Tätigkeitsbericht - hpi-hamburg.de · A duck hepatitis B virus strain with a knockout ... Diese neue Struktur gewährleistet eine Output-orientierte Förderung auf der Basis einer

Tätigkeitsbericht2003/2004

Heinrich-Pette-Institut

Leibniz-Gemeinschaft

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Heinrich-Pette-Institutfür Experimentelle Virologie und Immunologie

an der Universität Hamburg

Martinistraße 52 · 20251 Hamburg

Impressum

Verantwortlich Heinrich Hohenbergfür den Inhalt Joachim Hauber

Redaktion Heinrich Hohenberg

Redaktionsassistenz Rudolph Reimer, Alexa Bäns,Geno Hohenberg

Bildmaterial Ursula Müller, Angela Homfeld,Thomas Hauck, Rudolph Reimer

Grafik & Layout AlsterWerk MedienService GmbHHamburg

Druck Hartung Druck + Medien GmbHHamburg

Titelbild

Tiermodelle spielen eine wich-tige Rolle bei der Erforschungkomplexer systemischer Er-krankungen. Dazu zählen pa-thogenetische Veränderungennach Virusinfektion und Krebs-erkrankungen. Die abgebilde-ten transgenen Mäuse dienenim HPI als ein Modellsystem fürspezielle Tumorerkrankungen.

Heinrich-Pette-Institutfür Experimentelle Virologie und Immunologiean der Universität Hamburg

Martinistraße 52 · 20251 HamburgPostfach 201652 · 20206 Hamburg

Telefon: +49-40/4 80 51-100Telefax: +49-40/4 80 51-103Email: [email protected]: www.hpi-hamburg.de

Das Heinrich-Pette-Institut ist Mitgliedder Leibniz-Gemeinschaft (WGL)Internet: www.wgl.de

Tätigkeitsbericht2003/2004

Inhaltsverzeichnis

Allgemeiner ÜberblickVorwort .................................................................................................... 9

Struktur des Heinrich-Pette-Instituts ..................................................... 10

Einrichtung eines zentralen Bilddatenverarbeitungs- und Archivierungs-Systems .................................................................... 12

Ersatz- und Erweiterungsbau II .............................................................. 12

Personelle Veränderungen in den Jahren 2003/2004 ........................... 15

Mitglieder des Wissenschaftlichen Beirats des HPI .............................. 17

Die erste Begutachtung des HPI durch den neuen Wissenschaftlichen Beirat ....................................................................... 18

Retreats der wissenschaftlichen Mitarbeiter des HPI ........................... 18

Wissenschaftliche Highlights .................................................................. 19

Preisverleihungen .................................................................................... 20

In memoriam ............................................................................................ 22

Programmbereich „Virus-Wirts-Wechselwirkungen“Program Area „Virus-Host Interaction“Introduction ............................................................................................. 23

Research Fields ......................................................................................... 25

Research Projects ..................................................................................... 27■ Itinerary of hepatitis B viruses: Delineation

of restriction points critical for infectious entry ........................................ 27■ Spread of hepatitis B viruses in vitro requires

extracellular progeny and may be codetermined by polarized egress ................................................................................. 28

■ Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R ............................................................................. 29

■ Selection of a secretion-incompetent mutant in the serum of a patient with severe hepatitis B................................................................................................ 30

■ New hepatitis B virus of cranes that has an unexpected broad host range .................................................................. 31

■ Characterization of nonconventional hepatitis B viruses lacking the core promoter............................................................. 32

■ A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wildtype virus ................................. 33

■ Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies ........................................................................................ 33

■ Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70 ................................................... 35

■ Functional characterization of the interaction between human La and hepatitis B virus RNA ......................................... 35

■ The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization ...................................................................... 36

■ Nuclear trafficking of La protein depends on a newly identified nucleolar localization signal and the ability to bind RNA ........................................................... 36

■ Retroviral mRNA transport ...................................................................... 37■ Novel therapy strategies for the treatment

of HIV-1 infection .................................................................................... 40■ Restriction of HIV-1 replication in peripheral

blood mononuclear cells ......................................................................... 41■ APOBEC3G, a host cell defense system for HIV-1 ..................................... 43■ Subcellular localization of herpes simplex-virus

immediate early proteins ......................................................................... 44■ Intracellular transport of adenovirus regulatory

proteins ................................................................................................... 45■ SV40 large T-antigen induces global chromatin

re-organization ........................................................................................ 45■ Cellular receptors for retroviral entry ........................................................ 47■ Complex interplay of retroviral and cellular

determinants in disease specificity............................................................ 48■ Localization and dynamics of small circular

DNA in the nucleus of living cells ............................................................ 49■ The role of protein arginine methylation

in vivo ..................................................................................................... 51■ 3D liver organoid model system and analysis

of virus infection ...................................................................................... 52■ Cryo-virology on the ultrastructural level ................................................. 53■ Environmental preparation of infectious

suspensions: ultrastructural analysis of infected cells, virus particles, and cellular/viral components................................................................... 54

■ Exact measurement and 3D reconstructionof biological surfaces .............................................................................. 56

Programmbereich „Zelluläre Dysregulation“Program Area „Cellular Dysregulation“Introduction ............................................................................................. 57

Research Fields ......................................................................................... 58

Research Projects ..................................................................................... 63■ Transcriptional control of gene expression

by wildtype and mutant p53.................................................................... 63■ A novel human p53-isoform is an essential

element of the ATR-intra-S-phase checkpoint .......................................... 64■ Molecular response to DNA damage

depends on the cell cycle stage and p53 status........................................ 65■ Dissecting a cytoplasmic anchor for p53 .................................................. 66■ Crosstalk between p53 and retinoblastoma

proteins in cellular senescence ................................................................. 68■ Functional interactions of p53 with telomeres

and telomerase ....................................................................................... 69■ Sp100 is important for the stimulatory effect of

homeodomain-interacting protein kinase-2 on p53-dependent gene expression ......................................................... 69

■ PML is required for homeodomain-interacting protein kinase 2 (HIPK2)-mediated p53 phosphorylation and cell cycle arrest but is dispensable for the formation of HIPK domains .................................... 70

■ HIPK2 regulates transforming growth factor-beta-induced c-Jun NH(2)-terminal kinase activation and apoptosis in human hepatoma cells ................................. 71

■ The role of U-box proteins for ubiquitination and degradation of proteins .................................................................... 71

■ Pathophysiology of G-protein coupled receptors (GPCRs) .................................................................................... 72

■ SPOC1, a novel PHD-finger protein: association with residual disease and survival in ovarian cancer.................................. 74

■ Posttranscriptional mRNA processing in human dendritic cells .......................................................................................... 75

■ Disruption of the nuclear scaffold blocks genome replication ................................................................................. 77

■ Mechanisms of X chromosome inactivation: Contributions of the nuclear scaffold ....................................................... 79

■ Disruption of RUNX1 (AML1) function in acute leukemias ................................................................................... 81

■ Mutant forms of the C/EBPα transcriptionfactor inhibit myeloid differentiation ........................................................ 83

■ Retroviral insertional mutagenesis to identify novel hematopoietic regulators ......................................................................... 84

■ Dissecting the mechanism of apoptosis-resistance in hematopoietic malignancies expressing wildtype p53............................... 86

■ In vivo regulation of human and mouse telomerase TERT promoter during embryonal development and breast carcinogenesis / role of p53 ................................................... 87

■ Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice ............................................ 89

■ Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice .............................. 92

■ Proteomics: Analysis of the phosphorylation patter of the tumor suppressor p53 ................................................................... 93

■ Proteomics: Proteomic sequencing of a new p53 isoform, delta p53 ........................................................................... 94

■ New methods and micro-equipment for the fast and controlled cryo-processing of clinical material ................................... 95

Personelle Zusammensetzung der wissenschaftlichen Abteilungen und Arbeitsgruppen .......................................................... 97

Veröffentlichungen ................................................................................ 106

Diplomarbeiten, Dissertationen, Habilitationen ................................. 113

Lehrtätigkeit ........................................................................................... 117

Gutachtertätigkeit ................................................................................. 118

Mitherausgabe wissenschaftlicher Zeitschriften ................................ 120

Organisation von wissenschaftlichen Veranstaltungen, Vorsitzfunktionen .................................................................................. 121

Erfindungsmeldungen und Patente ..................................................... 122

Seminare ................................................................................................. 123

Drittmittelprojekte ................................................................................ 126

Finanzielle Förderung ............................................................................ 133

Verwaltung ............................................................................................. 135

Verwaltung /Besondere Ereignisseim Zeitraum 2003/2004 ......................................................................... 137

Personal der Verwaltung und Allgemeine Dienste ............................ 139

Zusammenfassung der Kostenträgerstruktur ..................................... 141

Organigramm ......................................................................................... 143

Aus der Geschichte des HPI

Prof. Heinrich Pette im Mai 1951 beim ersten Spatenstich für das neue Tier-stallgebäude des Heinrich-Pette-Instituts (rechts hinter ihm: Edith Pette)

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VorwortDieser Tätigkeitsbericht gibt eine ausführliche Darstellung der aktuellenEntwicklungen, der wissenschaftlichen Programme und der Aktivitäten derMitarbeiter des Heinrich-Pette-Instituts in den Jahren 2003 und 2004. Er stellt diewichtigsten Forschungsergebnisse, die zentralen Ziele und die zukünftigen Pläneim Rahmen unserer Forschungsprogramme dar und enthält nähere Infor-mationen zur Struktur, Mission und Vision unseres Instituts. Zudem soll derBericht dazu beitragen, die Ergebnisse unserer Arbeit, die uns bewegenden dring-lichen Fragestellungen und auch die Faszination wissenschaftlicher Grund-lagenforschung der Öffentlichkeit zu vermitteln.

Wir sind zuversichtlich, dass die in diesem Bericht aufgezeigten Leistungen dervergangenen zwei Jahre unsere Zuwendungsgeber, die Mitglieder des Kurato-riums und des Wissenschaftlichen Beirats sowie die Freunde und Förderer desHPI von der wissenschaftlichen und gesellschaftlichen Bedeutung unseresInstitutes und unserer Forschungsarbeiten überzeugen werden.

Hamburg, Februar 2005

Dr. Heinrich Hohenberg Vorsitzender des Kollegiums

Allgemeiner Überblick

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Struktur des Heinrich-Pette-InstitutsDas Heinrich-Pette-Institut (HPI) für Experimentelle Virologie und Immuno-logie an der Universität Hamburg ist eine gemeinnützige und selbstständigeForschungseinrichtung, die der Leibniz-Gemeinschaft (WGL) angehört.Zuwendungsgeber sind auf der Bundesseite das Bundesministerium fürGesundheit und Soziale Sicherung (BMGS), auf der Länderseite die Behörde fürWissenschaft und Gesundheit (BWG) in Hamburg.

Das HPI widmet sich laut Satzung der Erforschung der Biologie humanerVirusarten, der Pathogenese von Viruserkrankungen, der Abwehrreaktionen desOrganismus und damit zusammenhängender Probleme. Das HPI bewegt sichdamit im Zentrum aktuellster biomedizinischer Grundlagenforschung. DieErforschung der bei der Virusvermehrung wirkenden Mechanismen und ihrerDetails liefert kontinuierlich die wissenschaftlichen Daten und Ergebnisse, welchedie Grundlage zur Entwicklung neuartiger Strategien zur Therapie vonVirusinfektionen und Tumorerkrankungen schaffen. Wie bereits vom Wissen-schaftsrat 1996 festgestellt und durch eine externe unabhängige WGL-Evaluierungskommission im November 2002 bestätigt, gibt es in Deutschland keinInstitut mit vergleichbaren Arbeitsschwerpunkten und vergleichbarer Kompetenz.

Das HPI gliedert sich in permanente und auf Zeit eingerichtete Forschungsab-teilungen bzw. Forschungsgruppen. Die permanenten Forschungsabteilungenbilden die strukturellen Basiseinheiten, die in Bezug auf die zu untersuchendenhumanpathogenen Virusfamilien die jeweiligen Schwerpunkte setzen. Auf dieseWeise wird eine breite Technologiebasis als Kernstruktur am Institut etabliert undgesichert, die durch unabhängige Forschungsgruppen und ihre jeweiligen Zusatz-technologien ergänzt wird. Bei den Forschungsgruppen handelt es sich vermehrtum zeitlich begrenzt eingesetzte Nachwuchsgruppen. Deren projektbezogeneVerzahnung mit den Forschungsabteilungen soll den direkten und schnellenZugang zu den am HPI etablierten virologischen und zellulären Modellsystemengewährleisten und dadurch die notwendigen Voraussetzungen für eine hoheFlexibilität und Produktivität dieser Nachwuchsgruppen und ihrer Leiter/-innensicherstellen.

Der vorliegende Tätigkeitsbericht 2003/2004 folgt in seinem wissenschaftlichenTeil in Aufbau und Gliederung erstmals der im Berichtszeitraum neu konzipier-ten Forschungs-Programmsteuerung. Unter Beachtung der Empfehlungen derBund-Länder-Kommission für Bildungsplanung und Forschungsförderung(BLK) wurden auf der Basis der vorhandenen Kostenträgerstruktur am HPI zweiwissenschaftliche Programmbereiche definiert, die keine Ableitungen aus organi-satorischen Einheiten und Kostenstellen des Instituts sind. Es handelt sich viel-mehr um abteilungsübergreifende Programmbereiche, die mit den strukturellenOrganisationseinheiten (Abteilungen und Arbeitsgruppen) des HPI eine überge-ordnete Matrix bilden. Diese neue Struktur gewährleistet eine Output-orientierteFörderung auf der Basis einer vernetzenden Programmsteuerung (Programm-budgets). Die gesamte Forschungssteuerung des Institutes erfolgt im Rahmendieser beiden abteilungsübergreifenden Programmbereiche.

11Als Programmverantwortliche fungieren Prof. Hans Will (Abteilung AllgemeineVirologie) für den Programmbereich „Virus-Wirts-Wechselwirkungen“ undProf. Wolfgang Deppert (Abteilung Tumorvirologie) für den Programmbereich„Zelluläre Dysregulation“.

Dr. Heinrich Hohenberg (Arbeitsgruppe Elektronenmikroskopie und Mikro-Technologie) und Dr. Carol Stocking (Arbeitsgruppe Molekulare Pathologie)sind die jeweiligen Stellvertreter.

Während des Berichtszeitraumes wurde in stetiger Absprache mit dem Kura-torium eine neue Satzung für das Heinrich-Pette-Institut erarbeitet und imHerbst 2004 eingeführt. Ziel der Satzungserneuerung war unter anderem, dieEntscheidungslinien und Verantwortlichkeiten im HPI eindeutiger zu definierenund darüber hinaus durch verlängerte Amtszeiten für mehr Kontinuität auf derLeitungsebene des Institutes zu sorgen.

Seiner neuen Satzung folgend wird das Heinrich-Pette-Institut nun von einemVorstand geführt, der sich aus dem gewählten Wissenschaftlichen Direktor, demVerwaltungsleiter als permanentem Mitglied und den zwei jeweiligen Stell-vertretern zusammensetzt. Der Wissenschaftliche Direktor und die Stellvertreterwerden auf Vorschlag des Kollegiums vom Kuratorium bestellt, wobei dieAmtszeit der Wissenschaftlichen Vorstandsmitglieder fünf Jahre beträgt.Hervorzuheben ist, dass die neue Satzung es erstmals ermöglicht, auf Dauerangestellte Arbeitsgruppenleiter/-innen als Stellvertreter des WissenschaftlichenDirektors in den Vorstand des Institutes zu berufen.

Eine ähnlich wichtige strukturelle Änderung erfolgte bei der Besetzung des Kolle-giums. Das Kollegium – im Rahmen der wissenschaftlichen Selbstverwaltung fürdie wissenschaftliche Entwicklung des Institutes verantwortlich – erstellt das For-schungsprogramm und wirkt bei der Berufung von Abteilungsleiterinnen und -lei-tern mit. Erstmals gehören nun dem Kollegium satzungsgemäß und mit Stimm-recht auch die Leiter/-innen der Arbeitsgruppen an, wobei die Position der Leitungdes Kollegiums (Vorsitz) ebenfalls aus diesem Personenkreis besetzt werden kann.

Diese Ende 2004 bereits in die Praxis umgesetzten Satzungsänderungen habenein deutlich vergrößertes, längst dringend benötigtes Reservoir an entsprechen-den Kandidatinnen bzw. Kandidaten für die Leitungspositionen am HPI geschaf-fen. Zudem wird mit dieser Umstrukturierung auf institutioneller Ebene nunerstmals die langjährige berufliche Erfahrung der auf Dauer angestellten Arbeits-gruppenleiterinnen und -leiter, vor allem im Bereich der längerfristigen strategi-schen Leitung und Forschungssteuerung, für das HPI genutzt.

Diese Maßnahme führt nicht zuletzt zu einer erheblichen Entlastung der auf Zeitbeschäftigten Leiterinnen und Leiter von Nachwuchsgruppen, die auf diese Weisevon allgemeinen Instituts- und Verwaltungsaufgaben freigestellt werden. DieNachwuchsgruppen sollen sich in Zukunft – in dem ihnen zur Verfügung gestell-ten begrenzten Zeitraum – ganz auf die Durchführung ihrer wissenschaftlichenAufgaben konzentrieren können.


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Einrichtung eines zentralen Bild-datenverarbeitungs- und Archivierungs-Systems im HPIDie in der Forschung erarbeiteten Daten (Bilder, Texte etc.) liegen zunehmend indigitaler Form vor. Diese Daten müssen in überschaubarer und gut strukturierterForm erfasst, gespeichert und über längere Zeiträume sicher und jederzeit abruf-bar gelagert werden können. Das Institut hat sich deswegen 2004 entschlossen,eine zentrale Datenbank zur Erfassung und Archivierung digitaler oder digitali-sierter Daten (wie in der Verwaltung bereits vorhanden und bewährt) für dieWissenschaftler des HPI zu konzipieren und bereitzustellen.

Ein Projektteam (H. Hohenberg, N. Zangenberg und R. Reimer) hat im Auftragdes Kollegiums ein entsprechendes Konzept erarbeitet und bereits Ende 2004 alsBasisversion am Institut etabliert. In den nächsten zwei Jahren soll das System soweit ausgebaut und verfeinert werden, dass die Laborjournale der einzelnenLaborarbeitsplätze – unter Beachtung der Datenschutzvorschriften – in die zen-trale Datenbank eingespeist werden können.

Ersatz- und Erweiterungsbau IIInternational anerkannte biomedizinische Grundlagenforschung, besonders dieexperimentelle Handhabung humanpathogener Erreger, ist maßgeblich von einergeeigneten Infrastruktur abhängig, die einen adäquaten sicherheitstechnischenStandard garantiert. Da Teile der Gebäude des Heinrich-Pette-Instituts, und zwarAltbauten aus den 1950er und 1960er Jahren, nicht mehr diesen Anforderungenentsprachen, war der Erhalt und die Steigerung der wissenschaftlichen Leistungdes HPI von der positiven Entscheidung für den Neubau eines modernen Labor-und Verwaltungsgebäudes ab-hängig. Der entscheidende Schrittwurde vollzogen, als der Senat derFreien und Hansestadt Hamburgam 22. April 2003 eine Summevon 16,15 Mio. € für den Ersatz-und Erweiterungsbau II bewillig-te. Die Gesamtkosten des Bauswerden anteilig vom Bundes-ministerium für Gesundheit undSoziale Sicherung (BMGS) undder Behörde für Wissenschaft undGesundheit (BWG) der Freienund Hansestadt Hamburg getra-gen. Im Herbst und Winter 2003erfolgte zunächst der Umzug derArbeitsgruppen aus dem Altbau in

Abriss des ehemaligen Verwaltungs- und Laborgebäudes imSommer 2004

13den Erweiterungsbau I (aus dem Jahre 1996). Dies betraf insbesondere dieArbeitsgruppen Elektronenmikroskopie, Molekulare Pathologie und SomatischeStammzellgenetik. Sie waren bisher im Altbau untergebracht und mussten für dieBauphase umgesiedelt werden. Dabei wäre ohne das große Engagement allerBeteiligten vor allem der Umzug der Großgeräte und der Bibliothek nicht mög-lich gewesen. Die Verwaltung des Instituts, die ihre Büros bisher ebenfalls imAltbau hatte, war bereits im November 2003 in eine Außenstelle am LokstedterSteindamm umgezogen, so dass 2004 mit dem Abriss des Altbaus – des ältestennoch erhaltenen Gebäudes des HPI – begonnen werden konnte.

Seit Beginn der Bautätigkeit im August 2004 werden nun auf einer Fläche von5150 qm neben Räumlichkeiten für die Verwaltung auch modernste Laborräumefür die Virus- und Tumorforschung sowie für Nachwuchs- und Kooperations-gruppen geschaffen. Der Ersatz- und Erweiterungsbau II wird nach seinerFertigstellung eine weitere Vernetzung des HPI mit Forschungsgruppen desUniversitätsklinikums Hamburg-Eppendorf (UKE) bzw. der Universität Hamburgermöglichen. So beteiligt sich die Fördergemeinschaft Kinder-Krebs-ZentrumHamburg e.V. in Form einer Public-Private-Partnership mit zusätzlichen 3 Mio.€ ander Errichtung des Ersatz- und Erweiterungsbaus II und bezieht nach dessenFertigstellung auf einer Fläche von etwa 540 qm eigene Laborräume. Es entstehtdort ein Forschungsinstitut für pädiatrische Hämatologie und Onkologie. Dieräumliche Nähe und die organisatorische und inhaltliche Kooperation zwischenHPI und Fördergemeinschaft wird die Netzwerkbildung beschleunigen. Sie ist indieser Form in Hamburg einmalig und auf nationaler Ebene richtungweisend. DieGrundsteinlegung zum Ersatz- und Erweiterungsbau II fand am 11. November 2004in festlichem Rahmen statt. Wir sind der festen Überzeugung, dass sowohl die inter-nen strukturellen Veränderungen als auch die bauliche Erneuerung des HPI nichtnur neue Chancen programmatischer und räumlicher Vernetzung eröffnen, sondernein notwendiger Schritt sind, um den steigenden Anforderungen eines sich rapidevollziehenden Strukturwandels in der Grundlagenforschung gewachsen zu sein.


Ersatz- und Erweiterungsbau II. APB. Beisert, Wilkens, Grossmann-Hensel, Architekten BDA

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Bild links: Susanne Barkmann und Prof. Reinhard Schneppenheim vom Kinder-Krebs-Zentrum Hamburg e.V.Bild rechts, oben: Personalleiter Jörg Schinkel im Gespräch mit dem Vorsitzenden des Kollegiums, Dr. HeinrichHohenberg. Bild rechts, unten: Der Vorsitzende des Wissenschaftlichen Beirates des HPI, Prof. Hans Wolf, imGespräch mit Prof. Hans Will

Feier zur GrundsteinlegungVon links nach rechts: HerrGrossmann-Hensel (Architekt),Prof. Reinhard Schneppen-heim (Leiter der Kinderklinikfür pädiatrische Hämatologieund Onkologie am UKE), Prof.Joachim Hauber (Wissen-schaftlicher Direktor des HPI),Dr. Holger Iversen (Vorstandder Fördergemeinschaft Kin-der-Krebs-Zentrum Hamburge.V.), Prof. Jörg Debatin (ärzt-licher Direktor des UKE), HerrWolfgang Beuss (Vorsitzenderdes Wissenschafts-Ausschussesder Bürgerschaft Hamburg),Senator Jörg Dräger PhD

15Personelle Veränderungen in den Jahren 2003/2004Für die Steuerung und Kontrolle des Heinrich-Pette-Instituts sind zwei externeOrgane von entscheidender Bedeutung: das Kuratorium und der externeWissenschaftliche Beirat. Im Berichtszeitraum haben sich personelle Verän-derungen sowohl in der Zusammensetzung des Wissenschaftlichen Beirats alsauch des Kuratoriums ergeben (siehe Seite 143).

Das Kuratorium berät den Vorstand und überwacht dessen Geschäftsführungebenso wie die Wahrung des Stiftungszweckes. Im Jahr 2003 schieden derVorsitzende, Herr Eberhard Vater, und die weiteren Mitglieder, Herr Franz J.Bindert, Herr Prof. Bernd Groner und Herr Reinhard Hollunder nach langjähri-ger ehrenamtlicher Tätigkeit aus dem Kuratorium des HPI aus.

Es sei hier besonders erwähnt, dass der Ersatz- und Erweiterungsbau II in weiteFerne gerückt wäre ohne die beharrliche Arbeit von Herrn Vater, der von März1994 bis August 2003 das Amt des Vorsitzenden mit großem Engagement ausüb-te, und dem nicht nachlassenden Einsatz von Herrn Ministerialdirigent Bindert.Dafür danken wir beiden sehr herzlich.

Herr Wilfried Mohr, Geschäftsführer und Gesellschafter der medac GmbH inWedel bei Hamburg, übernahm im September 2003 den Vorsitz des Kuratoriums.Ebenfalls traten Frau Dr. Clara Schlaich, Herr Dr. Michael Kramer und Herr Prof.Hans Wolf diesem Gremium bei. Unsere besten Wünsche begleiten sie.

