterizationof two novel gene transfer systemsfrom Quiagen, SuperFect™ andEffectene™. SuperFect™ is anactivated dendrimer and Effectene™ anovel cationic lipid with an additionalDNA-condensing agent. The aim ofthese studies is the characterisation ofthe electrostatic and colloidalproperties which give rise to effectivegene transfer. These data will providea deeper insight into the structure-activity relationships of gene vectors.

To further advance the gene therapyfor cancer, we are cooperating withProf. Winter’s group in Dortmund todevelop an implantable drug depotencapsulating clinically wellestablished cytostatics with knowndose-limiting toxicity, such asCarboplatin and Taxol. This novelsystem represents a cubic phasestructure which releases bothencapsulated drugs with differentsustained phamacokinetics. It will beused for the local chemo-treatment ofglioblastomas after surgery. To gainmore genetic and basic molecularinformation about the growthcharacterisation and invasiveness ofrecurrent glioblastoma, we arecooperating with the neurosurgeryunit of Berlin-Buch to develop andexpand a brain tumor bank. Thisresource includes an extensivecatalogue of CNS tumors, includingdifferent stages of glioblastoma andastrocytoma (grade II, III, IV).

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Interaction of pharmacologicallyactive substances and differenttypes of drug carriers

To obtain a detailed knowledge of themolecular regulation of phospholipidassymetry in tumor cells aftertreatment with pharmacologicallyactive substances, as an indicator ofapoptosis or growth regulation, wehave studied calcium signalingprocesses in different cell lines.

Two dimensional polyacrylamide gelelectrophoresis (2-D PAGE) has beenestablished to determine the plasmaprotein adsorption patterns ofliposomes as a determinant for organdistribution. Information about thecorrelation between protein adsorptionand in vivo organ distribution can beused to achieve drug delivery to thedesired target sites.

For the pulmonary administration ofliposomes, we have developed a newtechnique to generate a liposomal drypowder aerosol by spray evaporation.Using this mild method, the particlesize of the aerosol can adjusted to therequirements of the particular disease,resulting in an improved depositionrate and, consequently, better

therapeutic effect with reduced side-effects. Furthermore, a new targetingconcept will be established based onprotein-receptor interactions toincrease drug concentrations in thetarget tissue.

New magnetic particles could beisolated and processed from thebacterium, Magnetospirillumgryphiswaldense. These so-called“magnetosomes” are now beingdeveloped as NMR-diagnostic andtherapeutic formulations and asconventional and gene-transfer tools.

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Figure 37: Lac Z expression in the marginalliver tumor zone in rats after a singleapplication of 10µg LacZ gene (pUT 651)(Treatment schedule: 105 CC 531 cells wereinoculated directly subcapsularly into the liverof male Wag/Rij rats at day 0, treatment was atday 10 with the LacZ DCES, and rats weresacrificed at day 15, histochemistry of the tissuesections).

Selected Publications

Groth, D., Keil, O., Schneider, M.,and Reszka, R. (1998) Transfectionassay for dual determination oftoxicity and gene expression. Anal.Biochem. 258, 141-143.

Schulz, J.G., Megow, D., Reszka, R.Villringer, A., Einhäupl, K.M., andDirnagl U.(1998) Evidence thatglypican is a receptor mediating β-amyloid neurotoxicity in PC12 cells.Eur. J. Neurosci. 10, 2085-2093.

Bucke, W.E., Leitzke, S., Diederichs,J.E., Borner, K., Hahn, H., Ehlers, andMüller, R.H. (1998) Surface-modifiedamikacin-liposomes: Organdistribution and interaction withplasma proteins. J. Drug Targ. 5, 99-108.

Lisdat, F., Ge, B., Ehrenteich-Förster,E., Reszka, R., and Scheller F.W.(1999) SOD activity measure-mentusing cytochrome c modifiedelectrode. Anal. Chem. 71, (7), 1359-1365

Mastrobattista, E., Storm, G., vanBloois, L., Reszka, R., Bloemen,P.G.M., Crommelin, D.J.A., andHenricks, P.A.J. (1999) Cellularuptake of liposomes targeted tointercellular adhesion molecule-1(ICAM-1) on bronchial epithelialcells. Biochim. et Biophys. Acta 1319(2), 353-363.

Patent Applications

Diederichs, J.E., Koch, W., Lödding,H., Reszka, R., and Windt H.Druckluftinhalator zur pulmonalenApplikation eines liposomalen Pulver-Aerosolssubmitted: 3.2.1999Reference: 199 05 285.9-41

Reszka, R., and Schlüter R.Implantierbares Wirkstoffdepotsubmitted: 6.8.1999Reference: 199 38 331.6

Structure of the Group

Group leaderDr. Regina Reszka

ScientistsDr. Jutta BergmannDr. Julia Eva DiederichsDr. Katrin EngelMathias LangDr. Denise van Rossum

Graduate and undergraduate studentsOliver BraunSandra GromelskiDetlef GrothCathleen LehmannJörg LodeStefanie SeemannHolger StraussKajetan von EckardsteinDaniela Wegner

Technical assistantsIngrid BergerGudrun HartmannBärbel PohlJana Richter

SecretariatSinaida Rink

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Molecular Basis ofCongestive HeartFailureRainer Dietz

Regulation of cardiac cellgrowth and death

Frequently, congestive heart failure isthe clinical consequence of astructural remodeling of the cardiacphenotype, which is characterized bytwo major components: maladaptivegrowth and excessive death ofcardiomyocytes. Therefore, in order tounderstand the molecular basis ofcongestive heart failure one has toelucidate the signaling cascadecontrolling both the growth and deathof cardiac cells and describe theirinterrelationship.

In contrast to cardiac growth wherenumerous stimulating factors havebeen identified, almost nothing isknown about the factors inducingprogrammed death of cardiac cells.Oxidative stress induced by oxygenfree radicals (ROS) is intimatelyinvolved in the development of thephenotype of the failing heart,particularly since cardiomyocytes arecharacterized by a high amount ofoxidative phosphorylation. Notably,the lack of scavenging enzymes leadsto severe and lethal dilativecardiomyopathy in knock-out mice. In cell culture models we have beenable to characterize the intracellularsignaling cascade in cardiomyocytesor vascular smooth muscle cellsexposed to oxidative stress ultimatelyleading to apoptosis.

Furthermore, the role of ROS in p53-induced apoptosis has beeninvestigated. The results show that notBax, but ROS, are the downstreammediators of p53-induced apoptoticsignaling.

It is of great importance to understandhow terminally differentiated andpost-mitotic cells like cardiomyocytes,can undergo programmed cell death,since it is generally believed thatapoptosis is restricted to proliferatingcell types due to the fact that it onlycan occur during a specific andlimited phase of the cell cycle. Thisindicates that there has to be a tightfunctional interrelationship betweenthe control of cell death, cell growthand the cell cycle in cardiomyocytes.Therefore, we recently investigatedthe effect of overexpression of E2F-1, which is a key factor in cellcycle control, on the function ofcardiomyocytes. Cultured ratcardiomyocytes infected with anadenovirus harboring the E2F-1cDNA start to initiate the cell cyclemachinery, as reflected by anincreased expression of S-phasespecific genes. However, the vastmajority of these cells undergoapoptosis before entering the S-phase.In contrast, cardiomyocytesoverexpressing E2F-1 overcome theapoptotic signaling cascade andinitiate DNA-synthesis when insulin-like growth factor I (IGF-I) is addedto the culture medium. This is ofparticular interest since, in the heart,IGF-I functions as the exclusivedownstream mediator of growthhormone, which is currently used inclinical trials in patients withcongestive heart failure.

In order to elucidate the cardiac cellcycle control more systematically, wehave delevoped a myocardial cell-freesystem where nuclei of terminallydifferentiated cardiomyocytes areexposed to cellular and/or nuclearextracts of proliferating cells, leadingto the reinduction of cardiomyocytenuclear DNA synthesis.

Selected Publications

von Harsdorf, R., Hauck, L., Mehrhof,F., Wegenka, U., Cardoso, C., andDietz, R. (1999) E2F-1overexpression in cardiomyocytesinduces downregulation of p21CIP1and p27KIP1 and release of activecyclin-dependent kinases in thepresence of insulin-like growth factorI. Circ. Res. 85, 128-136.

von Harsdorf, R., Li, P.-F., and Dietz,R. (1999) Signaling pathways inreactive oxygen species-inducedcardiomyocyte apoptosis. Circulation99, 2934-2941.

Li, P.-F., Dietz, R., and von Harsdorf,R. (1999) p53 regulates mitochondrialmembrane potential through reactiveoxygen species and inducescytochrome c-independent apoptosisblocked by Bcl-2. EMBO J. 18, 6027-6036.

Li, P.-F., Maasch, C., Haller, H.,Dietz, R., and von Harsdorf, R. (1999)Requirement for protein kinase C inreactive oxygen species-inducedapoptosis of vascular smooth musclecells. Circulation 100, 967-973.

Engel, F., Hauck, L., Cardoso C.,Leonhardt, H., Dietz, R., and vonHarsdorf, R. (1999) A mammalianmyocardial cell-free system to studycell cycle reentry in terminallydifferentiated cardiomyocytes. Circ.Res. 85, 294-301.

Structure of the Group

Group leaderProf. Dr. Rainer Dietz

ScientistsDr. Rüdiger v. HarsdorfDr. Ludger HauckDr. Pei-Feng Li

Graduate studentsFelix EngelFelix Mehrhof

Technical assistantsMarlies Grieben

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Immunology ofCardiovascularDiseasesGerd Wallukat

Our laboratory is engaged in clinicaland basic research mainly focused on immunological processes incardiovascular diseases. We haveobserved autoantibodies againstadrenergic receptors and AT1-receptorsin the sera of patients withmyocarditis, dilated cardiomyopathy,and hypertension. These autoantibodiesrecognize epitopes on the first orsecond extracellular loop of thereceptors and act like the correspondingpharmacological agonists. In patientswith myocarditis and dilatedcardiomyopathy, but also in Chagas’disease, the autoantibodies recognizethe β1-adrenoceptor and muscarinicM2 receptor as an antigen. In patientswith hypertension, the autoantibodiesare directed against the α1-adrenergicreceptor and/or AT1-receptor.

In recent years, we have investigatedin more detail the effects of β1-adrenoceptor autoantibodies. Thesehuman autoantibodies cross-react withthe rat β1-adrenoceptor and exhibittheir effects via the ß-adrenoceptor -adenylate cyclase - protein kinase A -cascade. Long-term treatment ofcultured rat cardiomyocytes with thisantibody leads to a subtype specificreduction of the expression of the β1-adrenoceptor on mRNA and proteinlevels and to upregulation of theinhibitory G-protein Giα.

Autoantibodies in myocarditisand dilated cardiomyopathy

The suggestion that the anti-β1-adrenoceptor autoantibody might playa role in the pathogenesis of DCM issupported by similar findings inpatients with myocarditis, a diseasewidely held to be a precursor ofDCM. It is, therefore, also of interestin the present context that, in a patientwith acute myocarditis, the healingprocess, as reflected by a normalizationof the ejection fraction and the heartrate, correlates with disappearance ofthe anti-β1-adrenoceptor autoantibodiesfrom the blood.

Based on our autoimmune hypothesis,we have proposed new therapeuticpossibilities to treat patients withendstage dilated cardiomyopathy. Oneof them is immunoadsorption usingTherasorb columns to removeimmunoglobulins from the patient’splasma. After this treatment, a markedimprovement in cardiac function isobserved. This improvement is notonly observed immediately aftertreatment, but for a long periodthereafter. In these patients treatedwith standard therapy plus β-blockingagents, after immunoadsorption,autoantibodies were not detectableand the ejection fraction (EF)increased from 22.3 % beforetreatment to 37.0 % after a period of12 months.

The strong correlation observedbetween the reduction in the amountof circulating autoantibodies to the β1-adrenoceptor and the improvementin the function of the heart just describedcan be interpreted as supporting thehypothesis that the anti-β1-adrenoceptorantibodies play a part in thepathophysiology of myocarditis andDCM.

To confirm this hypothesis, we havenow developed an immunoadsorptioncolumn that selectively removes onlythe anti-β1-adrenoceptorautoantibodies.

Autoantibodies in hypertension

Furthermore, we have investigated therole of autoantibodies in essential andmalignant hypertension. In some seraof patients with this disease we havedetected autoantibodies directedagainst the α1-adrenergic receptor.These autoantibodies and anti-peptideantibodies generated against peptidescorresponding to the first or secondextracellular loop of the α1-adrenergicreceptor recognize both theseextracellular loops and act like an α-adrenergic agonist.

In a special type of hypertension –preeclampsia – we have observedautoantibodies against the angiotensinII AT1-receptor. This antibody isdetectable after the 20th week ofpregnancy and disappears afterdelivery. The anti-AT1-receptorantibodies act like the agonistangiotensin II and induce theformation of the AP1- complex. Thesefunctional autoantibodies are found inall preeclamptic women investigatedand may play a role in elevatingvascular resistance and promotinghypertension and cardiac hypertrophyin these patients.

Role of mast cells in the heart

Another of our research topics iscardiac mast cells. Because the heart of patients with dilatedcardiomyopathy contain four timesmore mast cells and more histaminethan controls, we wish to discover therole of these cells. Using a monoclonalantibody against surface determinantsof rat connective tissue mast cells, wehave been able to identify a greatnumber of, mostly undifferentiated,mast cells in the neonatal rat heart andin cell cultures prepared from thisorgan. In cell culture, we have beenable to differentiate the mast cells.These differentiated mast cells, mostlylocated in intimate contact withcardiomyocytes, synthesize themediators histamine, serotonin andtumor necrosis factor α (TNFα). Inheart tissue of DCM patients, we haveinvestigated the degranulation ofTNFα and tryptase from mast cells.Both mediators may be involved inthe development of fibrosis in thefailing heart.

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Selected Publications

Schneider, G., Schrödel, W., Wallukat,G., Müller, J., Nissen, E., Rönspeck,W., Wrede, P., and Kunze, R. (1998)Peptide design by artifical neuralnetworks and computer-basedevolutionary search. Proc. Natl. Acad.Sci. USA 95, 12179-12184.

Podlowski, S., Luther, H.P.,Morwinski, R., Müller, J., andWallukat, G. (1998) Agonistic anti-β1-adrenergic receptor autoantibodiesfrom cardiomyopathy patients reducethe β1-adrenergic receptor expressionin neonatal rat cardiomyocytes.Circulation 98, 2470-2476.

Wallukat, G., Fu, H.M., Matsui, S.,Hjalmarson, Å., and Fu, M.L. (1999)Autoantibodies against M2 muscarinicreceptors in patients withcardiomyopathy display non-desensitizing agonist-like effects. Life Sci. 64, 465-469.

Wallukat, G., Homuth, V., Fischer, T.,Lindschau, C., Horstkamp, B., Jüpner,A., Baur, E., Nissen, E., Vetter, K.,Neichel, D., Dudenhausen, J.W.,Haller, H., and Luft, F.C. (1999)Patients with preeclampsia developagonistic autoantibodies against theangiotensin AT1 receptor. J. Clin.Invest. 103, 945-952.

Müller, J., Wallukat, G., Dandel, M.,Bieda, H., Brandes, K.,Spiegelsberger, S., Nissen, E., Kunze,R., and Hetzer, R. (2000)Immunoglobulin adsorption inpatients with idiopathic dilatedcardiomyopathy. Circulation 101, inpress.

Structure of the Group

Group leaderDr. Gerd Wallukat

ScientistsDr. Rosemarie Morwinski*Dr. Eberhard Nissen**

Graduate and undergraduate studentsDajana Neichel

Technical assistantsKarin KarczewskiHolle SchmidtMonika Wegener

SecretariatVerona Kuhle

* supported by BBB** supported by Affina GmbH

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MolecularImmunology andGene TherapyThomas Blankenstein

Interleukin 4-deficient micereconstituted with wild-typebone marrow fail to producenormal immunoglobulin E levels

The ability to reconstitute interleukin(IL)-4-/- mice with bone marrow fromIL-4+/+ mice was investigated. Theabsence of the IL-4-/- gene in donor orrecipient cells did not impair thereconstitution. All immunoglobulin(Ig) subsets occurred at normal serumlevels, except for IgE and to someextent IgG1. IgE production did notrecover in the reconstituted mice overa long period. However, these micewere competent for IgE production,because a single intrasplenic injectionof IL-4 restored IgE levels, whichthen remained constant. Wild-typemice reconstituted with wild-typebone marrow maintained IgE serumlevels comparable with untreatedanimals. In wild-type micereconstituted with IL-4-/- bone marrow,IgE levels decreased gradually anddisappeared after 12 weeks. We havemade three unrelated, but nonethelessimportant, conclusions: (a) (immunoregulation) the tightlyregulated IL-4 gene is expressedcontinuously in low amounts (andwith apparent absence of antigenstimulation) to maintain the normalthreshold of IgE; (b) (ontogeny of theimmune system) an early unidentifiedsource of IL-4 is postulated which islost in adult mice; and (c) (bonemarrow transfer/gene therapy) undercertain circumstance, the genotype ofthe recipient influences thereconstitution.

B cells inhibit induction of T cell-dependent tumorimmunity

Cytotoxic T lymphocyte (CTL)mediated tumor immunity against

major histocompatibility antigen(MHC) class I+ but class II- tumorsoften requires help from CD4+ T cells.These CD4+ T cells are activated byMHC class II+ cells that present tumorderived antigens. Considering thatdifferent antigen presenting cells(APC), such as B cells, macrophagesand dendritic cells, compete forantigen and influence the outcome ofan immune response, we haveexamined tumor immunity in B celldeficient mice and showed that thelow immunogenicity of tumors iscaused by B cells whose presence inthe priming phase results in disabledCD4+ T cell help for CTL-mediatedtumor immunity. Instead, in thepresence of B cells, a non-protectivehumoral immune response is induced.Our results may explain the enigmaticobservation that tumor-reactiveantibodies occur frequently in cancerpatients.

Direct and indirect T cellpriming by dendritic cellvaccines

The mechanisms by which dendriticcell (DC) vaccines prime host T cellsin vivo has been examined. Mice wereimmunized with syngeneic bonemarrow-derived DC and β-galactosidase (β-gal) was used as asurrogate antigen. DC, either pulsedwith peptide, loaded with β-galantigen or gene-modified, induced β-gal-specific CTL and moderaterejection of an in vivo challenge withβ-gal expressing tumors. In addition,β-gal-specific CTL lysed thesyngeneic DC that were used asvaccines. Using SCID micereconstituted with F1 lymphocytes,direct priming by gene-modified DCvaccines was demonstrated by thepresence of β-gal-specific CTL of thehaplotype exclusively expressed byDC, while indirect priming by hostAPC was shown by the detection ofCTL of the haplotype exclusivelypresent on host APC and absent onDC vaccines. DC in vitro bylymphokine-activated killer cells, DCvaccines appear to interact with hostnatural killer cells as well as withantigen-specific T cells. These effectorcells, in turn, may lyse DC vaccines,thereby, leading to the release ofantigens that can be taken up by hostAPC.