Der international besetzte Wissenschaftliche Beirat evaluiert in regelmäßigenAbständen die wissenschaftliche Leistung des Instituts. Der jetzige Beirat ist inseiner neuen Besetzung (siehe Seite 17) im Jahr 2004 zum ersten Mal zusam-mengetreten. Ihm gehören neben Prof. Dr. Hans Wolf (1. Vorsitzender)


Feierliche Verabschiedung von Herrn Vater (Mitte) im HPI,Kuratoriumsvorsitzender von 1994 bis 2003. Hinter ihm links: Prof.Hans Wolf, rechts: Frau Hanna Fangohr (BWG, Hamburg)

Wilfried Mohr, neuer Vorsitzender desKuratoriums seit 2003

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und Prof. Dr. Axel Rethwilm (stellvertretender Vorsitzender) die Professoren FrauDr. J. Valerie Bosch, Dr. Ueli Aebi, Dr. Wolfram Gerlich und Dr. Dean Jackson an.Als einziges Mitglied aus dem bisherigen Beirat ist Prof. Dr. Jonathan Yedwellauch im neuen Beirat vertreten. Wir freuen uns auf die Zusammenarbeit mit demjetzigen Beirat und danken an dieser Stelle allen Mitgliedern des bisherigenBeirates für ihr Engagement und ihre Unterstützung, die sie dem Institut habenzukommen lassen.

Der jetzige Beirat hat, über seine bereits erwähnte Rolle hinaus, im Rahmen derNeustrukturierung des Instituts im Wissenschaftsbereich eine entscheidendeFunktion in Bezug auf die Bewertung dieser Strukturmaßnahmen und ihrerAuswirkungen auf die Qualität und Effizienz der wissenschaftlichen Arbeit imInstitut. Dies drückt sich unter anderem in der engen Zusammenarbeit zwischendem HPI und dem neuen Vorsitzenden des Beirats Herrn Prof. Wolf aus. Fürseine stete Präsenz bei wichtigen wissenschaftlichen Entscheidungen undEreignissen, die das Institut betreffen, möchten wir ihm an dieser Stelle ganzbesonders danken.

Im Sommer 2003 wurde Dr. Heinrich Hohenberg eine Abteilungsleiterstelle amRobert-Koch-Institut in Berlin im Bereich „Biologische Sicherheit, FachgebietBildgebende Verfahren“ in Nachfolge von Prof. Hans Gelderblom angeboten.Dem Direktorium und dem Kollegium des HPI ist es in Verhandlungen gelungen,diesen Ruf erfolgreich abzuwehren und Herrn Hohenberg mit seinerArbeitsgruppe am HPI zu halten.

Dr. Thomas Hofmann aus der Abteilung für Allgemeine Virologie verließ imHerbst 2004 das Heinrich-Pette-Institut. Er leitet seit November 2004 amDeutschen Krebsforschungszentrum (DKFZ) in Heidelberg eine Nachwuchsfor-schungsgruppe zur Molekularen Altersforschung (Molecular Research on Aging).Wir gratulieren Thomas Hofmann zu seiner neuen Position.

Herr Dr. Schultz, Verwaltungsleiter in der Zeitvom 1.9.2000 bis 31.8.2004, verließ das HPI nach4-jähriger Tätigkeit und wechselte an die Hoch-schulrektorenkonferenz (HRK) nach Bonn.Schwerpunkte seiner Arbeit am HPI, neben derLeitung der Verwaltung, waren die Einführungder Kosten-Leistungs-Rechnung und die Pla-nung des Ersatz- und Erweiterungsbaus II. Wirwünschen ihm in seinem neuen Wirkungskreisviel Erfolg.

Sein Nachfolger, Herr Dr. Volker Uhl, nahm am3.1.2005 seine Tätigkeit im HPI auf.

Herr Jörg Schinkel wurde Anfang 2004 neuer Per-sonalleiter in der Verwaltung. Er hat sich zumZiel gesetzt, trotz angespannter Personalsituation ein junges, engagiertesPersonalteam im HPI zu etablieren, das den stetig steigenden Anforderungen anSchnelligkeit und Flexibilität der Personalabteilung gerecht wird.

Dr. Volker Uhl,neuer Leiter derVerwaltung

17Mitglieder des Wissenschaftlichen Beirates des HPI

Institut für med. Mikrobiologie und Hygiene Universität RegensburgFranz-Josef-Strauß-Allee 11 93053 Regensburg

Universität WürzburgInstitut für Virologie und ImmunologieVersbacher Str. 797078 Würzburg

Biozentrum, M.E. Müller-InstitutUniversität BaselKlingelbergstr. 70 CH-4056 Basel

ATV F0200, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 242 69120 Heidelberg

Institut für Medizinische Virologie, FB 20Universitätsklinikum GiessenFrankfurter Str. 107 35392 Giessen

University of ManchesterDept. of Biomoleculare SciencesJacksons MillSackville Street, P.O. Box 88Manchester M60 1QD, U. K.

National Institute of Allergy and Infectious DiseasesBuilding 4, Room 229, 4 Center DriveBethesda, MSC 0445, USA


Prof. Dr. rer. nat. Hans Wolf

(Vorsitzender)

Prof. Dr. med. Axel Rethwilm

(stellv. Vorsitzender)

Prof. Dr. phil. Ueli Aebi

Prof. Dr. J. Valerie Bosch

Prof. Dr. Wolfram Gerlich

Dr. Dean A. Jackson

Dr. JonathanWilson Yewdell

18


Die erste Begutachtung des HPI durch den neuen Wissenschaftlichen BeiratDie wissenschaftlichen Leistungen des HPI wurden im Rahmen einer zweitägigenVeranstaltung in den Räumen des HPI im Mai 2004 durch den jetzigen Beiratbegutachtet. Dabei stellten die Abteilungs- und Gruppenleiter in Kurzbeiträgenihre momentanen Arbeitsgebiete und die für die Zukunft geplanten Projekte vor.Zudem besuchten die Beiratsmitglieder die einzelnen Abteilungen undArbeitsgruppen und informierten sich vor Ort über einzelne wissenschaftlicheProjekte. Der Beiratsvorsitzende fasste die Ergebnisse dieser intensivenEvaluierung in einem Bericht zusammen. Der Beiratsbericht bestätigt, dass die imHPI stattfindende Grundlagenforschung im Bereich der Virus- und Tumor-forschung extrem wichtig sei und in ihrem jetzigen Themenspektrum und ihrerinternational anerkannten Qualität fortgeschrieben werden sollte. Dies insbeson-dere angesichts der Situation, dass ein Institut wie das HPI mit seinen wissenschaftlichen Schwerpunkten einzig in der deutschen Forschungslandschaftsei und auch im internationalen Vergleich ein exzellentes Themenspektrum bearbeite.

Retreats der Wissenschaftlichen Mitarbeiter des HPIIm März 2003 trafen sich alle Wissenschaftler des HPI auf einem zweitägigenRetreat in Bad Segeberg, um sich über ihre aktuellen Forschungsprojekte undderen Ergebnisse auszutauschen. Das wissenschaftliche Programm des Retreatshat – in Verbindung mit der ungezwungenen Atmosphäre – die institutsinterneKommunikation und Vernetzung vor allem zwischen den jungen Mitarbeiterndes HPI weiter verbessert. Aus diesem Grund wurde der Retreat am 6. Mai 2004in der alten Kaffeebörse in der Hamburger Speicherstadt wiederholt und war wie-der ein voller Erfolg. Es ist geplant, das nächste Treffen dieser Art im Herbst die-ses Jahres ebenfalls an diesem traditionsreichen Ort stattfinden zu lassen.

19WissenschaftlicheHighlights„Rezeptoren als Bindeglied zwischen Arteriosklerose und Hochdruck“

Bluthochdruck geht mit Arteriosklerose einher,einer krankhaften Veränderung der Gefäßwände.Neue Forschungsergebnisse aus dem HPI liefernjetzt eine Erklärung für diesen Zusammenhang.Heinz Lother, Said Abdalla, Andreas Langer undYasser el Faramawy vom HPI haben zusammenmit Ursula Quitterer von der UniversitätWürzburg ein Verbindungsglied zwischen denbeiden Krankheiten entschlüsselt. Es handelt sichdabei, wie sie im hoch renommierten Wissen-schaftsjournal „Cell“ berichten (Bd. 119, S. 343,

2004), um den Angiotensin-II-Rezeptor. Er steigert über die Bindung desAngiotensins II den Blutdruck und leistet offensichtlich über eine Vernetzung vonRezeptorpaaren auf Monozyten der Arteriosklerose Vorschub. Diese Befundewurden zudem in einem Preview zu genanntem Artikel kommentiert.

„Selbstorganisation organischerMoleküle in Wasser“

Bisher war umstritten, ob die Grundformen desLebens im Süß- oder Salzwasser entstanden sind.Neue Daten aus der Grundlagenforschung derUniversitäten Hamburg und Kiel konnten zeigen,dass die Selbstorganisation von organischenMolekülen und die Entstehung von immer kom-plexeren partikulären (micellären) Organisa-tionsformen in Süßwasser stattfindet, und zwarauch ohne die Mitwirkung von Mikroorga-nismen. Mit speziellen elektronenmikroskopi-schen Methoden konnten Heinrich Hohenberg(HPI), Martin Kerner, Siegmund Ertl, AlejandroSpitzy (Universität Hamburg) und Markus

Reckermann (Universität Kiel) erstmals bildlich darstellen, wie sich diese kom-plexen Nanostrukturen Schritt für Schritt aus molekularen Grundbausteinenselbst aufbauen. Veröffentlicht wurden die Forschungsergebnisse in derZeitschrift „Nature“ (Band 422, S.150, 2003). Auf der Basis der erarbeitetenMethoden lassen sich erstmals autopoetische Prozesse auf zellulärer Ebenelebensnah abbilden (Bild links).


Die Veröffentlichungin „Cell“ wurde im Hin-blick auf ihre klinischeRelevanz auch im re-nomierten „New Eng-land Journal of Medi-cine“ diskutiert

20


PreisverleihungenAm 24. Januar 2004 erhielt Dr. Julia Röglin denJoseph-Kimmig-Förderpreis 2003 der Alfred-Marchioni-Stiftung für ihre hervorragendenForschungsarbeiten zur Kryokonservierung undzum gezielten Lagern einzelner menschlicherSamenzellen, die für eine künstliche Befruch-tung benötigt werden. Julia Röglin entwickeltedie Methoden im Rahmen ihrer Doktorarbeit,die sie bei Prof. Wolfgang Schultze (Universitäts-klinikum Hamburg-Eppendorf) und in derArbeitsgruppe für Elektronenmikroskopie undMikro-Technologie des HPI unter der Leitungvon Dr. Heinrich Hohenberg anfertigte.

In beiden Jahren, 2003 wie auch 2004, wurdenWissenschaftler des Heinrich-Pette-Instituts mitdem Georg-Ernst-Konjetzny-Preis der Hamburger Krebsgesellschaft e.V. ausge-zeichnet. Im Jahre 2003 überzeugten Dr. Carol Stocking, Dr. Maike Täger undDr. Jürgen Löhler aus der Arbeitsgruppe Molekulare Pathologie die Verleiher desPreises mit ihrer Arbeit zur Entstehung akuter myeloischer Leukämien (AML).Den Preisträgern gelang es, mit Hilfe eines Mausmodells die Entstehung der aku-ten myeloischen Leukämie bei Menschen zu beschreiben. Neben der Bildung desHybrideiweißes AML1-ETO, das in 15 % der bösartigen Blasten nachzuweisen ist,charakterisierten die HPI-Wissenschaftler einen Schaden im Gen für das EiweißICSBP (Interferon Consensus Sequence Binding Protein). Beide Schäden wirken zusammen und führen gemeinsam zur Entstehung bösartiger Leukämiezellen.

Mit dem Georg-Ernst-Konjetzny-Preis 2004 wurden Dr. Kristijana Milovic undDr. Thomas Hofmann für ihre Beschreibung eines neuen Signalweges beim pro-grammierten Zelltod ausgezeich-net. Die Wissenschaftler ent-deckten im Rahmen ihrer Arbeitin der Abteilung für AllgemeineVirologie die Wechselwirkungzwischen dem Sp100-Protein, ei-nem Eiweiß der PML-Kerndo-mänen, und dem FLASH-Protein,einer bereits bekannten Signal-komponente im programmiertenZelltod. Die ausgezeichnete Arbeitkönnte wichtige Erkenntnisse fürdie Diagnose und Therapie vonErkrankungen liefern, die miteiner gestörten Apoptose einher-gehen.

Dr. Julia Röglin

Von links nach rechts: Dr. Maike Täger, Dr. Jürgen Löhler, Dr. CarolStocking

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Im November 2004 würdigte dieErich und Gertrud Roggenbuck-Stiftung Prof. Wolfgang Deppert,Leiter der Abteilung für Tumor-virologie, für sein herausragendesGesamtwerk zur Wirkung desTumorsuppressors p53 bei derKontrolle der Krebsentstehung. Ererhielt anlässlich des 25-jährigenJubiläums der Erich und GertrudRoggenbuck-Stiftung den För-derpreis 2004. Sowohl die Schutz-funktion des p53-Tumorsuppres-sors, als auch die durch WolfgangDeppert beschriebene krebsför-dernde Wirkung von mutiertemp53 bieten viel versprechendeAnsätze für die Krebstherapie und -diagnostik.


Von links nach rechts: Dr. Thomas Hofmann, Dr. Kristijana Milovic, HPI, und Dr. Matthias Zeiss, Abteilung fürHämatologie des AK St. Georg

Professor Dr. Wolfgang Deppert (links) wird die Preisurkunde vondem Vorsitzenden der Roggenbuck-Stiftung, Prof. Heinz-PeterLeichtweiss, überreicht

22In memoriam

Das Heinrich-Pette-Institut verliert in Prof. Dr.Dr. Dr. h.c. Peter Hans Hofschneider einen lang-jährigen Freund, der die Entwicklung desInstituts stets mit Wohlwollen und großem Inter-esse verfolgt und aktiv unterstützt hat. ProfessorHofschneider war einer der international führen-den Virologen und Begründer der Moleku-larbiologie in Deutschland. Von 1979 bis zu seinem Tode am 23. Juli 2004 war ProfessorHofschneider Mitglied des Kuratoriums desHeinrich-Pette-Instituts. In dieser Funktion setz-te er sich stets und bis zuletzt mit größtem Enga-gement für die Wissenschaft am Heinrich-Pette-Institut ein.

Wir bleiben Peter Hans Hofschneider in großerAnerkennung und Dankbarkeit verbunden.

Der Vorstand, das Kollegium und die Mitarbeiter

des Heinrich-Pette-Instituts für Experimentelle Virologie und Immunologie

an der Universität Hamburg

Prof. Dr. Dr. Dr. h.c. Peter Hans Hofschneider

23

IntroductionViruses are obligatory parasites which can only propagate with the help of hostcells. This implies a plethora of interactions between viral and cellular compo-nents as well as the exploitation of many cellular functions and machineries bythe virus. The aim of this research program is to decipher in detail these interac-tions in terms of the types of cellular and viral components involved, when andwhere do they exactly take place, and how important are these interactions forvirus propagation. Last but not least, we hope to be able to exploit this know-ledge for the development of novel therapeutic strategies which can prevent virusinfections, eliminate viruses from chronically infected persons or animals, andcan be used for medical treatment of virus-associated diseases. In order to facili-tate the understanding of the research projects outlined below, the followingchapter gives a brief overview of the various steps in the life cycle of viruses.

The early steps of viral infection include specific and unspecific interactions withthe surface of the outer membrane of the host cells. After binding to specific cellsurface receptors viruses can enter the cells by different mechanisms. Many viru-ses contain a lipid membrane into which virus-encoded proteins are embedded.One or several of these envelope virus proteins often bind to specific receptors onthe host cell membrane. During the entry process the viral and cellular membra-nes can fuse and consequently lead to the release of the nucleocapsids into thecytoplasmatic compartment of the host cell. Alternatively, viruses can enter byendocytotic processes and end up in lipid-containing vesicles from which they arereleased by different mechanisms into the cytoplasm. Once inside the cytoplasm,naked viral capsids or virus particles without an envelope are transported to spe-cific intracellular cytoplasmic or nuclear compartments where the viral genomesencounter ambient or unfriendly host conditions and cellular factors for amplifi-cation of their genomes. The genomes of plus strand RNA viruses can be useddirectly as template for synthesis of the viral proteins, while for DNA and minusstrand RNA viruses the mRNA must be produced by transcription or the corres-ponding genomes. Both, amplification of the genomes as well as mRNA synthe-sis, stability and its intracellular transport strongly depend on a large variety ofhost cell functions and components. Synthesis of the viral proteins from viralmRNA, their posttranslational modification, appropriate folding and eventualdegradation depend almost or completely on the host cell protein synthesis,modification, folding and degradation machineries. De novo assembly of viralparticles, export of progeny particles towards the host cell membrane and theirfinal release into either the extracellular space or to neighbouring cells againdepend on host cell components and machineries.

Cellular components and functions which are crucial for propagation of virusesor infection-associated diseases are potential targets for the development of novelantiviral drugs and for new therpeutic strategies. Major technical advances in avariety of research fields in the past and the recent wealth of information derivedfrom human genome research, provide excellent opportunities to identify morecellular components crucial for virus propagation and associated disease than

Programmbereich„Virus-Wirts-Wechselwirkungen“Program Area „Virus-Host Interaction“Head: Prof. Hans Will

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Virus-Host Interaction

known so far. Functional analysis of these proteins and their role in host-cell inter-actions can lead to a detailed understanding of the life cycle of viruses and asso-ciated mechanisms of pathogenesis. Based on the fact that the basic concepts forinfection of a cell by viruses of different families are similar, it is well conceivablethat cellular proteins essential for a broad range of different viruses can be iden-tified in the future. Such cellular proteins represent potential targets for the deve-lopment of broad range virostatic drugs, which may eventually even block propa-gation of newly emerging virus infections of unknown identity.

The type and strength of naturally existing or inducible host defense mechanismsas well as the potency of antiviral drugs available for a given virus or a group ofviruses will codetermine whether or not a virus will be able to establish an acuteor chronic infection in a permissive host. The emergence of drug- and vaccineresistant viruses upon application of currently used antiviral drugs/vaccines,which almost all target viral proteins, is an increasing global problem.Furthermore, the recent emergence of novel, highly pathogenic viruses (and pre-dictably more to come in the future), for which neither drugs nor vaccines areavailable, represents another global challenge for virologists and the public healthsystems. The development of novel antiviral strategies for the most frequentlyoccuring devastating human virus infections, as well as for newly emergencingviruses is therefore of utmost importance.

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Research FieldsType of Viruses Studied

During the past 2 years, members of almost all major human pathogenic virusesor related animal viruses were the subject of our studies on virus-host interac-tions. These studies included human immunodeficiency virus type 1 (HIV-1),human T-cell leukemia virus type I (HTLV-I) and related murine leukemia viruses (MuLV), Hepatitis B and C Viruses (HBV and HCV) and related animalviruses in ducks, herons and cranes (DHBV, HHBV, CHBV), human papillomaviruses (HPV) as well as SV40 which is also a member of the papovavirus family,human herpes viruses (HSV-1 and -2), adenovirus, as well as influenza A virus.As a result of space limitations only a brief and selective overview on some of theprojects successfully performed or initiated during the past 2 years are givenbelow. More detailed information on the projects can be obtained from our web-site (www.hpi-hamburg.de) and by reading of the papers published and cited inthis bi-annual report.

Cellular Components Critical for Virus Propagation and Targets for Therapy

One of our major focuses of this research was directed towards elucidation of thelife cycle of HBV, mainly through identification of critical cellular and viral com-ponents involved in infection and associated diseases. HBV infection represents amajor global health problem with an estimated 350 million people infectedworldwide, about five percent of the world’s population. HBV is responsible forup to 80% of the global incidence of liver cancer and is the ninth leading cause ofdeath. Studies on HBV are hampered by the absence of permissive cell lines.Moreover, infection of cell lines in vitro may not reflect all aspects of an in vivoinfection. To circumvent this in the future, major efforts have been undertaken toestablish a novel system for growth of microliver organs permissive for infectionand to analyse infections under life-like conditions. This includes the applicationof novel micro-preparation methods, kryoembedding of 3D tissue, and the use oflight-, confocal- as well as of electron microscopy techniques. Note, a great deal ofknowledge on HBV stems from animal hepatitis B viruses and some of these wereused for the studies reported below. A novel hepadnavirus was discovered.

The second focus of our research was on HIV-1, the causative agent of AIDS, andon related human and murine retroviruses causing leukemias. According to aWHO report (UNAIDS Epidemic Update 2004), there will be at the end of 2004about 40 million people infected with HIV. Experts consider this a pandemic withnegative socio-economic effects of unprecedented degree. The number of infec-tions is still steadily increasing globally, including Germany. Antiviral therapywith the currently used drugs is frequently associated with severe side effects anddoes not lead to virus elimination. Research presented below has led to the iden-tification of a cellular protein critical for the propagation of HIV-1 as well as to


26


the identification of an experimental drug which interferes with the correspon-ding virus-protein interaction. This approach appears promising for the develop-ment of more effective advanced antiretroviral therapies, particularly for theinhibition of multidrug-resistant viruses. Studies on murine retrovirus infec-tions, which can be associated with a wide variety of diseases, including leukemia,immunodeficiency, and neurological disorders, similar to some human retro-viruses, have provided interesting new insights into host cell determining factors,mechanisms of retroviral pathogenesis, evolution and selection.

Type and Function of Cellular Components with Antiviral Activity

An important line of research is the characterization of natural existing mecha-nisms involved in inactivating or eliminating viruses after entry, without harmingor destroying the host cell. For instance, the cellular protein APOBEC3G was pre-viously shown to possess antiretroviral activity which is blocked by the virus-encoded Vif protein. Studies reported below indicate significant differences inHIV-1/AIDS patients with respect to APOBEC3G and/or the interacting Vif pro-tein activities. Another example concerns the cell-encoded multifunctional RNAbinding protein La which is known to interact with a large variety of RNAs of dif-ferent viruses. Cytokine-induced processing of La was previously reported to beassociated with the complete elimination of HBV propagation mediated by post-transcriptional degradation of the viral RNA. Detailed functional studies on La,which are presented below support its importance in HBV RNA metabolism, sug-gest its potential as an antiviral target, and provide novel insights into its cellularfunctions. The nuclear multiprotein complex of PML-bodies as well as similarcomplexes containing MxA-protein, are additional examples of cellular compo-nents with antiviral activities which are capable of killing viruses in infected cellsin a non-cytotoxic manner. Studies on the modulation of the composition ofthese and related nuclear complexes with antiviral activity by infuenza A andHPV are reported below.

Modulation of Cellular Gene Expression by Viruses

Studies with the oncogenic SV40 and with murine leukemia viruses are interes-ting examples for virus-host interactions in the nucleus which lead to majorchanges in the host cells such as global remodelling of chromatin, changes in hostcell differentiation and cancer (see below). The fate of viral genomes after inva-sion into the nuclear compartments and the ensuing molecular and dynamicchanges of the nuclear architecture are so far incompletely understood. Majorprogress in this field will come from the identification of the cellular componentsinvolved, including their changes in subnuclear localisation, which is in part trig-gered by posttranslational modifications. This is evident from the research de-scribed below using cell culture model systems.

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Research ProjectsItinerary of hepatitis B viruses: Delineation of restriction points critical for infectious entry

A. Funk, M. Mhamdi, L. Lin, H. Will, H. SirmaHeinrich-Pette-Institut

Little is known about cellular determinants essential for human hepatitis B virusinfection. Using the duck hepatitis B virus as a model, we first established a sen-sitive binding assay for both virions and subviral particles and subsequently elu-cidated the characteristics of the early viral entry steps. The infection itinerarywas found to initiate with the attachment of viral particles to a low number ofbinding sites on hepatocytes (about 104 per cell). Virus internalization was fully

accomplished in less than 3 h but wasthen followed by a period of unprece-dented length, about 14 h, until com-pletion of nuclear import of the viralgenome. Steps subsequent to virusentry depended on both intact micro-tubules and their dynamic turnover butnot on actin cytoskeleton. Notably,cytoplasmic trafficking of viral particlesand emergence of nuclear covalentlyclosed circular DNA requires micro-tubules during entry only at and forspecific time periods. Taken together,these data disclose for the first time aseries of steps and their kinetics that areessential for the entry of hepatitis Bviruses into hepatocytes and are diffe-rent from those of any other virusreported so far.

Supported by Deutsche Forschungsge-meinschaft (DFG), BMBF and DAAD


Infectious entry and trafficking of hepatitis B viruses in hepatocytes. The hepadnaviral life cycle starts withthe attachment of virions to specific binding sites on the surface of hepatocytes mediated by the preS regionof the large envelope protein. Following internalization and fusion of viral and cellular membranes at theplasma membrane or endosomes, cores are released into the cytosol. The incoming viral genomes are de-livered by a dynein-mediated retrograde transport along microtubules to the nucleus. Subsequently, the viralgenome, the rc-DNA, is converted into cccDNA to complete the infectious entry. All transcripts function asmRNAs and the longest of them, the pregenomic RNA, encodes for core and polymerase protein. The sametranscript is subsequently encapsidated together with polymerase into capsid particles and reverse tran-scribed into partially double stranded DNA. These mature capsids are re-transported into the nucleus usingmicrotubules or they are secreted after their interaction with envelope proteins in a post-ER compartment.

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Spread of hepatitis B viruses in vitro requires extracellular progenyand may be codetermined by polarized egress

A. Funk, H. Hohenberg, M. Mhamdi, H. Will, H. SirmaHeinrich-Pette-Institut

Viruses can spread by different mechanisms: via intracellular particles throughcell junctions to neighboring cells or via secreted virions to adjacent or remotecells. The observation of clusters of hepadnavirus-infected cells both in vivo andin primary hepatocytes neither proves the first mechanism nor excludes thesecond. In order to test which mechanism, if not both, is used by hepatitis B viru-ses in order to spread, we used primary duck hepatocytes and duck hepatitis Bvirus (DHBV) as an infection model. If extracellular progeny virus alone deter-mines spreading, neutralizing antisera or drugs blocking virus binding to hepa-tocytes should abolish secondary infection. In order to test this, we used DHBVenvelope-specific neutralizing antisera, as well as suramin, a known inhibitor ofinfection. Both reagents strongly reduced hepatocellular attachment of viral par-ticles and almost completely abolished primary infection, whereas an ongoing

Viral intercellular spread can occur through three different mechanisms: A) By progeny virus secreted into theextracellular space which is able to infect also remote cells. B) Infection of neighbouring cells only throughtight junctions. C) Polarized secretion of virus into limited extracellular space leading to infection predomi-nantly of adjacent cells.

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intracellular infection was not affected as long as no progeny virus was released.In contrast, incubation of infected primary hepatocytes with these reagentsduring release of progeny virus completely prevented secondary infection.Moreover, the combination of electron and immunofluorescence microscopyanalyses revealed the residence of viral particles in cytoplasmic vesicles preferen-tially located near the basolateral membrane of infected hepatocytes. Taken to-gether, these data strongly suggest that hepatitis B viruses mainly spread by secreted,extracellular progeny and point to polarized egress of viral particles into intercel-lular compartments, which restricts their diffusion and favors transmission ofvirus to adjacent cells.

Supported by Deutsche Forschungsgemeinschaft (DFG) and BMBF

Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

T. Kalinina (1,2), A. Iwanski (1), H. Will (1,2), M. Sterneck (1,2)(1) Heinrich-Pette-Institut (2) Universitätsklinikum Hamburg-Eppendorf

Hepatitis B virus with a G145R mutation in the small surface protein is considered thequintessential immune escape mutant because it frequently is found in vaccinated


Model of DHBV spread. DHBV speads between neighbouring primary duck hepatocytes through synapticrelease of virions into the extracellular space at the basolateral membrane. This restricts free diffusion ofvirions, but does not protect them from neutralizing antibodies or drugs.

30


individuals with breakthrough infections and liver transplant recipients under anti-hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis. Nowadaysthe prevalence of the variant progressively increases. However, because spread of avirus depends not only on immune pressure but also on the viral phenotype, weinvestigated the biologic properties of the G145R variant. The G145R mutation wasintroduced into wild-type (Wt) virus genome by in vitro mutagenesis. After trans-fection into human hepatoma cells, the DNA, RNA, and protein synthesis and viralsecretion ability of the mutant were studied. Furthermore, cotransfection studieswere performed with the G145R variant and a Wt virus S-protein expressing con-struct and vice versa. Production and stability of viral messenger RNAs (mRNAs),DNA, and proteins were not affected by the G145R mutation. In contrast, secretionof mutant virions was reduced significantly. Only 20% of virions were found in themedium of G145R variant transfected cells compared with Wt virus. Furthermore,mutant virions were more sensitive to detergent treatment suggesting a diminishedstability. In cotransfection studies, Wt virus S-protein rescued secretion of mutantvirions, whereas mutant S-protein had a transdominant negative effect on secretionof Wt virus. Both mechanisms may support persistence of the defective mutant ina mixed population with Wt virus. In conclusion, the significant defect of theG145R mutant for secretion of infectious virions and the diminished stability ofmutant virions may limit global spread of the mutant.

Supported by the DFG and BMBF

Selection of a secretion-incompetent mutant in the serum of a patient with severe hepatitis B

T. Kalinina (1,2), A. Riu (1), L. Fischer (1,2), T. Santantonio (3), H. Will (1), M. Sterneck (1,2)(1) Heinrich-Pette-Institut(2) Universitätsklinikum Hamburg-Eppendorf (3) University Bari, Italy

A secretion-incompetent, highly replicating hepatitis B variant was previouslyfound as the dominant viral population in the serum of a liver transplant reci-pient with severe hepatitis B reinfection. The secretion block resulted from muta-tions in the S protein, including the Gly145Arg substitution known to emergeunder antibody to hepatitis B surface antigen immunoglobulin treatment. Weinvestigated the mechanisms that allow selection of a secretion-incompetentvirus as the predominant strain in the serum. To reproduce the interaction ofviral quasispecies occurring in vivo, cotransfection experiments were performedwith full-length genomes containing wild-type or mutant sequences. In addition,the relevance of mutations in the common S part of the surface proteins for thecompetence of L and S protein to support viral secretion was studied. A smallamount of wild-type virus or of a wild-type S protein-expressing variant rescued

31

secretion of the defective mutant. In the secreted virions, the high-replicatingmutant genome was predominant. Selection of the defective mutant was furthersupported by a transdominant negative effect of mutant S protein on wild-typevirion secretion. In contrast, mutant L protein with the same c-terminal muta-tions as mutant S protein efficiently supported virion formation and secretion.Interaction of the variant with a small amount of wild-type virus can reverse itssecretion-defective phenotype. Mutations in the common region of S and L pro-tein have different consequences for the ability of the envelope proteins to sup-port virion assembly and secretion.

Supported by the DFG and BMBF

New hepatitis B virus of cranes that has an unexpected broad host range

A. Prassolov (1), H. Hohenberg (1), T. Kalinina (1), C. Schneider (1), L. Cova (2), O. Krone (3), K. Frolich (3), H. Will (1), H. Sirma (1)(1) Heinrich-Pette-Institut(2) INSERM U271, Lyon, France(3) Institute of Zoo and Wildlife Research, Berlin

All hepadnaviruses known so far have a very limited host range, restricted to theirnatural hosts and a few closely related species. This is thought to be due mainlyto sequence divergence in the large envelope protein and species-specific differen-ces in host components essential for virus propagation. We show infection ofcranes with a novel hepadnavirus, designated CHBV, that has an unexpectedlybroad host range and is only distantly evolutionarily related to avihepadnaviru-ses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well asequencing of cloned viral genomes revealed that CHBV is most closely related to,although distinct from, Ross’ goose hepatitis B virus (RGHBV) and slightly lessclosely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are verydistant from geese and ducks and are most closely related to herons and storks.Naturally occurring hepadnaviruses in the last two species are highly divergent insequence from RGHBV and DHBV and do not infect ducks or do so only margi-nally. In contrast, CHBV from crane sera and recombinant CHBV produced fromLMH cells infected primary duck hepatocytes almost as efficiently as DHBV did.This is the first report of a rather broad host range of an avihepadnavirus. Our dataimply either usage of similar or identical entry pathways and receptors by DHBVand CHBV, unusual host and virus adaptation mechanisms, or divergent evolutionof the host genomes and cellular components required for virus propagation.


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Characterization of nonconventional hepatitis B viruses lacking the core promoter

S.F. Chang (1,2), S.H. Chang (2), B.C Li (1), H. Will (1), H. J. Netter (1,3)(1) Heinrich-Pette-Institut (2) Industrial Technology Research Institute, Hsinchu, Taiwan(3) Department of Microbiology, Monash University, Melbourne, Australia

The core gene (C-gene) promoter and regulatory sequences play a central role inthe hepatitis B virus (HBV) life cycle. They are essential for the synthesis of thepregenomic and precore mRNA. The pregenomic RNA is the template requiredfor replication and also the template for the synthesis of the core protein andpolymerase. Here, we report the in vivo existence and functional characterizationof HBV variants that lack the C-gene promoter region and the regulatory sequen-ces located therein. HBV promoter fragments were isolated by PCR from sera ofchronic carriers and characterized. Truncated promoter elements were identified,and then tested in the context of wild-type genomes in the HuH-7 cell line. Theexpression of the recombinant HBV genome resulted in the synthesis of surfaceproteins, and low level of core protein as well as a transcript pattern similar to,but smaller in size to wild-type virus. The recombinant HBV genome with thetruncated promoter region produced pregenomic RNA-like transcripts. Thesetranscripts were encapsidated and reverse transcribed when complemented bysufficient core and polymerase protein. These date provide an explanation as to

Phylogenetic relation-ship of all known avianhepadnaviruses basedon preS/S gene sequen-ces. The correspondingnatural hosts are alsoindicated. STHBV standsfor stork HBV, HHBV forheron HBV, DHBV forduck HBV, SGHBV forsnow goose HBV, RGHBVfor Ross’ goose HBV, andCHBV for crane HBV.

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why such deletion mutants of HBV can be produced at all, they highlight thefunctional potentials of viral sequences activated by mutations and may be ofrelevance for viral evolution and persistence.

Supported by BMBF

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wildtype virus

P. Meier (2), C.A. Scougall (2), H. Will (1), C.J. Burrell (2), A.R. Jilbert (2) (1) Heinrich-Pette-Institut(2) University of Adelaide, Adelaide, Australia.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis Bvirus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx andDHBx are transcriptional activators and modulate cellular signaling in in vitroassays. To test whether the DHBx protein plays a role in virus infection, we com-pared the in vivo infectivity and growth characteristics of a DHBV3 strain with astop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3strain. We demonstrate that the two strains showed no significant difference in(i). their ability to induce infection that resulted in stable viraemia measured byserum surface antigen (DHBsAg) and DHBV DNA, and detection of viral pro-teins and replicative DNA intermediates in the liver; (ii). the rate of spread ofinfection in liver and extrahepatic sites after low-dose virus inoculation; and (iii).the ability to produce transient or persistent infection under balanced age/doseconditions designed to detect small differences between the strains. Thus, none ofthe infection parameters assayed were detectably affected by the X-ORF knockoutmutation, raising the question whether DHBx expression plays a physiologicalrole during in vivo infection with wild-type DHBV.

Supported by BMBF/NGNF

Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies

O.G. Engelhardt (2), H. Sirma (1), PP. Pandolfi (4), O. Haller (4)(1) Heinrich-Pette-Institut (2) University of Oxford, UK(3) Sloan-Kettering Institute, USA(4) Universität Freiburg

The interferon-induced murine Mx1 GTPase is a nuclear protein. It specificallyinhibits influenza A viruses at the step of primary transcription, a process knownto occur in the nucleus of infected cells. However, the exact mechanism of inhi-


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bition is still poorly understood. The Mx1 GTPase has previously been shown toaccumulate in distinct nuclear dots that are spatially associated with promyelocy-tic leukaemia protein (PML) nuclear bodies (NBs), but the significance of thisassociation is not known. We could show that, in cells lacking PML and, as a con-sequence, PML NBs, Mx1 still formed nuclear dots. These dots were indistinguish-able from the dots observed in wild-type cells, indicating that intact PML NBs are not required for Mx1 dot for-mation. Furthermore, Mx1 retain-ed its antiviral activity againstinfluenza A virus in these PML-deficient cells, which were fullypermissive for influenza A virus.Nuclear Mx proteins from otherspecies showed a similar sub-nuclear distribution. This was alsothe case for the human MxAGTPase when this otherwise cyto-plasmic protein was translocatedinto the nucleus by virtue of aforeign nuclear localization signal.Human MxA and mouse Mx1 donot interact or form heterooligo-mers. Yet, they co-localized to alarge degree when co-expressed inthe nucleus. Taken together, thesefindings suggest that Mx1 dotsrepresent distinct nucleardomains (‘Mx nuclear domains’)that are frequently associatedwith, but functionally indepen-dent of, PML NBs.

Supported by BMBF/NGNF

Topological relationship between Mx1 dots and other nucleardomains. (a)–(d) Vero cells were transfected with plasmid pEGFP-Mx1, fixed 1 day later and immunostained with a polyclonal rab-bit antiserum against PML (blue) and a mouse monoclonal anti-body against SC35 (red) (a, b) or a mouse monoclonal antibodyagainst p80 coilin (red) (c, d). Red, green and blue channel imageswere recorded separately and overlaid electronically. Panels label-led with letters and numbers (e.g. a1) are enlargements of theareas indicated in the corresponding images (a)–(d). White linessignify the shortest distance between the centroid positions ofstained domains and numbers next to the lines indicate the dis-tance in mm. (e)–(f). Means±SD are plotted. Asterisks indicatestatistically significant differences.

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Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70

L. Florin (2), K.A. Becker (2), C. Sapp (2), C. Lambert (2), H. Sirma (1), M. Muller (2), R.E. Streeck (2), M. Sapp (2) (1) Heinrich-Pette-Institut(2) Johannes Gutenberg-Universität Mainz

Minor capsid protein L2 of papillomaviruses plays an essential role in virus assemblyby recruiting viral components to PML bodies, the proposed sites of virus morpho-genesis. We demonstrate that the function of L2 in virus assembly requires the cha-perone Hsc70. Hsc70 was found dispersed in naturally infected keratinocytes and cul-tured cells. A dramatic relocation of Hsc70 from the cytoplasm to PML bodies wasinduced in these cells by L2 expression. Hsc70-L2 complex formation was confirmedby coimmunoprecipitation. The complex was modulated by the cochaperones Hipand Bag-1, which stabilize and destabilize Hsc70-substrate complexes, respectively.Cytoplasmic depletion of Hsc70 caused retention of wild-type and N-terminallytruncated L2, but not of C-terminally truncated L2, in the cytoplasm. This retentionwas partially reversed by overexpression of Hsc70 fused to green fluorescent proteinbut not by ATPase-negative Hsc70. Hsc70 associated with L1-L2 virus-like particles(VLPs) but not with VLPs composed either of L1 alone or of L1 and C-terminallytruncated L2. Moreover, displacement of Hsc70 from L1-L2 VLPs by encapsidation ofDNA, generating pseudovirions, was found. These data indicate that Hsc70 transient-ly associates with viral capsids during the integration of L2, possibly via the L2 C ter-minus. Completion of virus assembly results in displacement of Hsc70 from virions.

Supported by Deutsche Forschungsgemeinschaft (DFG) and Deutsche Krebshilfe

Functional characterization of the interaction between human La and hepatitis B virus RNA

I. Ehlers I (1), S. Horke (1), K. Reumann, A. Rang (1), F. Grosse (2), H. Will (1), T. Heise(1)(1) Heinrich-Pette-Institut (2) Institut für Molekulare Biotechnologie, Jena

The La protein is a multifunctional RNA-binding protein and has also been sug-gested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Herewe demonstrate that antibodies against the human La protein specifically preci-pitate HBV RNA from HBV ribonucleoprotein-containing mammalian cellextracts, providing evidence for the association between human La and HBVRNA. Moreover, we report that the turnover of HBV RNA depends on structuralfeatures and less on the primary sequence of the La-binding site on the viral RNA.In addition we show that the interaction between human La and HBV RNA invitro is modulated by accessory factor(s) in a phosphorylation-dependent man-ner. Taken together these data indicate that both structural features, the compo-sition of La/HBV ribonucleoprotein particles as well as interacting cellular fac-tors, are critical determinants in the regulation of the stability of the HBV RNA.



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The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization

S. Horke (1), K. Reumann (1), C. Schulze (2), F. Grosse (3), T. Heise (1)(1) Heinrich-Pette-Institut(2) Zentrum für Neurobiology, Hamburg(3) Institute for Molecular Biotechnology, Jena

The human La autoantigen (hLa) protein is a predominantly nuclear phospho-protein that contains three potential RNA binding domains referred to as the Lamotif and the RNA recognition motifs RRMs 1 and 2. With this report, we differ-entiated the contribution of its three RNA binding domains to RNA binding bycombining in vitro and in vivo assays. Also, surface plasmon resonance techno-logy was used to generate a model for the sequential contribution of the RNAbinding domains to RNA binding. The results indicated that the La motif maycontribute to specificity rather than affinity, whereas RRM1 is indispensable forassociation with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucialfor the interaction with various RNAs in vivo, although needed for full-affinitybinding in vitro. Moreover, earlier studies suggest that RNA binding by hLa maydirect its subcellular localization. As shown previously for RRM1, deletion ofRNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed afterdeletion of the La motif. Here we discuss a model for precursor RNA bindingbased on a sequential association process mediated by RRM1 and the La motif.

Nuclear trafficking of La protein depends on a newly identified nucleolar localization signal and the ability to bind RNA

S. Horke (1), K. Reumann (1), M. Schweizer (2), H. Will (1), T. Heise (1)(1) Heinrich-Pette-Institut(2) Zentrum für Neurobiologie, Universität Hamburg

Here we provide evidence for an interaction-dependent subnuclear trafficking ofthe human La (hLa) protein, known as transient interaction partner of a varietyof RNAs. Among these, precursor transcripts of certain RNAs are located in thenucleoplasm or nucleolus. Here we examined which functional domains of hLaare involved in its nuclear trafficking. By using green fluorescent-hLa fusion pro-teins, we discovered a nucleolar localization signal and demonstrated its functio-nality in a heterologous context. In addition, we revealed that the RRM2 motif ofhLa is essential both for its RNA binding competence in vitro and in vivo and itsexit from the nucleolus. Our data imply that hLa traffics between different sub-nuclear compartments, which depends decisively on a functional nucleolar locali-zation signal as well as on RNA binding. Directed trafficking of hLa is fully con-sistent with its function in the maturation of precursor RNAs located in differentsubnuclear compartments.

Supported by Deutsche Forschungsgemeinschaft (DFG) and BMBF (NGFN)

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Retroviral mRNA transport

D. Schwarck (1), I. Hauber (1), K. Wiegers (1), C. Ehlers (1), K. Harbers (1), U. Aebi (2), J. Hauber (1)(1) Heinrich-Pette-Institut(2) Universität Basel - Biozentrum

Replication of human retroviruses, such as the human immunodeficiency virustype 1 (HIV-1) depends on the transport of subgenomic mRNAs and unsplicedgenomic RNA from the nucleus to the cytoplasm. This nuclear RNA export ispromoted by an essential viral regulatory protein, termed Rev. Rev is a shuttleprotein that constantly moves between the nuclear and cytoplasmic compart-ment of infected cells. In the nucleus, Rev binds directly to the viral Rev response


HIV-1 Rev-mediated viral RNA Transport. Transcriptional activation of the HIV-1 long terminalrepeat (LTR) promoter results in the synthesis of primary full-length RNA transcripts. TheseRNAs are either completely spliced or retained in the nuclear compartment. Completely-splicedtranscripts are constitutively transported from the nucleus to the cytoplasm for subsequentsynthesis of the viral regulatory proteins Tat, Rev and Nef. Rev is a nucleocytoplasmic shuttle-protein that travels back into the nucleus and directly binds to incompletely-spliced or unsplicedviral RNA via the Rev response element (RRE) sequence. Upon interaction with various criticalcellular cofactors, including eukaryotic initiation factor 5A (eIF-5A) and the general exportreceptor CRM1, Rev-containing ribonucleoprotein (RNP) complexes are translocated across thenuclear envelope. In the cytoplasm, the incompletely-spliced and unspliced viral transcriptsserve as templates for the synthesis of the viral structural proteins and enzymes Gag, Pol andEnv. In addition, the full-length unspliced RNA is packaged as genome into newly formed viralparticles. In addition to mediating the nuclear export of viral transcripts, activities of the Revtrans-activator have also been described on the level of stability, splicing and translation ofHIV-1 mRNAs.

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element (RRE) RNA element, which is present on all unspliced and singly-splicedHIV-1 transcripts. Subsequently, Rev interacts with multiple cellular factors thatmediate the translocation of the Rev-bound viral RNA across the nuclear envelope.A functionally equivalent regulator, termed Rex, is encoded by human T-cell leukemia virus type I (HTLV-I). Both trans-activators, HIV-1 Rev and HTLV-IRex, have been shown to exploit the same cellular pathway for nuclear export ofthe respective retroviral RNAs.

The translocation of Rev or Rex-containing ribonucleoprotein (RNP) complexesacross the nuclear envelope is mediated by the general export receptor CRM1,which interacts in presence of RanGTP with a specific domain in Rev or Rex thathas been termed the nuclear export signal (NES). However, efficient Rev/Rex-mediated nucleocytoplasmic transport of incompletely-spliced viral mRNAs also requires factors in addition to CRM1 and Ran. These include, for example,eukaryotic initiation factor 5A (eIF-5A), the Src-associated protein in mitosisSam68 and DEAD Box RNA Helicase DDX3. The cellular Rev/Rex cofactor eIF-5Ais the only known cellular protein which contains the amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)-lysi-ne], a modification that is requiredfor eIF-5A activity. The hypusineformation is a highly-specific,spermidine-dependent post-trans-lational reaction that is catalyzedby two enzymes. In the first step,the aminobutyl moiety of spermi-dine is transferred to the ε-NH2

group of a single lysine residue atposition 50 within the human 154amino acid eIF-5A protein. Thisreaction is catalyzed by deoxyhy-pusine synthase (DHS). The resul-ting eIF-5A intermediate is thenfurther modified by deoxyhypusi-ne hydroxylase, generating theactive form of eIF-5A. Therefore,inhibition of the essential Revcofactor eIF-5A by interferencewith hypusine-formation shouldprovide a novel strategy to blockthe HIV-1 life cycle.

By using RNA interference (RNAi)technology we knocked-down inhuman cells gene expression of theeIF-5A modifying enzyme DHS.The subsequent infection of thesecells with HIV-1 demonstratedthat the viral replication capacity

Schematic representation of the pathway of eIF-5A biosynthesis.Eukaryotic initiation factor 5A (eIF-5A) is the only known proteinin mammalian cells that contains the amino acid hypusine, a modi-fication that is required for eIF-5A activity. The hypusine formationis a highly specific, spermidine-dependent posttranslational modi-fication that is catalyzed by two enzymes. In the first step, the ami-nobutyl moiety of spermidine is transferred to the ■■ -NH2 group ofa single lysine residue at position 50 within the human 154-aminoacid eIF-5A protein. This reaction is catalyzed by deoxyhypusine-synthase (DHS). The resulting eIF-5A intermediate is then furthermodified by deoxyhypusine-hydroxylase (DHH), generating theactive form of eIF-5A, which is a critical cellular cofactor of the HIV-1 Rev trans-activator. The guanylhydrazone CNI-1493 is an experi-mental drug that blocks DHS and thereby, indirectly, Rev activityand virus replication.

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was indeed drastically reduced upon DHS silencing. Therefore, inhibition of DHSappears to be a promising approach to provide novel antiretroviral therapies.

It is important to note that in addition to mediating nuclear export of HIV-1mRNA, also Rev activities on the splicing, half-life and translation of these trans-cripts has been reported in the literature. In that respect an important and oftenoverlooked feature of Rev is the ability of this viral regulatory protein to formhomo-multimers. Various studies have demonstrated that Rev binds as a mono-mer to its high-affinity RRE binding site and subsequently multimerizes on itsRNA target. Although it has been unambiguously established that multimer for-mation is indeed required for Rev trans-activation, it is to date unclear which Revactivity depends on Rev multimer-formation. To address this question we gene-rated biologically inactive multimerization-deficient Rev mutant proteins. Byfusing these mutants to heterologous oligomerization structures we were able toreconstitute Rev biological activity. By exploiting this system we will now be in aposition to dissect the various aspects of Rev-mediated posttranscriptional pro-cessing of retroviral mRNA in detail, thereby integrating the various Rev activi-ties into a comprehensive model of trans-activation.

Supported by Deutsche Forschungsgemeinschaft (DFG)


Inhibition of HIV-1 by siRNA targeted against DHS-specific sequences. HeLa cells, which express the CD4 surfacemolecule, were transfected with either human DHS-specific (DHS) or control scramble II duplex (SDII) siRNAs. Ofnote, the SDII siRNAs lack complementary sequences in the human genome. As shown in panel A by RT-PCR analy-sis, the DHS-specific siRNA clearly downregulated the corresponding transcripts in the transfected cells while, incontrast, control SDII siRNAs did not negatively affect the abundance of DHS messages. Importantly, the HIV-1 repli-cation capacity was significantly reduced in the cultures transfected with DHS-specific siRNA compared with the cul-tures transfected with control siRNA (B, left panel). Analysis of cytotoxicity as measured by MTT-assay revealedcomparable levels of viable cells in these cultures (B, right panel). These data demonstrated that DHS is a valuablecellular target for interference with the HIV-1 life cycle.

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Novel therapy strategies for the treatment of HIV-1 infection

I. Hauber (1), B. Schäfer (1), A. Bunk (1), S. Frankenberger (1), H.Hohenberg (1), D. Bevec (2), T. Harrer (3), F. Krätzer (4), A. Choidas (4)(1) Heinrich-Pette-Institut(2) mondoBIOTECH SA, Gentilino (Schweiz)(3) Universität Erlangen – Medizinische Klinik III(4) Axxima Pharmaceuticals AG, München

The efficient inhibition of the HIV-1 life cycle by highly active antiretroviral the-rapy (HAART) has been shown to profoundly improve the morbidity and mor-tality among HIV-1 infected patients. Current routine drug regimens typicallyconsist of various combinations of compounds that target the viral proteinsreverse transcriptase, protease and gp41. Unfortunately, in a growing number ofpatients long-term HAART is accompanied by significant adverse side-effectsincluding mitochondrial toxicity, lipodystrophy, diabetes mellitus and osteoporo-sis. In addition, HIV-1 can acquire resistance to all known inhibitors of reversetranscriptase, protease and gp41 and transmission of multidrug-resistant HIVstrains is becoming a growing problem among newly infected persons. Therefore,in order to develop advanced therapies for the treatment of HIV-1 infections weinvestigated various novel strategies to block the retroviral replication cycle.