TH1 associated and cytotoxic Tlymphocyte-mediated tumorimmunity is impaired in IL-4deficient mice

Cellular immune responses areinduced by CD4+ T helper 1 (Th1)cells secreting interleukin (IL)-2 andinterferon (IFN)-γ. Tumor immunity isoften mediated by CTLs whoseactivation is supported by Th1cytokines. Since IL-4 directs Th2development, and has been shown toinhibit Th1-dominated responses, wehave assumed that IL-4-deficient (IL-4-/-) mice would develop vigorousCTL-mediated tumor immunitycompared with IL-4-competent (IL-4+/+) mice. Surprisingly, IL-4-/-

mice exhibited a severely impairedability to develop tumor immunity.The lack of tumor immunity in IL-4-/-

mice was associated with reducedIFN-γ production, diminished levelsof tumor-reactive serum IgG2a, andundetectable CTL activity, indicatinga defective Th1 response in theabsence of endogenous IL-4. Anti-IL-4 monoclonal antibody blocked tumorimmunity in IL-4+/+ mice whenadministered at the time ofimmunization but not at the time ofchallenge. Additionally, tumorimmunity could be induced in IL-4-/-

mice, if IL-4 was provided by gene-modified cells together withimmunizing tumor cells. These resultsdemonstrate that tumor immunityrequires IL-4 in the priming phase forthe generation of effector cells ratherthan for their maintenance. Together,our results demonstrate a novel, andpreviously unanticipated, role of IL-4in the generation of Th1-associated,CTL-mediated tumor immunity.

Retroviral gene transfer

We have constructed retroviral vectorscarrying marker genes such as β-galactosidase and green fluorescentprotein and have optimized retroviralgene transfer into different cell types.We have analyzed the retrovirusreceptor expression on differenthuman tissues and cell lines andshown that the amount of receptorexpression does not correlate with thetransduction efficiency of threeretrovirus vector pseudotypes (A-MuLV, GALV, 10A1) using thesereceptors for cell entry. We havegenerated retrovirus vectors carrying‘suicide’ genes encoding cytosinedeaminase and HSV thymidine kinaseand transferred these genes into

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different murine and human tumorcells. We have found that the ‘suicide’gene/prodrug effect depends on thetumor model and that a double‘suicide’ gene approach is superior tosingle suicide gene activation both invitro and in vivo. Successful ‘suicide’gene/prodrug treatment requires hostimmune competence.

Selected Publications

Uckert, W., Kammertöns, T., Haack,K., Qin, Z., Gebert, J., Schendel, D.J., and Blankenstein, Th. (1998)Double suicide gene (cytosinedeaminase and herpes simplex virusthymidine kinase) but not single genetransfer allows reliable elimination oftumor cells in vivo. Hum. GeneTherapy 9, 855-865.

Lange, C., Schüler, T., andBlankenstein, Th. (1998) Interleukin 4gene-defective mice reconstituted withwild-type bone marrow fail to producenormal immunoglobulin E levels. J.Exp. Med. 187, 1487-1493.

Qin, Z., Richter, G., Schüler, T., Ibe,S., Cao, X., and Blankenstein, Th.(1998) B cells inhibit induction of Tcell-dependent tumor immunity.Nature Med. 4, 627-630.

Uckert, W., Willimsky, G., Pedersen,F. S., Blankenstein, Th., and Pedersen,L. (1998) RNA levels of humanretrovirus receptors Pit1 and Pit2 donot correlate with infectibility by threeretroviral vector pseudotypes. Hum.Gene Therapy 9 (17),2619-2627.

Cayeux, S., Richter, G., Becker, C.,Pezzutto, A., Dörken, B., andBlankenstein, Th. (1999) Direct andindirect T cell priming by dendriticcell vaccines. Eur. J. Immunol. 28,225-234.

Schüler, T., Qin, Z., Ibe, S., Noben-Trauth, N., and Blankenstein, Th.(1999) TH1 associated and cytotoxicT lymphocyte mediated tumorimmunity is impaired in IL-4 deficientmice. J. Exp. Med. 189, 803-810.

Structure of the Group

Group leaderProf. Dr. Thomas Blankenstein

ScientistsDr. Christian BeckerDr. Zhihai QinDr. Thomas SchülerDr. Wolfgang UckertDr. Gerald Willimsky

Graduate and undergraduate studentsMonika GladowJens HemmeSabrina IbeThomas KammertönsLiang-Ping LiMariette MohauptSusanne Preiß

Technical assistantsKatja BeckerAngelika GärtnerIrmgard KüttnerMarion RöschChristel Westen

SecretariatSylvia Klahn

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Cellular ImmunologyKirsten FalkOlaf Rötzschke

The newly established group will startoperation in spring 2000. One of themajor areas of interest is theinvestigation of mechanisms relevantfor the induction or suppression ofimmune tolerance.

The thymal selection of T cells, inconcert with peripheral tolerancemechanisms, generates and maintainsa T cell repertoire, which responds toforeign antigens but not to peptidesderived from self-proteins. Undercertain circumstances, however, thesetolerance mechanisms can fail.Autoreactive T cells escape theselection-process and cause theinduction of chronic autoimmunediseases, such as multiple sclerosis,diabetes mellitus or rheumatoidarthritis. In most of these autoimmunediseases the typical tissue-specificdamage is caused by the activity ofautoreactive CD4+ T cells (and not byCD8+ T cells). In the case ofexperimental autoimmuneencephalomyelitis (EAE) the CD4+ Tcells are responsible for the removalof the isolating myelin sheath fromthe axons of the CNS. They inflict thedamage either by a direct attack of themyelin sheath or indirectly byactivating B cells, which producemyelin-specific antibodies. While inthese chronic autoimmune diseasesthe effect of autoreactive CD4+ T cellsis extremely harmful, it could be verybeneficial if the response could bedirected against self-proteinsexpressed in transformed tissue.

In order to break tolerance it isnecessary to increase the sensitivity ofthe T cells to the autoantigen. Oneway is to manipulate co-stimulatorysignals. For instance, the blockade ofCTLA-4, an attenuator of the T cellactivation cascade, can significantlylower the threshold for the T cellactivation. However, a general

disadvantage of this approach is thecomplete lack of selectivity. Ourefforts, therefore, concentrated onways to address autoreactive T cells inan antigen–specific manner. Inprevious studies we showed thatmultimers containing repeats of apeptide antigen derived from theinfluenza hemagglutinin protein cantrigger an antigen-specific T cellresponse at almost 1000 fold lowerconcentrations than the peptide. Theenhancement was strictly antigen-specific and appeared to result fromthe cross-linking of MHC/peptide/TCRcomplexes. To adapt this approach toa true autoimmune model systemmultimerized forms ofencephalitogenic peptides derivedfrom the myelin basic protein (MBP)or proteolipid protein (PLP) weregenerated and tested in the EAEsystem. The trials revealed that themultimerization increased the in vivopotency of these epitopes to such anextent that EAE was induced even instrains of mice normally not effectedby the monomeric peptides.Furthermore utilizing immunizationprotocols, which aim at a tolerizationrather than the activation of the Tcells, relatively small amounts ofthese multimers were found to besufficient to suppress the disease (themonomeric peptides did not show anysuppressive effect). In vitroexperiments in the hemagglutininsystem indicated that this suppressionresults from the apoptotic eliminationof overstimulated CD4+ T cells (‘high-zone tolerance’), one of the mainmechanisms of peripheral tolerance.

Another tool to enhance the sensitivityof CD4+ T cells was found duringbiochemical studies of twoconformational variants of MHC classII/ligand complexes. Bindingexperiments revealed that the on-ratefor the formation of the peptide/MHCcomplex was significantly increased ifcertain small molecular compoundswere present during the bindingreaction. Subsequent studies revealedthat these compounds catalyze theligand exchange in mechanism similarto HLA-DM molecules. In contrast toHLA-DM, however, these compoundsfacilitated the peptide exchangedirectly on the surface of antigenpresenting cell, increasing thesensitivity of an antigen-specific Tcell response by almost 2 logs. Thisproject is still at an early stage andwill be continued utilizingcombinatorial chemistry. The controlof autoimmune reactions is crucial for

the treatment of autoimmune diseasesas well as for the development oftumor immunotherapies. To achievethis goal the group will continue toinvestigate the underlyingmechanisms in vitro, in vivo as wellas on the molecular level.

Selected Publications

Rötzschke, O., Falk, K., andStrominger, J. L. (1997) Super-activation of an immune responsetriggered by oligomerized T cellepitopes. Proc. Natl. Acad. Sci. U S A.94, 14642-14647.

Rötzschke, O., Falk, K., Mack, J.,Lau, J. M., Jung, G., and Strominger,J. L. (1999) Conformational variantsof class II MHC/peptide complexesinduced by N- and C-terminalextensions of minimal peptideepitopes. Proc. Natl. Acad. Sci. U S A.96, 7445-7450.

Falk, K., Rötzschke, O.,Santambrogio, L., Dorf, M. E.,Brosnan, C., and Strominger, J. L.(2000) Induction and suppression ofan autoimmune disease: Enhanced invivo potency of oligomerized T cellepitopes. J. Exp. Med. 191, 1-14.

Structure of the Group

Group leadersDr. Kirsten FalkDr. Olaf Rötzschke

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Molecular and CellBiology ofHematopoietic CellsMartin Zenke

The focus of research of this group isthe molecular and cell biology ofhematopoietic cells. Two hematopoieticcell types are studied in detail:erythroid cells and antigenpresentingdendritic cells (DC). Both cell typesare analysed in experimental modelsystems (mouse and chicken); humanerythroid cells and human DC are alsobeing investigated. Additionally, genetransfer methods are being developedto generate gene-modifiedhematopoietic cells for therapeutic usein medicine.

The thyroid hormonereceptor/c-erbA (TR/c-erbA)acts as a binary switch in redblood cell development

P. Bartunek, G. Blendinger, M. F. Heikenwälder, and S. M. Kurz

The c-erbA protooncogene productrepresents a high affinity receptor forthyroid hormone (thyroid hormonereceptor, TR). Our previous work hasestablished that TR/c-erbA inducesred cell-specific gene expression andeffectively accelerates erythroid celldifferentiation when activated byligand (Zenke et al., Cell 61, 1035).We have now found that additionallyunliganded TR/c-erbA affectserythroid cell development; it supportssustained growth of erythroidprogenitor cells in vitro by blockingdifferentiation (Bartunek and Zenke,1998) and, therefore, exhibits anactivity very similar to its oncogenicversion v-erbA (Zenke et al., Cell 52,107). Thus, TR/c-erbA acts as a binaryswitch in determining the fate of theerythroid cell: unligated TR/c-erbAsupports growth while ligand-activatedTR/c-erbA induces differentiation.Our activities are now being directedtowards the identification of TR/c-erbA target genes (in collaborationwith P. Pajer and M. Dvorak, IMG,Prague, Czech Republic). To this end,several potential erbA target geneshave been isolated and are currentlybeing analysed.

Determining the geneexpression repertoire of redblood cells.

N.P. Koritschoner, B. Anzinger, G. Blendinger, S. Knespel, B. Lemke, and P. Bartunek

In initial studies, the tyrosine kinasegene expression profiles in erythroidprogenitors and differentiated cellswere determined by employing genefamily PCR and targeting the highlyconserved tyrosine kinase domain.Several receptor and non-receptortyrosine kinases have been identifiedthat undergo specific changes inexpression when cells differentiate(Koritschoner et al., 1999). Theseexperiments led to the identificationof fibroblast growth factor receptor-4(FGFR-4) as a new ligand-dependentregulator of erythropoiesis.

To extend these studies, we have nowestablished an in vitro differentiation

system for human red blood cells(Panzenböck et al., 1998; incollaboration with M. Mapara,Charite, Robert-Rössle-Klinik,Berlin). Erythroid precursor cells fromcord blood, CD34+ stem cells or bonemarrow are amplified in vitro in thepresence of stem cell factor (SCF),erythropoietin (Epo), dexamethasoneand estrogen, and differentiated byEpo and insulin treatment. Specificchanges in gene expression duringdifferentiation are monitored. Thisexperimental system provides thebasis for determining the entire geneexpression repertoire of human redblood cells by DNA chip technology.These studies are currently beeingperformed.

Gene expression in antigen-presenting dendritic cells (DC)

J. Madruga, S.M. Kurz, S.S.Diebold, N.P. Koritschoner, S. Knespel, M. Meyer, and X.-S. Ju

Dendritic cells (DC) are professionalantigen-presenting cells that areunique in that they can initiateprimary immune responses. However,so far, many of the functional andmolecular properties of DC are poorlyunderstood. We previously describedan in vitro differentiation system forDC based on the conditional,hormone-inducible v-rel estrogenreceptor fusion gene v-relER(Boehmelt et al., Cell 80, 341). Usingthis system, molecular mechanisms ofDC motility were investigated(Madruga et al., 1999). Severalcomponents of focal adhesioncomplexes are expressed in v-relERDC that are, however, not organizedin classical focal adhesion plaques,but rather exhibit a polarizedexpression pattern and colocalize withactin. Additionally, the expression andfunction of receptor tyrosine kinasesin DC are being assessed.

To gain further insight into theunderlying mechanisms that determineDC differentiation, an in vitro systemfor differentiation of human DC fromhematopoietic stem/progenitor cellshas been developed. Cells are grownwith a stem cell factor cytokinecocktail that maintains the progenitorphenotype, and induced to undergosynchronous differentiation into DCby administration of GM-CSF and IL-4. Differentiated cells express allthe hallmarks of DC, as judged bymorphology, surface marker

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expression, functional activities andtheir gene expression profile, and canbe induced to further mature by TNFαor CD40L. Additionally, upondifferentiation induction DC ceaseproliferation and effectively undergocell cycle arrest. The expression ofvarious cell cycle regulators andtranscription factors in differentiatingDC is now being investigated.

Gene transfer into antigen-presenting dendritic cells (DC)

Diebold, S. S., Esslinger, C. andGust, T. C.

Given their unique properties inantigen-specific T cell activation, DCrepresent a particularly attractive celltype for use in the immunotherapy ofdiseases such as cancer. In peripheralorgans (for example in skin), DC areexposed to a variety of pathogens,such as viruses and bacteria, whichthey capture through specific cellsurface receptors. To develop DC formedical therapy, gene-modified DChave been generated that capitalize onusing such surface receptors for genedelivery into DC by receptor-mediatedendocytosis (in collaboration with M.Cotton, IMP, Vienna, Austria; E.Wagner, Boehringer Ingelheim AustriaR&D, Vienna, Austria.; J. Westermannand A. Pezzutto, Charite, Robert-Rössle-Klinik, Berlin).

DC abundantly express mannose andadenovirus receptors. Accordingly,mannose polyethylenimine (ManPEI)conjugates were synthesizedconsisting of the receptor bindingmoiety mannose and the polycationPEI that binds and condenses DNAand, following uptake into cells,facilitates exit from the endosomalcompartment. Additionally, Ad/PEI/DNA transfection complexes havealso been generated that containplasmid DNA bound to the outside ofadenovirus particles by PEI, withadenovirus particles serving as theligand for receptor-specific uptake.Both ManPEI/DNA and Ad/PEI/DNAtransfer complexes are effective indelivering DNA into human andmouse DC and eliciting specific T cellresponses (Diebold et al., 1999,1999a; 1999b). The activity of gene-modified mouse DC is being studiedboth in vivo and in vitro. The Ad/PEIand ManPEI gene delivery systemsare particularly versatile and shouldbe useful for the generation of gene-modified DC to be employed in

medical therapy and to study DCbiology and function.

Selected Publications

Bartunek, P., and Zenke, M. (1998)Retinoid X receptor and c-erbA/thyroid hormone receptor regulateerythroid cell growth anddifferentiation. Mol. Endo. 12, 1269-1279.

Panzenböck, B., Bartunek, P., Mapara,M., and Zenke, M. (1998) Growth anddifferentiation of human stem cellfactor/erythropoietin-dependenterythroid progenitor cells in vitro.Blood 92, 3658-3668.

Koritschoner, N. P., Bartunek, P.,Knespel, S., Blendinger, G., andZenke, M. (1999) The fibroblastgrowth factor receptor FGFR-4 as anew ligand dependent modulator oferythroid cell proliferation. Oncogene18, 5904-5914.

Madruga, J., Koritschoner, N. P.,Diebold, S. S., Kurz, S. M., Knespel,S., and Zenke, M. (1999a) Polarisedexpression pattern of focal contactproteins in highly motile antigenpresenting dendritic cells. J. Cell. Sci.,112, 1685-1696.

Diebold, S. S., Lehmann, H., Kursa,M., Wagner, E., Cotten, M., andZenke, M. (1999) Efficient genedelivery into human dendritic cells byadenovirus polyethylenimine (Ad/PEI)and mannose polyethylenimine(ManPEI) transfection. Hum. GeneTher. 10, 775-786.

Diebold, S.S., Kursa, M., Wagner, E.,Cotten, M., and Zenke, M. (1996b)Mannose polyethylenimin (ManPEI)conjugates for targetted DNA deliveryinto dendritic cells. J. Biol. Chem.274, 19087-19094.

Structure of the Group

Group leaderDr. Martin Zenke

ScientistsDr. Petr BartunekDr. Christoph Esslinger*Dr. Nicolas P. Koritschoner*Dr. Xin-Sheng Ju*

Graduate StudentsBirgit Anzinger (nee Panzenböck)Sandra S. DieboldSteffen M. KurzTatjana C. Gust*Britt Lemke*Jaime MadrugaMirjam Meyer*

Diploma StudentsMathias F. Heikenwälder*

Laboratory TechniciansGitta BlendingerSiegne Knespel

SecretariatIrene GallagherPetra Haink

* part of the period reported

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PhospholipidsDietrich Arndt

Cytotoxic effects of antitumoragents induced at the plasmamembrane level

We are investigating the anticancerproperties of special phospholipids.These compounds, ether lipids andalkylphospholipids (APLs), representa new class of non-DNA-interactivecompounds for cancer therapy. Theyact as growth factor antagonists,growth factor receptor blockers, andinterfere with mitogenic signaltransduction, modulate phospholipidturnover, induce differentiation andapoptosis and activate macrophages toa tumoricidal state.

The aim of our research is toinvestigate the correlation betweenantineoplastic activity and thesupramoleculare structure ofphospholipids with antitumorproperties. Thus, we are concentratingmainly on the investigation,characterization and use of liposomesprepared from phospholipids withinherent antineoplastic activity

Antineoplastic activity ofalkylphospholipid liposomes inhuman breast carcinomas

We have developed stericallystabilized APL liposomes which avoiduptake by the reticuloendothelialsystem and can be targeted passivelyto tumor tissue by increasedmicrovascular permeability in thetumor area. The bilayer of suchsterically stabilized liposomes consistsof hexadecylphosphocholine,cholesterol and polyethylene glycol-linked phosphoethanol-amine. Thereduced uptake of sterically stabilizedAPL liposomes correlates in vitro(J774 cells) with an increasedthickness of the fixed aqueous layeraround these liposomes and supports

the hypothesis that the thickness ofthis aqueous layer is an importantfactor responsible for preventingopsonization, thereby resulting inreduced macrophage uptake. Thepharmacokinetics of free and differentliposomal APLs is in agreement withthese assumptions; the serum levels ofAPL obtained with stericallystabilized liposomes are consistentlyhigher than with conventionalvesicles and free APL. In xenograftedMaTu carcinoma, the differences inAPL content between the differentgroups are unexpectedly low and donot reflect the high therapeuticactivity of sterically stabilized APLliposomes. Detailed analysis showsthat the liposomal drug displaysmodified pharmacokinetics whichmay also involve lymphaticabsorption of the liposomal APL.