Triggered by our finding that the hypusine-containing protein eIF-5A is an essen-tial cellular cofactor of the HIV-1 Rev trans-activator (see above) we identified themultivalent guanylhydrazone CNI-1493, an experimental drug that is currently

Inhibition of HIV-1 Rev trans-activation by CNI-1493. The effect of the DHS inhibitor CNI-1493 on Rev trans-activation was analyzed. In panel A cytoplasmic (C) and total (T) RNAs from HIV-1 infected cells were analyzedby Northern analysis. As shown, three classes of HIV-1 specific RNAs, corresponding to unspliced, singly-spliced, and multiply-spliced transcripts were detectable in total RNA isolated from solvent (DMSO)-treatedcells (lane 3). However, analysis of cytoplasmic RNA samples revealed reduced levels of the Rev-regulatedunspliced and singly-spliced HIV-1 mRNA species in DHS inhibitor (CNI-1493)-treated cells (compare lane 1 and2). The detection of the exclusively nuclear U6 small nuclear RNA (snRNA) served as control for the purity ofthe cytoplasmic samples in these experiments. As an additional test of whether DHS inhibition by CNI-1493represses Rev activity, specific trans-activation analyses were performed by using established Rev- and Tat-dependent reporter plasmids in transient transfection experiments. As shown, CNI-1493 clearly inhibited Revtrans-activation (panel B), while no negative effect on Tat activity was observed (panel C), thereby indicatingspecificity of this inhibitor for the Rev pathway.

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in phase II clinical trials for Crohn’s disease, to be a small molecular weight inhi-bitor of human Deoxyhypusine Synthase (DHS) in vitro. We were able to demon-strate that inhibition of DHS by CNI-1493 efficiently suppressed Rev activity andblocked the HIV-1 replication cycle in cell culture and primary cells. We showedthat CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratorystrains, clinical isolates and viral strains with high level resistance to inhibitors ofviral protease and reverse transcriptase. Moreover, no measurable drug-inducedadverse effects on cell cycle transition, apoptosis and general cytotoxicity wereobserved. Therefore, human DHS represents a novel and promising drug-targetfor the development of advanced antiretroviral therapies, particularly for theinhibition of multidrug-resistant viruses.

Supported by Axxima Pharmaceuticals AG

Restriction of HIV-1 replication in peripheral blood mononuclear cells

K. Wiegers, D. Schwarck, W. BohnHeinrich-Pette-Institut

Unstimulated peripheral blood mononuclear cells (PBMCs) are restricted for theestablishment of a productive infection of HIV-1 and simian immunodeficiencyvirus (SIV). However as a result of differentiation after stimulation, PBMCs be-come susceptible for infection, suggesting that gene expression during differentia-tion is important for the establishment of the permissive phenotype. In addition,the role of the glucocorticoid receptor (GR) on the regulation of HIV-1 geneexpression is still controversial and probably cell type dependent.

It had been previously shown that the HIV-1 accessory protein Vpr is importantfor virus replication in non-dividing cells and in unstimulated PBMCs. Moreover,it was shown that Vpr can interact with GR and is able to transactivate both, cel-lular and viral promoters. Therefore we analyzed whether the known restrictionof HIV-1 in unstimulated PBMCs correlates with a distinct intracellular localiza-tion of GR and whether activation of GR affects the localization of Vpr and virusreplication.

When we analyzed the subcellular localization of GR in unstimulated PBMCs, wefound that GR is exclusively localized in the cytoplasm. However, after additionof dexamethasone the GR is translocated into the nucleus, where it can fulfill itsrole as an activator of gene expression by interaction with the cis-active gluco-corticoid responsive element (GRE) and transcription factors. As shown in ourexperiments, nucleocytoplasmic translocation of GR also resulted in nuclear accu-mulation of coexpressed HIV-1 Vpr.

In order to study the influence of the intracellular localization of GR on HIVreplication in more detail we made use of two clones of a rat glioma cell linewhich differed in their phenotypes regarding in their constitutive intracellular


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localization of GR. In the C6D8 clone, GR is exclusively sequestered in the cyto-plasm. After addition of hormone, GR is transported to the nucleus as observedin PBMCs. In the clone C6D10, GR is constitutively localized in the nucleus andaddition of dexamethasone had no effect on its intracellular distribution.

We next tested whether the different phenotypes of these cell clones could alsoaffect the ability to support HIV replication. After infection with pseudotypedHIV-1, synthesis of viral proteinswas severely impaired in the C6D8cell clone (characterized by cyto-plasmic GR), whereas in the cellclone C6D10 (characterized bynuclear GR), increased amounts ofviral capsid protein was detected.Interestingly, Northern analysisdemonstrated that in the permissi-ve cell clone C6D10 the amount ofvirus specific RNA is much moreabundant as compared to the non-permissive clone C6D8.

The combined data suggest that aconcerted action of GR and Vpr isneeded to attract the appropriateset of transcription factors andcofactors to the HIV LTR promo-ter in order to allow unstimulatedPBMCs to become permissive forHIV replication. Moreover, ourcell system could be useful to ana-lyze the cellular factors involved inthe sequestration of GR and Vpr.

Supported by Deutsche Forschungs-gemeinschaft (DFG)

Subcellular localization of the glucocorticoid receptor and Vpr.Images show the localization of the glucocorticoid receptor (upperpanels) and a Vpr-GFP fusion protein (lower panels) without (-Dex)or after addition of dexamethasone (+Dex) in human PBMCs.

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APOBEC3G, a host cell defense system for HIV-1

I. Hauber (1), A. Bunk (1), C. Steininger (2), H.-J. Stellbrink (2), J. Hauber (1)(1) Heinrich-Pette-Institut(2) Universitätsklinikum Hamburg-Eppendorf – Medizinische Klinik I

The genome of HIV-1 contains a sequence that encodes the accessory protein Vif.Vif is required for effective viral spread in primary CD4+ T cells, which are natu-ral targets of HIV-1 infection. Recently, it has been shown that Vif blocks theaction of the antiviral host cell factor apolipoprotein B mRNA-editing enzyme,catalytic polypeptide-like 3G (APOBEC3G). In the absence of Vif, APOBEC3G isincorporated into HIV-1 particles and acts in de novo infected cells to disruptprovirus formation by deaminating cytidine to uridine residues on the retroviralDNA minus-strand (synthesized by reverse transcription of the viral RNA ge-nome). This reaction eventually introduces G to A hypermutations in the DNAplus-strand, resulting in lethal mutations (by introducing translation terminationsignals), cDNA degradation or in defective reverse transcription. The HIV-1 Vifprotein counteracts APOBEC3G action by inducing its ubiquitination and sub-sequent degradation via the proteasome pathway. Thus, APOBEC3G and poten-tially other APOBEC family members are part of a novel antiviral host defensemechanism that mediates innate resistance to retroviral infection. It remains to

be determined how APOBEC3Gand Vif affect the course of diseasein HIV-1 infected patients.

In order to analyze the geneticdeterminants that underly theAPOBEC3G-Vif interaction inHIV-1 infected individuals weestablished various assay systemsto analyze APOBEC3G and Vifactivity with respect to APO-BEC3G degradation and HIV-1infectivity. Moreover, we isolatedvif and APOBEC3G sequencesfrom a cohort of HIV-1 infectedindividuals with establishedimmunological and virologicalparameters. In particular, subjectsfrom the cohort were selectedaccording to their ability to control virus replication (e.g.Controllers vs. Progressors).These analyses revealed that signi-ficant differences exist in HIV-1/AIDS patients with respect toAPOBEC3G and/or Vif activities.


Effects of APOBEC3G on HIV-1 replication. In nonpermissive cellsinfected with HIV-1▲▲vif virus, cellular APOBEC3G (A3G) is incorpo-rated into viral progeny. Upon infection of new cells these virionsundergo the early steps of reverse transcription. Subsequently,A3G deaminates cytosines in nascent minus-strand DNA to uracilat specific mutagenic hot-spots. The uracil-containing viral DNA ishighly labile. Copying the mutated base into plus-strand DNA andrepair results in permanent mutations and creates hypermutatedviruses. In cells infected with wildtype (wt) virus, the viral acces-sory protein Vif binds to A3G, thereby preventing its encapsida-tion. Furthermore, Vif triggers A3G’s degradation by the cellularproteasome.

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Subcellular localization of herpes simplex-virus immediate early proteins

N. Albrecht (1), B. Keyser (1), K. Milovic (1), I. Hauber (1), M.M. Eibl (2), J. Hauber (1)(1) Heinrich-Pette-Institut(2) Biomedizinischen Forschungsgesellschaft mbH, Wien (Österreich)

Herpes Simplex-Virus (HSV) gene expression is regulated in a distinct, highlycoordinated and sequential fashion. The first genes to be expressed are the imme-diate-early (IE) genes, which encode largely multifunctional regulatory proteins,termed infected cell proteins (ICPs). These govern the subsequent expression ofthe delayed-early (DE) genes. Both, IE- and DE-gene products are then requiredfor expression of the late (L) genes that mainly encode structural proteins.

Here we investigated the intracellular localization and trafficking of various ICPs.We were able to show, that distinct regions in these proteins mediate active nuclearimport of these multifunctional viral regulators. In particular, we compared thesubcellular localization of ICP0 from HSV Type 1 and Type 2. For this, we transi-ently expressed ICP0-GFP gene constructs in mammalian cells and, in addition,performed microinjection experiments into somatic cells using recombinant HSV-2 GST-ICP0 fusion protein. These data demonstrated that both ICP0 can be exclu-sively detected at steady state within distinct speckles in the cell nucleus. Moreover,we functionally identified a nuclear localization signal (NLS) of the SV40 T-anti-gen type that maps to a short stretch of amino acids (aa) at position 510-521 in the825 aa HSV-2 ICP0. Furthermore, the mutational analysis of this NLS revealed thatthe amino acids at position 513-516 are functionally essential for nuclear import ofthis HSV-2 regulatory factor. These data will provide the basis for the constructionof nuclear import-deficient mutants and their detailed functional analysis.

Supported by Biomedizinische Forschungsgesellschaft mbH

Subcellular localization of HSV-2 ICP0. The subcellular localization of recombinant expressed wildtype HSV-2ICP0 (A) and a nuclear localization signal (NLS)-deficient ICP0 mutant (B) after microinjection into VERO cellsis shown. Following nuclear injection of wildtype ICP0 (right cell in panel A), the protein remains in the nucle-ar compartment. Following cytoplasmic injection (left cells in panel A), the wildtype protein can also be detec-ted in the nucleus, indicating active nuclear import. In contrast, the NLS-deficient ICP0 mutant always remainsin the compartment, in which the protein was injected (panel B). Insets show the IgG injection controls.

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Intracellular transport of adenovirus regulatory proteins

J. Hauber (1), T. Dobner (2)(1) Heinrich-Pette-Institut(2) Universität Regensburg – Medizinische Mikrobiologie und Hygiene

Regulation of Adenovirus (Ad) gene expression involves the intracellular trans-port of Ad immediate early proteins. In collaboration with Dr. Thomas Dobner(University Regensburg) we analyzed the nuclear export of the 55-kDa earlyregion 1 protein (E1B-55kDa) of Ad Type 5 (Ad5). E1B-55kDa belongs to a groupof adenovirus regulatory proteins that are individually capable of cooperatingwith Ad early region 1A protein (E1A) to oncogenically transform primaryrodent cells in culture.

Ad5 E1B-55kDa contains a leucine-rich nuclear export signal (NES), which con-fers rapid nucleocytoplasmic transport via the CRM1 pathway. Here we demon-strated that nuclear export-deficient E1B-55kDa augments oncogenic transfor-mation of primary rat cells. Moreover, a nuclear export-deficient E1B-55kDamutant protein accumulated together with p53 in subnuclear dot-like structuresthat contained cellular promyelocytic leukemia protein (PML). Taken together,these data indicated that NES-deficient E1B-55kDa promotes oncogenic trans-formation by combinatorial mechanisms that involve modulation of p53 in thecontext of PML nuclear bodies.

SV40 large T-antigen induces global chromatin re-organization

G. Tolstonog, F. Krepulat, A. Hermannstädter, W. DeppertHeinrich-Pette-Institut

Chromosomal instability is a hallmark of neoplastic transformation, and is in-duced by dysregulation of cellular proteins controlling or modulating chromatinstructures, or by viral oncogenes mimicking their activity. To understand theimpact of the SV40 large T-antigen, a multifunctional viral protein that belongsto a class of hexameric DNA/RNA helicases, on global chromatin organization,we established a cellular model based on mammary epithelial cell cultures from amurine ductal mammary carcinoma that had developed in bi-transgenic WAP-mutp53 H22 x WAP-T mice. A chromatin remodeling activity of SV40 large T-antigen so far has been documented only indirectly, through modulation ofCBP histone acetyltransferase and elevating DNA methyltransferase activity.Therefore, we analyzed the intranuclear organization of centromeric and telome-ric heterochromatin domains by in situ hybridization in cells expressing or notexpressing T-antigen after induction of the cells with lactotrophic hormones.Using a mouse pan-centromeric (γ-satellite) probe, we found that the centro-meres in the cells without or with very low T-antigen expression are physically incontact with each other, forming so-called chromocenters, with numbers varyingfrom 10 to 20. In sharp contrast, in the nuclei of all cells expressing high levels of


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T-antigen the numbers of centromere clusters increased by 2-3 times, but theirsize was reduced, indicating partial dissociation of the chromocenters resultingfrom a global change in chromatin structure. The notion of a global chromatinremodelling function of T-antigen was further substantiated by investigating thespatial arrangement of telomeres. In cells expressing T-antigen both proximal anddistal telomere ends of acrocentric mouse chromosomes seem to be in close con-tact with the centromere clusters. However, T-antigen negative nuclei exhibitednumerous telomeres distant to centromere clusters. On the basis of these obser-vations, we conclude that T-antigen contributes to de-clustering of centromericheterochromatin as well as physical association of proximal with distal telomeres.Further studies are required to better characterize this activity and to understandits physiological role in SV40 infection and cellular transformation.

SV40 T-antigen induced global chromatin reorganization in hormone treated murine mammary carcinoma cellsderived from WAPT1 x WAP-mutp53R270H-H22 bi-transgenic mice as revealed by immunofluorescence doublelabeling (panels A, B1, B2) and in situ hybridization with centromeric and telomeric DNA probes (panels C, D,E). A: nuclear localization of SV40 T-antigen (red) and cell membrane association of EpCam (green), revealing theepithelial origin of the tumor cells. B1, B2: SV40 T-antigen and mutp53 (HAtag) double labeling of carcinoma cells.All tumor cells exhibit nuclear T-antigen immunoreactivity (red, B1), only a portion of these cells show also posi-tive mutp53 (HAtag) staining (green, B2) C: in situ hybridization of a centromere DNA probe (red) and immuno-staining of SV40 T-antigen (green). T-antigen positive nuclei (green) reveal increased numbers of centromeres,which appear smaller and more condensed. The double labeling was merged with a differential interferencecontrast (DIC) micrograph to show the outlines of tumor cells. D, E: double in situ hybridization of centromericand telomeric DNA in nuclei of SV40 T-antigen non-expressing (D) and expressing (E) carcinoma cells. In T-anti-gen positive nuclei (E, T-antigen staining not shown) proximal as well as distal telomeres (blue) appear to be inclose contact with the centromeres, whereas in T-antigen negative nuclei (D) many of the telomeres have a posi-tion distant from the centromeres. Positive and negative T-antigen immunolabeling is not depicted. Panels A,B1, B2 represent equatorial confocal sections and panels C, D, E the 3D projections. Scale bars A-E: 10 µm.

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Cellular receptors for retroviral entry

S. Hein (1),V. Prassolov (2), Y. Zhang (3), D. Ivanov (2), J Löhler (1), S. Ross (3), C. Stocking (1)(1) Heinrich-Pette-Institut(2) Engelhardt-Institute of Molecular Biology, Moscow (Russia)(3) University of Pennsylvania, Philadelphia, PA (USA)

Retrovirus infection is initiated by binding of the surface (SU) portion of the viralenvelope glycoprotein (Env) to specific receptors on cells. This binding triggersconformational changes in the transmembrane (TM) portion of Env, followed bysecondary events that lead to fusion of the viral and cellular membranes, either atthe cell surface or after trafficking through acidic endosomal compartments. Thisinitial interaction is a major determinant of the tissue and species tropism ofretroviral infections. Mutations in the viral env gene that result in altered recep-tor specificity is an important factor in the evolution of endogenous viruses,cross-species infection, and viral pathogenesis. Important examples of this havebeen revealed in studies of the human immunodeficiency virus, but invaluableinsight has also been gained in the analysis of receptor-Env interactions of otherretroviruses.

The M813 murine leukemia virus (MuLV) is a highly fusogenic gamma retrovirusisolated from Mus cervicolor, whose host range is limited to mouse cells. To delin-eate the molecular mechanisms of its restricted host range and its high fusogenicpotential, we initiated studies to characterize the cell surface protein that media-tes M813 infection. Screening of a mouse/hamster radiation hybrid panel forM813 infectivity localized the receptor gene to the distal end of mouse chro-mosome 16. Expression of one of the likely candidate genes (Slc5a3) within thisregion in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. Slc5a3 encodes the sodium myo-inositol transporter 1(SMIT1), thus adding another sodium-dependent transporter to the growing listof proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologuesin different species identified several amino acid variations within two extracel-lular loops (ECL) that may be essential for susceptibility to M813 infection.Recombinant Env proteins have demonstrated the importance of amino acids inECL5 and ECL6 for its functions as a viral receptor.

A striking characteristic of M813 is its high fusogenicity both in vitro and in vivo.Mice infected with M813 develop a peripheral T-cell lymphoma, which is associ-ated with large multinucleated cells. Identifying mSMIT as the receptor for M813will enable further studies to determine the critical Env-receptor interactions thatregulate fusion.



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Complex interplay of retroviral and cellular determinants in disease specificity

M. Rodenburg, M. Fischer, A. Engelmann, C. Stocking, J. LöhlerHeinrich-Pette-Institut

Retroviruses are associated with a wide variety of diseases, including leukemia,immunodeficiency, and neurological disorders. Study of these diseases has un-covered a wide variety of pathogenic mechanisms used by the virus, includingtranscriptional activation or deregulation of cellular genes by either the long-terminal repeat (LTR) or via viral accessory proteins (e.g. Tax), induction of syn-cytium by Env-receptor interactions, or activation of signaling pathways via pro-tein-protein interactions. Signi-ficantly, however, the complexinterplay of host and cellular fac-tors that modulate the latency,severity, and specificity of thedisease is poorly understood. Weare particularly interested in defi-ning the parameters that influencethe lineage specificity (e.g. lym-phoid vs myeloid) of leukemiainduced by murine leukemia virus(MuLV) variants in differentmouse strains. Earlier work hasdemonstrated the importance ofsequences within the LTR in speci-fying disease specificity. However,as the LTRs between several vari-ants with distinct pathologies arepractically identical, we sought todelineate other important sequen-ces. In addition, we wanted todetermine the importance ofreceptor usage (dictated by Envsequences) in disease specificity.

The 10A1-Murine Leukemia Virus (MuLV) was isolated from tumor tissue afterserial infections of amphotropic 1504-MuLV. In NIH/3T3 or NIH/Ola mice,10A1-MuLV induces a „stem-cell“-like leukemia (Sca1+/Kit1+) with 100% corre-lation of the disease phenotype with integration upstream of the Fli1 oncogene,encoding the Ets-like transcription factor. In contrast, NIH mice develop eitherB-cell or T-cell lymphomas when infected with the closely related amphotropic4070A-MuLV or the ecotropic Moloney (Mo)-MuLV, respectively. Interestingly,4070A and 10A1 share the same LTR, but show striking differences in sequencesdirectly downstream of the 5`LTR and in the 5´ env sequences. Indeed, 10A1-MuLV contains a recombinant env gene harboring sequences from both ampho-tropic and endogenous polytropic env sequences, enabling it to bind to either the

Analysis of recombinant MuLVs that use different receptors showthe importance of receptor usage in the pathogenecity of theretrovirus. Newborn mice infected with retrovirus were monitoredfor disease symptoms over a six month period. FACS analysis wasused to determine the hematopoietic lineage transformed in leu-kemic mice. Mice infected with 10A1-4070env demonstrated a highfrequency of degenerative odontropathy. Histochemical analysisconfirmed a fibrotic alteration of dental pulp with extensive virusexpression. (A) Broken incisor with fibrotic pulp; (B) detail of A; (C)normal dental pulp observed in 10A1-infected mice.

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amphotropic Pit2 receptor or the related Pit1 molecule. To determine what viralsequences were responsible for the unique pathogenicity of 10A1, a series ofrecombinants were made. Results from these experiments demonstrate that a10A1 recombinant carrying the amphotropic env is unable to induce a stem-cellleukemia, suggesting that utilization of the Pit1 receptor is important for trans-formation of the stem-cell compartment. This is consistent with earlier studiesshowing that Pit1 but not Pit2 is expressed at high levels in early hematopoieticcells.

Interestingly, however, the ecotropic env gene, mediating infection via the Cat1receptor, can also mediate a stem-cell leukemia within the 10A1-backbone. Again,this is consistent with the observed high expression levels of this molecule withinthe hematopoietic compartment. The T-cell tropism can be regained by eitherreplacing the 10A1 LTR with that of MoMuLV or exchanging a small fragmentspanning the U5 LTR and immediate downstream sequences, termed NP. We havepreviously shown that NP sequences derived from 10A1 increase virus spread invivo. This work demonstrates that sequences that enable efficient infection of aparticular cell compartment (e.g. env) or sequences known to facilitate virus spread during early stages of development (e.g. LTR or NP) are important deter-minants of viral pathogenicity. Interestingly, the viral recombinant containing theamphotropic env gene within a 10A1-backbone (and thus now unable to use Pit1but exclusively Pit2 as a receptor) induces osteopetrosis in infected NIH mice.These results underline the complex interaction between host and viral factorsthat are in play during viral evolution and selection.


Localization and dynamics of small circular DNA in the nucleus of living cells

G. Mearini (1), P.E. Nielsen (2), F.O. Fackelmayer (1)(1) Heinrich-Pette-Institut(2) Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen (Denmark)

Small circular DNA molecules invade the nucleus of eukaryotic cells during viralinfection or the transfection of plasmids, such as vectors for gene therapy. Eventhough this is a common event, surprisingly little is know about the fate of thesemolecules in the nuclei of living cells. Import into the nucleus was recently shownto depend on transport through the nuclear pore complex piggybacked on pro-teins with nuclear localization signals. Once inside the nucleus, the incomingforeign DNA acquires a state that facilitates the expression of the enclosed genes.For many viruses, this allows the expression of „early” genes that stabilize theinfection and prepare the host cells for virus replication. Likewise, genes on trans-fected plasmids are expressed efficiently without requiring a prior integrationinto the genome. Thus, functional interactions of regulatory sequences on the


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incoming DNA with nuclear components in the host cells can be inferred. How-ever, the mechanism by which this occurs is unclear, as is the question whetherthese initial contacts can be modified or abolished e.g. for therapeutic use.

To address this point, we have created traceable model substrates by direct labe-ling of plasmid DNA with fluorescent PNA, and have investigated their fate aftermicroinjection into living cells. With this model system, we were able to show thatforeign DNA rapidly undergoes interactions with intranuclear structural sitesthat strongly reduce its mobility, and restrict the DNA to regions excluding nuc-leoli and nuclear bodies such as PML bodies. The labeled plasmids partially colo-calize with SAF-A, a well characterized marker protein for the nuclear „scaffold“or „matrix“, and are resistant towards extraction by detergent and, in part, ele-vated salt concentrations. We show that the localization and the low mobility ofplasmids is independent of the plasmid sequence, and does neither require thepresence of a scaffold attachment region (SAR) DNA element nor of a functionalpromoter.

Thus, attachment to nuclear substructures does not appear to result directly fromgene expression, where active, matrix-bound RNA polymerase II could tether theplasmid to the matrix or transcription „factories“. Importantly, we also failed todetect a requirement for SAR DNA elements, which are thought to represent thegenomic attachment sites to the nuclear matrix. It is therefore unlikely that SARbinding proteins such as SAF-A or SAF-B are predominantly responsible forimmobilizing plasmid DNA. This is particularly noteworthy because one of thevectors used in the present paper, pEPI-1, was recently demonstrated to be boundto SAF-A in vivo after many generations of episomal replication. In this case, theSAR DNA element on the vector, and its interaction with SAF-A, was essential forepisomal replication and for blocking its integration into the genome.

Future experiments will now address the nature of the proteins that affect thelocalization and mobility of plasmid DNA. Tagged PNA clamps will be a valuabletool for interaction screening of nuclear proteins, and will allow specific enrich-ment for the involved factors that can then be identified by mass spectrometry.Understanding the molecular mechanism that tethers plasmids to the nuclearmatrix, and thereby create a permissive environment for the expression of theenclosed genes, might allow the rational design of compounds to block this inter-action. Possibly, this will later enable inhibiting the expression of some viralgenes, and attenuate viral infections.

Supported by Deutsche Forschungsgemeinschaft (DFG) and a personal fellow-ship of the Istituto Pasteur, Fondazione Cenci Bolognetti, University of Rome „La Sapienza“ to G.M.

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The role of protein arginine methylation in vivo

F. Herrmann (1), A. Schwander (1), M. Bossert (1), E. Akgün (1), J. Lee (2), M.T. Bedford (2), F.O. Fackelmayer (1)(1) Heinrich-Pette-Institut(2) Department of Carcinogenesis, M. D. Anderson Cancer Center,University of Texas Smithville (USA)

Methylation of arginine residues in proteins is a posttranslational modificationimplicated in transcriptional regulation, signal transduction, viral genome repli-cation and subcellular trafficking of RNA and proteins, but the mechanism bywhich these functions are achieved has remained undetermined. In human cells,arginine methylation primarily occurs on nucleic-acid binding proteins, and iscatalyzed by a family of protein arginine methyltransferases (PRMT), the pre-dominant member of which is PRMT1. Despite its major role in arginine methy-lation of nuclear proteins, surprisingly little is know about the subcellular locali-zation, interaction partners and dynamics of PRMT1.

In a project to determine the role of PRMT1 and other members of the PRMT family, we were able to show for the first time that PRMT1 is associated with hetero-geneous nuclear ribonucleoprotein (hnRNP) complexes, where it methylates itspreferred substrates in situ. Important new insights into the enzymatic mechanismof PRMT1 came from our observation that binding to hnRNP complexes dependson the methylation status of the cell, and is strongly enhanced in presence of inhi-bitors of methylation. Binding of PRMT1 to hnRNP particles occurs through phy-sical interaction with Scaffold Attachment Factor A (SAF-A), also known ashnRNP-U, which is quantitatively methylated by PRMT1 in all investigated celllines as determined by a novel highly specific, methylation-sensitive antibody.

Even though most of its substrates are nuclear, only a fraction of PRMT1 is loca-ted in nucleus, but the protein is predominantly cytoplasmic. Fluorescence reco-very after photobleaching (FRAP) experiments revealed that PRMT1 is highlymobile both in the cytoplasm and the nucleus. However, inhibition of methyla-tion leads to a significant nuclear accumulation of PRMT1, concomitant with theappearance of an immobile fraction of the protein in the nucleus, but not thecytoplasm. Both the accumulation and immobility of PRMT1 is reversed whenre-methylation is allowed, suggesting a mechanism where PRMT1 is trapped byunmethylated substrate proteins in the nucleus in live human cells until it hasexecuted the methylation reaction.

Interestingly, the related enzyme PRMT6, which has recently been shown to beimportant for replication of the HIV, does neither change its subcellular localiza-tion nor its mobility in presence of unmethylated substrates. This supports theidea that PRMT6 does not form complexes with other polypeptides, in contrastto PRMT1 which has been reported to associate with a large variety of proteins.Maybe, the enzymatic mechanism of PRMT6 is different from that of PRMT1and does not include a step where the enzyme forms a stable intermediate withthe substrate. This hypothesis can now be tested in structure/function experi-ments by switching regions of PRMT1 and 6. It will be interesting for future expe-


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riments to also characterize the dynamics and distinct interactions of other mem-bers of the growing PRMT family. These investigations will shape our understan-ding of PRMT enzymatic function, will help to determine the physiological roleof the individual family members, and will hopefully elucidate novel pathwaysinvolving arginine methylation.

Supported by Deutsche Forschungsgemeinschaft (DFG), and in parts by NIHgrant DK62248 and institutional grant ES07784 to M.T.B.

3D liver organoid model system and analysis of virus infection

H. Hohenberg, H. Sirma, H. Will, A. FunkHeinrich-Pette-Institut

Our new in vivo-like liver organoid and 3D cell culture model offers the possibi-lity to cultivate, manipulate and analyse liver tissue and heterogeneous liver cellcultures. Tissues and cells are integrated into a transparent tubular filter matrix,preserving the life-like complexity of the cellular information network.

One of our goals – applying „Systemic Electron Microscopy“ – is to obtain andanalyse high resolution images of liver biopsies in situ, cultivated as organoidsunder diverse physiological and pathophysiological conditions by combiningnovel micro-biopsy, fast cryofixation and cryopreparation techniques. This syste-mic preparation chain is perfected with correlative light microscopy and highresolution immuno-electron microscopy. The applied 3D tubular filter matrix isbiocompatible and highly porous, with a molecular cut-off of about 10 kDa and

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was already successfully applied in cryo-electron microscopic studies on cultiva-ted plant tissues, virus-infected cells and electron tomographic reconstruction ofC.elegans structures. The tubes have a diameter of about 200 µm and a high ratioof surface area to volume and can be sealed at both ends. Both the hepatic andsurrounding non-parenchymal cells prepared from duck liver or intact duck liverbiopsies were transferred into these micro-containments and maintained for upto 4 weeks under „life-like“-conditions.

To study e.g. the mechanisms of intracellular assembly of hepadnaviruses we haveapplied this new 3D cell culture system, using also conventional EM-preparationmethods.

Cryo-virology on the ultrastructural level

H. Hohenberg (1), H. Sirma (1), H. Will (1), S. Fuller (2), A. Vogt (3)(1) Heinrich-Pette-Institut (2) University of Oxford (United Kingdom) (3) Bal-Tec AG (Liechtenstein)

Virus-infected liver organoid systems, cryopreparation techniques and electrontomography are excellent tools to analyse life-like cell and virus interaction andthe different steps of hepatitis virus assembly and maturation on the ultrastruc-tural level.


Adding the third dimension to the hepadnaviral life cycle: Duck hepatitis B virus infected primary hepatocytesgrown in 3D cultures were frozen at high pressure, cryo-processed and analysed in the TEM. Left: two capsidparticles in the lumen of rER, decorated with ribosomes (1) outside the nucleus (Nu) near a nuclear pore (3).Right: one of the numerous cytoplasmic vesicles (CV), formed upon virus infection, containing preformedmature viral particles (inset: higher magnification of a virion) and represent hepadnaviral „factories” in whichthe distinct steps of viral assembly and budding occur. The different steps of virus maturation in situ are inves-tigated by EM tomography, inclusive 3D reconstruction

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The best available approach so far to immobilize and preserve the life-like archi-tecture of infected cells in situ is a chain of cryo-preparation steps starting withfreezing, followed by freeze-substitution, low-temperature embedding and polymerisation. In the case of virus-infected liver organoid systems, only high-pressure freezing (HPF) permits adequate cryoimmobilisation up to a thicknessof 200 µm that corresponds to the approximately 40 cell layers of the organoid.Ultrathin sections of these infected cells, investigated by electron tomographyallow for the first time the 3D analysis and reconstruction of intact virus particlesin the life-like state. These different virus assembly and maturation states are neither altered by chemical treatment nor by isolation and centrifugation pro-cedures and are fully integrated into the structural and biochemical environmentof the intact infected cell.

This novel holistic morphological approach is very helpful to investigate some ofthe unresolved mechanisms of hepatitis B virus infection and very suitable fortherapeutic screening of antiviral drugs interfering with assembly, budding andegress of hepatitis B viruses.

Environmental preparation of infectious suspensions: ultrastructural analysis of infected cells, virus particles, and cellular/viral components

H. Hohenberg (1), H. Sirma (1), H. Will (1), I. Hauber (1), B. Schäfer (1), S. Behrens (2)(1) Heinrich-Pette-Institut (2) Fox Chase Cancer Centre, Philadephia (USA)

The basic principle of this environmental preparation method is the micro-encapsulation of suspended biomaterial inside of the transparent filter matrix ofour tubular CarboCell micro-containments. The encapsulation process is accom-plished by sealing both ends of the micro-tube. The molecular filtration proper-ties of the tube walls prevent the passage of molecular structures exceeding 5-20kDa during all preparative and analytical steps for EM analysis. Therefore onceencapsulated, all biological components can be processed „blind“ without loss ofany structural elements. Only 0.2 µl of suspended material is required and harshcentrifugation steps can be avoided.

This allows a controlled processing of virus-infected cells for TEM investigationsinclusive the cultivation of virus-infected cells inside of the CarboCell micro-containments, simulating in vivo-like virus production with no loss of any releasedvirus, which is advantageous in the case of low virus-production rates. The virusproduction can be quantified and the extracellular virus maturation can be inves-tigated under sterile conditions.

Moreover, cell and virus suspension, anti-viral substances etc. can be analysedseparately with the same preparation method, testing novel therapy strategies forthe treatment of HIV-1 infection.

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Using the same environmental preparation principle, pure viral particles fromsera can be investigated and quantified in the transmission electron microscopeeven at low virus concentrations. In the first step of this immuno-capture tech-nique, the viral particles are specifically and selectively captured from sera andadsorbed to micro-carriers. Thereafter the suspended carriers are concentrated in


Visualization of de novo infection of PM1 cells with HIV-1 in presen-ce of antiretroviral peptides.

Ultrastructure of the flavivirus BVDV,immun-adsorbed to a micro-carrier (MC).

Suspension of IgG-molecules + 5 nm pAg in a cellulose carbonate micro-tube (CCT): Overview (left), x 400.000(right).

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the lumen of the micro-tubes, circumventing multiple ultracentrifugation stepsand resulting in the preservation of viral particles in a „life-like state“. On thebasis of this technique we analysed the ultrastructure of two new members of theavian hepadnavirus family infecting White Storks (STHBV) and Cranes (CHBV)as well as two members of the flavivirus family namely human hepatitis C andbovine viral diarrhea virus by transmission electron microscopy.

Exact measurement and 3D reconstruction of biological surfaces

M. Ritter (1), M. Hemmleb (2), O. Sinram (2), J. Albertz (2), H. Hohenberg (1)(1) Heinrich-Pette-Institut(2) Photogrammetry and Cartography, TU Berlin

The exact 3D analysis and measurement of surface details or nano-particles likeviruses in the micro- and nano-range is still a problem and requires adequate sensors and micro-range measurement methods. A most suitable micro-rangesensor is the electron microscope. Furthermore modern techniques in scanningelectron microscopy like ESEM-technology offer the possibility of imaging evenhydrated microstructures while maintaining their original 3D topography. Theapplication of photogrammetric methods for the analysis of electron micro-scopic data has a long tradition and has become the method of choice for thequantitative 3D reconstruction of SEM or (ESEM) images. However, quantitativephotogrammetric reconstruction of electron microscopic data requires a set ofbasic components. We recently presented a micrometer-sized 3D calibrationstructure that allows the calibration of the SEM. Yet, also optical errors of alter-native micro-range measurement methods, e.g. ESEM or Confocal LaserScanning Microscopy (CLSM) can be detected. The 3D microstructure was fabri-cated using gas-assisted focused ion (FIB) beam technique. Based on this tech-nique, an optimally designed 3D micro-object was created. The subsequent highprecision spatial measuring with atomic force microscopy (AFM) revealed thecalibration object qualities of the fabricated structure. We are now able to build3D calibration structures of various size with a flexible design in order to fit spe-cific applications. Microscopes with different resolutions (CLSM, AFM, SEM)can now be calibrated based on one high-precision nano-object, allowing thecomparative analysis of the quantitative microscopic data of identical biologicalobjects.

Supported by Deutsche Forschungsgemeinschaft (DFG) and FEI, Eindhoven,Netherlands

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IntroductionThe cell is the smallest functional unit of a multi-cellular organism and buildingblock of highly organized functional units, the organs. The multiple functions ofa cell are strictly controlled by a variety of cellular regulatory mechanisms. Cellgrowth and cell division, cell differentiation and cell removal by programmed celldeath (apoptosis) must be regulated by a multitude of signaling pathways in co-operation with other cells within an organ and the whole organism. Additionalsignals and pathways will control its physiological performance.

It is obvious that the multitude and complexity of the regulatory mechanismsoperating within and in-between cells will make a cell vulnerable to endogenousand exogenous insults that could disturb its function. A number of endogenouscellular stress conditions, like e.g. hypoxia, can lead to cellular damage at the levelof individual organelles, and at level of the genome. Exogenous stress conditions,like the influence of DNA damaging agents, or, for example, infection with viru-ses will threaten the fragile functional balance of a cell. It therefore is of vitalimportance that cells have developed a variety of protective pathways that copewith endogenous or exogenous stress situations, leading either to repair of thedamage incurred, or to elimination of the damaged cell by apoptosis. However,despite the elaborate protective arsenals of a cell, there always will be conditionsthat lead to either transient or permanent non-physiological alterations in cellu-lar signaling characteristic for pathologic effects. Important examples are viralinfections and tumor development. In both conditions cellular safeguards havefailed, leading to the re-programming of cellular pathways for the needs of viralreplication or for successful tumor evolution. To understand these and otherpathologic conditions, it therefore is necessary to study in detail the basic mecha-nisms of cellular signaling, as well as the safeguard pathways insuring their pro-per functioning. Often the study of a dysregulated pathway will lead to the under-standing of the regular pathway, and vice versa.

Clearly, „cellular dysregulation“ is a very broad topic. However, it is becomingmore and more evident that many pathologic conditions lead to alterations insimilar major cellular pathways, or violate similar protective actions of the cell. Inanalyzing these pathways at the cellular level, the distinction between pathwaysdysregulated by e.g. viral infection or tumor evolution no longer seems reason-able. On the contrary, attacking the problems of cellular dysregulation from dif-ferent angles will clearly have synergistic effects. Thus the seemingly broad spec-trum of projects addressing this topic serves the major goal of understanding theregulatory mechanisms that govern the appropriate functioning of a cell, with thehope that understanding such mechanisms and how they might become dysregu-lated under various pathologic conditions well help us to find handles for eitherpreventing dysregulation or for curing the effects of cellular dysregulation at thelevel of the organism.

Programmbereich „Zelluläre Dysregulation“Program Area „Cellular Dysregulation“Head: Prof. Wolfgang Deppert

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Cellular Dysregulation

Research FieldsDissecting p53 pathwaysThe first section describes important results obtained in understanding the func-tion and regulation of the tumor suppressor p53. The p53 protein is the centralplayer in coordinating and executing cellular defense and protection mechanismsagainst endogenous and exogenous cellular stress situations. The major molecu-lar activity of p53 is that of a sequence-specific transactivator of genes that willinitiate repair, growth arrest, or apoptosis. The p53 response is very variable,depending on several factors, including the kind of stress and the severity of thedamage incurred, cell type and cellular environment. Clearly, damage repair is themost desired physiological response, but when the damage incurred is too severe, the damaged cell must be eliminated either by apoptosis, or by inducing a per-manent growth arrest, cellular senescence. The plethora of genes transactivatedby p53 (> 200 genes, so far) requires a tight regulation of p53 activity in order toensure that p53 activates the pathways appropriate for the given damage situationin a given cellular environment. To ensure such regulation, p53 is modified byvarious posttranslational events and interacts with a variety of proteins. Last notleast, it is becoming more and more evident that epigenetic events, like chroma-tin remodeling, strongly modulate p53 activity. Similarly, subnuclear compart-mentalization of p53, or of p53 modifying or interacting proteins, plays animportant role in the regulation of p53.

A novel and important finding in deciphering p53 pathways is the identificationand functional characterization of a novel p53 isoform, delta p53. Delta p53mRNA is generated by exon-exon splicing in primates and some other species,but not e.g. in mice and rat. Delta p53 plays an important role in the intra-S-phase checkpoint and promotes attenuation of the S-phase after S-phase dama-ge. Attenuation allows uncoupling of repair and DNA synthesis, i.e. repair ofdamaged DNA before DNA synthesis is resumed. The fact that delta p53 isexpressed in humans, but not in mice, explains several discrepancies regardingp53 action in the literature, when analyses were performed with cells form diffe-rent species. In addition and probably more important, the finding raises theintriguing possibility that the higher fidelity of DNA repair observed in humancells compared to mouse cells is at least in part due to the expression of delta p53.

Mutant p53 (mutp53) not simply is an inactivated tumor suppressor, but at leastseveral „hot spot“ mutations confer oncogenic properties to the ensuing mutp53proteins. Thus single point mutations in the p53 gene may serve two purposes,elimination of the tumor suppressor function of wild type p53, and activation ofmutp53 as an oncoprotein. Further analysis of the oncogenic functions ofmutp53 proteins at the molecular level will be an important task which holdssome promise for the development of novel, mutp53 based therapies.

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Switch controlsThe section on p53 is followed by a number of projects describing the role of pro-teins that regulate important cellular pathways.

HIPK2 is a kinase involved in transcriptional regulation and induction of apop-tosis. Mechanistically, HIPK2 participates in the TGF-b signaling pathway thatleads to the activation of another kinase (JNK) and to apoptosis.

Degradation of proteins by proteasomes is an important process in the regulationof cellular homeostasis that requires ubiquitination of the proteins by ubiquitin-ligases, like the UIP5 protein. Interestingly, analysis of UIP5 function demonstrat-ed that ubiquitination of proteins not only is a signal marking proteins for pro-teolytic degradation, but also a signal in so far non-identified non-proteolyticprocesses.

G protein-coupled receptors comprise the largest family of cell surface membra-ne receptors and mediate multiple physiological functions. Many G protein-coupled receptors form dimmers in cells. However, underlying mechanisms werebarely understood. AbdAlla and coworkers demonstrated in an article in the pre-stigious journal Cell that the intracellular factor XIIIA transglutaminase cross-links agonist-induced AT1 receptor dimers. The crosslinked dimers displayedenhanced signaling. Elevated crosslinked dimers are present on monocytes ofpatients with the common atherogenic risk factor hypertension. The finding thusputs a handle on the genetic base of hypertension and might open new ways forhypertension therapy, stirring a lot of interest not only in the scientific commu-nity. The Cell paper was found worthy of a comment in Cell, and the data werealso discussed in the newspapers, e.g. in the science pages of the FrankfurterAllgemeine Zeitung. The paper thus constitutes a scientific highlight of the HPIresearch during this reporting period demonstrating that starting from the ana-lysis of cellular regulatory mechanism by insertional mutagenesis one might endup deciphering a cellular dysregulation of importance for a common humandisease.

SPOC1 is a novel PHD-finger protein that was found to be overexpressed in unre-sectable carcinomas. Overexpression was found to be associated with shorter sur-vival in ovarian cancer. The discovery of SPOC1 thus may have important impli-cations for prognostic diagnosis of ovarian cancer and possibly also for the deve-lopment of novel therapies.

CD83 is the so far best known marker for fully mature dendritic cells (DC) thatis required for efficient T cell activation by these potent antigen presenting cells.Interestingly, CD83 expression is down-regulated by virus infection (e.g. HSV),indicating a novel virus-induced mechanism for immune evasion. CD83 seems tobe controlled, at least in part, by transport of its mRNA. Therefore the investiga-tion of the posttranscriptional processing of CD83 mRNA will improve ourunderstanding with respect to the activation and virus-induced reprogrammingof DC.


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The nuclear scaffoldDetailed knowledge of subnuclear structure and function is of vital importancefor understanding the complex processes associated with DNA replication,recombination, RNA transcription and processing, as well as for RNA transport.Considering the multitude and complexity of nuclear processes, the question ari-ses of how these processes are organized at the structural level. Since many yearsa controversy exists of whether nuclear processes require a structural organiza-tion through a nuclear scaffold (synonymous to the „nuclear matrix“), or whe-ther self-organization of soluble partners is sufficient for proper organization.Especially the complexity of DNA replication, requiring spatial and temporalregulation, has fostered the idea that structural organization by a nuclear scaffoldis required. Analysis of the role of the nuclear scaffold protein SAF-A now hasprovided the first convincing evidence that the nuclear scaffold is required forDNA replication in vivo. The finding will be important both for basic science aswell as for a variety of applications, e.g. understanding of viral replication anddeveloping safe vectors for gene therapy, respectively.

The SAF-A protein, and hence the nuclear scaffold, has further roles in control-ling gene expression. Probably the most impressive example is its role in silencingthe expression of X-linked genes in female mammals, in order to ensure equalexpression of these genes in both genders. Silencing is induced by a long non-coding RNA, the Xist RNA, followed by a series of chromatin remodeling events,leading to silencing. SAF-A then seems to play an important role in maintainingthe silenced state of the second X-chromosomes in females.

Animal modelsAnimal models for the analysis of processes leading to hematopoetic disorders tradi-tionally are a strong research topic at the HPI. Results described in the „animalmodels“ section contribute to clarify the function of key molecules in leukemia deve-lopment, like RUNX1, a transcription factor at the crossroad of proliferation and dif-ferentiation, as well as of mutant forms of the C/EBP-alpha transcription factor thatinhibit myeloid differentiation. Retroviral insertional mutagenesis is used to identifynovel hematopoetic regulators and genes responsible for apoptosis resistance inhematopoetic malignancies expressing wild-type p53. It would not have been possibleto obtain any of the important results reported here in cell culture alone, highlightingthe importance of animal models for the dissection of the complex pathways whichrequire the interplay of tumor cells with the organism.

Although by far not all problems in leukemia research have been resolved, hema-topoetic tumors are much better studied then solid tumors, owing to the higherdegree of structural and genetic complexity of such tumors. It therefore is impor-tant that during the last years, also models to analyze ductal mammary carcino-genesis have been established at the HPI. Ductal mammary carcinoma is the mostcommon carcinoma in women in the Western world, with about 45,000 new cases

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per year in Germany alone. The models meanwhile have allowed addressingimportant questions in ductal mammary carcinogenesis. One important issue isthe deregulated expression of telomerase in tumors, an enzyme responsible formaintaining the ends of chromosomes, the telomeres. Telomerase activity is com-pletely shut down in differentiated mammary epithelial cells, but becomes reacti-vated during tumor development. Activation is required for replication of thetumors cells, because they otherwise would die due to telomere-shortening.Crossing transgenic mice destined for mammary tumor development with micetransgenic for the indicator gene lac-Z, driven by the promoter of the humantelomerase gene TERT, allows expression analyses of the human TERT geneduring mammary carcinogenesis.

The models are also suited to analyze the postulated gain of function of mutp53proteins in vivo. To this end several transgenic mouse lines have been established,carrying mutp53 transgenes under the control of the WAP-promoter, whichallows the spatially and temporally controlled expression of the transgene in ductal mammary epithelial cells upon induction with lactotrophic hormones.Characterization of the individual mouse lines revealed that transgene expressionis controlled at two distinct epigenetic levels: the first level probably is determi-ned by position effects, leading to a variegated, but mouse line specific expressionof the transgenes. Variegated expression encompassed mouse line with no, low(<2% of induced mammary epithelial cells [MECs]), intermediate (30-50% ofinduced MECs), and high (>90% of induced MECs) expressing lines. At a secondlevel it was found that secondary events, like number of parturitions, or co-expression of the chromatin remodeling protein SV40 T-antigen strongly affecttransgene expression. Aside from studying the oncogenic effects of mutp53 pro-teins, these mice therefore provide a useful model for the study of the often varie-gated expression of mutp53 in many human tumors.

Another novel model established at the HPI is an animal model for liver regene-ration. Detailed knowledge of the molecular processes driving the differentiationof liver stem or progenitor cells is a prerequisite for using such cells in therapiesfor liver diseases. Using quantitative gene expression profiling, the study reportedhere shows that gene expression of fetal liver progenitor cells after transplantationchanges rapidly from fetal to adult phenotype despite ongoing proliferation.

Technical advancementsScientific progress is strictly coupled to technical advancements. Although tech-nical advancements achieved usually are incorporated into the individual projectreports, the projects reported in this section constitute novel methodologicaldevelopments that will be of interest for a wider audience.

The HPI has established a small proteomics unit that provides state of the art pro-teomic services to cooperating partners and is actively engaged in developingnovel or improving existing technologies. Currently, a major objective is to estab-


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lish methods that allow the determination of posttranslational modifications onregulatory proteins, using p53 as a model substrate of high biological relevance.As outlined in the section „dissecting p53 pathways“, regulatory modifications ofp53 are important for governing p53 function. Thus, correlating p53 posttransla-tional modification with different functional states of the protein is an importanttask. The report on this study demonstrates the technical problems associatedwith this project.

An important technical requirement that already has been achieved is that map-ping of posttranslational modifications requires high sequence coverage of theprotein in mass spectrometry. The availability of this methodic arsenal helped toprovide stringent evidence by proteomic sequencing that delta p53, already des-cribed in the section „dissecting p53 pathways“ is indeed generated by alternati-ve exon-exon splicing.

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Research ProjectsDissecting p53 pathwaysTranscriptional control of gene expressionby wildtype and mutant p53

W. Deppert (1), E. Kim (1,2), G. Tolstonog (1)(1) Heinrich-Pette-Institut(2) Universität Lübeck, Neurochirurgie

Transcriptional control of geneexpression is the most importantfunction of the tumor suppressorp53 and indispensable for its rolein the regulation of the cellularresponses to genotoxic insults.Despite the fact that mechanisticaspects of transcriptional activa-tion or repression mediated bywtp53 are well delineated, theunderstanding of how these acti-vities integrate into diverse pro-grams that may lead to differentfunctional outcomes is far frombeing complete. The turning pointthat marked the field within pasttwo years was the realization thatthe transcriptional response ofp53 can not be understood if con-sidered independently from thedynamic changes of the chroma-tin structure and DNA topology.


Interaction of p53 proteins withradioactively labeled DNAs using p53Express-Array containing 49 mutant p53forms identified in human cancers.Autoradiograph of radioactively labeledDNAs bound to p53 proteins immobili-zed on a solid support (p53-ExpressArray). p53 arrays were incubated withDNA present either in linear or in stem-loop conformation, washed and expo-sed to X-ray film for autoradiographicdetection. Positions of wtp53 andmutp53 proteins 273H and 245S areindicated. Many mutant p53 proteinsexhibit strong selectivity for stem-loopDNA binding (marked by the dottedlines in the left panel) while beingimpaired for binding to linear DNA(right panel).

Non-linear DNA binding of mutant p53: Implications for mutantp53 activities.

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Our original findings that p53 is a DNA topology-dependent transcription factorformed the basis of the concept that transcriptional activation mediated by wtp53is promoter-selective and that such selectivity is determined by topological feat-ures of the DNA within p53 response elements from individual promoters. Wefound that wtp53 is unable to distinguish specific binding sites from unspecificDNA in linear DNA, but it binds with the highest efficiency and with a high degreeof specificity to specific DNA in a non-linear conformation. The findings withwtp53 inspired us to test the idea whether non-linearity of DNA may be importantfor the interaction of mutp53 proteins with DNA. We found that mutp53 proteinsare capable of binding to DNA specifically and with a specificity that is differentfrom sequence-specific binding of wtp53. Mutp53 binds DNA in a DNA topology-selective manner, which does not require the presence of a specific sequence, butstrictly requires non-linear DNA in a specific configuration. Furthermore, wefound that the high selectivity of mutp53 to non-linear DNA is supported by thecomplete loss of a high affinity linear DNA binding, an activity inherent to wtp53.Our findings uncover for the first time the existance of the previously unknowncommon denominator such as DNA non-linearity, which underlies DNA recogni-tion by mutp53 proteins. One of the directions explored currently in our groupconcerns the identification of mutp53 specific target sites in genomic DNA andfunctional characterization of mutp53 non-linear DNA binding.