The physical properties andpharmacological activity of liposomesmade from a new, highly activealkylphospholipid (OPP) have beenoptimized with special reference tothe composition of the vesicles. Thestrongest antitumor effect onxenotransplanted human breast cancerMT-3 on nude mice was obtained withsterically stabilized OPP liposomeswith a low cholesterol content. Thebeneficial therapeutic effect of thesevesicles was accompanied by bettertolerance and a significant inhibitionof hemolysis, compared with micellarOPP.

Immunoliposomes fromalkylphospholipids

For active targeting, we haveinvestigated the preparation ofimmunoliposomes usinghexadecylphosphocholine and themonoclonal antibody fragment 4D5,specific against the p185HER2 protein, agrowth factor receptor-tyrosine kinase.Conjugation was achieved bycoupling the protein via a thioetherlinkage to the liposomal surface. Toinvestigate the cellular uptake andendocytosis by tumor cells, a pH-sensitive fluorescence marker wasencapsulated into the liposomes.Experiments in vitro demonstrated adifference in binding of liposomes,with and without antibody, to cellswith different receptor expressions.For active targeting of theendothelium of the tumorneovasculature, we are investigatingthe preparation of immunoliposomesusing APL and peptides that

specifically target distinct bloodvessels. Each of these peptides bindsto different receptors that areselectively expressed on thevasculature of the target tissue. Thetumor-binding peptides, e.g peptidescontaining an integrin-binding Arg-Gly-Asp motif or the Asn-Gly-Argmotif, bind to receptors that areupregulated in tumor angiogenicvasculature.

Selected Publications

Zeisig, R., Arndt, D., Stahn, R., andFichtner, I. (1998) Physical propertiesand pharmacological activity in vitroand in vivo of optimised liposomesprepared from a new cancerostaticalkylphospholipid. Biochim. Biophys.Acta 1414, 238-248.

Arndt, D., Zeisig, R., and Fichtner, I.(1998) Alkylphospholipid liposomes:preparation, properties and use incancer research. Drugs of Today 34,83-96.

Arndt, D., Zeisig, R., Fichtner, I.,Teppke, A.D., and Fahr, A. (1999)Pharmacokinetics of stericallystabilized hexadecylphosphocholineliposomes versus conventionalliposomes and freehexadecylphosphocholine in tumorfree and human breast carcinomabearing mice. Breast Cancer Res.Treat. 58, 71-80.

Patent Application

DE 198 55 953.4. Mittel zurTumortherapie. Erfinder: Zeisig, R.,Fichtner, I., and Arndt, D.DE 198 55 952.6 Mittel zumnichtviralen Transfer von DNS ineukaryotische Zellen. Erfinder: Zeisig,R., and Walther, W.

Structure of the Group

Group leaderDr. Dietrich Arndt

Scientists Dr. Reiner Zeisig

Technical assistantsAnne-Dorothee TeppkeAdnan Ibikli*

* part of the period reported

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RNA ChemistryEckart Matthes

Two target inhibitors of humantelomerase (hTERT)

Telomerase is an uniqueribonucleoprotein polymerase usingits RNA component as a template forthe synthesis of multiples of telomericrepeats onto the end of replicatingchromosomes. The extensionmechanism of telomerase compensatesfor the loss of telomeric DNAassociated with each round of DNAreplication. However, most somaticcells lack telomerase and so thetelomere length reduction is thoughtto limit their proliferative capacity andto lead to cellular senescence.

On the other hand, an activation oftelomerase seems to be required forthe sustained growth potential ofmalignant tumor cells, stem cells ofrenewable tissues and germ cells.Telomerase activity was detected in85-95 % of advanced malignanttumors.

These findings make telomerase anattractive target for anti-neoplasticdrugs. One promising target might bethe RNA molecule of telomerasewhich is an intregral part of theenzyme. Indeed, it has been shownthat oligonucleotides (ODNs)covering the template site of RNA areable to control the growth of tumorcells.

Telomeric DNA has been suggestedto bind not only to the template RNAbut seems also to be attached via its5´-end to a telomerase protein sitecalled the primer binding site. Wehave found that this protein site is anappropriate target for inhibition oftelomerase. This possibility emergedfrom our investigations of differentlymodified ODNs. Of these,phosphorothioate-modified ODNs(PS-ODNs) were found to be the most

efficient inhibitors compared withother oligomers including peptidenucleic acids (PNA). We found thattelomerase protein, rather than itsRNA, is the target of PS-ODNs, aproperty which has proved to morelength- than sequence-dependent. Thismode of action seems to lead to ahigher efficiency of PS-ODNscompared with antisense oligomerstargeting telomerase RNA. Theconcentration required for 50 %inhibition of telomerase in HL-60cell-lysates was found to be in thenanomolar range.

To increase the selectivity of PS-ODN, we designed chimeric ODNs(cODNs) which are extended at the3´-end by an oligomer hybridizingeffectively with the subsequenttemplate region of RNA. Furthermore,such cODNs address two differenttargets of telomerase and might bemore efficient. Most of these cODNshave been synthesised by our groupand optimized by length-variations(10-20mers) of the PS-part and bylength- (5 and 11 mers) and structural-modifications of of the antisense part (e.g. 2´-methoxy, 2´-methoxyethoxy,phosphoramidate). PS-PNA chimericoligomers were provided by Dr. E.Uhlmann, Hoechst Marion Roussel. Our results show that cODN are moreeffective than pure PS-ODN andinhibit human telomerase in thesubnanomolar range. Complexed withlipofectin, cODNs can be taken up byU87 glioblastoma cells and effectivelyinhibit telomerase. The concentrationrequired for 50 % inhibition oftelomerase inside U87 cells is 0.05 -0.3 µM.

Therefore, we consider our cODNs tobe useful candidates for in vivoapplications to investigate theconsequences of permanent inhibitionof telomerase on the growth of humantumors in nude mice.

Selected Publications

von Janta-Lipinski, M., Costisella, B.,Ochs, H., Hübscher, U., Hafkemeyer,P., and Matthes, E. (1998) Newlysynthesized L-enantiomers of 3´-fluoro-modified β-2´-deoxynucleoside5´-triphosphates inhibit hepatitis BDNA polymerases but not the fivecellular DNA polymerases α, β, γ, δ,and ε nor HIV-1 reverse transcriptase.J. Med. Chem. 41, 2040-2046.

Theil, F., Ballschuh, S., Flatau, S., vonJanta-Lipinski, M., and Matthes, E.(1998) Chemoenzymatic synthesis ofa new type of enantiomerically purecarbocyclic nucleoside analogues withstrong inhibitory effects on terminaldeoxynucleotidyl transferase. Bioorg.& Med Chem. 41, 701-706.

Matthes, E., and Lehmann, Ch. (1999)Telomerase protein rather than itsRNA is the target ofphosphorothioate-modified oligo-nucleotides. Nucleic Acids Res. 27,1152-1158.

Krayevsky, A. A., Dyatkina, N. B.,Semizarov, D. G., Victorova, L. S.,Shirokova, E. A., Theil, F., von Janta-Lipinski, M., Gosselin, G., andImbach, J.-L. (1999) Reasons andlimits of substrate activity of modifiedL-dNTP in DNA biosynthesis.Nucleosides & Nucleotides 18, 863-864.

von Janta-Lipinski, M., Gaertner, K.,Lehmann, C., Scherr, H., Schildt, J.,and Matthes, E. (1999) Protein andRNA of human telomerase as targetsfor modified oligonucleotides.Nucleosides & Nucleotides 18, 1719-1720.

Patent Application

Matthes, E., and von Janta-Lipinski,M. Chimeric oligonucleotides and theuse of thereof PCT-Application:PCT/DE98/01216; 4th May, 1998.

Structure of the Group

Group leaderDr. Eckart Matthes

ScientistsDr. Martin von Janta-LipinskiDr. Dieter Bärwolff

Technical assistantsMike AntoniusKlaus GaertnerChristine LehmannHelga ScheerJürgen SchildtMarianne Stulich

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Regulation andDeregulation of CellProliferation andGene Therapy

Research Group of the HumboldtUniversity of Berlin at the MDC

Regulation of cell proliferation

S. Boeckh, S. Schlisio

The cell cycle research in our grouphas so far focused on the role of theretinoblastoma protein (pRb) in theG1-checkpoint, the so-calledrestriction point, controlling theswitch between alternative cellularfates. Referring to its role as atranscriptional regulator, variousextracellular matrix genes,thrombospondin and cyclin D1,among others, have been identified aspRb-responsive genes. Dissecting thecyclin D1 promotor in more detail, weprovided evidence for a direct linkbetween NF-κB activity and cell cycleregulation by demonstratingtranscriptional activation of cyclin D1by NF-κB (work by Michael Hinz).

In order to also evaluate theintegration of antimitogenic pathwaysin the pRb pathway, we have recentlystarted to investigate the relationshipbetween TGF-β signalling and cellcycle control. TGF-β's are potentgrowth suppressors in many differentnormal cell types whereas, in contrast,many cancers are resistant to TGF-β.In many cancer cell lines, especiallythose of the pancreas and colon,impairment of the TGF-β pathway, asmanifested by genetic alterations ofTGF-β's, their receptors, ordownstream targets, in addition to aderegulated pRB pathway, has beenobserved. In order to understand theacquisition of a malignant phenotypein pancreatic carcinoma we are aimingto identify alterations of geneexpression induced in pancreatic andcolon cancer cells by TGF-β,especially at early time points. We areusing “high density cDNA filters”,provided by the Resource Center ofthe German Human Genome Project.So far, our screening experimentshave identified more than 25 distinctcDNA clones as potential TGF-βtarget genes. However, many of themare regulated in response to the TGF-β induced cell cycle arrest, asconfirmed by Northern Blot andFACS analysis. To select the ‘real’TGF-β targets we are now screeningcells with a reconstituted TGF-βpathway. Judging from our results sofar, this approach is completelyreproducible in our hands and mayhelp us understand the effect of asignalling cascade on the expressionprofile of cancer cells.

Gene therapy of familialhypercholesterolemia

G. Cichon in collaboration with P.M. Schlag, T. Benhidjeb and K. Engelmann

A second research project of our groupis the development of gene transfersystems for the correction ofmonogenetic diseases affecting normalliver function. In animal models forFamilial Hypercholesterolemia (FH), a disease caused by an inbornmalfunction of the low densitylipoprotein receptor (LDL-R), theefficiency of viral vector systems andvector-related side- effects are underinvestigation. The application ofrecombinant adenoviruses, carrying afunctional LDL-R, leads tonormalisation of serum cholesterollevels in Watanabe rabbits (animalmodel for FH), but the therapeuticeffect is only short-term (10-14 days)and accompanied by acutehematological changes(thrombocytopenia, anemia,erythroblastosis). The hematologicalside-effects are a result of a rapidsystemic distribution of viral vectors,which cannot be controlled by localvector administered via the portalvein. Beside acute hematologicalchanges, adenoviral vectors induceadverse immunological reactions inmammals which interfere withtransgene expression and could induceinflammatory changes in the liver andother organs. Pharmacologicalimmunosuppression leads toprolongation of the therapeutic effectsbut is not a preferred solution as far asthe induction of lymphoproliferativedisorders and the permanentlysuppressed immune state areconcerned. Less immunogenic vectorsproviding long-term gene expressionare required. We are currentlyfocussing on the use of lentiviralvectors for liver gene transfer. Toovercome the current titer problems inlentiviral vector technology, we aredeveloping chimeric adenovirus-lentivirus vectors for in vivo release ofrecombinant lentiviruses.

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Transient immunosuppression with15-deoxyspergualin prolongs reportergene expression and reduces humoralimmune response after adenoviralgene transfer. Cichon G., Strauss M.Gene Ther. 1998 Jan; 5(1): 85-90

Intravenous administration ofrecombinant adenoviruses causesthrombocytopenia, anemia anderythroblastosis in rabbits. Cichon G.,Schmidt H.H., Behdidjeb T., Loser P.,Ziemer S., Haas R., Grewe N.,Schnieders F., Heeren F., Manns M.P.,Schlag P.M., Strauss M. Journal ofGene Medicine Sept-Oct 1999; 1(5):360-371

Gene therapy of malignanttumors

Karsten Brand, Sefer Eleskurtaj,Martina Geheeb, Christina Montag,Ansiah Shakeri-Garakani

This project group deals mainly withthe development of new methods forthe virus-based gene therapy oftumors, especially colorectal livermetastases. We are pursuing threeapproaches:

1. Gene therapeutic chemotherapy byintratumoral gene transfer of theHerpes Simplex Virus thymidinekinase gene which toxifiesintravenously administeredGanciclovir.

In previous studies, we have describedthe toxicitiy of this approach and thepossible underlying mechanisms. We then demonstrated how toovercome this toxicity with a markeddegree of anti-tumor efficacy by usingthe tumor tissue-specific CEApromoter instead of the ubiquitouslyexpressed CMV promoter. Currently,we are constructing gutless adenoviralvectors which are less toxic than firstgeneration adenoviruses. We areexamining diverse ameliorated tumortissue specific promoters in thecontext of this new vector generationwith the aim of obtaining a clinicallyrelevant therapy with high efficacybut low toxicity.

2. The transfer of cell cycle-inhibitoryand apoptosis-inducing genes.

In our previous work, we were able toshow the therapeutic relevance of thesimultaneous expression of more thanone gene of these classes of proteins.We are currently examining theinteractions of the apoptosis inducer,p53, and the cell cycle modulators,pRb and E2F. The nature of theseinteractions can in turn be exploitedfor gene therapeutic applications.

3. The inhibition of the invasion ofmicrometastases by the transfer ofprotease inhibitors into theunaffected tissue of host organs toconfer a defensive function.

We have recently shown that theadenoviral gene transfer of inhibitorsof tumor cell-asssociated proteasesleads to a dramatically reducedgrowth of metastatic deposits in theliver of mice after injection of highlymetastatic cells into the spleens ofthese animals. We are currently tryingto construct modern viral vectors(AAV, gutless Ads) with proteaseinhibitor genes with the aim ofcombining this very efficient approachwith the lowest possible toxicity.

Selected Publications

Brand, K., Löser, P., Arnold, W.,Bartels, T., and Strauss, M. (1998)Tumor cell-specific transgeneexpression prevents liver toxicity ofthe adeno-HSV-tk/GCV approach.Gene Therapy 5, 1363-1371.

Brand, K., Klocke R., Poßling, A.,Paul, D., and Strauss, M. (1999)Induction of G2/M arrest andapoptosis by replication deficientadenovirus. Gene Therapy 6, 1054-1063.

Structure of the Group

Group leaderProf. Dr. Michael Strauss(deceased in 1999)

Project leadersDr. Susanne BoeckhDr. Karsten BrandDr. Günther Cichon

Postdoctoral fellowDr. Ansiah Shakeri-Garakani

Graduate studentsMartina Geheeb Christina Montag

Medical studentSefer Eleskurtay

TechniciansUta Fischer Beate Goldbrich Kordelia Hummel Heidrun Peter Alexandra Poßling Heidi Riedel Michael Sacharjat

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Evolution, Regulationand GeneticApplications ofTransposableElements inVertebrates

Zoltán Ivics

Transposable elements are mobilesegments of DNA that are ubiquitousin most living organisms. Theseelements can be significantly amplifiedin genomes, which may have aprofound impact on genomeorganization and are, therefore, thoughtto play a role in speciation. In contrastto viruses, transposons are permanentinhabitants of genomes. Thus, therelationship between transposon andthe host genome is delicately controlled.The types, numbers, conservation andgenetic locations of transposableelements provide important clues aboutthe evolution of transpositionalmechanisms and their regulation, andthe effects of transposons on theexpression of host genes. In addition,transposable elements can be harnessedas useful experimental tools for thecharacterization and geneticmanipulation of genomes.

Accordingly, we have embarked on anintensive search to identify andcharacterize transposable elements inzebrafish (Danio rerio), a powerfulmodel system for vertebrateembryogenesis. As a result of ourongoing efforts, approximately 20 %of the complex genome of thezebrafish is now relatively wellcharacterized.

DANA is a composite, tRNA-derivedretroposon, which is amplifiedthrough an RNA intermediate. Someof the sequence modules that make upthe DANA element are apparentlycapable of forming new groups ofmobile, composite transposons. Oneof these sequences, called MER-6, isan abundant repeat found in thehuman genome. Both DANA andMER-6 elements contain polymorphicmicrosatellite CA repeats, raising thepossibility that these elements aresources of genomic instability invertebrate genomes.

Angel is an abundant miniatureinverted-repeat transposable element(MITE), dispersed in the zebrafishgenome. Angel elements arepalindromic sequences with thepotential to form stem-loop structuresin vitro. Despite considerablesequence divergence, the invertedrepeat structures of these elementshave been maintained, implyingfunctional importance. We haveproposed a model in which MITEstake advantage of a basic cellularmechanism, DNA replication, for theiramplification, which is dependent onthe characteristic inverted repeatstructures of these elements.

Both DANA and Angel elements areparticularly suitable as geneticmarkers because they have high copy-numbers and random distribution inthe genome, and segregate in aMendelian fashion. There are extensiveDNA polymorphisms betweenzebrafish populations and strainsdetected by PCR amplification usingprimers specific to DANA and Angel.Database searches indicate a highassociation of Angel elements withzebrafish EST’s; thus, these elementsare excellent markers for geneticmapping.

Tc1/mariner-like transposableelements spread through a DNAintermediate, and this process iscatalyzed by the element-encodedtransposase. Members of thistransposon family have been found inseveral vertebrate genomes; however,all of the transposon copies isolated todate are clearly relics of once activetransposons that, after successfullycolonizing genomes, have becomeinactivated by mutations. This inactivestate of these elements greatly hindersinvestigations into the mechanisms,regulation and evolution of DNAtransposition in vertebrate species.

Based on a comparative phylogeneticapproach, we have reconstructed anactive Tc1-like transposon from bitsand pieces of inactive elements foundin the genomes of teleost fish, andnamed this transposon SleepingBeauty (SB). SB mediates efficientand precise cut-and-paste transpositionin cells of a variety of vertebratespecies, including humans. SB is thefirst active member of the Tc1 familyof transposons in vertebrates, andcould be identical or equivalent to anancient element that dispersed inteleost genomes, in part by horizontaltransmission between species.

We are concentrating our efforts onthe following main research areas.1) Developing high copy number and

polymorphic elements (especiallyDANA and Angel) as molecularmarkers, and establishing arepetitive element database for thezebrafish to facilitate mapping,identification and sequenceanalysis of genes.

2) Transposons have learned how tocoexist peacefully for millions ofyears with their host cells byminimizing the mutational damagethey inflict on the host genome.Down-regulation of transpositioncan be achieved by factors andmechanisms provided by the hostcell or by self-limiting regulatoryfeatures intrinsic to the transposonitself. We are investigating howtransposition is regulated invertebrates, and the molecularinteractions that allow this peacefulrelationship to be maintainedduring evolution.

3) Sleeping Beauty has a number ofadvantages as a gene vector whencompared with current viral andnon-viral gene transfertechnologies. Our goal is toevaluate and develop SB as amolecular tool for human genetherapy, so that it will become auseful vector for insertingtherapeutic genes into humanchromosomes.

4) Exploiting transposons todetermine the identity, function andbiological relevance of genes thatare associated with vertebrateembryonic development andhuman disease, by developinginsertional mutagenesis screens inmodel organisms such as fish,frogs, and mice.

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Selected Publications

Izsvák, Zs, Ivics, Z., Garcia-Estefania,D., Fahrenkrug, S.C., and Hackett,P.B. (1996) DANA elements: a familyof composite, tRNA-derived shortinterspersed DNA elements associatedwith mutational activities in zebrafish(Danio rerio). Proc. Natl. Acad. Sci.USA 93, 1044-1048.

Ivics, Z., Izsvák, Zs., Minter, A., andHackett, P.B. (1996) Identification offunctional domains and evolution ofTc1-like transposable elements. Proc.Natl. Acad. Sci. USA 93, 5008-5013.

Ivics, Z., Izsvák, Zs., and Hackett,P.B. (1997) Molecular reconstructionof Sleeping Beauty, a Tc1-like trans-poson in fish, and its transposition inhuman cells. Cell 91, 501-510.

Luo, G., Ivics, Z., Izsvák, Zs., andBradley, A. (1998) Chromosomaltransposition of a Tc1/mariner-likeelement in mouse embryonic stemcells. Proc. Natl. Acad. Sci. USA 95,10769-10773.

Ivics, Z., Izsvák, Zs., and Hackett,P.B. (1999) Genetic applications oftransposons and other repetitiveelements in zebrafish. IN (Detrich II,H., Westerfield, M., and Zon, L.I.eds). The zebrafish: Genetics andGenomics. Methods Cell Biol. 60, 99-131.

Plasterk, R.H., Izsvák, Zs., and Ivics,Z. (1999) Resident Aliens: TheTc1/mariner superfamily oftransposable elements. Trends Genet.15, 326-332.

Patent Application

DNA-based transposon system for theintroduction of nucleic acid into DNAof a cell Tc1-based transposon vectors

Structure of the Group

Group leaderDr. Zoltán Ivics

ScientistsDr. Zsuzsanna Izsvák

Technical assistantsDora FiedlerEva Stüwe

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127

Molecular and DevelopmentalNeurosciences

As the average life expectancycontinues to increase in the Westernhemisphere, it is anticipated that theincidence of age-related disorders,such as Alzheimer’s disease, will alsorise. This expectation has stimulatedrenewed interest in the neurosciences.In the past few decades, significantadvances have been made in ourunderstanding of the functional basisof the nervous system. Nevertheless,despite the rapid growth inneuroscience research at theinternational level – the Americangovernment has indeed declared thenineties the “Decade of the Brain” –considerable progress remains to bemade in the elucidation of thosemolecular events that are responsiblefor brain disorders.

It is due to the immense complexity ofthe brain that progress inunderstanding the molecular processesthat govern its function is slow.However, two different approacheshave been developed to combat thisproblem: positron emissiontomography (PET) and nuclearmagnetic resonance imaging (NMRI)enable neuroscientists to visualize andmap active centers of the brain inrelation to specific functions, andmolecular and cell biological tools arebeing employed to study theproperties and behavior of single braincells. The combination of these twoapproaches should lead to animproved understanding of higherbrain function, and will help to designnew treatments for the specificdegenerative mechanisms which lie atthe root of many brain diseases.

The research groups of the MDC’sneuroscience program are usingmolecular and cellular approaches.Cellular Neurosciences, headed byHelmut Kettenmann, is investigatingthe role of glial cells in health anddisease, while DevelopmentalNeurobiology, under the direction ofFritz G. Rathjen, is analyzing themolecular aspects of axonal growthduring the development of the centralnervous system. Gary R. Lewin andhis coworkers are focussing onidentifying novel genes responsiblefor mechano-transduction and theirregulation by neurotrophins, whileFrank W. Pfrieger is interested infactors which control synaptogenesis.

The central theme of the MDC is tolink basic and clinical research and wehave, therefore, establishedcooperations with the NeurosurgeryDepartment in Berlin-Buch and theCharité with a focus on brain tumorsand with the Department ofNeurology at the Charité with a focuson brain inflammation. Theseinteractions are being fostered sincewe are part of the CollaborativeResearch Center (Sonderforschungs-bereich) at the Charité established tostudy the role of non-neuronal cells inthe pathogenesis of CNS diseases.

Helmut Kettenmann

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Molecular and DevelopmentalNeurosciences

CellularNeurosciences

Helmut Kettenmann

The central nervous system containstwo major cell populations, neuronsand glial cells. The neurons areregarded as the elements mediatingelectrical activity in the brain. As aconsequence, previous neuroscienceresearch has focused on this cell type.The functional role of glial cells is notas obvious: while they were firstdescribed as cells providing onlystructural support to neurons, recenttechnical advances have brought thesecells into the arena of neuroscience. It is now evident that glial cells areessential for the proper functioning of the brain and different types ofglial cells fulfill distinct tasks.Oligodendrocytes are the myelin-forming cells of the central nervoussystem and ensure rapid signalconduction in the white matter. Therole of astrocytes is less well defined;they provide guiding structures during development and are importantelements for controlling thecomposition of the extracellular space,mediating signals between brainendothelium and neuronal membrane.Microglial cells in the brain areimmunocompetent and theirfunctional role is best defined as thefirst elements to respond to pathologicevents. While in recent years thegroup has studied aspects of all threetypes of glial cells, the presentresearch program is focussed on threetopics: (1) the role of astrocytes ininformation processing (2) theresponse of microglial cells to braininjury and (3) the cellular propertiesof gliomas. Each of these topics isintergrated in, and funded by, arespective group grant from theGerman Research Council(Sonderforschungsbereich 507, 515,Schwerpunktprogramm on Microglia).

1. Do astrocytes expressreceptors for neurotransmittersand neuromodulators?

In recent years, we have learned thatastrocytes in cell culture have thecapacity to express almost allreceptors known to mediate synaptictransmission. When we analyzed thereceptor repertoire in more intactsystems, i.e. in freshly isolated brainslices, we observed that defined typesof astrocytes express only a restrictedpattern of neurotransmitter receptors.One of our best studied examples isthe Bergmann glial cell in thecerebellum, a morphologicallyspecialized astrocyte. We have foundthat these cells express a receptorrepertoire similar to the correspondingneurons of that brain region. Tofacilitate studies in other areas of thecentral nervous system which do notcontain morphologically distinctastrocytes, we have generated atransgenic mouse in which allastrocytes express a green fluorescentprotein. Using this approach, we haverecently shown that cortical astrocytesexpress the NMDA-type glutamatereceptor, a receptor which has notbeen found in cultured astrocytes.

2. Do glial cells perceiveneuronal activity?

To test whether glial cells have thecapability to sense neuronal activity,we have used a cerebellar slicepreparation and confocal microscopy,combined with ion concentrationimaging, to test for such neuron-gliainteractions. We have found thatstimulation of parallel fibres, theaxons of the granule cells, triggerscalcium elevation in Bergmann glialcells. These increases are confined tomorphological units which are 5-10micrometers in length. These units,termed ‘microdomains’, can evenexhibit spontaneous intrinsic calciumactivity. We believe that these unitscould be involved in the feedback ofinformation on a defined populationof synapses, i.e. those which areenclosed by a given microdomain.

3. What are the controllmechanisms of microglialactivation?

Microglial cells are the majorimmunocompetent cells in the brain.We have studied the signals whichlead to the activation of microglia andhave also analyzed the cellular andmolecular consequences of thisactivation. We have found thatdifferent stimuli, e.g. confrontationwith Gram-positive or Gram-negativebacteria, can lead to differentactivation patterns in these cells. Wehave recently developed an in situmodel which allows us to study thephysiological responses of resting andactivated microglia. This has enabledus to characterize the funtionalreceptors and the physiologicalphenotype of these cells. Using thisapproach we have recently reportedthat resting microglia expresses aphysiological phenotype which isdistinct from all other CNS cell typesand also shows differences withregard to macrophages.

4. What are the physiologicalproperties of gliomas and howdo they compare with normalglia?

The majority of tumors of the centralnervous system are thought tooriginate from glial cells. These include astrocytomas,oligodendrogliomas and the mostmalignant (and untreatable) braintumor, the glioblastoma multiforme.We are studying the cellular propertiesof these tumor cells and comparingthem with normal glial cells withrespect to their physiologicalproperties, their ability to proliferateand migrate. The cells are analyzed inliving brain slices from surgicallyobtained human material. One of thenew interesting aspects of this line ofresearch is the finding that cells inoligodendrogliomas do not exhibit thephysiological properties ofoligodendrocytes, but have propertiesmore in common with neurons – theyare able to generate action potentials.This similarity extends to the findingthat the main excitatoryneurotransmitter of the centralnervous sytem, glutamate, triggerselectrical excitability in the tumorcells. Recently, we have focused onthe expression of GABA receptors byglioma cells since we have found avery strong correlation between theexpression pattern and malignancy of

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the tumor. Only cells from tumorswith low malignancy express GABAreceptors, and all tumor cell lineswhich are selected for highproliferative activity lack thisreceptor. We now have evidence thatthe brain environment triggers theinduction of this receptor and we willtest whether interference with receptoractivity influences the behavior of thetumor cells.

Selected Publications

Labrakakis, C., Patt, S., Hartmann J.,and Kettenmann H. (1998) FunctionalGABAA receptors on human gliomacells, Eur. J. Neurosci. 10, 231-238.

Lyons, S. A., and Kettenmann H.(1998) Oligodendroglia and microgliaare particularly susceptible tocombined hypoxia/hypoglycemiainjury in vitro, J. Cerbr. Blood FlowMetabolism, 18, 521-530.

Draheim H.J., Prinz M., Weber J.R.,Weiser T., Kettenmann H., andHanisch U.-K. (1999) Induction of K+

channels in mouse brain microglia:cells aquire responsiveness topneumococcal cell wall componentsduring late development. Neurosci.89, 1379-1390.

Grosche, J., Matyash, V., Möller,Verkhratsky, A., Reichenbach A., andKettenmann H. (1999) Microdomainsfor neuron-glia interaction: parallelfiber signaling to Bergmann glialcells. Nature Neurosci. 2, 139-43.

Prinz, M., Kann, O., Draheim, H.Schumann, R., Kettenmann, H.,Weber, J. and Hanisch U-K. (1999)Microglial activation by componentsof Gram-positive and –negativebacteria: Distinct and common routesto the induction of ion channels andcytokines. J. Neuropath andExperimental Neurology. 58, 1078–1089.

Structure of the Group

Group leaderProf. Dr. Helmut Kettenmann

Assistant to the group leaderMeino Gibson

ScientistsDr. Uwe-Karsten HanischDr. Anja HoffmannDr. Frank KirchhoffDr. Christiane NolteDr. Vitali MatyashDr. Katharina MertschDr. Carsten OhlemeyerDr. Michaela Schaenke

Guest ScientistsDr. Susanne KuhnDr. Michael Synowitz

Graduate and undergraduate studentsClemens BoucseinGeorg HäuslerWolfgang KresseMarina MatyashAngelika RappertCarola SchipkeDaniel SirtesAnke Witting

Technical assistantsSilke FleischhauerChristiane GrasBrigitte GerlachGerda MüllerHorst Kagelmaker

SecretariatBirgit Jarchow

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Figure 38: A small section of a Bergmann glialprocess has been reconstructed based on EMdata. Within these structures the neuronalelements are embedded and constitute the siteof neuron-glia interaction.

Growth Factor andRegeneration

Gary R. Lewin

We are using sensory neurons of thedorsal root ganglia as a model systemto study neuronal specification.Sensory neurons subserve sensationssuch as touch and pain and we haveshown that different members of theneurotrophin family interact withfunctionally-distinct sensory neuronsto regulate the survival and specificfunctional properties of sensoryneurons. We have also established that brain-derived neurotrophic factor (BDNF) regulates themechanotransduction properties ofslowly-adapting sensory neurons, butnot their survival (Carroll et al. 1999).

Molecular basis ofmechanotransduction

Our finding that neurotrophinsregulate mechanotransduction has ledus to concentrate on elucidating themolecular mechanisms underlyingmechanotransduction in mammals.Based on a molecular model ofmechanotransduction in C.elegans, wewant to establish whether mammalianhomologues of genes essential fortouch sensitivity in C.elegans (the‘Mec’ genes) also function as part of amechanotransduction complex inmammals. In my lab, we have carriedout in situ hybridization, Northernblotting and immunocytochemicalstudies that have established that somespecies homologues are appropriatelyexpressed in dorsal root ganglionneurons (DRG) (Mannsfeldt et al.,1999). Two mammalian homologuesof MEC proteins are stomatin, anintegral membrane protein, and mdeg,a sodium channel (MEC-2, and MEC-4 respectively in C.elegans). To testwhether such genes are functionallyinvolved in mechanotransduction, weare presently making transgenic micethat overexpress these putativemechanotransduction genes in sensoryneurons. In addition to these functionalstudies, we have recently isolated twonovel cDNAs encoding new membersof the stomatin family and have foundthat both are highly expressed bymammalian sensory neurons. We alsoplan to establish whether these novelgenes have a functional role inmechanotransduction.

Development of physiologicallydistinct sensory neurons

Using an in vitro electrophysiologicalpreparation, where one can recordfrom identified sensory neuronsinnervating skin, it is possible toquantify and characterizephysiologically distinct sensoryneurons. We have taken advantage ofthis preparation to ask whetherdifferent molecules are involved inspecifying the numbers of thesedifferent receptor types or theirproperties during development. Bytaking mice with targeted deletion orreplacement of neurotrophins or theirreceptors, we have been able to showthat individual receptor types requireindividual neurotrophins during theirdevelopment. In our most recent studycarried out in collaboration with Dr. Rüdiger Klein from the EMBL(Heidelberg), we have shown thatneurotrophin-4 exclusively supportsthe survival of one skin receptor type,the D-hair receptor, by activatingsignalling pathways downstream ofthe shc binding site on the trk Breceptor (Minichiello et al. 1998). Inaddition to these studies, using patchclamp techniques, we have alsorecently shown that nociceptiveneurons (pain sensing) with differentneurotrophic requirements arefunctionally distinct (Stucky andLewin, 1999). These studies may beimportant as we were also able toshow that NGF directly regulates thenoxious heat sensitivity of someneurons, a mechanism that might beresponsible for injury-inducedhyperalgesia in humans. Interestingly,the ability of these NGF- and GDNF-dependent populations to sprout isalso dramatically different(Belyantseva and Lewin, 1999).

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Figure 39: Photomicrograph of a cultured adult sensory neuron. The cell has been stained with an antibody directed against theectodomain of the mdeg channel, a putativemechanotransducing channel. Note that thischannel is localized in microdomains on axonalmembranes.

Phase contrast Mdeg immunofluorescence

Selected Publications

Mannsfeldt A.G., Carroll P., StuckyC.L., and Lewin G.R. (1999)Stomatin, a MEC-2 like protein, isexpressed by mammalian sensoryneurons. Mol. Cell. Neurosci. 13, 391-404.

Stucky, C.L., and Lewin G.R. (1999)Isolectin-B4-positive and negativenociceptors are functionally distinct. J Neurosci. 19(15), 6497-6505.

Belyantseva I.A., and Lewin G.R.(1999) Stability and plasticity ofprimary afferent projections followingnerve regeneration and centraldegeneration. Eur. J. Neurosci. 11,457-469.

Minichiello L., Casagranda F., TatcheRS, Stucky C.L., Postigo A., LewinG.R., Davies A.M., and Klein R.(1998) Mutation in trkB causing lossof NT-4 dependent neurons withoutmajor effects on diverse BDNFresponses. Neuron 21, 335-345.

Carroll, P. Lewin, G.R., KoltzenburgM, Toyka K.V., & Thoenen, H. A rolefor BDNF in mechanosensation.(1998) Nat. Neurosci. 1(1), 42-46.

Structure of the Group

Group leaderDr. Gary R. Lewin

ScientistsDr. Paul Heppenstall*Dr. Andreas Eilers*Dr. Hans Lucius*Dr. Cheryl L. Stucky

Graduate StudentsAnne Mannsfeldt*Sabrina McIlwrath*Jung-Bum Shin*

Technical AssistantAnke Kanehl

* part of the period reported

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Synapse Formationand Function

Frank W. Pfrieger

A major aim of neurobiologicalresearch is to understand theformation and function of chemicalsynapses, highly specializedintercellular connections that mediatethe dynamic exchange of electricalsignals between neurons. Our groupfocuses on the identification of thesignals and mechanisms that controlthe formation and stabilisation ofsynapses in the mammalian centralnervous system (CNS).

Identification of signalscontrolling synapse formationand maturation in themammalian CNS

Up to now, the signals that control theformation of synaptic connectionsbetween CNS neurons are largelyunknown. Recently we presentedevidence that macroglial cells secretea activity that specifically promotesthe formation of efficient synapses incultured CNS neurons. We now aim toidentify these signals and characterizetheir mode of action using primarycultures of purified neurons and awide range of techniques includingelectrophysiology, microfluorometryas well as biochemical and molecularbiological methods. During the lastyear, we have been able to take thefirst steps towards the biochemicalpurification of these factors and haveshown that the synapse-promotingactivity is carried by soluble glialproteins which may interact withcomponents of the extracellularmatrix. In order to learn more aboutthe neuronal signaling pathways thatmediate the glial effects on synapseformation, we have establishedmicrocultures of purified CNSneurons, where neurons are culturedon small drops of substrate thatconfine neuronal outgrowth to a smallcircular area and, thus, force neuronsto make synapses onto themselves(see figure). Using these cultures, wecan now study the number,localization and efficacy of synapsesin individual neurons and underdifferent culture conditions.Furthermore, we have establishedpurification procedures for differenttypes of CNS neurons and can nowask whether glial factors controlsynapse development throughout theCNS, or whether different types ofneurons have different signalingrequirements to form efficientsynapses. In the long-term, we wouldalso like to explore the potential of“synaptogenic” factors to repairsynaptic connections that have beenlost in the wake of neurodegenerativediseases or brain injury.

Mechanisms controlling the lifeand death of synapses

The selective stabilization andelimination of synaptic contacts areimportant processes controlling thedevelopment, plasticity and functionalintegrity of the CNS. The uncontrolledloss of synapses is largely responsiblefor the devastating deficits in brainfunction caused by neurodegenerativeor injury-induced lesions. Despite theimportance of synaptic stability, weknow very little about the signalscontrolling the life-time of synapsesand the mechanisms leading to theirelimination during development andin the adult. We are currentlyaddressing these questions using twotestable hypotheses: namely, a) thatsynapses have an intrinsic ‘expirationdate’ that is modified by external andinternal signals and b) that synapsesare eliminated by apoptotic processeswhich, under pathologic conditions,may ultimately lead to cell death. Weare testing these hypotheses bymeasuring the life-time of synapses insuitable experimental model systemsand determining whether apoptoticsignals play a role during synapseelimination. The identification ofsignals and mechanisms that controlthe synapse stability and initiate theirelimination may help us identify newtargets to treat the pathologic loss ofsynapses.