Supported by the Deutsche Forschungsgemeinschaft and the EC (fifth and sixthframework)

A novel human p53-isoform is an essential element of the ATR-intra-S-phase checkpoint

G. Rohaly, J. Chemnitz, S. Dehde, A. Nunez, J. Heukeshoven, I. DornreiterHeinrich-Pette-Institut

The tumor suppressor p53 is considered to have unique structural and transacti-vation properties in human cells. The assumption is based on the fact that thebiological functions of additional identified p53-isoforms are unknown and therefore considered to be of marginal importance. However, we provide eviden-ce for the existence of a novel p53-isoform, designated as ∆p53 that is generatedby alternative exon splicing. In contrast to p53, ∆p53 transactivates the endoge-nous p21- and 14.3.3�- but not the mdm2-, bax- and PIG3-promoter. Unlike p53,the novel p53-isoform transactivates only p53-target genes that are involved incell cycle arrest functions; therefore, ∆p53 displays differential transcriptionalactivity per se. The first evidence that the novel p53-isoform is of physiologicalrelevance derived from p21-promoter binding studies performed with synchroni-zed wtp53 cells that were UV-irradiated at discrete stages in the cell cycle. Resultsdemonstrate that ∆p53 bound and transactivated the p21-promoter exclusively indamaged S-phase cells, indicating that the differential transcriptional activity of∆p53 is regulated in a cell cycle dependent manner. Moreover, cell cycle depend-ent p21-promoter binding was also observed for p53 since promoter bound p53was retrieved from irradiated G1- and G2-, but not from S-phase cells.

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The physiological implication of ∆p53 during replication blockade was studied inmore detail by activating the intra-S-phase checkpoint with UV-irradiation. Ourinvestigations revealed a novel pathway that operates in the UV-induced ATR-mediated intra-S-phase checkpoint and depends on functional ∆p53. The fea-tures of this new pathway are ATR-dependent phosphorylation of the noveltranscription factor ∆p53, ∆p53-mediated up-regulation of p21 and accordinglydown-regulation of cyclin A-Cdk2 activity. While the response of the well knownATR-Chk1-Cdc25A-cyclin E-Cdk2 pathway is rapid and lasts for only 2 h, theATR-∆p53-p21-cyclin A-Cdk2 pathway affects S-phase progression 2 h after UV-irradiation and attenuates S-phase progression for additional 6 h. In addition,∆p53-mediated down-regulation of cyclin A-Cdk2 activity is necessary to preventDNA replication for 6 h to allow sufficient time for repair of damaged DNA.Therefore, in response to UV-irradiation, activation of ATR triggers two parallelbranches of the DNA damage-dependent intra-S-phase checkpoint that coopera-te by inhibiting the activities of cyclin E- and cyclin A-Cdk2, which are involvedin distinct steps of DNA replication. Thus, the novel ATR-∆p53-p21-cyclin A-Cdk2 pathway is crucial to allow coordinated repair and replication events. InDNA damaged S-phase cells inhibition of DNA replication is mandatory, sinceduplication of damaged DNA is considered responsible for acquiring genelesions, which promote tumor development.

Supported by Bundesamt für Strahlenschutz (BMU StSch4213)

Molecular response to DNA damage depends on the cell cycle stage and p53 status

G. Rohaly, A. Nunez, I. DornreiterHeinrich-Pette-Institut

Our investigations revealed the existence of two ATM-mediated G2-checkpoints,which are activated according to the cell cycle position and the p53 status of thedamaged cell. The transition from the replicative S-phase to G2-phase requiresthe sequential activation of the G2-kinases cyclin A- and cyclin B-Cdk1.Activation of cyclin A-Cdk1 is accomplished by Cdc25A/B-dependent dephos-phorylation of Tyr15 on its catalytic subunit Cdk1 in S-phase; whereas activationof cyclin B-Cdk1 requires both dephosphorylation of Cdk1 by Cdc25C andnuclear import in early G2-phase. Wtp53, but not mutp53 cells, which are damagedin late S-phase or at the S/G2-transition, activate the ATM-Chk2-phosphoryla-tion cascade resulting in stabilization and activation of wtp53; consequently, thetranscription factor p53 promotes transactivation of the Cdk-inhibitor p21,which binds and inactivates cyclin A-Cdk1. In view of the fact that cyclin A-Cdk1is essential for the S/G2-transition and its activity is required to activate the mitosis-promoting factor cyclin B-Cdk1, damaged cells attenuate cell cycle pro-gression in early G2. Cells that were damaged in early G2 have already passed thecyclin A-Cdk1 activating step, but still can block the activation of the second mit-otic regulator cyclin B-Cdk1. Activation of cyclin B-Cdk1 is prevented by Chk2-


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catalyzed inhibiting phosphorylation of the Cdk1-activating phosphataseCdc25C. Thus, in response to DNA damage in early G2, cells attenuate late G2progression by accumulating inactive cyclin B-Cdk1. In conclusion, the p53-dependent and p53-independent ATM-Chk2-Cdc25 pathways lead to attenua-tion of G2 progression allowing additional time for DNA repair as demonstratedby Comet assay. While the repair of damaged DNA takes about 2 h after activationof the p53-dependent early G2-checkpoint, it takes 2 h more to repair damagedDNA following activation of the p53-independent late G2-checkpoint. We assumethat the density of the chromatin structure, which increases during G2-phase pro-gression, determines the time for repair.

Upon severe genotoxic stress, the ATM-mediated p53-dependent early G2- andp53-independent late G2-checkpoints are abrogated and replaced by apoptosis.So far, the mechanisms promoting apoptosis in G2-arrested cells are unknown.However, the proteolytic degradation of growth inhibiting hypophosphorylatedpRb appears to be essential for triggering the programmed cell death. In contrast,severely damaged mutp53 cells that express functional wt∆p53, escape apoptosisvia polyploidization.

Supported by Bundesamt für Strahlenschutz (BMU StSch4359)

Dissecting a cytoplasmic anchor for p53

R. Reimer, H. Helmbold, W. BohnHeinrich-Pette-Institut

Cytoplasmic accumulation of wtp53, detectable in undifferentiated tissue andhuman tumors, is regarded as a mechanism to block entry of p53 into the nucleusand to override p53 growth control. There is ample evidence that cytoplasmicsequestration of p53 is determined by its association with cytoplasmic proteins.In rat glioma cells we found p53 to be anchored to vimentin, a class III interme-diate filament protein. In human neuroblastoma cells wtp53 was described tointeract with the glucocorticoid receptor. Now we show that binding of p53 tovimentin and to the receptor are not independent varieties, but are part of thesame sequestration mechanism.Using rat glioma cells transfected with a tempe-rature sensitive mutant p53 we show that p53 bound to vimentin filaments is incomplex with the glucocorticoid receptor. Formation of the p53-receptor com-plex itself is not sufficient to sequester p53, but vimentin is required to anchor thecomplex in the cytoplasm. In the absence of vimentin, p53-glucocorticoid recep-tor complexes accumulate in the nucleus. The complex of p53, receptor, andvimentin is not static, but its composition can be modulated. p53 can be dis-placed from the complex and can be induced to translocate separately into thenucleus, while the receptor remains bound to the vimentin filaments and stays inthe cytoplasm. A subsequent addition of a receptor agonist displaces the receptorfrom the cytoskeleton and induces its accumulation in the nucleus. The data

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assign vimentin a new physiological role in regulating the activity of the twomajor stress response proteins p53 and glucocorticoid receptor. They suggest thatanchoring to vimentin is used to coordinate the crosstalk between p53 and thesteroid receptor in growth control.

Supported by Deutsche Krebshilfe


Immunofluorescence micrographs (upper panels) and corresponding scattergrams (lower panels) of a rat gliomacell treated with cytochalasin B and triple stained with antibodies to actin (upper panel A, red signal), gluco-corticoid receptor (GR; upper panel A and B, green signal), and vimentin (upper panel B, blue signal). No sig-nificant correlation between GR (x-axis) and actin (y-axis) signals (lower panel A), strong correlation betweenGR (x-axis) and vimentin (y-axis) signals (lower panel B).

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Crosstalk between p53 and retinoblastoma proteins in cellular senescence

A. Kapic (1), H. Helmbold (1), R. Reimer (1), B. Szalay (1), Ch. Hagel (2), W. Bohn (1)(1) Heinrich-Pette-Institut(2) Institut für Neuropathologie, Universitätsklinikum Hamburg-Eppendorf

p53 functions are central to apoptosis and cellular senescence, pathways that areactivated to withdraw cells irreversibly from proliferation. In tumor cells loss ofthese growth control mechanisms frequently coincides with inactivation of p53,leaving pathways downstream of p53 intact. Thus, restoration of p53 function intumor cells should be a suitable approach to block tumor cell growth. In p53 nulltumor cells ectopic expression of p53 has proven to induce directly apoptosis orcellular senescence. To determine pathways interconnected with p53 in thisresponse we supplemented a p53 deficient rat glioma cell line with a temperaturesensitive mutant p53. The cells lack expression of p16INK4a, indicating that theG1/S checkpoint control by pRb/p16INK4a is defunct.

When the temperature sensitive p53 was activated, the cells were stalled with highlevels of cyclin E in late G1, early S-phase and adopted a senescence phenotype.Maintenance of the arrest was dependent on functions of the pocket proteinp130/Rb2, whereas expression of the other pocket proteins pRb and p107 wasreduced to hardly detectable levels. Our data indicate that p53 can selectively co-operate with p130/Rb2 to induce a sustained arrest, when the major arrest path-way in cellular senescence controlled by pRb and p16INK4a is defunct. Functionsof p53 and p130/Rb2 have both to be maintained to prevent escape of senescentcells from the arrest. Inactivation either of p53 or p130/Rb2 proved to be sufficientto abrogate the arrest and to initiate entry into and passage through the S-phase.Senescent cells escaping the arrest pass through the S-phase, but do not resumeproliferation. Entry into the proliferative status seems to be hampered by DNAstrand breaks, which accumulate while the cells stay in the arrest and evoke apop-tosis subsequent to the release from growth suppressive functions by p53 and pro-gression into the S-phase. The accumulation of DNA damage seems to be a majordeterminant for the change from the reversible to the irreversible arrest status.

Supported by Roggenbuck-Stiftung and Werner Otto-Stiftung

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Functional interactions of p53 with telomeres and telomerase

K. Iwanski, B. Wittek, W. Deppert, Ç. GünesHeinrich-Pette-Institut

p53 is involved in transcriptionalregulation of human TERT geneexpression, post-translationalfunction of telomerase activityand telomere function by directbinding telomeric DNA. More-over, p53 is involved in signalingof telomere defects and inducesapoptosis as a result of severe telo-mere damage.

We are interested in definingmolecular mechanisms underly-ing these functions. Initial experi-ments show that p53 interactswith telomerase components invivo and in vitro.

In vitro studies with stably trans-fected and inducible cell lines withwild type and mutant p53 as wellas isogenic cell lines with differentp53 status are used to further elu-cidate the p53, telomerase andtelomere connections.

Sp100 is important for the stimulatory effect of homeodomain-interacting protein kinase-2 on p53-dependent gene expression

A. Möller (2,3), H. Sirma (1), T. G. Hofmann (1), H. Staege (1), E. Gresko (3), K. Schmid-Lüdi (3), E. Klimczak (2), H. Will (1), W. Dröge (2), M.L. Schmitz (3)(1) Heinrich-Pette-Institut(2) German Cancer Research Center, Heidelberg.(3) University Bern (Switzerland)

HIPK2 shows overlapping localization with p53 in promyelocytic leukemia(PML) nuclear bodies (PML-NBs) and functionally interacts with p53 to increasegene expression. We demonstrate that HIPK2 and the PML-NB resident proteinSp100 synergize for the activation of p53-dependent gene expression. Sp100 andHIPK2 interact and partially colocalize in PML-NBs. The cooperation of HIPK2and Sp100 for the induction of p21(Waf1) is completely dependent on the pre-sence of p53 and the kinase function of HIPK2. Downregulation of Sp100 levelsby expression of siRNA does not interfere with p53-mediated transcription,


p53 is associated with the telomerase-complex in vivo: combinedimmunoprecipitation and TRAP assay: IP-TRAP. Hela cells were trans-fected with the following plasmid vectors (a, GFP-Vector as a nega-tive controle, b, p53-vector only, c, hTERT-vector only and d, p53 andhTERT-vectors together) and cell lysates were obtained at the secondday of transfection. Immunpreciptitation was carried out with p53specific antibodies. Aliquots (2µl or 4µl) from the immunprectipta-tion step were used in TRAP-assay (which allows detection of theenzymatic activity of telomerase). Only extracts from p53 and hTERTcotransfected cells (arrows) exhibit telomerase activity, indicating apotential interaction of the telomerase-complex with p53.

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but obviates the enhancing effect of HIPK2. In summary, these experiments re-veal a novel function for Sp100 as a coactivator for HIPK2-mediated p53 activation.


PML is required for homeodomain-interacting protein kinase 2(HIPK2)-mediated p53 phosphorylation and cell cycle arrest but isdispensable for the formation of HIPK domains

Möller (2, 3), H. Sirma (1), T. Hofmann (1), S.Rueffer (2), E. Klimczak (2),W. Dröge (2), H. Will (1), A.M.L. Schmitz (2,3)(1) Heinrich-Pette-Institut(2) German Cancer Research Center, Heidelberg(3) University Bern (Switzerland)

We demonstrate that endogenous human homeodomain-interacting proteinkinase (HIPK) 2 and the highly homologous kinase HIPK3 are found in a novelsubnuclear domain, the HIPK domains. These are distinct from other subnucle-ar structures such as Cajal bodies and nucleoli and show only a partial colocali-zation with promyelocytic leukemia (PML) nuclear bodies (PML-NBs). A kinaseinactive HIPK2 point mutant is localized in the nucleoplasm. The occurrence ofHIPK domains in PML-/- fibroblasts reveals their independence from the PMLprotein. HIPK2 can be almost completely recruited to PML-NBs by the PML iso-form PML IV, but not by PML-III. PML IV-mediated recruitment of HIPK2 doesnot rely on its kinase function and also occurs in PML-/- fibroblasts, showing thatthis PML isoform is sufficient for recruitment of HIPK2. Whereas the architectu-re of HIPK domains is PML independent, HIPK2-mediated enhancement of p53-dependent transcription, p53 serine 46 phosphorylation and the antiproliferativefunction of HIPK2 strictly rely on the presence of PML.


PML-bodies: A growingnumber of componentsand putative functions.Components in the fo-cus of our study are in-dicated in red.

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Switch controlsHIPK2 regulates transforming growth factor-beta-induced c-Jun NH(2)-terminal kinase activation and apoptosis in humanhepatoma cells

T. Hofmann (1), N. Stollberg (1), M.L Schmitz (2), H. Will (1)(1) Heinrich-Pette-Institut(2) University Bern (Switzerland)

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. We demonstrate thatHIPK2 regulates transforming growth factor (TGF) beta-induced c-Jun NH(2)-terminal kinase (JNK) activation and apoptosis. HIPK2 colocalizes with Daxx, aprotein acting in TGF-beta-induced JNK activation and apoptosis, in promyelo-cytic leukemia (PML) nuclear bodies, and triggers PML-nuclear body disruptionand release of Daxx. HIPK2 interacts in vitro and in vivo via its kinase domainwith Daxx, and a fraction of Daxx coprecipitates with HIPK2 under physiologi-cal conditions. Moreover, overexpression of HIPK2 leads to Daxx phosphoryla-tion, and ectopic expression of HIPK2 activates the JNK signaling pathway, whichis enhanced by coexpression of Daxx. HIPK2 signals to JNK via a pathway usingDaxx and the mitogen-activated protein kinase kinases MKK4/SEK1 and MKK7.Ectopic expression of HIPK2 and Daxx potentiates TGF-beta-induced apoptosisin human p53-deficient hepatocellular carcinoma cells. Finally, we demonstratethat knockdown of endogenous HIPK2 using RNA interference inhibits TGF-beta-induced JNK activation and apoptosis. Taken together, our findings indicatethat HIPK2 participates in the TGF-beta signaling pathway leading to JNK acti-vation and apoptosis.


The role of U-box proteins for ubiquitination and degradation of proteins

U. Müller (1), K. Harbers (1)(1) Heinrich-Pette-Institut

Modification of proteins by the attachment of ubiquitin is a universal signalresponsible for controlling many key biological processes. Although ubiquitin-conjugation is mainly thought to tag proteins for degradation, more recent resultsshow that it is also involved in non-proteolytic processes. Because of its impor-tance, abberation in the ubiquitination pathway has been implicated in thepathogenesis of several diseases, including cancer. Fundamental to understandingthe ubiquitin system is the question of how specificity is achieved. Evidence isaccumulating that the E3 ubiquitin ligases play a major role in the specificity ofsubstrate recognition. E3 enzymes constitute a large family of diverse proteinsincluding RING finger proteins. Structurally related to the RING finger motif isthe so-called U-box domain where the metal coordinating cysteines and histidi-


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nes of the RING finger have been replaced with other amino acids. Known U-boxcontaining proteins have been implicated in the ubiquitin conjugation system.However, very little is known about their function. We have recently identified anovel U-box containing protein, termed UIP5. Previous results, based mainly onin vitro experiments, suggested that UIP5 is an E3 ubiquitin ligase that is invol-ved in the ubiquitination of PLZF, a transcriptional repressor, that plays animportant role in myeloid differentiation. Several experiments were performed toanalyze the in vivo role of UIP5. Cotransfection experiments in HEK293 cellsconfirmed that UIP5 can ubiquitinate PLZF also in vivo. However, no evidencewas found that UIP5 is involved in the degradation of PLZF. Taken together, theseresults suggest that UIP5-mediated ubiquitination of PLZF has most likely a non-proteolytic function.

To further analyze the biological role of UIP5 the gene was inactivated by target-ed mutagenesis via homologous recombination. Exon 3 of the UIP5 gene, con-taining the U-box domain, was deleted in ES cells and these cells were used toestablish mutant mice. Using this approach we have obtained mice homozygousfor the mutated UIP5 gene. These mice do not synthesize UIP5 specific mRNA orprotein. Results obtained so far show that inactivation of the UIP5 gene does notinterfere with viability and reproduction of the animals. Further studies, especi-ally of the hematopoeitic system, are necessary to detect possible changes associ-ated with loss of UIP5.


Pathophysiology of G-protein coupled receptors (GPCRs)

S. AbdAlla (1), A. Langer (1), Y. el Faramawy (1), H. Abdel-Baset (2), A. el Massiery (2), U. Quitterer (3), H. Lother (1)(1) Heinrich-Pette-Institut(2) Ain Shams University Hospital Cairo (Egypt)(3) Universität Würzburg - Institut für Pharmakologie

G protein-coupled receptors (GPCRs) comprise the largest family of cell surfacemembrane receptors and mediate a panoply of different physiological functions.Although GPCRs have long been considered to function as monomers, increasingevidence suggests that homo- and heterodimerization are characteristic featuresof this receptor family. Homo- and heterodimerization of GPCRs regulate manyreceptor-specific functions such as ligand binding, signaling, desensitization, andcell surface targeting. Yet, little is known about the pathophysiological relevanceof GPCR dimerization in vivo.

We previously detected increased levels of AT1/B2 receptor heterodimers on pla-telets and vessels of preeclamptic women. Preeclampsia is a pregnancy-specifichypertensive disorder with unknown etiology, which affects 5% to 10% of allpregnancies. These dimers seem to contribute to the enhanced angiotensin II

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responsiveness of this hypertensive disorder. Our data reveal a potential mecha-nism for the increased angiotensin II responsiveness: increased levels of heterodi-mers between the vasopressor receptor AT1 and the vasodepressor receptor B2.The receptor heterodimers display increased sensitivity toward angiotensin II andare found in platelets and in omental vessels of preeclamptic women. Moreover,AT1/B2 receptor heterodimers are resistant to inactivation by reactive oxygen spe-cies, which is elevated in normal and preeclamptic pregnancies. Thus, a majorsymptom of preeclampsia is the result of complex formation between two G-pro-tein-coupled receptors.

Since the angiotensin II resonsiveness of AT1 receptors can be specifically modi-fied by AT1/B2 dimerisation, we determined the AT1 receptor dimerization statusin an experimental model of hypertension. AT1/B2 receptor heterodimers wereabundant on renal mesangial cells isolated from spontaneously hypertensive rats compared to cells from normotensive controls. Heterodimerisation of AT1

with B2 receptors was correlated with high levels of B2 receptor protein kinase onkidneys and on mesangial cells of hypertensive rats as determined in immonoblot with receptor-specific antibodies. Specific inhibition of AT1/B2

receptor heterodimers revealed that AT1/B2 receptors mediated an enhancedangiotensin II-stimulated Gαq/11 activation and an increased endothelin 1 secre-tetion of mesangial cells from hypertensive rats. Thus, AT1/B2 receptor hetero-dimierisation contributes to the angiotensin II hyperresponsiveness of mesangialcells from hypertensive cells in experimental hypertension.

An enhanced angiotensin II responsiveness is also a prominent feature of essen-tial hypertension and of related cardiovascular disorders. Moreover, the involve-ment of the angiotensin II-AT1 system in the pathogenesis of essential hyper-tension and of vascular disease is well established. Remarkably, hypertensivepatients display an enhanced angiotensin II-dependent monocyte activation andadhesion to endothelial cells that are still not understood. Since an enhancedmonocyte adhesiveness may sensitize hypertensive patients to the development ofatherosclerosis, we investigated mechanisms accounting for the enhanced AT1

responsiveness of monocytes.

We found that intracellular factor XIIIA transglutaminase crosslinks agonist-induced AT1 receptor homodimers via glutamine315 in the carboxyl-terminal tailof the AT1 receptor. The crosslinked dimers displayed enhanced signaling anddesensitization in vitro and in vivo. Inhibition of angiotensin II release or of fac-tor XIIIA activity prevented formation of crosslinked AT1 receptor dimers. Inagreement with this finding, factor XIIIA-deficient individuals lacked crosslinkedAT1 dimers. Elevated levels of crosslinked AT1 dimers were present on monocytesof patients with the common atherogenic risk factor hypertension and correlatedwith an enhanced angiotensin II-dependent monocyte adhesion to endothelialcells. Elevated levels of crosslinked AT1 receptor dimers on monocytes couldsustain the process of atherogenesis, because inhibition of angiotensin II genera-tion or of intracellular factor XIIIA activity suppressed the appearance ofcrosslinked AT1 receptors and symptoms of atherosclerosis in ApoE-deficientmice.


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SPOC1, a novel PHD-finger protein: association with residual disease and survival in ovarian cancer

G. Mohrmann (1,2,3), J.G. Hengstler (4,5), T.G. Hofmann (1), S.U. Endele(2,3),B. Lee (6), C. Stelzer (5), B. Zabel (5),J. Brieger (5), D. Hasenclever (4), B. Tanner (4), J. Sagemueller (3),

J. Sehouli (7), H. Will (1), A. Winterpacht (2,3)(1) Heinrich-Pette-Institut (2) Universität Erlangen-Nürnberg(3) Universität Hamburg(4) Universität Leipzig

We report the identification of a novel human gene (SPOC1) which encodes a proteinwith a PHD-finger domain. The gene is located in chromosome region 1p36.23, aregion implicated in tumor development and progression. RNA in situ hybridizationexperiments showed a strong SPOC1 expression in some rapidly proliferating celltypes, such as spermatogonia, but not in non-proliferating mature spermatocytes.

SPOC1, a novel PHD-finger protein: association with residual disease and survival in ovarian cancer (A) Domainsof the SPOC1 protein. The blue box indicates an evolutionary conserved domain of unknown function. Nuclearlocalization signal (NLS), Plant homeodomain (PHD). (B) Influence of SPOC1 mRNA expression on survival timevisualized by Kaplan-Meier analysis for primary and recurrent epithelial ovarian carcinomas. Expression of SPOC1mRNA was measured in tumor tissue of 84 patients with primary ovarian cancer and 19 patients with recurrentdisease by quantitative RT-PCR (Taqman-analysis). Expression levels were calculated relative to the housekeepinggene huPO (human phosphoprotein) expression levels.

(5) Universität Mainz(6) Baylor College of Medicine,Houston (USA) (7) Charité, Berlin

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In addition, a high SPOC1 mRNA expression was observed in several ovarian cancercell lines. This stimulated us to systematically examine SPOC1 expression in ovariancancer in relation to prognosis. SPOC1 mRNA expression was quantified in tumortissue of 103 patients with epithelial ovarian cancer. Interestingly, SPOC1 was associ-ated with residual disease, whereby patients with unresectable tumors showed higherlevels of SPOC1 compared to patients without residual tumor tissue after surgery(p=0.029). The univariable proportional hazards model showed an association bet-ween SPOC1 expression and survival (p=0.043, relative risk: 1.535). Survival time was1596 days for patients with low SPOC1 expression versus only 347 days for patientswith high expression using Kaplan-Meier analysis. However, SPOC1 was not associa-ted with survival, when multivariable analysis was adjusted for residual disease. Thiscan be explained by the correlation between residual disease and SPOC1 expression.In conclusion, SPOC1 is a novel PHD-finger protein showing a strong expression inspermatogonia and ovarian cancer cells. SPOC1 overexpression was associated withunresectable carcinomas and shorter survival in ovarian cancer.

Supported by the BMBF and the Deutsche Krebshilfe

Posttranscriptional mRNA processing in human dendritic cells

J. Chemnitz (1), A. Prechtel (1), S. Schirmer (1), C. Grüttner (1), B. Fries (1), B. Abel (1), M.-C. Dabauvalle (2), A. Steinkasserer (3), J. Hauber (1)(1) Heinrich-Pette-Institut(2) Universität Würzburg – Biozentrum(3) Universität Erlangen-Nürnberg – Dermatologische Universitätsklinik

Dendritic cells (DC) are the mostpotent antigen presenting cells(APC) of the immune system andare specialized to sensitize helperand killer T cells. Immature DCcapture antigens in the peripherybut lack full T cell-stimulatorycapacity. In the presence of appro-priate stimuli (e.g. inflammatorycytokines), the DC then mature.DC upregulate T cell adhesionand costimulatory molecules aswell as selected chemokine recep-tors that guide DC migration intosecondary lymphoid organs forpriming of antigen-specific Tcells. DC are defined by theirpotent T cell-stimulatory capacityas well as a characteristic morpho-


BIACORE measurements of HuR – CD83 mRNA interaction. Surfaceplasmon resonance using a BIACORE X optical sensor that allowsthe observation of association and dissociation interactions in real-time and the determination of binding affinities (Kd-values) wasemployed. The analysis of binding kinetics using sequential dilu-tions of a distinct cis-acting CD83 mRNA sequence and HuR isshown. A Kd of 2.0 x 10-9 M was determined for this specific RNA-protein interaction.

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logy (nonadherent cells with motile veils) and phenotype (upregulation of CD86and de novo expression of CD83). For example, the CD83 molecule is to date thebest known marker for fully mature DC, since CD83 is predominantly expressedon the surface of dendritic lineage cells and cannot be detected on immature DCprecursors. Functional studies revealed that CD83 expression is important forefficient DC-mediated T cell activation.

We previously identified a novel cis-acting RNA element in the CD83 mRNA.Our laboratory subsequently demonstrated that this RNA element is a specifictarget of HuR, a cellular RNA-binding shuttling factor, that appears to mediatethe nucleocytoplasmic transport of specific transcripts via the CRM1 pathway. Inparticular, we were able to determine the kinetics and affinities of this RNA-pro-tein interaction in real-time by plasmon surface resonance analyses (BIACOREmeasurements).

By using RNA interference (RNAi) technology and cellular fractionationmethods we demonstrated that this CD83-specific RNA element indeed media-tes the cytoplasmic accumulation of CD83 transcripts. Moreover, the CRM1 inhi-bitor Leptomycin B blocked the nucleocytoplasmic translocation of CD83 trans-cripts. In sum these data suggested that selected cellular mRNAs, such as CD83mRNA, are exported from the nucleus to the cytoplasm via the CRM1 pathway.Therefore, inhibition of this specific nuclear export pathway provides a novelstrategy to block DC function, which could possibly allow the modulation ofT cell-mediated immunity.

Supported by Wilhelm Sander-Stiftung

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The nuclear scaffoldDisruption of the nuclear scaffold blocks genome replication

R. Helbig (1), A. Schwander (1), F. Herrmann (1), D. Speidel (1), F.O. Fackelmayer (1)(1) Heinrich-Pette-Institut

The faithful duplication of the genome is one of the most important processes ofa living cell. Especially in multi-cellular organisms, this process must be understringent regulation to ensure that the genome is replicated exactly once duringeach cell cycle. The relevance of the involved control mechanisms is evident formthe fact that a deregulation of DNA replication and the cell cycle cause a multi-tude of human diseases, such as the different forms of cancer. DNA replication andits integration into the lifecycle of the cell are controlled by a complex network ofcellular pathways, many of which are understood in great detail and determinethe temporal regulation of these processes. Much less is known about the spatialregulation, although it is well established that DNA replication depends on chro-matin structure and global nuclear organization. In fact, intact nuclear structureappears to be a prerequisite for DNA replication in nuclei reconstituted fromXenopus egg extracts as well as in human and yeast cells. On the other hand, thebiochemical reactions of the replication process can also occur in absence ofnuclear structures. Thus, it has been proposed that nuclear architecture is involvedin regulation rather than in the enzymatics of DNA replication. The regulatoryrole of nuclear structure may be exerted by affecting the local concentration ofprotein factors required to initiate DNA replication, by facilitating the assemblyor activity of replication forks, and/or by determining where in the genome initi-ation of replication will occur. All three ways could guarantee that the genome isreplicated exactly once in the cell cycle, while retaining the flexibility to adapt thenumber and location of initiation sites to the particular needs of a cell, e.g. duringdevelopment and differentiation.

The architecture of the nucleus is notoriously difficult to investigate in the con-text of cultured cells. We therefore started by using a simpler model system togain insight into fundamental structure-function relationships in the nucleus,and, in a second step, advanced into aspects of nuclear architecture in humancells to test whether the results obtained in the model system are applicable to livehuman cells. In the first step, we reconstituted nuclei in extracts from Xenopuslaevis eggs, and investigated the role of a specific DNA binding protein, ScaffoldAttachment Factor A (SAF-A), for the assembly and maintenance of a nucleo-skeleton. SAF-A and related proteins also investigated in our department arecomponents of a putative „nuclear scaffold“ in human cells, and are functionallyinvolved in the regulation of gene expression and DNA replication. The majorgoal of the project was to elucidate whether SAF-A is necessary for nuclear archi-tecture and function, and to obtain a molecular understanding of how DNAreplication and transcription are regulated in the three-dimensional context ofthe nucleus.


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Scaffold Attachment Factor A (SAF-A) is well-characterized as a multifunctionalprotein involved in DNA replication, gene expression and proliferation. Manyexperiments had suggested that SAF-A exerts these functions by tethering chro-matin to a proteinaceous nuclear substructure, the „nuclear scaffold“. However, ithad been difficult to demonstrate beyond doubt that such a structure exists in livecells. In the first part of the project, we therefore used the Xenopus homologue ofSAF-A, to follow the assembly of a putative nuclear scaffold and test for its func-tional relevance. We were able to show that SAF-A is essential for the assembly ofa scaffold-like structure during the in vitro reconstitution of simple artificialnuclei, and provide evidence that this scaffold acts as a platform on which DNAreplication occurs. Simultaneous visualization by confocal microscopy of replica-ting DNA and proteins essential for DNA replication allowed, for the first time,to directly address the role of nuclear substructures in DNA replication withoutinterference by gene expression, complex genome structure, cell cycle, or epigene-tic effects. This was an important advance in our understanding of spatial andtemporal coordination of DNA replication.

In a second part of the project, the functional implications of ScaffoldAttachment Factors were investigated in live human cells. Mobility measurementsof SAF-A in vivo allowed us to demonstrate, for the first time, that a stable pro-teinaceous nuclear scaffold also exists in living cells. Moreover, mapping the pro-tein domains that are responsible for the specific DNA binding activity and thescaffold attachment of SAF-A enabled us to develop a dominant negative con-struct that leads to a collapse of nuclear architecture. Global changes of chroma-tin organization were observed in vivo, resulting in a block of DNA replicationand cell proliferation.

Investigations in both the Xenopus nuclear reconstitution system and in livehuman cells provided first-time evidence that SAF-A is essential for the assemblyand maintenance of the nuclear scaffold, as well as the spatial regulation of DNAreplication in vivo. Our results do not only add to the fundamental understan-ding of the cell nucleus in terms of basic science, but have already allowed – incollaboration with the group of Hans Lipps at the University of Witten – therational design of a new class of episomally replicating vectors that will be usefulfor gene therapy in mammalian cells.

Supported by the Deutsche Forschungsgemeinschaft (DFG)

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Mechanisms of X chromosome inactivation: Contributions of the nuclear scaffold

R. Helbi (1), F.O. Fackelmayer (1) (1) Heinrich-Pette-Institut

The equal expression of X-linked genes in male and female mammals is achievedby transcriptional silencing of the second copy of the X chromosome in females.Silencing is a complex multistep process that leads to a heterochromatization ofmost of the inactive X chromosome, which becomes microscopically evident asthe Barr body. On the microscopical level, the Barr body has been widely used asa cytogenetic marker to identify cells with an aberrant number of X chromo-somes, and is usually located close to nucleoli or the nuclear periphery togetherwith other heterochromatic regions. On the molecular level, the processes thatlead to silencing of the second X chromosome are induced by expression of alarge, non-coding RNA that is essential for inactivation. The gene encoding thisRNA, called Xist, is the only known gene that is expressed exclusively from theinactive X chromosome. Expression of Xist RNA is the first detectable event in Xinactivation, later followed by large scale chromatin remodeling, a series of epige-netic modifications of chromatin such as the lysine methylation of histone H3,hypoacetylation of histone H4, the enrichment of the peculiar histone variantmacroH2A, and finally methylation of the DNA itself. Importantly, even thoughXist is present on inactive X chromosomes throughout the life of an organism, itis directly involved in transcriptional silencing only in early embryogenesis. Later,silencing becomes Xist independent, and the inactive state can be perpetuated byredundant epigenetic mechanisms even in absence of Xist. Apparently, Xistimparts a chromosomal memory early in development („initiation phase“),which facilitates histone methylation in Xi during the „maintenance phase“ atlater times. During maintenance, the chromosomal memory allows Xist to effi-ciently establish histone methylation, but Xist is no longer necessary for silencingitself. In both phases, Xist RNA is locally constrained to the territory of the inac-tive X chromosome, in contrast to almost any other RNA species in the nucleusthat freely diffuse and can roam the entire nucleus. Xist remains in the vicinity ofits expression site, exclusively spreading in cis to neighboring regions of the samechromosome (but not in trans to non-X chromatin regions that may even be closer), until it essentially „coats“ the entire inactive X chromosome.

The molecular basis of the very low mobility of Xist, and the mechanism thatimparts its local constraint, are currently unclear. Earlier experiments have sug-gested that diffusion of Xist is prevented by binding to components of a stableproteinaceous substructure of the nucleus. Indeed, we were able to demonstratethat Scaffold Attachment Factor A (SAF-A) is enriched in the territories of theinactive X chromosome. This protein has earlier been identified and characteri-zed as a DNA- and RNA-binding component of a putative nuclear scaffold, andappears to be involved in the regulation of gene expression and DNA replication.As the enrichment of SAF-A in inactive X territories depends on an intact RNAbinding domain on the protein, and the protein can be extracted from Xi by mildRNase (but not DNase) treatment, SAF-A is a plausible candidate for a structural


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protein that might contribute to Xist immobilization. In fact, we have recentlybeen able to provide first in-vivo evidence in favor of this hypothesis, demonstra-ting the presence of a stable, locally constrained substructure in inactive X terri-tories.

Combined with our earlier findings that SAF-A requires its RNA binding domainfor enrichment in Xi, and the strong nucleic-acid induced self-assembly of SAF-A to large complexes, our recent results are compatible with a role of SAF-A forthe self-organization of a stable structure that immobilizes Xist RNA. Indeed, thelocalization of Xist RNA results from cooperative binding of protein factors toredundant low-affinity sequence elements, in exactly the same way in which SAF-A interacts with nucleic acids. While SAF-A is certainly not needed for coating Xichromatin with Xist, which can bind to chromatin by itself, such a structure couldprevent spreading of Xist RNA in the nuclear interior to confine it to the Xi ter-ritory. In addition, the proteinaceous structure formed by SAF-A might act as aplatform on which epigenetic modifications occur, especially during the main-tenance phase of X inactivation. On first sight, a direct involvement of SAF-A inthe initiation process would also appear plausible because SAF-A has recentlybeen shown to physically interact with proteins involved in epigenetic chromatinmodifications, such as the histone acetylase p300 and protein arginine methyl-transferase 1 (PRMT1). However, these proteins have been implied in gene(co)activation rather than silencing, a function clearly incompatible with X inac-tivation. Importantly, though, these proteins are not co-enriched with SAF-A inXi (unpublished observations), and the methylation/acetylation status of histo-nes in Xi indicates that p300 and PRMT1 are not active there. Unless an interac-tion of SAF-A with a component of the silencing cascade that leads to transcrip-tional inactivation of on X chromosome is convincingly demonstrated, we tendto believe that the structure described in this paper may be important for themaintenance of the silenced state, rather than for its induction. In this scenario,the stable proteinaceous structure containing SAF-A might be part of the „chro-mosomal memory“ mechanism that ensures efficient histone methylation speci-fically in the territory of Xi. Future experiments will now focus on the possiblerole of SAF-A in this „memory“, and should allow a more detailed understandingof the dosage compensation in mammalian cells.


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Animal modelsDisruption of RUNX1 (AML1) function in acute leukemias

M. Fischer (1), M. Schwieger (1), B. Niebuhr (1), S. Horn (1), J. Cammenga (1), U. Bergholz (1), S. Roscher (1), T. Ford (2), M. Greaves (2), J. Löhler (1), C. Stocking (1)(1) Heinrich-Pette-Institut(2) Leukemia Research Fund Centre, London (United Kingdom)

RUNX1 is one of the most frequently mutated genes in human acute leukemiasand hematopoietic disorders. Located on chromosome 21, it is directly targetedby several chromosomal translocations in AML, with the t(8;21) being the mostcommon, occurring at a frequency of approximately 15%. Moreover, 20% ofpediatric B-cell acute lymphoblastic leukemia (ALL) carry the t(12;21), juxtapo-sing the RUNX1 gene with the gene encoding the ETS transcription factor TEL.In addition to translocations, inactivating or dominant-negative mutations in theRUNX1 gene have been identified in 15 to 25% of the relatively rare, minimallydifferentiated M0 AMLs, up to 25% of myelodysplastic syndromes associatedwith AML development, and in pedigrees of familial platelet disorder with a pro-pensity to develop AML. The RUNX1 gene product (RUNX1 or often calledAML1) is one of three members of the RUNX family of transcription factors thatplay pivotal roles in normal development. RUNX proteins bind DNA at sequence-specific motifs through a conserved Runt homology domain, which is also theinteracting domain of a shared b-subunit that modulates nuclear localization andDNA binding. Significantly, disruption of the gene encoding the b-subunit is alsoobserved in 5% of AMLs.

Although RUNX1 has been shown to regulate a number of genes relevant to mye-loid and lymphoid development, it is a relatively weak activator of transcriptionby itself and probably functions as an organizing protein that facilitates assemblyof transcriptional activation or repressor complexes. The importance of RUNX1in establishing and the HSC compartment in adult (murine) hematopoiesis, aswell as regulating T-cell and megakaryocyte differentiation has been establishedby the analysis of conditional knock-outs. However, how its deregulation contri-butes to AML and ALL remains unclear. Both the t(12;21) associated with B-cellALL and the t(8;21) associated with AML fuse RUNX1 with a transcriptionalrepressor, with the net result being the active inhibition of RUNX1-regulated pro-moters. The dissimilar leukemias that they induce thus likely reflects distinctinteractions with other transcriptional regulators due to either differences in theirexpression pattern and/or their distinct composition. Furthermore, the disrup-tion of the normal function of the fusion partner may also contribute to thepathogenic consequences. Nevertheless, common pathways may be activiated (orinactivated) by these fusion proteins, and identifying these pathways may revealits pivotal role in leukemogenesis.

We have previously established an in vivo mouse model system to define the roleof RUNX1/ETO, the fusion product of t(8;21) in AML. We demonstrated thatexpression of RUNX1/ETO in murine hematopoietic progenitors has a profound


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effect on the proliferation and differentiation capacity of the myeloid compart-ment in vivo, consistent with its association with AML, although more subtleeffects were also observed in terminal stages of B- and T-cell differentiation. Inmore recent studies, we sought to establish why the t(12;21) translocation, gene-rating the TEL/RUNX1 fusion protein, induces a B-cell leukemia. By introducingTEL/RUNX1 into murine bone marrow cells enriched for early progenitors andsubsequent transplantation into irradiated mice, we demonstrated thatTEL/RUNX1 impairs hematopoietic differentiation, leading to the accumulationand persistence of an early progenitor compartment with a Sca1+/Kithi/CD11b+

phenotype.

Differentiation of these cells is not blocked, but the frequency of mature cells ari-sing from TEL/AML1-transduced progenitors is quite low, possibly reflecting ashift in the balance between self-renewal and differentiation, as evidenced by invitro cloning assays. Impaired differentiation is prominently observed in the pro-B cell compartment, consistent with the t(12;21) ALL phenotype. The conse-quence of TEL/RUNX1 expression on myeloid differentiation was much less pro-nounced. This subtle effect is in striking contrast to the profound effect thatRUNX1/ETO has on expanding the early myeloid compartment. Further studiesare necessary to determine if this reflects an inability of TEL/RUNX1 to down-regulate C/EBPa, an important regulator of granulocytic differentiation, whichhas been postulated to be an important target of RUNX1/ETO. Importantly, thedistinct effects of TEL/RUNX1 and RUNX1/ETO on hematopoietic differentia-tion in these studies, where expression is regulated by the same promoter ele-

RUNX1 (encoding the transcription factor AML1) is a common target of acute leukemias. Shown are thefusion proteins generated by the translocation (8;21) found at a high incidence in acute myeloid leukemia(AML) and the translocation (12;21) found in 20% of acute lymphoblastic leukemia in children. Importantdomains of the proteins or indicated. HSR, homology-sequence region; RHD, runt homology domain; AD,transactivation domain; Ptd, pointed domain. Repressors are recruited by the Ptd of TEL and the HSR2 andHSR3 domains of ETO to downregulate RUNX1 (AML1) target genes.

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ments and targeted to the same cell types, indicates that the distinct leukemiaphenotypes are dictated by intrinsic differences in the fusion protein themselves.Experiments have been initiated to determine the importance of specific domainsin the TEL/RUNX1 fusion protein for impairing differentiation, as well as theimportance of TEL1 disruption in ALL.

Despite the accumulation of an early progenitor compartment and the impaired(but not blocked) B-cell differentiation, no leukemia induction was observed.These results are consistent with the hypothesis developed from clincial data sug-gesting that TEL/RUNX1 is alone insufficient to induce leukemia, but establishesa preleukemic stage. Further studies are underway to determine the mechanismby which TEL/RUNX1 leads to the accumulation of early (Sca1+/Kithi/CD11b+)cells and to determine if a similar effect is observed in human hematopoietic cells.We postulate that these additional hits in these early cells may render them leu-kemogenic and thus may be the precursors of the leukemic stem cell (LSC) com-partment, which feeds a leukemia. We are currently testing if anti-apoptotic orpro-proliferation factors (e.g. Bcl2 and FLT3) will cooperate with TEL/RUNX1 toinduce an acute leukemia.

Supported by the Deutsche José Carreras Leukämie Stiftung and Amgen, Inc.

Mutant forms of the C/EBPα transcription factor inhibit myeloid differentiation

M. Schwieger (1), J. Löhler (1), B. Niebuhr (1), M. Fischer (1), G. Iwanski (1), M. Ziegler (1), U. Herwig (2), D. Tenen (3), C. Stocking (1)(1) Heinrich-Pette-Institut(2) Albertinen-Krankenhaus, Hamburg(3) Harvard Institutes of Medicine, Boston, MA (USA)

The CCAAT/enhancer binding protein alpha (C/EBPα) is an essential transcrip-tion factor for granulocytic differentiation. Recent studies have reported C/EBPαmutations in approximately 8% of acute myeloid leukemia (AML) patients. Mostof these mutations occur in the N-terminal coding region, resulting in a frame-shift and the enhanced translation of a 30 kDa protein lacking the transactivatingenhancer 1. In vitro assays have demonstrated that the 30 kDa protein acts in adominant-negative fashion to inhibit DNA binding and transactivation by wild-type C/EBPα and may thus be responsible for the differentiation block observedin AML. To test this hypothesis, we introduced a cDNA encoding a N-terminalmutated C/EBPα (mut10) into primary human cord blood (CB) cells and murinebone marrow (BM) cells using a retroviral vector. Expression of mut10 in humanCD34+ CB cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated the co-expression of both myeloid and erythroid markers in the immature transformedcells.


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Surprisingly, mut10 did not blockgranulocytic differentiation inmurine BM cells, but resulted rat-her in a decrease in self-renewaland relative increased levels ofgranulopoiesis. This was not dueto an inability to infect relevantcells, because a differentiationblock was observed when theCebpa gene was excised after retro-viral expression of the CRE recom-binase in BM cells derived frommice carrying floxed Cebpa alleles.

Taken together, these results pro-vide strong evidence that mutationsin CEBPA are critical events in thedisruption of myeloid differentia-tion in AMLs. Moreover, theyunderline the intrinsic differencesbetween hematopoietic controls inmouse and human, demonstratingthe need for caution in usingmouse models to recapitulateevents in human AMLs. Currentwork focuses on defining themechanism by which truncated C/EBPα interferes with the action of wild-typeC/EBPα in human myelopoiesis and to more closely define differences betweenthe role of C/EBPα in regulating murine and human myelopoiesis by comparingthe effect of C/EBPα down-regulation in human cells using siRNA technology.

Supported by Deutsche José Carreras Leukämie Stiftung

Retroviral insertional mutagenesis to identify novel hematopoietic regulators

M. Forster (1), M. Ziegler (1), M. Fischer (1), E. Olsen (2), C. Stocking (1)(1) Heinrich-Pette-Institut(2) University of Texas Southwestern Medical Center, Dallas TX (USA)

Several pivotal regulators of hematopoietic development have been identified,including transcription factors and cytokine signalling pathways, that are disrup-ted during leukemogenesis. Nevertheless, in the majority of leukemias the gene-tic mutations necessary for the progression to an acute and overt leukemia areunknown. A powerful tool to identify novel genes that may be involved in humanleukemogenesis is retroviral insertional mutagenesis. Although the etiological

Point mutations in the coding region of the N-terminus of C/EBPαare often found in AML and lead to the expression of a p30 pro-tein that inhibits wild-type function in vitro. Expression of such aC/EBPα mutant in human CD34+ cells blocked normal myeloid anderythroid differentiation. Cytospins of day 12 colony assays fromhuman cord blood cells infected with a control or mut10 vectorswere subjected to immunocytochemical staining. Mut10 transdu-ced cells with an abnormal promyelocytic morphology exhibit apromiscuously positive immunoreactivity for myeloperoxidase(99% positive), lysozyme (76% positive), and glycophorin A(71.6% positive), whereas the control cell show a lineage restrictedimmunoreactivity of the same antigens in promyelocytic, monocy-tic, and erythroid cells, respectively. Shown is the myeloperoxida-se staining.

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agent differs, the mechanisms and target genes are often the same or closely rela-ted. We have previously shown that infection of mice susceptible to myeloiddisorders succumb to an acute myloid leukemia after infection with Moloneymurine leukemia virus (MoMuLV). Analysis of the retroviral integration sites ofthese tumors have confirmed deregulation of genes previously shown to be invol-ved in human acute myeloid leukemias and/or in mouse models for myeloid leu-kemia (e.g. NFI1, HOXA7, and HOXA10), thus validating our approach. Morerecently, we have detected integration and transcriptional activation of theMEF2C gene in several tumors, making this MADS-box transcription factor acandidate oncogene. Our results are reinforced by the recent observation thatMEF2C is highly expressed in distinctive subsets of AML with an immature phe-notype, and that its closely related family member, MEF2D, is a target of trans-locations in ALL.

It is well established that MEF2C is a critical regulator of cardiac and skeletalmuscle, as well as neuronal differentiation, but its role in hematopoiesis has not

been examined, although it isexpressed at high levels in hema-topoietic cells. In addition, it hasbeen found to be a critical regula-tor of several mitogenic activatedprotein kinases (MAPKs), whichare important regulators of extra-cellular stimuli. We are currentlytesting the role of MEF2C in leu-kemia using our in vivo modelsystem, as well as in hematopoieticdifferentiation using conditionalMEF2C knock-out mouse strains.

Supported by Deutsche Forschungs-gemeinschaft (DFG)


Panel A: A common retroviral integration site was found upstreamof the Mef2C gene in acute myeloid tumors arising in ICSBP-/- miceinfected with 10A1NB-MuLV. Viral integrations were clustered wit-hin 1kb of the 1st coding exon of Mef2C (arrowheads). Panel Bshows the various isoforms of MEF2C that are generated by alter-native splicing. The γ sequences have been shown to containrepressor sequences that can be reversed by phosphorylation.Panel C depicts the expression pattern and level of Mef2c in diffe-rent tumors. An asterik denotes tumors in which integration wasobserved in the Mef2c locus. As a control, RNA was isolated frombone marrow of ICSBP-/- mice.

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Dissecting the mechanism of apoptosis-resistance in hematopoieticmalignancies expressing wildtype p53

A. Engelmann (1), W. Deppert (1), G. Bornkamm (2), C. Stocking (1)(1) Heinrich-Pette-Institut(2) Gesellschaft für Strahlenforschung (GSF), München

p53 is a central player in a rather complex network that protects genomic integri-ty by inducing apoptosis or growth arrest in response to DNA damage, hypoxia,or aberrant oncogene expression. Thus it is not surprising that inactivation of p53is a common occurrence in human cancers. However, in striking contrast to solidtumors, wild-type p53 expression is detected in 80 to 95% of tumors of acute lym-phoblastic leukemias (ALL) and high grade Non-Hodgkin’s Lymphomas (NHL),a fact that correlates well with the observation that these tumors respond well toclassical chemo- or radiation therapies. However, despite this initial good re-sponse, long-term survival rates are generally poor in adult ALL and NHL patientsdue to the outgrowth of therapy resistant clones and subsequent relapse of thedisease. Defining the mechanism by which these tumors escape apoptosis in thepresence of intact p53 is an important endeavor essential to the development ofnovel and effective therapies for treatment of these diseases. Significantly, the availa-bility of multiple ‘death’ circuits in p53-mediated apoptosis provides complemen-tary and independent pathways to target key ‘nodes’ in the network. The critical circuit or circuits needed to activate apoptosis will thus depend on the cellularmilieu, dictated by the particular cell type, differentiation and proliferation status– or by genetic alterations that have occurred during tumor progression. Thus themechanisms by which tumors escape apoptosis after genotoxic insults in the pre-sence of wild-type p53 may vary greatly between different tumor types. The chal-lenge is to determine how contextual factors influence the apoptosis network andcan they be exploited for therapeutic purposes to restore the apoptotic program.