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Automatization of cellpurification

Primary cultures of highly purifiedneurons and glial cells provide anideal model to study differentiationprocesses since they permit strictlydefined cultures conditions. A majordrawback of these models is the ratherlaborious procedures of cellpurification. In collaboration withpartners in the biotechnology industry,we aim to develop an integratedsystem for the automated purificationof brain cells. As a first step, we havedesigned a platform that performs thedifferent steps from tissue dissociationto cell selection. Next, we aim todevelop a system prototype thatallows functionality tests and designimprovements to be carried out.

Selected Publications

Pfrieger, F.W., and Barres, B.A.(1996) New views on synapse-gliainteractions. Curr. Opin. Neurobiol. 6,615-621.

Pfrieger, F.W., and Barres, B.A.(1997) Synaptic efficacy enhanced byglial cells in vitro. Science 277, 1684-1687.

Structure of the Group

Group leaderDr. Frank W. Pfrieger

Graduate and Undergraduate StudentsChristian Göritz*Jens Hjerrling-Leffler*Daniela Mauch*Karl Nägler*

Technical AssistantsIrene HauptJacqueline Klewer

*part of the period reported

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Figure 40: Figure depicts a retinal ganglioncell, purified from postnatal rats and culturedon a substrate microisland in defined mediumfor 14 days.

DevelopmentalNeurobiology

Fritz G. Rathjen

Molecular analysis of axonalgrowth and pathfinding duringembryonic development of thenervous system

An interesting and long-standingquestion is how axons are guided totheir target region to establishsynaptic connections. Duringembryonic development and axonalregeneration after injury, neuronsrespond to an array of molecularsignals that are present in themicroenvironment of extending axons.These signals activate axonal cellsurface receptors and elicit specificgrowth cone responses. Severalclasses of proteins have been shownto be implicated in these processesincluding neural members of theimmunoglobulin superfamily (IgSF),semaphorins, netrins, ephrins and theirreceptors, as well as tenascins and theEGF family of growth anddifferentiation factors. The mostdiversified class of proteins that isimplicated in contact-dependentregulation of neurite outgrowth andaxon guidance are the neural membersof the IgSF which can be categorizedinto several structural subclassesincluding the L1-, F11- and IgLON-subgroups. Functional in vitro studieshave been supported by intriguing invivo observations in mice and humansindicating that this class of proteins isimportant for the correct wiring of thenervous system. Currently, ourresearch is focussing on the in vitroand in vivo function of members ofthe IgSF and tenascins, as well on amember of the EGF family ofdifferentiation factors using differentmodel systems.

Neurofascin exerts its functionthrough interactions withmultiple heterophilic ligands

The L1 subgroup of the IgSF invertebrates consists of four members:L1 itself, neurofascin, NrCAM andCHL1. They are transmembraneproteins that have been localized togrowth cones and processes ofpostmitotic neurons where theymediate cell adhesion, neuriteoutgrowth and axon bundling.Currently we are focussing on twomembers of this subgroup,neurofascin and L1 and their ligands.In contrast to L1, neurofascin isexpressed as a complex population ofisoforms during development. Toanalyse the function of this extensivealternative splicing in the extracellularregion of neurofascin, we havequantified the binding of differentisoforms of neurofascin to its ligands.While the IgSF members NrCAM andF11, as well axonin-1 were found tobind to all isoforms of neurofascin,the ECM component, tenascin-R,interacts only with a subset ofneurofascin isoforms. Surprisingly,insertion of short amino acid chainsinto the neurofascin polypeptideresults in a modulation of binding.

The functional consequences of thisregulation of binding, by inclusion oromission of specific segments withinthe neurofascin polypeptide, has beeninvestigated using in vitro neuriteoutgrowth assays. These investigationsindicate that neurofascin-mediatedneurite extension can be regulated bythe presence and binding of interactingproteins.

Disease-associated mutationswithin the human L1 gene affectheterophilic and homophilicinteractions

Sue Kenwrick and Patrick Willemshave shown that the L1 protein, which is a key member of thissubfamily of IgSF, is involved in an X chromosome-linked humanhereditary brain disorder. This diseasehas been termed X-linkedhydrocephalus, MASA syndrome(mental retardation, aphasia, shufflinggait, adducted thumbs) or spasticparaplegia type I. A prominent featureof this disease is a relatively broadspectrum of symptoms which includesmental retardation, lower limbspasticity, hydrocephalus, flexiondeformities of the thumbs, hypoplasiaof the corticalspinal tract and anunderdeveloped corpus callosum.Mutations linked to this disease aredistributed over all domains of L1,both extracellular and intracellular. Asa first step towards understanding themolecular aspects of this disease weinvestigated how these mutations inthe L1 protein influence binding ofdifferent ligands, in particular, thoseligands which are functionally linkedto neurite elongation and fasciculation(in collaboration with S. Kenwrick,Cambridge).

These binding analyses have shownthat different disease-associatedmutations have distinct effects onheterophilic ligand binding. Severalmutations result in a loss or reductionof binding while other mutations leadto increased binding. The heterophilicbinding profile of the mutations doesnot mirror that of homophilic binding.Our findings indicate an involvementof extensive extracellular regions ofL1 in interactions with axonin-1 andF11 (see figure). In summary, the factthat pathological mutations can affecteither homophilic or heterophilicinteractions alone suggests that bothforms of L1 binding activity areimportant in vivo and that someaspects of patient pathology are due todisturbances in cell-surfaceinteractions.

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Tenascin-R modulates neuriteextension on F11 in vitro

F11 forms another subgroup of IgSFrecognition molecules that delineatessubpopulations of axons in the centraland peripheral nervous system and isimplicated in axonal fasciculation andextension in vitro. In contrast to theL1 subgroup of proteins, F11 and theother members of this subclass areanchored to the plasma membrane viaa glycosylphosphatidyl inositol (GPI)moiety. F11 appears to exert itsfunction via interactions with multipleheterophilic ligands, including otherIgSF members, tenascins and proteintyrosine phosphatases. To gain insightinto how these interactions modulatethe activities of F11, we haveanalyzed F11-mediated neuriteextension in the presence of tenascin-R or tenascin-C in detail. Our studiesindicate that tenascin-R increases cellattachment and neurite outgrowth onimmobilized F11. These tenascin-R-induced changes are accompanied bya shift in receptor usage by tectal cellsfrom NrCAM to β1 integrins.Furthermore, tenascin-R inducesmorphological changes in tectalneurons including enlargement ofgrowth cones and increased collateralbranching of neurites.

IgLON subfamily: identificationof neurotractin

To examine the complex biology ofneural IgSF proteins further, we arelooking at the identification andfunctional characterization of novelmembers of this superfamily. Using asystematic PCR approach, we haveidentified a novel GPI-linked IgSFmember, termed neurotractin, that isexpressed on subsets of commissuraland longitudinal axon tracts in thedeveloping chick brain. Molecularcharacterization indicates thatneurotractin is a member of theIgLON subgroup of the IgSF whichhas been created by the limbicsystem-associated membrane proteinimplicated in hippocampal circuitformation. Its binding characteristics,histological distribution together within vitro neurite outgrowth studiessuggest that neurotractin plays a rolein the development of central nervoussystem axon tracts.

CALEB – a member of the EGFfamily of differentiation factorsin the developing nervoussystem

Another group of proteins implicatedin neuronal differentiation duringnervous system development are themembers of the EGF family of growthand differention factors. Bycombining binding assays withimmunological screening, we recentlyidentified a novel member of thisfamily, which we termed CALEB, and which is expressed exclusively inthe nervous system. cDNA cloningindicates that CALEB is a multidomainprotein that consists of an N-terminalglycosylation region, a leucine-proline-rich segment, an acidic box, asingle EGF-like domain, atransmembrane domain, and a shortcytoplasmic stretch. In the developingnervous system, CALEB is associatedwith glial and neuronal surfaces and isdownregulated in the adult nervoussystem. CALEB binds to theextracellular matrix glycoproteins,tenascin-C and –R, and in vitroantibody perturbation experimentsindicate the participation of CALEBin neurite formation in a permissiveenvironment.

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Figure 41: Schematic representation of thehomophilic L1 binding and heterophilicinteraction of L1 with F11 or axonin-1. Ig-likedomains (circles) and fibronectin-relateddomains (ellipses) of L1, which carry disease-associated mutations interferring with themolecular interactions, are shown in blue (fordetails please see De Angelis et al., 1999).

Selected Publications

Volkmer, H., Zacharias, U.,Nörenberg, U., and Rathjen, F.G.(1998) Dissection of complexmolecular interactions of neurofascinwith axonin-1, F11 and tenascin-Rwhich promote attachment and neuriteformation of tectal cells. J. Cell Biol.142, 1083-1093.

Brümmendorf, T., Kenwrick, S., andRathjen, F.G. (1998) Neural cellrecognition molecule L1: from cellbiology to human hereditary brainmalformations. Current Opin.Neurobiology 8, 87-97.

Treubert, U., and Brümmendorf, T.(1998) Functional cooperation of β1-integrins and members of theimmunoglobulin superfamily inneurite outgrowth induction. J.Neurosci. 18, 1795-1805.

Zacharias, U., Nörenberg, U., andRathjen, F.G. (1999) Functionalinteractions of the immunoglobulinsuperfamily member F11 aredifferentially regulated by theextracellular matrix glycoproteinstenascin-R and tenascin-C. J. Biol.Chem. 274, 24357-24365.

Marg, A., Sirim, P., Spaltmann, F.,Plagge, A., Kauselmann, G., Buck, F.,Rathjen, F.G., and Brümmendorf, T.(1999) Neurotractin, a novel neuriteoutgrowth-promoting Ig-like proteinthat interacts with CEPU-1 andLAMP. J. Cell Biol. 145, 865-876.

De Angelis, E., MacFarlane, J., Du J.S., Yeo, G., Hicks, R., Rathjen, F.G.,Kenwrick, S., and Brümmendorf, T.,(1999) Pathological missensemutations of neural cell adhesionmolecule L1 affect homophilic andheterophilic binding activities. EMBOJ., 18, 4744-4753.

Structure of the Group

Group leaderProf. Dr. Fritz G. Rathjen

ScientistsDr. Margret Moré*Dr. Ursel Nörenberg*Dr. Hannes Schmidt*Dr. Stefan SchumacherDr. Ute Zacharias*

Guest scientistProf. Dr. Michael Hortsch*(University of Michigan)

Graduate studentsArmin Dorner*Kamal Sharma*Michael Koroll*

Technical assistantsHannelore Drechsler*Mechthild Henning*Frank-Peter Kirsch*

SecretariatBirgit Cloos

* present collaborators

Associated Research GroupGroup leader

Dr. Thomas Brümmendorf

ScientistDr. Andreas Marg

Graduate studentsHarald Horst Khier*Michael Schäfer*Luzi Sendtner-Voelderndorff*

Technicial assistantDieter Jobsky*

* present collaborators

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Structure and Organization

As provided by § 7 of its Charter theorgans of the Foundation of the MaxDelbrück Center for MolecularMedicine (MDC) are the following:• the Board of Trustees with the

Scientific Committee, • the Management Board.

The Board of Trustees

As provided by § 8 of the Charter ofthe Foundation, the Board of Trusteesensures that the transactions of theFoundation are conducted in a lawful,expedient and financially responsiblemanner. The Board determines withinthe framework of the law the broadresearch objectives and the main research policy and financial mattersof the Foundation, lays downprinciples of management and theprinciples for evaluating results,intervenes appropriately within thedecisions of the Board ofManagement, and directs the Board ofManagement in special matters ofresearch policy and finances.Furthermore, the Board of Trusteesapproves annual and extended budgets(including expansion and investmentprograms), draws up the Charter anddecides upon amendments to it,decides upon the dissolution of theFoundation, and takes decisions inother cases provided for in the Lawand the Charter.

Members of the Board of Trustees

Parliamentary State Secretary Wolf-Michael Catenhusen (Chair)Federal Ministry of Education andResearch, Bonn/Berlin (since March1999)

Parliamentary State Secretary Elke Wülfing (Chair)Federal Ministry of Education, Science,Research, and Technology, Bonn*

State Secretary Prof. Dr. Ingolf Hertel(Vice-Chair) Senate Administration for Science,Research and Culture, Berlin (since October 1998)

State Secretary Prof. Dr. Erich Thies(Vice-Chair) Senate Administration for Science,Research and Culture, Berlin*

Dr. Jürgens BehrensMax Delbrück Center for MolecularMedicine (MDC) Berlin-Buch, Berlin

Prof. Dr. Hans R. Brunner C.H.U. Vaudois, Division ofHypertension, Lausanne, Switzerland

Dietmar BürgenerFederal Ministry of Finances,Bonn/Berlin

Dr. Reinhold FörsterMax Delbrück Center for MolecularMedicine (MDC) Berlin-Buch, Berlin

Prof. Dr. Peter GaehtgensPresident of the Free University ofBerlin, Berlin (since June 1999)

Prof. Dr. Johann W. GerlachPresident of the Free University ofBerlin, Berlin*

Dr. Michael HackenbrochFederal Ministry of Education andResearch, Bonn/Berlin

Prof. Dr. Dr. Falko Herrmann Institute for Human Genetics,University of Greifswald, Greifswald

Senator Beate HübnerSenate Administration of Health,Berlin

Prof. Dr. Georg W. KreutzbergMax Planck Institute for Psychiatry,Department of Neuromorphology,Martinsried

Prof. Dr. Eckart KöttgenDirector of the Clinical Board of theCharité Medical Faculty of theHumboldt University of Berlin, Berlin

Prof. Dr. Fritz Melchers*Basel Institute for Immunology, Basel,Switzerland

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Organs of the Foundation

Figure 42 (left): Wolf-Michael Catenhusen from the Federal Ministry of Education andResearch, has become Chairman of the Boardof Trustees at the MDC at the end of 1998.

Figure 43 (right): Fritz Melchers from the Basel Institute for Immunology (Basel,Switzerland), has shaped the MDC as chairmanof the Scientific Committee for almost eightyears. He left this post at the MDC in 1999.

Prof. Dr. Hans Meyer President of the Humboldt Universityof Berlin, Berlin

Prof. Dr. Mary OsbornMax Planck Institute for BiophysicalResearch, Göttingen (since October 1999)

Dr. Helmut SchühslerTVM Techno Venture Management IIIGmbH, München (since October 1999)

Prof. Dr. Peter C. ScribaInner City Medical Clinic, Munich

Dr. Albert Statz Federal Ministry of Health,Bonn/Berlin (since November 1999)

Prof. Dr. Günter StockSchering Aktiengesellschaft, Berlin*

Prof. Dr. Volker ter MeulenInstitute of Virology, UniversityWürzburg, Würzburg (since October1999)

Prof. Dr. Thomas A. TrautnerMax Planck Institute for MolecularGenetics, Berlin

Prof. Dr. Ernst-Ludwig WinnackerGene center of the LudwigsMaximilians University Munich,Munich*

Dr. Stefan Winter Federal Ministry of Health, Bonn*

*part of the time reported

Members of the ScientificCommittee

Prof. Dr. Fritz Melchers (chair)Basel Institute for Immunology, Basel,Switzerland*

Prof. Dr. Thomas A. Trautner (chair,since October 1999)Max Planck Institute for MolecularGenetics, Berlin

Prof. Dr. Günter Breithardt Medical Clinic, University ofMünster, Münster (since April 1999)

Prof. Dr. Hans R. Brunner C.H.U. Vaudois, Division ofHypertension, Lausanne, Switzerland

Prof. Dr. Dr. Falko Herrmann Institute for Human Genetics,University of Greifswald, Greifswald

Prof. Dr. Georg W. KreutzbergMax Planck Institute for Psychiatry,Department of Neuromorphology,Martinsried

Prof. Dr. Klaus MüllerHoffmann-La Roche & Co., Basel,Switzerland*

Prof. Dr. Mary OsbornMax Planck Institute for BiophysicalChemistry, Göttingen (since October1999)

Prof. Dr. Lennart PhilipsonKarolinska Institut, Stockholm,Sweden (since April 1999)

Prof. Dr. A. Günter Riegger University Medical Clinic II,Regensburg*

Dr. Helmut SchühslerTVM Techno Venture Management IIIGmbH, München (since October 1999)

Prof. Dr. Martin SchwabInstitute for Brain Research,University Zurich, Zurich,Switzerland

Prof. Dr. Peter C. ScribaInner City Medical Clinic, Munich

Prof. Dr. Kai SimonsEuropean Molecular BiologyLaboratory (EMBL), Heidelberg

Prof. Dr. Günter StockSchering Aktiengesellschaft, Berlin*

Prof. Dr. Volker ter MeulenInstitute of Virology, UniversityWürzburg (since October 1999)

Prof. Dr. Alex J. van der EbDepartement of MolecularCarcinogeneses, Sylvius Laboratories,Leiden, The Netherlands

Prof. Dr. Ernst-Ludwig WinnackerGene center of the LudwigsMaximilians University Munich,Munich*

*part of the time reported

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Figure 44: Christa Thoben, Senator for Scienceand Cultural Affairs at MDC’s New Year‘sReception on January 28, 2000.

Figure 45: Gudrun Erzgräber, BusinessDirector of the BBB Biomedical ResearchCampus Berlin-Buch Inc. (3rd from right), andErwin Jost, Administrative Director of the MDC(2nd from left), chatting with a guest at theMDC’s New Year’s Reception on January 15,1999.

The Management Board

As provided by § 13 of the Charter,the Management Board directs theFoundation. The Board ofManagement consists of one or morescientific members and oneadministrative member, and is chairedby a scientific member. The Board ofManagement is appointed by theBoard of Trustees. Since January 1,1992, the chairman and scientificmember has been Prof. Dr. DetlevGanten. Dr. jur. Erwin Jost has beenthe administrative member of theManagement Board.

Scientific Council

According to §14 of the Charter of theMDC, the Scientific Council advicesthe Management Board in matters offundamental scientific importance.The Scientific Council is consulted inall scientific appointments andformulates suggestions for thedevelopment of new researchprograms of the MDC.

During the past two years theScientific Council adviced theManagement Board particularly inmatters concerning the establishmentof core facilities and the rules andregulations governing permanentemployment of scientists. In addition,the Scientific Council maderecommendations on working ethicsand publication policy, theimprovement of the graduate students’education program, as well as onestablishing a department for thespecial requirements of scientists andguests from abroad.

Elections were held in November1998 and the Scientific Council hasbeen reconstituted.

Members of the Scientific Council (as of November 1998)

Dr. Martin Lipp (Chair)Dr. Wolfgang Uckert (Vice-chair)Prof. Dr. Walter BirchmeierProf. Dr. Thomas BlankensteinDr. Kurt BommertDr. Iduna FichtnerDr. Hannelore HaaseProf. Dr. Udo HeinemannDr. Uta HöpkenDr. Uwe KarstenProf. Dr. Helmut KettenmannDr. Frank KirchhoffDr. Gary R. LewinProf. Dr. Friedrich C. LuftDr. Thomas MüllerDr. Frank W. PfriegerDr. Claus ScheidereitProf. Dr. Peter M. SchlagDr. Ruth Schmidt-UllrichDr. Gerd Wallukat

Staff Council

The Staff Council at the MaxDelbrück Center for MolecularMedicine has a legal right toparticipate in matters associated withthe Center. This includes participatingin decisions and collaborating inmatters involving employment,grouping, and conversion, as well asin resolving internal problems andparticipating in staff discussions.

In addition, the Staff Councilparticipates in the Canteen Committeeand the chairmanship of the MDCWorks Safety Committee is taken by amember of the Staff Council.

In the context of the national“Association of Work and StaffCouncils of Extra-university ResearchInstitutes” (AGBR), Staff Councilmembers take part in the followingcommittees, “Questions of Principle”,“Data Protection”, “Works-, Health-and Environmental Protection” and“Staff and Compensation” as well as“Further Education and Training”.

With direct collaboration by the StaffCouncil, other projects have beeninitiated in which scientists andtechnicians, depending on theirprevious experience, have theopportunity to acquire new techniquesand skills in order to be able to workin new research fields beinginvestigated at the Max DelbrückCenter on the Berlin-Buch Campus. Incollaboration with BBB GmbH(Biomedical Research Campus Berlin-Buch), these projects are financed bythe Berlin Senate sub-group forEmployment, Professional Trainingand Women and by the Ministry forEmployment, Women, Health andSocial Affairs with help from theEuropean Structural Fund (ESF).

Members of the Staff Council1998/1999

Dr. Dieter BärwolffInge BeyerdörferMarion Bimmler (Chair)Lutz ElseIngrid GrunewaldDr. Peter KonzerBernd LemkeWerner LiebigChristel Westen

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Safety

The diversity and quality of scientificresearch carried out at the MDCrequires adherence to a wide range oflaws, regulations, guidelines, andstandards. Measures to ensure notonly the quality of science, but alsothe security of researchers include thenew Working Protection Law, theGenetic Engineering Law, theRadiation Protection Decree, theChemicals Law and the Waste Law.The Safety Group regularly inspectsfacilities and compiles internaloperating instructions to preventworking accidents, work-relatedillness, and emergencies. Safetyinstructions for working groups,identification of possible sources ofdanger, as well as equipment testingand the training of safety proceduresare all important functions of thesafety engineers. The Committee forWorking Safety at MDC consists ofscientifically trained safety officersand specialists for technical mattersand radiation protection. The SafetyGroup regularly discusses topicalproblems concerning health and safetyat work in close cooperation with themedical officer and the staff council.

Head: Dr. Peter Stolley / Dr. Regina Möhring

Building CoordinationEngineering and Reconstruction

Renovation of the main working areasin the MDC’s buildings wascompleted by the end of 1998.

Following agreement with donors, anadditional working area of 362 m2 wasconstructed in the Max DelbrückHouse (MDH) for use in connectionwith GMP activities. This facilityallows integration of the basicconcepts of quality assurance, goodmanufacturing practice and qualitycontrol of the development andproduction of sterile drugs.

As far as the planned construction of anew combined facility, intended foranimal experimentation andtheoretical studies for the MaxDelbrück Center (MDC) and theForschungsinstitut für MolekularePharmakologie )FMP), is concerned,agreement on planning the concepthas been granted and a date ofSeptember 1999 set. The MDCintends to start the project in the year2000.

Scientific congresses and seminars arekey components in raising the nationaland international profile of the MaxDelbrück Center. With this in mind, aCommunications Center will be built,incorporating a lecture theatre with acapacity of 500, on the Berlin-BuchCampus.

The building plans have beenapproved and construction has beenscheduled to start in March 2000. Thefacility is scheduled to be ready foruse by October 2001.

Part of the funding for theCommunications Center andrenovation of the outside of Building31.1 – MDH – are being met bymoney from the European Fund forRegional Development (EFRE).

Head: Sören-Peter Plöhn

Auditing and Legal Affairs

The duties of Auditing and LegalAffairs are to plan and implement theinspection of and adherence to legalissues. The Auditing Office bears theresponsibility for overseeing whetherlaws, practices, regulations and thedirectives of the Management Boardare properly observed and whetherallocated public resources areappropriately, economically andproductively utilized. For thesepurposes, examination plans, byapproval of the Management Board,are instituted yearly to check forregulatory compliance oforganizational activities, such thatindividual actions are inspected on acase-by-case basis for full proprietywith regard to both form and content.

All checks of regulatory complianceare undertaken with respect for eco-nomic soundness and productiveness,so as to ensure that proper judgment isexercised in establishing regulationsand in the execution of businessmatters. These checks result in theannouncement of recommendations as to how detected oversights mightbest be alleviated and avoided in thefuture.

In the area of legal affairs specialattention is given to the administrationof cooperative research contracts withclose support from the Finance De-partment. The majority of such con-tracts are established with partners inIndustry. In addition, jurisdictionalproceedings must be prepared, and inmany individual cases legalconsultation must be provided.

Head: Christine Rieffel

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Supporting Divisions

Patents/Licences

The total number of German patentsand patent applications from the MDCin 1999 amounted to 98, comparedwith 86 in 1998 and this included 3 patents which had been awardedbefore the MDC was established. In1999, for the first time, the annualnumber of patent applications reached20, compared with 19 in 1998. Thisshows that there is no sign of theinnovative ability of MDC researchersreaching a plateau.

The first two US patents fordiscoveries originating from the MDCwere awarded in 1999. There werealso 12 PCT (Patent CooperationTreaty) patent applications. In 1999, 3 contract options and 2 licensingcontracts were awarded. In addition,the first sale of an MDC patent wasconcluded. The license income for1999 amounted to 145,207 DM,compared with 111,711 DM for 1998.This represents a significant year-on-year increase.

The MDC has set up a LegalProtection Committee with Dr. IdunaFichtner, Prof. Walter Birchmeier andDr. Martin Lipp as members. It is theresponsibility of this committee toinvestigate whether application shouldbe made for foreign patents, inaddition to the primary patentprotection sought in Germany. Thisinvolves the inventors submitting anapplication to the MDC board and,following a checking procedure by theattorney, Dr. Fritz Baumbach, it isthen submitted to the Legal ProtectionCommittee for their consideration.

Head: Dr. Fritz Baumbach

Technology Transfer

The MDC strongly supports allactivities concerning the potentialcommercialization of research results.Therefore, a Technology TransferCommittee headed by Iduna Fichtnerhas been set up to manage andoptimize all activities and improvecooperation among research institutes,small and medium enterprises (SME),and the clinics.

In recent years, these activities haveled to an increased number of filedand approved patents and this processhas been markedly improved byexternal evaluation of submittedmanuscripts for patentable researchfindings before publication. Activitiesinvolving the commercialization ofpatents have been intensified by directcontact with licensing agencies (e.g.Fraunhofer Patentstelle, BritishTechnology Group). In addition, aTechnology Transfer Conference hasbeen held to give scientists theopportunity for direct contact withpotential sponsors for theirinnovations.This conference will beheld once a year.

Within the MDC, a “BiomedicalResearch Transfer” initiative has beenset up comprising several groups withlong-standing and successfulexperience in applied research. Theprocess of technology transfer will befacilitated by mutual support in grantapplications, intensified cooperationand joint presentation of scientificresults.

Since 1997, six companies have beenfounded on the campus Berlin-Buchby outsourcing of scientific resultsfrom the MDC. Among them areATUGEN AG, developed by a jointventure with Ribozyme, GenProfileAG, involved in the search fordisease-related genes, Kelman GmbH,focussing on the prediction of protein-ligand interactions and EPO GmbH,offering support in the development ofnovel anticancer agents.

A joint venture with Schering AGrecently led to the foundation of GTBGene Therapeutics, engaged in theGMP-certified production of viral andnonviral vectors.

Head: Dr. Iduna Fichtner

144

Figure 46: Ceremony laying the foundationstone for the second new laboratory building ofthe BBB’s Biotechnology and BusinessDevelopment Center for start-up companies onDecember 17, 1999 with Wolfgang Branoner,Berlin Senator for Economic Affairs (on theleft), Detlev Ganten, Scientific Director of theMDC, and Gudrun Erzgräber, BusinessDirector of the BBB Biomedical ResearchCampus Berlin-Buch GmbH (front row, fromleft to right).

Research at the MDC is conducted atthe forefront of biomedical science -in the promising field of molecularmedicine. The MDC’s researchactivities need to be communicated tothe general public in a way that can bereadily understood. In addition, theMDC’s expenses must be justified tothe German tax payer. The PressDepartment initiated various activitiesto serve this need which are outlinedbelow.

In 1998 and 1999 the MDC PressDepartment organized more than 30guided tours for almost 500 visitors -university students, high schoolstudents, international delegations,and the general public. These toursincluded lectures and visits tolaboratories of the MDC, thecooperating university affiliatedclinics, Robert-Rössle-Cancer-Clinicand Franz Volhard Clinic forCardiovascular Diseases, and theHands-on Laboratory at theBiomedical Research Park.

In 1998 and 1999 the MDC PressDepartment also continued the seriesof popular scientific lectures in theCity Hall of Berlin Pankow, initiatedin the MDC’s first year of existence,with 18 “Sunday Lectures“ given byscientists from the MDC and otherscientific institutions in Germany. Italso presented MDC’s research at theHannover Industrial Fair and at theBerlin Science Fair.

A total of 43 news releases publishedin German and English by the MDCPress Department in 1998 and 1999were the basis for many reports in themedia. About 2 000 newspaperarticles with a circulation of well over230 million copies were published onthe MDC, the Robert Rössle Clinicand Franz Volhard Clinic, and theBiomedical Research Park. Inaddition, 16 television and 16 radioproductions, including the BBCTomorrow’s World and Arte, theFrench/German program, were airedon the research conducted at theMDC, the clinics and the BiomedicalResearch Park.

In the time-span reported, three pressconferences were initiated, organizedand moderated by the MDC PressDepartment. One of these pressconferences was held at the “6thInternational Gene TherapySymposium” in Berlin-Buch in 1998,the other at the Grand Opening of theBiotechnology Business andDevelopment Center of the BBBBiomedical Research Campus GmbH,also in 1998. The third pressconference was initiated to informabout genome research with theAmerican Nobel laureate Paul Berg.He had given a talk in the series of the“Berlin Lectures on MolecularMedicine” in 1999 and had receivedthe Max Delbrück Medal of the MDCand other Berlin research institutions,and the Schering Forschungs-gesellschaft at that occasion.

In 1998 and 1999 the MDC PressDepartment prepared and organizedmore than 160 interviews for themedia in Germany and abroad,including newspapers, magazines,television and broadcasting stationsand scientific journals such as Nature,Science and The Lancet. It alsopublished four press reportssummarizing the coverage of theMDC, the clinics, the BiomedicalResearch Park, and related topics inthe printed press as well as two issuesof the MDC-Report, an in-housemagazine.

Head: Barbara Bachtler

145

Press and Public Relations

Figure 47: The MDC is an internationalresearch institute, attracting scientists from allover the world

Personnel Department

The department is responsible for allmatters relating to staff, wages,salaries, separation allowances,removal and travel expenses etc.

During the last eight years, MDC hasnot only managed to functionefficiently as a unified body, but staffalso work in close harmony. During1992, a total of 382 staff wereemployed by MDC and, by December1999, this figure had risen to 653,including those (199) paid by third-party funding. As before, most (89 %)of the scientists’ contracts are limitedto a maximum of five years.

MDC is currently financing 30graduate students studying for a PhD,who are not included in the list ofemployees. In addition, at MDC, thereare 68 part-time, third-party financed,young scientists and 2 graduatestudents studying for a PhD, who arealso third-party financed.

Head: Dr. Hans-Joachim Seehrich

Finances

The Finance Department concernsitself with all matters relating toMDC’s financial funding, includingaccounting. The primary source (90 %) of MDC’s annual fundingcomes from the Federal budget(Federal Ministry of Education andResearch). The remaining 10 % isprovided by the State of Berlin(Senate Administration for Science,Research and Culture). Within theframework of its basic funding, MDCwill receive 99 million DM for theyear 2000; approx. 18 million DM (as of December 1999) will be madeavailable in 2000 from third-partyfinancial sources.

Increases have also taken place in thestaff sector, due to general wage rateincreases and, in particular, increaseswithin Berlin. A decline in spending isseen in investments in equipment aftera period of heavy investment duringMDC’s first years. Extensivemeasures are necessary to maintainthe MDC laboratories in their presentstructural state. Another positivedevelopment has been in attractingthird-party financial resources and, in1996, the MDC was able to spend 17million DM of third-party money.

As mentioned above, approval forapprox. 18 million DM of extra-muralfunding has already been received forthe year 2000.

Head: Wolfgang Kühlewind

146

Administration

MDC MAX DELBRÜCK CENTERFOR MOLECULAR MEDICINEBERLIN-BUCH Personnel status

0

100

200

300

400

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1996 1997 1998 1999

669

141

174

47

307

675

175

146

63

291

652

159

145

52

296

653

144

148

54

307

/

/

/

/

Scientific staff(third party funded)

Scientific staff(MDC funded)

Technical andadministrativ staff(third party funded)

Technical andadministativ staff(MDC funded)

Figure 48: Personnel status. Distinctions according to financial sources.

Purchasing and MaterialsManagement

The tasks of the Purchasing andMaterials Management Departmentare focused on three main areas:

• rapid and efficient supply ofquality laboratory materials,auxiliary and consumables, andequipment at cost-effective rates

• step-by-step introduction of adecentralized ordering department,to implement an effective andtransparent form of purchasing

• revision and compilation of new,up-to-date rules of procurement.

Over 17,700 orders are processedyearly. Compared to MDC’s earlyyears, this is an increase of 2 percent.In particular, the number of orders forchemicals has risen (about 4 percent).

The step-by-step introduction of adecentralized ordering system willease the workload of the department.After hooking up to the company’sinternal data processing network,employees will be able to log in theirorders themselves. This will guaranteefast processing in purchasing. Linkingthe scientist’ work places with thepurchasing department is also animportant requirement for the futuretransfer of data to appropriatelaboratory and specialist suppliers,upon conclusion of generalagreements and calling-forwardcontracts.

Head: Dr. Peter Konzer

147

MDC MAX DELBRÜCK CENTERFOR MOLECULAR MEDICINEBERLIN-BUCH Extra mural funding

(in thousends of DM)

0

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10,000

15,000

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1692

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682 14

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2113

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894 17

1337

44

21325

8538

7454

816 15

1530

02

1833

065

40

6391

1386

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1996 1997 1998 1999 2000(estimated) (planned)

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Total

BMBF

DFG

Industry

EU

others

Figure 49: Approved third-party financedprojects and other financial funds.

Third-party funds are represented according totheir individual sources, the 2000 estimate isbased on the scope of approvals received, datedDecember 10, 1999. BMBF = Federal Ministryof Education and Research; DFG = DeutscheForschungsgemeinschaft.

MDC MAX DELBRÜCK CENTERFOR MOLECULAR MEDICINEBERLIN-BUCH

0

20,000

40,000

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80,000

100,000

98,554

31021

5820

20503

36389

1996

96,897

28031

5700

21100

40291

1997

97,691

29230

8100

24530

33631

1998

92,653

22450

8628

24040

35550

1999

98,554

1325

29000

6500

22300

39637

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1985220017754821

Budget(in thousends of DM)

/

/

/

/

/

Special programsfor investments andpersonnel

Investments

Allocations forclinical research

Expenditures inmaterial assets

Expenditures forpersonell

Figure 50: MDC’s budget development in individual expenditure sectors between 1996and 2000 (estimated).

Library

The MDC library is a specializedscientific library. Its work involvesproviding and supplying informationin all research areas of the MDC andits associated clinics. The library actsas a modern information centre usingadvanced network structures andallowing access to informationsources around the world. Moderninformation networks offer theincreased possibility of supplyingusers with specific literature andinformation resources at their workplace. The collection contains morethan 40,000 media items and 280periodicals, many of which areadditionally provided in electronicform, and different kinds of non- printmaterials. Campus-wide provision ofmajor local databases include Medline(starting from 1966 ), CurrentContents (5 Science Editions) as wellas Science Citation Index withabstracts (starting from 1980) andCD-ROM databases via an internalMDC-network with a range ofscientific options.

The open area provides 26 readingdesks, 5 computer workstations withInternet access. The library operateswith the local library computer systemSISIS. The OPAC (Online PublicAccess Catalogue) lists all collections,starting from 1994, and is alsoavailable via the internet. Printedcatalogues describing older holdingswith alphabetical and classifiedindices are also available while thelibrary and its service are all availablevia the internet. The client-serverarchitecture provides fast access,regardless of the user’s operatingsystem. The library offers a selectionof main links related to research at thecampus.

Head: Dr. Dorothea Busjahn

Animal Facilities

Animal experiments make a majorcontribution to biomedical research,particularly in such complex areas ascardiovascular, cancer andneurological diseases. Animalexperiments are especially concernedwith the development of methods forimproved diagnosis and treatment ofhuman tumors and cardiovasculardiseases. They are essential forestablishing gene therapy strategies.With the recent development ofpowerful new technologies formanipulating genes, scientistsworldwide have produced thousandsof transgenic animals and knock-outmodels. Both approaches have becomeinvaluable in studies of gene functionin disease. In 1993, a transgenic unitwas set up to allow the production oftransgenic rats and mice and mousechimeras. Since then, more than 70transgenic rat strains have beendeveloped to model hypertension. Inaddition, mice have been reconstructedby ES-cell injection and have beensuccessfully bred since 1995. Morethan 250 strains of knock-out mice arenow available as experimental modelsin cancer research, as well ascardiovascular and neurologicaldiseases. Of all the research institutesin Berlin, the animal facility of theMDC has the greatest number ofgenetically engineered rat strains andmice stocks.

Four animal houses support transgenicand animal experimentation at theMDC and mice, rats and rabbits arebred. Occupying 1,260 square meters,the facilities include animal rooms(636 m2), operating theatres, storagerooms and cage-washing facilities andanimals are bred in a disease-freeenvironment. Nevertheless, thenumber of available animal rooms andsurgical facilities in and around the

MDC does not meet presentrequirements.

The rapidly growing number ofgenetically engineered models ofsevere human illness means thatfurther animal facilities for breedingand experimentation are needed. TheMDC, therefore, plan to construct anew central animal house. The projecthas been approved by the Board ofTrustees and will, hopefully, beopened by the year 2003.

Head: Dr. Karin Jacobi

Campus Net Management

The Campus Net Management of theBBB Biomedical Research CampusBerlin-Buch GmbH is responsible forthe operation of the high-speedcampus network to supply of campus-wide internet server services (mail-, WWW-, FTP-, and phone-servers),and for communication with othernetworks, e.g. B-WiN (Science Net)and BRAIN (Berlin Research AreaNetwork). Further services for theCampus, like video-conferencing andbilling/accounting are underconstruction.

Head: Hans Mitulla

148

Central Facilities

Figure 51: The sculpture “Großer Nagelkopf”by the artist Rainer Kriester was exposed on theBerlin-Buch Campus from September 1998 toFebruary 1999 as part of a changing exhibitionof sculptures, expressing the link betweenscience and art on the Campus.

Computing

The computing group of the MDCmanages the central computerfacilities of the MDC (RemoteAccess-, File-, and Backup-servers).

The group is responsible for theclient/server operation of the MDC’sAdministration and Executive Board,and is responsible for the system- anduser-support of the SAP-R/3-administration system.

The group focuses its activities onuser-oriented support of data andimage processing at the MDC. Thegroup supports users if there are anyhardware and software problems,connects PC, Macintosh and local netswith the MDC net, and installs clientsoftware for different computerplatforms for the usage both thecentral computer facilities of theMDC and the campus-wide computerfacilities of the BBB GmbH. In addition, the group organisesstandard software courses in ourcomputer laboratory, which isequipped specifically for suchpurposes.

Our computer laboratories for imageprocessing provide support for theresearch groups if there are anyscientific problems involving image-processing, data-analysis and -visualisation as well as in thepresentation of scientific results(graphics, slides, posters). The latesttechnology, such as digitalphotography, video-digitalisation and-processing has been installed.

Head: Bernd Lemke

Technical Affairs

During the period covered by thisreport, the Technical/WorksDepartment has carried out key workassociated with the takeover oftechnical operations for the GMPsector.

In conjunction with this, a number oftechnical safety systems have beeninstalled, functions checked andreflected in the Latest BuildingTechniques (GLT). In the researchbuildings of the MDC, air-conditioning equipment has beenwidely installed in the areas that haveundergone reconstruction and has nowbeen taken over from theTechnical/Works Department. Here,too, the switch to GLT was ofparticular importance.

The telephone equipment of the MDChas been upgraded to a highlysophisticated level and a voice mailsystem has been added.This now means that the telephoneequipment meets ISDN standards. AllMDC buildings have been fitted withmodern fire alarms and the buildingare linked to one another andconnected to the Berlin FireDepartment via a CSN computer.

Head: Harry Schenk

149

Figure 52: Walter Friedrich House, laboratorybuilding of the MDC. The building is namedafter Walter Friedrich, Head of the Institute ofMedicine and Biology of the Academy ofSciences in Berlin-Buch from 1948 to 1960. He was renowned for his contributions withMax von Laue to the electromagnetic nature of X-rays and to X-ray cristallography of proteinsand their structure.

The following events organized underthe auspices of the MDC and itsclinical partners took place in 1998and 1999

Staging Laparoscopy(March 20-21, 1998)

4th MDC Graduate Students’Symposium(March 26, 1998)

6th Symposium on Gene Therapy“Towards Gene Therapeutics”(May 4-6, 1998)

2nd Congress of Molecular Medicine(May 6-9, 1998)

Forum of European Neuroscience (June 27 - July 1, 1998, Inter-Continental, Berlin)

2nd Cell Biology Symposium of theMDC: Protein Transport and Stability(September 5-9, 1998)

120. Jahrestagung der GesellschaftDeutscher Naturforscher und Ärzte(September 19-22, 1998, HumboldtUniversity, Berlin)

Jahrestagung der Hermann-vonHelmholtz-Gemeinschaft DeutscherForschungszentren(November 4-5, 1998, HumboldtUniversität zu Berlin)

MDC Symposium MolecularMedicine(December 18-19, 1998)

Bucher Symposium “MolecularGenetics and Genome Analysis”(March 4, 1999)

5th MDC Graduate Students’Symposium(March 10, 1999)

Berliner Seminar “Moderne Peptid-und Proteinanalytik: Anwendungenund Limitierungen”(March 19, 1999)

9th European Congress of ClinicalMicrobiology and Infectious Diseases(March 21-24, 1999, ICC, Berlin)

Application of Molecular Methods forthe Development of New Therapies(March 29-31, 1999)

Informationsveranstaltung zum 5. EU-Rahmenprogramm für Forschung,technologische Entwicklung undDemonstration (1998-2002)(March 31, 1999)

BIO’99 (May 16-20, 1999, Seattle/USA)

Zukunft Biotechnologie(May 28, 1999)

Molekulare Kardiologie: NeueForschungsstrategien gegenHerzinsuffizienz(August 27, 1999)

Solution Structure and Interaction ofBiopolymers using AnalyticalUltracentrifugation (October 19,1999)

Berlin Lectures on MolecularMedicine with Prof. Paul Berg,Stanford University, School ofMedicine, Beckman Center forMolecular and Genetic Medicine,Stanford, USA(November 18, 1999 Charité MedicalFaculty of the Humbold University ofBerlin)

1st Technology Transfer Conferencein Berlin-Buch(November 22, 1999)

Jahrestagung der Hermann-vonHelmholtz-Gemeinschaft DeutscherForschungszentren(November 24-25, 1999, Bonn)

3rd Congress of Molecular Medicine(CMM) and VIth Franz-Volhard-Symposium “Molecular Mechanismsin Dilated Cardiomyopathy”(December 3-4, 1999)

Michael-Strauss Memorial Lecture,Jeffrey Leiden (Harvard School ofPublic Health, Boston/USA), “GeneticApproaches to Understanding andTreating Heart Failure” at the 3rdCongress of Molecular Medicine(CMM) and VIth Franz-Volhard-Symposium “Molecular Mechanismsin Dilated Cardiomyopathy”(December 4, 1999)

150

Meetings, Workshops and Symposia

Figure 53: Xu Zhihong (3rd from left), Vice-President of the Chinese Academy of Sciences(CAS), in a meeting with Detlev Ganten,Scientific Director of the MDC (2nd from left),and Chinese guest scientists at the MDC on theoccasion of his visit to Berlin-Buch inNovember 1998.

Thomas Biederer Boehringer-Mannheim-Förderpreisder Deutschen Gesellschaft fürZellbiologie, 1998

Jens Reich Urania-Medaille, 1998

Regina Reszka Innovationspreis des Landes Berlin-Brandenburg, 1998

Thomas E. Willnow Heinrich-Wieland-Preis, 1998

Jürgen Behrens Monika Kutzner-Preis zur Förderungder Krebsforschung der Berlin-Brandenburgischen Akademie derWissenschaften, 1999

Walter Birchmeier, Peter M. Schlag Deutscher Krebspreis 1999

Hermann Haller, Volker Homuth, Friedrich C. Luft, Gerd Wallukat Galenus-von-Pergamon-Preis, 1999

Max-Delbrück-Medal

Since 1992, oustanding scientists arebeing awarded the Max DelbrückMedal by Berlin research institutionsand the Schering Research Foundation.In 1998, the Swedish anthropologistSvante Pääbo received the MaxDelbrück Medal, and in 1999, theAmerican Nobel-Laureate Paul Berg(Stanford University, California). In2000, this medal has been awarded toFritz Melchers (Basel Institute forImmunology), head of the ScientificCommittee of the MDC till 2000.Recipients in the past years were:

1992 Günter Blobel (Rockefeller University New York ,USA; Nobel Laureate in 1999)

1994 Sydney Brenner(University of Cambridge, UK)

1995 Jean-Pierre Changeux (Institut Pasteur, Paris, France)

1996 Robert A. Weinberg(Whitehead Institute, MassachusettsInst. of Technology Cambridge, USA)

1996 Nihat Bilginturan (University of Hacettepe, Ankara,Turkey)

1997 Charles Weissmann(University of Zürich, Switzerland)

1998 Svante Pääbo(Ludwig-Maximilians-UniversitätMünchen and Max Planck Institute forEvolutionary Anthropology, Leipzig)

1999 Paul Berg(Nobel-Laureate 1980; StanfordUniversity, California)

2000 Friedrich Melchers (Basel Institute for Immunology,Basel/Switzerland)

151

Awards

Figure 54: Nobel Laureate Paul Berg fromStanford University, California, USA (in themiddle), on his visit to the Berlin-Buch Campuson November 18, 1999 with MDC-scientistMartin Lipp and Stefanie Korthals.

Addresses of Scientific Journalsat the Berlin-Buch Campus

Journal of Molecular Medicine(JMM)

Editorial OfficeCary Petersen (Assistant Editor)Max Delbrück Center for MolecularMedicine (MDC) Berlin-BuchRobert-Rössle-Strasse 10D–13122 Berlin; GermanyTel.: +49-30 9406 2222Fax: +49-30 9406 2266e-mail: jmmoff@mdc-berlin.dehomepage: http://link.springer.de/link/service/journals/00109/index.htm

Glia

Editorial Office (Europe)Helmut KettenmannMax Delbrück Center for MolecularMedicine (MDC) Berlin-BuchCellular NeurosciencesRobert-Rössle-Strasse 10D-13125 BerlinTel.: +49 30 94 06 33 25Fax: +49 30 94 06 38 19e-mail: hketten@mdc-berlin.dehomepage:www.interscience.wiley.com

Neuroforum

Editorial OfficeRedaktion NeuroforumMeino Alexandra GibsonMax Delbrück Center for MolecularMedicine (MDC) Berlin-Buch Robert-Rössle-Strasse 10D-13125 BerlinTel.: +49-30-9406 3133Fax: +49-30-9406 3819e-mail: gibson@mdc-berlin.dehomepage: http://nwg.glia.mdc-berlin.de

152

AAbdul, Yetunde 90Aguirre-Arteta, Ana M. 88Ahlers, Annette 68Al Kateb, Hussam 35Al-Yahyaee, Said Ali 35Alenina, Natalia 23Alexander, Gabriele 27, 96, 97Ambrosino, Concetta 43Ameln, Ingrid 83Andrade, Miguel 43André, Françoise 29Andreé, Christel 94Andreev, Dmitri 23Ansieau, Stephane 66Antonius, Mike 122Anzinger (nee Panzenböck), Birgit

119, 120Appel, Silke 29Arndt, Dietrich 102, 121, 160Asbrand, Christian 75, 77, 79Aumann, Jutta 109Axmann, Guido 23Aÿ, Jacqueline 50, 52Aydin, Atakan 35, 43

BBabu, Allampura M. 50, 52Bachtler, Barbara 145, 160Bader, Michael 18, 22, 23, 85, 160Badock, Volker 60, 103Bähring, Sylvia 35Baldy, Christina 109Baltas, Leonidas 85Baltatu, Ovidiu 23Balzereit, Daniela 85Barbosa-Sicard, Eduardo 94Bargou, Ralf 72, 74, 103, 104, 105Baron, Heike 35Bartel, Sabine 82, 83Barthel, Beatrix 31, 32Bartunek, Petr 119, 120Bärwolff, Dieter 122, 142Bauer, Lisa 90Baumbach, Fritz 144Becker, Christin 23, 107Becker, Michael 109Becker, Monika 109Becker, Christian 117Becker, Katja 117Becker-Follmann, Johannes 29Begay, Valerie 66Behlke, Joachim 19, 44, 45, 49,

54, 99, 160Behrend, Martin 77Behrens, Diana 109Behrens, Jürgen 13, 15, 62, 73, 75,

77, 78, 79, 89, 140, 151, 160Bengs, Marion 66Berger, Ingrid 15, 112Bergmann, Jutta 112Bergmann, Stephan 73, 74Bernhardt, Günter 71Beyerdörfer, Inge 83, 142Bienert, Ralf 52Bieringer, Markus 27Bigott, Jessica 107Bimmler, Marion 142, 160Binas, Bert 23Birchmeier, Carmen 18, 19, 23, 36,

37, 75, 76, 77, 160Birchmeier, Walter 15, 63, 75, 77,

78, 79, 89, 99, 142, 144, 151, 160Bischof, Dana 41Blachut, Susanne 21

Blankenstein, Thomas 8, 13, 102, 106, 107, 116, 117, 142, 160

Blendinger, Gitta 119, 120Boeckh, Susanne 123, 124Böckel, Thomas 35Bödner, Bärbel 45Böhm, Siegfried 56Boetzel, Joachim 60Bohlender, Jürgen 20, 21, 27, 82Bommert, Kurt 59, 60, 72, 74, 103,

105, 142Bönsch, Christian 41Bork, Peer 19, 35, 43Bosnar, Martina 90Böttger, Adelheid 23Boucsein, Clemens 130Brachwitz, Hans 160Brand, Karsten 74, 124Braun, Oliver 112Breitfeld, Dagmar 71Brett, David 35, 43Brinkmann, Volker 37, 75, 77Britsch, Stefan 36, 37Brohmann, Henning 37Brümmendorf, Thomas 137Buchert, Sven 37Burkhardt-Göttges, Elke 29, 31, 32Burgstahler, Ralf 71Burmeister, Regina 41Busjahn, Andreas 25, 34, 35Busjahn, Dorothea 148, 160Butschak, Günter 80, 81

CCalkhoven, Cor 66Cardoso, M. Cristina 86, 88, 113Cartier, Regis 90Carvalho Araujo, Ronaldo de 23Catenhusen, Wolf-Michael 140, 160Cayeux, Sophie 105, 107, 117Cayla, Cécile 23Cesare, Silvana Di 77Chaboissier, Marie-Christine 39Chai, Guixuan 23, 85Choli, Theodora 60Cichon, Günther 123, 124Cifire, Felix 71Claßen, Elvira 30, 32Cloos, Birgit 52, 137Contzen, Jörg 54Czychi, Jana 97

153

Index

DDaemen, Kerstin 107Dakos, Vassilios 90Damaschun, Gregor 19, 46, 47, 48,

160Damaschun, Hilde 47Dandekar, Thomas 43Daniel, Peter T. 59, 103, 105, 107Danneberg, Heike 23Debs, Pierre 72Dehapiot, Elyzabeth 54Dehmel, Bastian 27Dehmel, Anke 109Delbrück, Sebastian 25, 35, 43Delbrück, Heinrich 50, 52Dell’Oro, Andrea 75, 77Derer, Wolfgang 88Dettmer, Rudolf 68Diebold, Sandra S. 119, 120Diederichs, Julia Eva 112Diehl, Annette 50, 52Dietz, Rainer 8, 29, 35, 87, 88,

102, 113, 160Dong, Jiaxin 98Doerks, Tobias, 43Dörken, Bernd 8, 59, 68, 72, 73, 74,

102, 103, 105, 107, 117, 160Dorner, Armin 137Drab, Marek 97Drechsler, Hannelore 137

EEaswaran, Hariharan P. 88Eckardstein, Kajetan von 112Egorov, Tsezi 60Eilers, Andreas 132Eisenhaber, Birgit 43Eisenhaber, Frank 43Eleskurtay, Sefer 124Else, Lutz 142Emmerich, Florian 68, 74, 103, 105Engel, Felix 113Engel, Katrin 72, 112Epps, Amanda von 83Erdenechimeg, Chimge 27Erdmann, Bettina 75, 77, 93, 99, 100Esslinger, Christoph 120Eßmann, Frank 60Estevéz-Schwarz, Lope 30, 32, 89, 90

FFaass, Bettina 60Fabian, Heinz 19, 48, 49, 160Fädrich, Birgit 29Fälber, Katja 49, 51, 52Falk, Kirsten 13, 118, 160Faulhaber, Hans-Dieter 25, 34, 35Feldner, Anja 21Feller, Katharina 79Feske, Anette 52Fichtner, Iduna 90, 102, 108, 109,

121, 142, 144, 160Fiedler, Dora 126Fischer, Uta 124Flachmeier, Christina 25Fleischhauer, Silke 130Förster, Alexandra 29Förster, Reinhold 13, 71, 140Franke, Gudrun 90Franke, Renate 76, 77Frege, Renate 32Friedlander, Ruth 91, 92Fürst, Robert 43Fujita, Yasuyuki 77Fürstenau, Michael 96, 97

GGaertner, Klaus 122Gaio, Ursula 36, 37Gaiser, Olaf 50, 52Galat, Vassili 23Gallagher, Irene 120Galle, Renate 32Ganten, Detlev 6, 12, 18, 20, 21,

23, 26, 142, 160Ganten, Ursula 85, 100Ganzel, Karin 68Garratt, Alistair 36, 37Gärtner, Angelika 117Gast, Klaus 47, 48, 49Gaudet, François 88Geheeb, Martina 124Gehring, Niels H. 76, 77Geißler, Erhard 160Genersch, Elke 97Gerlach, Brigitte 130Gibson, Meino Alexandra 130, 152Gladow, Monika 117Goan, Silvia-Renate 109Goldbrich, Beate 124Goletz, Steffen 80, 81Gollasch, Maik 80, 96, 97Gong, Maolian 21Göritz, Christian 134Gösele, Claudia 21Gossen, Manfred 13, 72, 160Gottschling, Karin 37Gräler, Markus 71Gras, Christiane 130Graschew, Georgi 90Grasmo-Wendler, Unn-Hilde 31, 32Graßhoff, Peter 71Grelle, Gerlinde 60, 74Grieben, Marlies 113Grigull, Sabine 90Grimm, Jan 77, 79Grinstein, Edgar 74Grishkovskaya, Irina 51, 52Grittner, Christine 80, 81Gromelski, Sandra 112Gross, Volkmar 26, 27, 87, 93, 94Großmann, Katja 85Groth, Detlef 15, 112Grüger, Sabine 27Grunewald, Ingrid 88, 142Gunia, Wilfried 29Guo, Jiankan 39Gust, Tatjana C. 120

154

HHaase, Hannelore 85, 97, 100, 142Haase, Sabine 90Haefker, Antje 105Haensch, Wolfgang 89, 90Hagemann, Dirk 83Hahn, Mike 90Hahn, Stefanie 30, 32Haink, Petra 120Haller, Hermann 13, 15, 26, 27, 41, 93,

95, 96, 97, 98, 113, 115, 151, 160Hammes, Annette 39Hampig, Franziska 27Hanisch, Uwe-Karsten 130Hanke, Jens 42, 43Harder-d‘ Heureuse, Johanna 29Harsdorf, Rüdiger von 87, 88, 113Hartmann, Gudrun 77, 112Hatada, Eunice 68Hauck, Ludger 88, 113Hauke, Lisa 29Haupt, Irene 134Häusler, Georg 130Heere, Petra 25Heikenwälder, Mathias F. 119, 120Heinemann, Udo 19, 31, 50, 52,

142, 160Heißmeyer, Vigo 68Hemme, Jens 117Hempel, Petra 83Hennies, Hans Christian 29Henning, Mechthild 137Heppenstall, Paul 132Heringer-Walther, Silvia 23Hess, Dana 23, 39Hieke, Brigitte 21Hildebrandt, Kati 105Hildenbrand, Ulrich 83Hilpert, Jan 41Hinz, Britta 29Hinz, Michael 68, 123Hirano, Fuminori 68Hjerrling-Leffler, Jens 134Hoch, Brigitte 82, 83Hoehe, Margret 19, 24, 25, 34, 35, 160Hoffmann, Anja 130Hoffmann, Katrin 29, 34, 35Hofmann, Wera 30, 32Hofstätter, Maria 66Hohaus, Annette 85, 100Höhnemann, Dirk 105Homuth, Volker 15, 27, 96, 97,

115, 151Honeck, Horst 94Hong, Liu 21Höpken, Uta 71, 142Horn, Denise 30, 32Hortsch, Michael 137Hübner, Norbert 20, 21, 39Huijnen, Martijn 43Hülsken, Jörg 75, 76, 77, 99Hummel, Kordelia 124Hun Lee, Kang 106, 107

IIbe, Sabrina 117Ibikli, Adnan 121Isac, Corina-Mihaela 85Ivanov, Anton 60Ivics, Zoltán 13, 102, 125, 126, 160Izsvák, Zsuzsanna 126

JJacobi, Karin 148, 160Jäger, Katrin 73, 74Jan, Guillaume 54Jandrig, Burkhard 30, 31, 32Janke, Jürgen 32Janta-Lipinski, Martin von 97, 122Janz, Martin 73, 74Jarchow, Birgit 130Jarosch, Ernst 91, 92Jeblonski, Frank 71Jerchow, Boris 78, 79Jia, Wentao 74Jobsky, Dieter 137Johnen, Heiko 71Jorcke, Dierk 60Jordan, Jens 8, 26, 27Joschko, Simone 66Jost, Erwin 142, 160Jost-Reuhl, Brigitte 109Ju, Xin-Sheng 119, 120Jundt, Franziska 74, 103, 105Jung, Bories 29Jung, Martin 29Jung, Christiane 19, 53, 54, 160Jungblut, Peter 60Junghahn, Ilse 109Junghans, Christine 35Jungmann, Sabine 68Jürchott, Karsten 73, 74, 89, 90

KKaba, Rudolf Gabor 71Kagelmaker, Horst 130Kaiser, Frank 106, 107Kammertöns, Thomas 117Kanehl, Anke 132Kang, Ningling 23, 26, 27Kannen, Brunhilde 58Karawajew, Leonid 104, 105Karczewski, Karin 115Karczewski, Peter 82, 83Kärgel, Eva 94, 100Karsten, Uwe 80, 81, 89, 90, 142, 160Katzer, Andrea 105Kemmner, Wolfgang 80, 89, 90Kemsies, Christel 85Kettenmann, Helmut 13, 128, 129,

130, 142, 152, 160Kettritz, Ralf 95, 97Keyner, Daniela 68, 71Khier, Harald Horst 137Kirchhoff, Frank 130, 142Kirsch, Thorsten 97Kirsch, Frank-Peter 137Kistel, Heide 21Klahn, Sylvia 92, 117

Klamm, Ursula 85Klautza, Jeanette 29Klein, Eirien 35Klein, J. 60, 97Klein, Nicolai 90Klein, Rüdiger 131, 132Klewer, Jacqueline 134Knespel, Andreas 52Knespel, Siegne 119, 120Knoblauch, Margit 21Knoblauch, Hans 35, 43Knoblich, Maria 98Koberling, Annemarie 81Koch, Ina 43Köhler, May-Britt 94Köhler, Matthias 95, 97Kohrt, Christiane 81Kölble, Konrad 31, 32, 89, 90Köpke, Karla 25Konzer, Peter 142, 147, 160Kordes, Uwe 68Koritschoner, Nicolas P. 119, 120Körner, Ida-Johanna 105Koroll, Michael 137Kostka, Susanne 60, 85, 100Kotitschke, Erika 100Kott, Monika 85, 100Kowenz-Leutz, Elisabeth 62, 66Krafft, Christoph 58Kraft, Regine 60, 73, 74, 85, 100Kramer, Annett 57, 81Krämer, Jochen 88Krappmann, Daniel 68, 74, 105Krause, Ernst-Georg 82, 83, 160Krause, Julia 29Krause, Sabine 66Kresse, Wolfgang 130Kries, Jens-Peter von 75, 77Kröber, Reinhard 47Krüger, Kerstin 105Krukenberg, Ina 105Krusekopf, Solveigh 94Kühlewind, Wolfgang 146, 160Kuhle, Verona 83, 85, 115Kuhn, Susanne 130Kulbe, Hagen 71Kuring, Dirk 29Kurz, Steffen M. 119, 120Kurzchalia, Teymuras 160Kusch, Angelika 71Küttner, Irmgard 117

155

LLamounier-Zepter, Valeria 85Landrock, Danilo 39Lang, Mathias 112Lange, Christian 90, 117Lange, Robert 71Langenickel, Thomas 23Lankes, Wolfgang 36, 37Lathe, Warren 43Lättig, Stefan 58Lauble, Hanspeter 50, 52Lee, Young-Ae 20, 28, 29Leheste, Jörg-Robert 41Lehmann, Cathleen 112Lehmann, Christine 122Lehmann, Gerrit 35, 43Leistner, Werner 56Lemke, Bernd 142, 149, 160Lemke, Britt 119, 120Lemm, Margit 109Lenk, Uwe 91, 92Lenter, Klaus-Ulrich 25Leonhardt, Heinrich 63, 86, 88,

113, 160Leutz, Achim 62, 63, 64, 66, 160Lewin, Gary R. 128, 131,

132, 142, 160Li, Guo-Hua 25Li, Li 37Li, Liang-Ping 117Li, Pei-Feng 113Liang, Han 90Liebig, Werner 142Liebner, Iska 29Lin, Shaoqian 90Lindner, Tom 34, 35Lindschau, Carsten 85, 96, 97, 98,

100, 115Linkens, Hans-Josef 160Lipp, Martin 69, 71, 142, 144, 160Liu, Songjie 25Lode, Jörg 112Löffler, Anja 104, 105Löhn, Matthias 96, 97Lucius, Hans 132Ludwig, Wolf-Dieter 104, 105Luft, Friedrich C. 8, 15, 18, 19, 20,

25, 26, 27, 28, 35, 41, 42, 43, 60, 93, 94, 96, 97, 115, 142, 151, 160

Lutsch, Gudrun 85, 99, 100

MMaasch, Christian 96, 97Madruga, Jaime 119, 120Maetzig, Martin 49Maier, Alexandra 56Maier, Olaf 98Malcherek, Lieselotte 90Manasterski, David 29Mannsfeldt, Anne 131, 132Mapara, Markus 74, 105, 119, 120Marg, Andreas 137Margot, Jean B. 88Mathas, Stefan 68, 103, 105Matthes, Eckhart 102, 122, 160Matyash, Marina 130Matyash, Vitali 130Mauch, Daniela 134McIlwrath, Sabrina 132Meding, Jörg 88Meese, Carmen 71Mehrhof, Felix 68, 88, 113Meißner, Kathrin 21Mennier, Ulrike 29Menzel, Ralph 94, 100Mertsch, Katharina 130Metzner, Sylvia 74, 105Meusser, Birgit 92Meyer, Dagmar 71Meyer, Mirjam 119, 120Micheel, Burkhard 13, 31, 32Milia, Anna Franca 26, 27Misselwitz, Rolf 58Mitulla, Hans 148Möhring, Regina 143, 160Mohaupt, Mariette 117Mokros, Thilo 71Moldenhauer, Ferdinand 43Molweide, Andreas 106, 107Montag, Christina 124Monti, Jan 21Morano, Ingo L. 13, 84, 85, 97,

100, 160Mordmüller, Benjamin 68Moré, Margret 137Morkel, Markus 77Morwinski, Rosemarie 115Mouro, Corinne 54Mrowka, Christian 39Mühl, Astrid 35Muller, Yves A. 50, 51, 52Müller, Anita 21, 52Müller, Annekathrin 56Müller, Dominik 26, 27Müller, Eva-Christina 44, 45, 60, 98Müller, Gerd 71, 105Müller, Gerda 130Müller, Jürgen J. 50, 52Müller, Thomas 36, 37, 142Müller, Uwe 50, 52

NNägler, Karl 134Neff, Klaus 25Neichel, Dajana 115Neubauer, Helga 60Neuhaus, Yvette 35Neumann, Claudia 109Nguyen-Hoay, Tam 106, 107Niss, Knut 66Nissen, Eberhard 97, 115Nitschke, Ute 105Nitz, Monika 23Nolte, Christiane 130Nörenberg, Ursel 137Nothnagel, Anita 30, 43Nürnberg, Gudrun 29, 43

OÖczelik, Chemil 36, 37Ohl, Lars 25, 71Ohlemeyer, Carsten 130Otto, Albrecht 44, 45, 60, 98, 106

PPachale, Dietlind 29Pagel, Ines 85Pankow, Harald 43Pantelic, Liliana 21Papst, Marion 72Pecher, Gabriele 71Pei, Weidong 33, 35Peter, Heidrun 124Peter, Jens-Uwe 23Peters, Niels 103, 105Petretti, Tobias 90Pevzner, Veniamin 71Pezzutto, Antonio 80, 102, 106, 107,

117, 120, 160Pfitzmaier, Birgit 85Pfrieger, Frank W. 128, 133, 134,

142, 160Pidde, Heiko 31, 32, 89, 90Pietsch, Hubertus 66Plöhn, Sören-Peter 143, 160Pohl, Bärbel 112Pommeranz, Sascha 29Poppe, Brunhilde 43Poppe, Konstanze 30, 31, 32Posner, Steffen 71Poßling, Alexandra 124Preiß, Susanne 117Prokoph, Helmuth 31, 32

156

157

QQin, Zhihai 107, 117Quass, Petra 97Quensel, Christina 88

RRadau, Boris 98Räder, Charlotte 41Radtke, Susan 23Rahn, Hans-Peter 88Rappert, Angelika 130Rathjen, Fritz G. 128, 135, 137, 160Rechmann, Stefanie 25Reich, Jens 19, 35, 42, 43, 151, 160Reis, André 13, 19, 28, 29, 34, 160Reiterer, Philipp 71Reszka, Regina 15, 102, 110, 112,

151, 160Richter, Gerhild 77Richter, Günther 106, 107, 117Richter, Jana 15, 112Rickers, Anke 60, 103, 105Riechert, Freya 103, 105Ried, Christian 71, 97Riedel, Heidi 124Rieffel, Christine 143, 160Riege, Dorothea 83Riethmacher, Dieter 13, 36, 37Rietzke, Helga 100Rink, Sinaida 112Ristau, Otto 45Ritschel, Silke 105Roefzaad, Claudia 90Rohde, Klaus 30, 43Rohs, Remo 56Rösch, Marion 117Roske, Yvette 50, 52Rossum, Denise van 112Rötzschke, Olaf 13, 118, 160Royer, Hans-Dieter 73, 74, 105, 160Rücker, Karin 30, 32Rudolph, Cathrin 37Rudolph, Sabine 81Rüschendorf, Franz 29, 43Ruiz, Patricia 77

SSaar, Kathrin 29Sacharjat, Michael 124Sachs, Martin 76, 77Sawitzki, Bianca 29Schäfer, Michael 137Schäfer, Olaf 60, 97Schaenke, Michaela 130Schaeper, Ute 76, 77Scharschmidt, Erika 68Scharte, Gudrun 31, 32Schaup, Annette 58Schedl, Andreas 18, 38, 39, 160Scheel, Sabine 21Scheer, Helga 122Scheidereit, Claus 63, 67, 68, 74,

103, 105, 142, 160Schenk, Harry 149, 160Schenk, Jörg 31, 32Scherneck, Siegfried 19, 30, 32,

89, 90, 160Schildt, Jürgen 122Schipke, Carola 130Schirrmann, Thomas 71Schlag, Peter M. 8, 15, 19, 31, 32,

42, 78, 79, 80, 89, 90, 123, 124,142, 151, 160

Schlegel, Wolfgang 83Schlisio, S. 123Schmid, A. 73, 74Schmidt, Folke 27Schmidt, Hannes 137Schmidt, Holle 115Schmidt, Mathilde 27Schmidt, Susanne 29Schmidt, Sabine 39Schmidt, Steffen 43Schmidt, Willy 43Schmidt-Ullrich, Ruth 68, 142Schmitz, Christian 41Schneider, Frank 90Schneider-Mergener, Jens 57, 58Schnitzer-Ungefug, Jutta 160Schnitzler, Mark 107Schüler, Thomas 117Schultz, Jörg 43Schulz, Hannelore 41Schulz, Wolfgang 56Schulze, Bettina 90Schumacher, Karin 90Schumacher, Stefan 13, 137Schümann, Margitta 60Schunck, Wolf-Hagen 26, 93, 94,

100, 160Schuster, Herbert 19, 25, 33, 35, 160Schuster, Stefan 43Schütz, Gunnar 77Schwabe, Holger 89, 90Schwartz, Arnfried 31, 32Schwarz, Dieter 94Schwarz, Monika 29, 106, 107Schwarz-Romond, Thomas 78, 79Schwenkenbecher, Jan 104, 105Seeger, Michaela 29Seehrich, Hans-Joachim 160Seelow, Dominik 29Seemann, Stefanie 112

Seibert, Patric 103, 105Seidensticker, Martin 77Seitz, Susanne 30, 31, 32Sendtner-Voelderndorff, Luzi 137Shakeri-Garakani, Ansiah 124Shan, Ying 73, 74Sharma, Kamal 137Shin, Jung-Bum 132Sieber, Martin 37Siemer, Ansgar 47Silva Junior, José-Antonio 23Siouniaev, Chamil 43Sirtes, Daniel 130Sklenar, Gisela 51, 52Sklenar, Heinz 19, 55, 56, 160Skorna, Madeleine 29Sochnikova, Natalia 75, 77Sommer, Thomas 13, 91, 92, 99, 160Sonnenberg-Riethmacher, Eva 36, 37Spillmann, Freia 29Spingies, Christine 29Sporbert, Anje 88Stade, Katrin 92Stahn, Renate 80, 81, 121Stein, Ulrike 89, 90Steinert, Fabian 29Steinhusen, Ulrike 105Sternke, Anne 94Stoffels, Mandy 27Stolley, Peter 143, 160Strauss, Holger 112Strauss, Michael 13, 14, 68, 124,

150, 160Strehle, Michael 36, 37Stucky, Cheryl L. 131, 132Stulich, Marianne 122Sturm, Isrid 103, 105, 107Stüwe, Eva 126Sugimura, Keiichi 23Sümnich, Ingeborg 30, 32Sydow, Hanna 85Synowitz, Michael 130Szangolies, Inka 29Szelestei, Tamas 35

TTank, Jens 26, 27Tegethoff, Sebastian 68Teppke, Anne-Dorothee 121Theile, Michael 32Theuring, Sebastian 105Thiele, Verena 43Thierfelder, Ludwig 160Tian, Xiao-Li 21Timmermann, Bernd 25Tobias, Doerks 43Toka, Hakan 33, 35Tönjes, Michael 77Trautner, Thomas A. 160Trotier, Fabienne 29Tschapek, Alexej 43Tünnemann, Gisela 52

158

UUckert, Wolfgang 117, 142Uhlmann, Regina 35Ullrich, Oliver 31, 32, 89, 90Urban, Jörg 92

VVaron-Mateeva, Raymonda 29Vetter, Donathe 83Vidal, Valerie 39Vogel, Frank 94, 99, 100Vogel, Margit 99, 100Vogel, Regina 75, 76, 77Volkwein, Corinna 92

WWallukat, Gerd 15, 41, 96, 102, 114,

115, 142, 150, 160Walter, Jan 92Walther, Diego 23, 41Walther, Thomas 23, 85Walther, Ingrid 76, 77Walther, Wolfgang 89, 90, 121Wedekind, Brigitta 37Wegener, Monika 115Wegner, Daniela 112Weinert, Ingeborg 74Welfle, Heinz 19, 57, 58, 160Welfle, Karin 58Weller, Andrea 29Wellner, Maren 95, 97Wenzel, Katrin 25, 80Werner, Erik 50, 52Werner, Sabine 31, 32Wessel, Ralf 99, 100Westen, Christel 117, 142Westermann, Jörg 106, 107, 120Westermann, Peter 98, 160Wetzel, Katarina 85Wetzel, Ulrike 85, 100Wieske, Martin 99, 100Willimsky, Gerald 117Willnow, Thomas E. 15, 18, 26, 40,

41, 151Winbeck, Georgia 75, 77Winkler, Liselotte 23Wirtz, Ralph 78, 79Witting, Anke 130Wittmann-Liebold, Brigitte 19, 59,

60, 103, 106, 160Wittstruck, Angelika 92Wiznerowicz, Irmgard 77Woischwill, Christiane 73, 74Woldeyesus, Mas 36, 37Wolf, Edelgard 43Wolff, Gerhard 73, 74, 105Wuchter, Christian 104, 105Wunderlich, Heidrun 81Würtele, Martin 77, 78, 79

YYuan, Yanping 43, 49

ZZacharias, Martin 56Zacharias, Ute 137Zacharzowski, Udo 85Zastrow, Inga 35, 43Zechner, Dietmar 77Zeidler, Helga 30, 32Zeisig, Reiner 121Zenke, Martin 100, 102, 106, 119,

120, 160Zenker, Jana 39Zhaoqiang, Cui 21Zhou, Lepu 79Ziegler, Ulrike 39Zimdahl, Heike 21Zinke, Silke 35Zirwer, Dietrich 47Zummach, Ramona 94Zuske, Alexandra 90

159

Molecular TherapyCoordinator: Prof. Dr. Thomas Blankenstein

Rainer DietzZoltán IvicsEckart MatthesGerd Wallukat

Bernd DörkenThomas BlankensteinIduna FichtnerAntonio PezzuttoKirsten Falk/Olaf Rötzschke

Regina ReszkaMartin ZenkeMichael Strauss (deceased)Dietrich ArndtHans Brachwitz

Molecular and Developmental Neuroscience Coordinator: Prof. Dr. Helmut Kettenmann

Helmut Kettenmann Fritz G. Rathjen Gary R. Lewin Frank W. Pfrieger

Scientific CouncilChairDr. Martin Lipp

Scientific CoordinatorDr. Jutta Schnitzer-UngefugDr. Hans-Josef Linkens

Public RelationsBarbara Bachtler

Clinical and Experimental Research Programs

Cardiovascular DiseasesCoordinator: Prof. Dr. Friedrich C. Luft

CancerCoordinator: Prof. Dr. Peter M. Schlag

Genetics, Bioinformatics and Structural BiologyCoordinator: Prof. Dr. Udo Heinemann

Friedrich C. LuftHerbert SchusterCarmen BirchmeierDetlev GantenMichael BaderAndreas Schedl

Margret HoeheJens ReichAndré ReisThomas E. WillnowLudwig Thierfelder

Udo HeinemannSiegfried ScherneckGregor DamaschunChristiane Jung

Heinz SklenarHeinz FabianHeinz WelfleJoachim BehlkeBrigitte Wittmann-LieboldErhard Geißler

Cell Growth and Differentiation Coordinator: Prof. Dr. Walter Birchmeier

Ernst-Georg KrauseHeinrich LeonhardtTeymuras KurzchaliaIngo L. Morano

Wolf-Hagen SchunckHermann HallerPeter WestermannManfred Gossen

Walter BirchmeierAchim LeutzClaus ScheidereitMartin LippJürgen Behrens

Uwe KarstenHans-Dieter RoyerPeter M. SchlagThomas Sommer

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Scientific CommitteeChairProf. Dr. Thomas A. Trautner, Berlin

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Administrative DirectorDr. Erwin Jost

Directors

Animal FacilitiesDr. Karin Jacobi

ComputingBernd LemkeLibraryDr. Dorothea Busjahn

Construction DepartmentSören-Peter PlöhnTechnicsHarry Schenk

Central Facilities

PersonnelDr. Hans-Joachim Seehrich

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Administration

Auditind and Legal AffairsChristine Rieffel

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Employee´s RepresentativChairMarion Bimmler

Deputy Science DirektorProf. Dr. Walter Birchmeier

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