Defining the mechanisms by which lymphoid tumors escape the apoptotic program is best studied using primary cells in their natural environment. Wechoose to use a mouse model in which a lymphoid tumor is induced by Moloneymurine leukemia virus (MoMuLV). MoMuLV is a retrovirus that induces an earlystage of hyperproliferation in the hematopoietic system within the first two weeksof infection, followed by the clonal outgrowth of a malignant tumor due to theaccumulation of retroviral integrations in the vicinity of oncogenes and/or tumorsuppressors. The genetic loci disrupted in the tumor can be identified by isolationand characterization of the retroviral integration sites. Mouse strains that carryactivated oncogenes (e.g. Eµ-myc) or inactivated tumor suppressors (Trp53-/-)can be also used for such studies, providing the „first hit“ in tumorigenesis. Suchan approach can be used to model a particular malignancy associated with speci-fic genetic alterations, as well as defining the biological function of these coope-rating events during tumorigenesis.

Using a mouse model for Burkitt’s lymphoma, a NHL associated with activationof the c-myc oncogene by translocation to the IgH locus, we have demonstratedthat MoMuLV infection leads to acceleration of an aggressive malignant disease.The majority (circa 80%) of these tumors express a wild-type p53, consistent

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with the incidence found in Burkitt’s lymphomas. Significantly, g radiation trig-gers apoptosis in these cells, but cells that escape apoptosis can be detected intransplanted animals at a incidence of <10-6. Our goal is to define the criticalmutation in these radiation resistant clones by analysis of integration sites thatare distinct to these tumors.

Viral integration sites in ten independent tumor sets (radiation sensitive and resis-tant tumors) were isolated, sequenced and their chromosomal location mappedusing available data bases.

Significantly, integration upstream of the Bmi-1 locus was observed in circa 35%of the independent tumors. Bmi-1 encodes a bithorax protein, previously shownto interfere with p19ARF, and important upstream regulator of p53 after oncogenicexpression. Integrations in these loci were observed in both radiation resistantand sensitive tumors, and thus may abrogate p53 induction in response to over-expression of myc, but is not sufficient to block radiation induced apoptosis. Inaddition to a number of other loci shared by both tumor types, we have also iden-tified two loci at which integration correlated with the acquisition of radiationresistance. One loci mapped to the 1st intron of the Bcl-X gene, an important anti-apoptotic member of the Bcl-2 family. RNA analysis has confirmed that this geneis expressed in these tumors provided a satisfying „proof-of-principle“ to ourapproach. The second gene mapped upstream of the gene encoding GADD45β,and again high levels of expression was observed in γ-radiation-resistant tumors.GADD45β was originally implicated in both p53 dependent and independentnegative growth control. However, accumulating evidence has shown that undercertain conditions GADD45β mediates cell survival through NF-κB signalling.GADD45β blocks apoptosis at the level of the mitochondria, but using a pathwaydistinct to Bcl-x. Demonstrating the importance of this pathway in apoptosis inhi-bition in B-cell malignancies will provide novel targets for therapeutic approaches.


In vivo regulation of human and mouse telomerase TERT promoter during embryonal development and breast carcinogenesis / role of p53

J. M. Ritz (1), B. Wittek (1), O. Kühle (1), S. Riethdorf (2), B. Sipos (3),W. Deppert (1), C. Englert (4), Ç. Günes (1)(1) Heinrich-Pette-Institut(2) Universitätsklinikum Hamburg-Eppendorf, Institut für Tumorbiologie(3) Department for Pathology, University of Kiel(4) Institute for Molecular Biotechnology, Jena, Germany

The activity of telomerase, a cellular reverse transcriptase that is capable of syn-thesizing telomeric DNA de novo, is necessary to overcome the end replicationproblem. Telomerase activity can be detected in early human development but isabsent in most normal somatic cells except germ line cells and cells with prolife-rative capacity such as stem cells or cells of the mucosal lining of the gut. On the


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other hand, high telomerase activity can be detected in over 85-90% of humantumors and in vitro immortalized cells. The human telomerase enzyme is com-posed of two essential components, the RNA component (TERC: TelomeraseRNA component) which acts as a template for reverse transcription; and the cata-lytic subunit TERT (Telomerase reverse transcriptase) with the reverse transcrip-tase activity. Whereas TERC is constitutively expression in human cells, expres-sion of TERT is the crucial for telomerase activity and reactivation of TERT geneexpression is one of the key events during tumorigenesis. Therefore, understan-ding the mechanisms of TERT gene regulation is essential for the development ofdiagnostic and therapeutic strategies for human diseases.

We have generated transgenic mice with an 8.0 kbp human TERT (hTERT) pro-moter fragment driving expression of the bacterial lacZ gene (hTERTp-lacZtransgenic mice) to explore the physiological regulatory mechanisms involved inhTERT gene regulation during embryonal differentiation in vivo and to betterunderstand the role of telomerase in tumorigenesis and its potential for tumortherapy.

We find that the expression pattern of the lacZ reporter under the control of an8.0 kbp human TERT promoter fragment mostly recapitulates the expression pat-tern of the TERT gene found in normal human tissues and is stringently regula-ted, unlike the broader expressionpattern of the endogenous mouseTERT gene. Moreover, humanTERT promoter activity is up-regulated in mammary tumors ofhTERTp-lacZ x p53+/- bitrans-genic mouse mammary tumormodel. Thus, this transgenicmouse model provides a suitablein vivo system to analyze theexpression of the human TERTgene under physiological condi-tions and during tumorigenesis.


Human TERT promoter activity is up-regulated in the p53+/- mousemammary tumor model. β-gal immunostaining. A 2-µm paraffinsection of the mammary tumor tissue of hTERTp-lacZ x p53+/-

bitransgenic mouse was subjected to immunohistochemical stain-ing with β-gal-specific antibodies. Specific β-gal immunoreactivitywas detected in tumor cells (red arrows) but not in the surround-ing stroma cells (black arrows).

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Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice

F. Krepulat, J. Löhler, C. Heinlein, W. DeppertHeinrich-Pette-Institut

Around 90% of all mutations in the tumor suppressor gene p53 are missensepoint mutations resulting in the expression of a functionally altered protein withan exchange of a single amino acid. In vitro and cell culture experiments as wellas statistical correlation analysis of human tumor data bases indicate that suchmutations in p53 may not only result in a loss of wild-type (wt) p53 function, butalso in the generation of mutp53 proteins with gain-of-function properties.Furthermore, specific mutations are associated with poor prognosis in humanmammary carcinoma and with primary resistance to chemotherapy. In order tostudy the putative mutp53 gain-of-function in vivo, we generated WAP-mutp53transgenic mice expressing different mutp53 proteins in mammary gland epithe-lium. These mice carry a murine p53 minigene harbouring point mutations equi-valent to human tumor derived hot spot mutations (human mutp53-R248W =


>

WAP-mutp53 transgene constructs under the control of the murine whey acidic protein (WAP-) promoter con-taining mutp53R245W(*) and mutp53R270H(x) point mutations. Primers used in studies are shown by arrows.Transgene screening was performed by using murine p53 exon IX specific and HA-tag sequence-specific primers leading to a 1075 bp PCR product. A Southern DNA probe (762 bp) was amplified with primers in p53intron 3 and 4. Transcription of mutp53 was analyzed by RT-PCR using murine p53 exon VI specific and HA-tag sequence-specific primers leading to a 628 bp PCR product (for details see material and methods). (B)Tissue-specific transcription of mutp53 in lactating mammary glands of BALB/c WAP-mutp53 mice (F1 genera-tion, day 5 pp). Using a pair of primers complementary to murine p53 and HA-tag sequences, mutp53 trans-gene transcription was detected in mammary glands and not in other tissues. Control RT-PCR with murine p53specific primers (586 bp) reveals the integrity of RNA. Lanes: cervical (1), first thoracal (2), second thoracal (3),abdominal (4) and inguinal mammary gland (5), liver (6), kidney (7), spleen (8), heart (9), lung (10), water asnegative control (–), cDNA of mutp53 transcribing mammary gland as positive control (+) and 100 bp DNAstandard (M.W.).

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murine mutp53-R245W, human mutp53-R273H = murine mutp53-R270H).The transgene is under control of the murine whey-acidic-protein (WAP) promo-ter, which is induced by lactotrophic hormones in mammary epithelial cells allow-ing temporal and tissue-specific expression in WAP-mutp53 mice. In order todistinguish transgenic mutp53 from endogenous wtp53, a haemagglutinin (HA)-tag was fused to the C-terminus.

Analysis of 23 WAP-mutp53 transgenic mouse lines revealed that epigenetic mecha-nisms affect mutant p53 transgene expression in these mice. The mutp53 expressinglines showed a mosaic expression pattern for the transgenes, leading to a hetero-geneous, yet mouse line specific expression pattern for mutp53 upon induction. Onlyfew lines were obtained, in which the majority of the induced mammary epithelialcells expressed the mutp53 transge-ne, most of the transgenic lines didnot express mutp53, or expressedthe transgene in less than 2% of theinduced mammary epithelial cells.Hormone requirements for mutp53transgene expression from theWAP-promoter differed in highand low expressing lines, being lowin high expressing lines, and evenlower in multiparous mutp53 mice,where persistent expression of thetransgene occurred. Repeated induc-tion of mutp53 expression throughrepeated parturition resulted in theformation of expanding mutp53expressing foci within the mamma-ry alveolar epithelium.

The expression pattern was stablein about half of the analyzedmutp53 transgenic lines over the4–8 generations analyzed so far,suggesting that the specific expres-sion pattern is „programmed“ within the individual mouse line,

Morphology and transgene expression patterns in lactating mammary glands of BALB/c WAP-mutp53 trans-genic mice. A-C: Unchanged histology of lactating mammary glands. Panel A, line W4; panel B, line W15; panelC, line H8. H&E (F1 generation, day 5 pp). D-F: Representative examples of the variegated, mosaic transgeneexpression patterns of mutp53 are shown for a low (line W4, panel D), an intermediate (line W15, panel E)and a high expressing line (line H8, panel F). Mutp53 (HA-tag) immunostaining on paraffin sections (F1 gene-ration, day 5 pp). Scale bars A-C: 100 µm; D: 50 µm; E-F: 100 µm. Different magnifications were chosen forlines W4, W15 and H8 to clearly demonstrate the typical mosaic expression patterns. G-J: Morphology of glan-dular tissue during end stage of involution (day 50 pp) appears normal in mutp53 transgenic mice withheterogeneous mutp53 expression compared with a wild type control. Panel G, line W4 in generation F7;panel H, line W15 in generation F6; panel I, line H8 in generation F4; panel J, BALB/c wt. Whole mount tech-nology with carmine staining. Scale bars G-J: 1 mm.

>

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while the changes in the expression patterns of other lines over time was accom-panied by the loss of integrated copies.

The data suggest that epigenetic mechanisms play a role in modulating theexpression of the mutp53 transgene. To support this idea, we crossed a non-expressing WAP-mutp53 line with a strongly SV40 T-antigen expressing WAP-Tmouse line. In the bi-transgenic mice, T-antigen induced chromatin remodelingled to re-expression of epigenetically silenced mutp53 transgene(s). In these mice,mutp53 expression was much more variable compared to SV40 T-antigen expres-sion, and seemed to depend on the co-expression of SV40 T-antigen. The WAP-mutp53 mice described here mimic the often variegated expression of mutp53 inmany human tumors and thus may constitute a model for analyzing parametersaltering mutp53 expression during tumor development and progression whencrossed into WAP-T mice.

Generally, the mutp53 transgenic mouse lines provide per se or as crosses withother mouse lines an excellent experimental basis for the investigation of thepathogenetic role of mutated p53 during tumor initiation, progression, invasionand metastasis, especially of breast cancer.

Supported by SFB 545 (DFG) and the EC (fifth and sixth framework)


Double labelling of mutp53 and large Tantigen in lactating mammary tissue of the BALB/c WAPT1 x WAP-mutp53R270H-H22 bi-transgenic mouse line (day 5 pp). Panels A and D demonstrate the heterogeneous expres-sion of mutp53 (green), both in the cytoplasm (A) and nuclei (D) of a limited number of cells. Panels B and Eshow that the majority of the epithelial cells of the same sections stain positive for SV40 Tantigen (red). Thereare no mutp53 positive cells without Tantigen immunoreactivity. This becomes evident, when the micrographsof the two fluorescent signals are merged (panels C and F). Indirect immunofluorescence, double labellingtechnique on paraffin sections. Scale bars A-F: 10 µm.

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Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice

J. Petersen (1,2), D. Zuckerman (1), M. Burda (1,2),M. Dandri (1,2), M. Ott (3), M.P. Manns (3)(1) Heinrich-Pette-Institut(2) Universitätsklinikum Hamburg-Eppendorf(3) Medizinische Hochschule Hannover

Therapies for liver diseases with stem and progenitor cells will require a detailedknowledge of the molecular mechanisms driving the in vivo differentiation pro-cess toward adult hepatic tissue. We applied quantitative gene expressionmethods to analyze the differentiation process of fetal liver progenitor cells aftertransplantation into an animal model of liver regeneration. Enhanced green fluor-escent protein (EGFP)-transgenic liver progenitor cells were isolated from fetalmouse liver at stage embryonic day 13.5 and transplanted into uPA/RAG-2 mice.Two, 4, and 6 weeks after cell transplantation cryosections of liver tissue were ana-lyzed for EGFP-positive regeneration nodules. RNA from laser-microdissectedEGFP-positive tissue was isolated and used as template for quantitative real-timereverse transcriptase-polymerase chain reaction. Phenotypic differentiation wasanalyzed by staining of the canalicular marker enzyme dipeptidyl-peptidase IV.Proliferation in regenerative nodules and surrounding tissue was monitored withthe BrdU incorporation assay. Alpha fetoprotein gene expression had alreadydecreased 2 weeks after transplantation in EGFP-positive regeneration nodulescompared to pretransplantation values and was not detectable after 4 and 6weeks, whereas albumin slightly increased in transplanted cells indicating differ-entiation into a mature phenotype. The dipeptidyl-peptidase IV antigen wasassociated with some liver progenitor cells 2 weeks after transplantation and invirtually all cells after 4 and 6 weeks. Cell proliferation index in transplanted cellswas maximally increased (4.8% BrdU-positive cells) after 2 weeks and decreased(0.4%) after 6 weeks to normal levels. Our results demonstrate that gene expres-sion in liver progenitor cells changes from fetal to adult phenotype within 4 to 6weeks after transplantation despite ongoing proliferation of the transplanted cellsin a mouse model of liver regeneration. Quantitative gene expression profiles asshown here will have important implications in our understanding of the in vivodifferentiation process of stem cells.

Supported by the Deutsche Forschungsgemeinschaft (DFG)

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Technical AdvancementProteomics: Analysis of the phosphorylation patter of the tumor suppressor p53

J. Heukeshoven

According to literature data, the tumor suppressor p53 has 15 potential phospho-rylation sites and three acetylation sites, indicating that its multiple functions aretightly regulated by posttranslational modifications. It therefore is important tostudy the phosphorylation pattern of p53 in dependence of its function and thephysiological status of the cell.

In an approach to get insight into the complexity of p53 postranslational modi-fication, we separated human p53 expressed in insect cells by high-resolution 2D-electrophoresis. By using an extremely flat pH-gradient, we detected 35-40 diffe-rent spots in a Coomassie stained gel that were sufficiently separated to cut outsingle spots for further proteomic analysis.

Mapping of the precise location of a posttranslational modification on a proteinrequires that tryptic peptides are determined with a high sequence coverage. Thecommonly used MALDI matrix -cyano-hydroxy cinnamic acid is not suitable forthis type of mass spectrometric analysis. Therefore we had to develop a suitablematrix on our own and found that the best matrix is a mixture of dihydroxy ben-zoic acid (DHB) and diammonium citrate in 50% acetonitrile with a trace ofphosphoric acid. Using this matrix we obtained a maximal yield of phosphory-lated peptides, as shown with synthetic phosphopeptides, and received a sequencecoverage up to 95% for the extracted peptides of the p53 spots. This implies thatvirtually all p53 peptides were detected in the MALDI analysis, only very smallpeptides (<500 mu [mu == mass unit]) could not be recovered.

Surprisingly, mass spectrometric analysis of all major p53 spots revealed onlyminor differences regarding their phosphorylation status. In the peptide mass listare only 1-2 positions which were consistent with phosphopeptides. But they donot influence the pI of the spots. The same was found with acetyl groups. In all,the mass spectra of all spots are nearly identical.

We next substituted possibly existing phosphate groups by reaction with cystea-mine in the presence of barium hydroxide, which should result in a shift of theisoelectric points of the p53 spots after 2D-electrophoresis, but no such shiftsoccurred. A shift of the isoelectric points of the p53 spots after 2D-electrophore-sis should also occur after splitting off the phosphate groups by reaction with50% HF. But again no such shifts were detected.

The estimation of the mass of p53, alkylated with iodoacetamide, by MALDImass spectrometry resulted in average mol weight (from 5 measurements) of44238 mu, which is exactly the theoretical value of the non phosphorylated p53molecule.

All these findings indicate, that the majority of the p53 proteins expressed in thebaculovirus system is not phosphorylated.


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We next addressed the question regarding the apparent charge heterogeneity ofbaculovirus-expressed p53 in the pI-range 5.5 – 6.7. Our first assumption wasthat the different immobilines, added to the sample before isoelectric focusing,sticked to the p53 molecules and imposed a new pI value. Numerous 2D-gels withimmobilines which varied in type and concentration and at last also withoutimmobilines, however conviced us that there is no artefact caused by immo-bilines. The question what causes the charge heterogeneity of p53 remains openat the moment.

Proteomics: Proteomic sequencing of a new p53 isoform, delta p53

J. Heukeshoven

Delta p53 is a novel p53 isoform, discovered and characterized at the HPI. Deltap53 is generated by exon-exon splicing, deleting amino acids 257-322. Delta p53therefore is shorter and displays a lower molecular mass. In order to confirm thatthe observed truncated band seen in Western blots is identical with the 984-bpalternative p53-splice product, �p53 and p53 were immunoprecipitated fromnuclear extracts that were prepared from irradiated human wtp53 HSC93 cells. Flwtp53 was precipitated with anti-p53 antibody DO12, which recognizes only flp53. Subsequent to fl p53 depletion from the nuclear extract with antibodyDO12, remaining �p53 was enriched with anti-p53 antibody PAb1801. Followingone-dimensional gel separation the Coomassie G250 stained 53-kDa and 45-kDaprotein bands were excised, digested with trypsin and subjected to MALDI-TOFmass spectrometry using the newly composed matrix described above. The tryp-tic fragments were identified by peptide mass fingerprinting using database mat-ching procedures. The amino acid sequence of human p53 and the correspondingtryptic map assembled after MALDI-TOF analysis showed that the peptides oftatal fl p53 and Æp53 tryptic digests covered 98% and 88% of the p53 sequence,respectively.

Two peptides corresponding to amino acids 249-267 (2068.38 mu) and to 320-333 (1708.12 mu) were only detected in the mass spectrum of fl p53, but not inthe spectrum of �p53. The positions of the peptides are within the spliced-outregion (residues 257-323) of �p53. In addition, alternative splicing leads tofusion of residues 249-257 and 323-333 resulting in a new peptide (mu 2262.47)that can only be found in �p53. Thus, data provide hard proteomic evidence forthe splicing positions of �p53, and unequivocally prove that �p53 is expressed invivo.

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New methods and micro-equipment for the fast and controlled cryo-processing of clinical material

H. Hohenberg (1), V. Mutschler (1), R. David (2)(1) Heinrich-Pette-Institut(2) Leica Microsystems GmbH, Vienna (Austria)

The integration of cryopreparation and electron microscopy (EM) into clinicaldiagnostics was – amongst other reasons – restricted so far by the longsome time-frame (> 3 days) of the specific EM-preparation methods. To overcome this time-dependent restriction for EM-application and ultrastructural analysis of tissuesin clinical research and diagnostics we have – in cooperation with Leica – deve-loped a bundle of new cryopreparation methods combined with newly designedmicro-equipment. The goal, to shorten the necessary period for EM-preparationto a minimum (< 24 hours), preserving simultaneously the ultrastructural andimmuno-cytochemical quality of the clinical material, could be reached. This waspossible by combining new microbiopsy techniques, low temperature (PLT)dehydration, deep temperature embedding and UV-polymerisation techniques.

Moreover, special thin film embedding techniques allowed correlative LM- andEM-investigations of identical morphological features, the pinpoint preselectionof structures of interest in the LM inclusive.

Supported by NesTec (Lausanne).


Rat brain biopsy, cryo-processed and analysed by High-Resolution Immuno-Electron Microscopy within 24 honly. The left panel shows ultrathin sectioned and labelled myelin at the rim (arrows) of the 200µm thick biopsy. Myelin (My) was labelled with the myelin glycolipid-reactive mAb O1. Right panel: Compact labelledmyelin sheath at higher magnification.

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Personelle Zusammensetzungder wissenschaftlichenAbteilungen und Arbeitsgruppen

Abteilung Allgemeine Virologie

Hans Will, Prof. Dr. rer. nat.

Michael Bruns, PD Dr. med. vet.Tilman Heise, Dr. rer. nat.Thomas Hofmann, Dr. rer. nat.Sven Horke, Dr. rer. nat.Gerrit Mohrmann, Dr. rer. nat.Gunhild Sommer, Dr. rer. nat.Hüseyin Sirma, Dr. med.Hannah Staege, Dr. rer. nat.

Jorge Braz, Dipl.Biochem.Julia Dittmann, Dipl.Biol.Sandra Fleischer, Dipl.Biol.Claudia Franke, Dipl.Biol.Anneke Funk, Dipl.Biol.Mouna Mhamdi, Dipl.Biol.Kristijana Milovic, Dipl.Biol.Sarah Kinkley, Dipl.Biol.Li Lin, Dipl.Biol.Gritta Tettweiler, Dipl.Biol.

Johanna CroneJana KrügerMouna Mhamdi

Raluca FleischerStephan HäntschMathias HombauerKatharina HöflerAnn-Kathrin KnöfelStephanie MüllerAnnika RennenbergAnneli SagarNina SchmalstiegJohanna SchützBritta van Veen

Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten

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Personelle Zusammensetzung

Abteilung Allgemeine Virologie

Raluca FleischerAlicja IwanskiKerstin ReumannNicole Stollberg

Ourania Andrisani, Prof. PhD Martin Burda, Dr. rer. nat Maura Dandri-Petersen, Dr. rer. nat Jian-nan HuangTatjana Kalinina, Dr. rer. nat.Janine Kraunus, Dr. med.Jörg Petersen, PD Dr. med.Ana Riu-GarciaMartina Sterneck, Prof. Dr. med.Karsten Wursthorn, Dr. med.

Ina Haye

Ingrid Michalski

Assoziierte Heisenberg-Gruppe

Ulrich Schubert, Prof. Dr. rer. nat.

Felicitas Hornung, Dr. rer. nat.Evelyn Schubert, Dr. rer. nat.

Nicole Bedke, Dipl.Biol.Karsten Bruns, Dipl.Biol.Kerstin Zander, Dipl.Biol.

Uwe Tessmer, Dipl.Ing.

Technische Assistenten

Gäste

Spülküche

Sekretariat

Leiter

Wissenschaftler

Doktoranden

TechnischeAssistenten

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Abteilung Tumorvirologie

Wolfgang Deppert, Prof. Dr. rer. nat.

Wolfgang Bohn, PD Dr. rer. nat.Marie Brazdova, Dr. rer. nat.Irene Dornreiter, Dr. rer. nat.Cagatay Günes, Dr. rer. nat.Jochen Heukeshoven, Dr. rer. nat.Ella Kim, Dr. rer. nat.Frauke Krepulat, Dr. rer. nat.Gabor Rohaly, Dr. rer. nat.Daniel Speidel, Dr. rer. nat.Genrich Tolstonog, Dr. rer. nat.

Jan Chemnitz, Dipl.Biol.Thomas Göhler, Dipl.Chem.Sven Hanson, Dipl.Biol.Heike Helmbold, PharmazeutinAndrea Hermannstädter, Dipl.BioIng.Christina Heinlein, Dipl.Biol.Kamila Iwanski, cand.med.Amir Kapic, cand.med.Annette März, Dipl.Biochem.Simone Profittlich, Dipl.Biol.Rudolph Reimer, Dipl.Biol.Julia Ritz, Dipl.Biol.Beate Schmidt, cand.med.Lars Tögel, Dipl.Biol.Korden Walter, Dipl.Chem.Britta Wittek, Dipl.Biol.

Andrea Hermannstädter Alejandro Mena Nunez

Matthias BötheSabrina Köcher

Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten

Abteilung Tumorvirologie

Silke DehdeAndrea DiesterbeckKathrin FrahmGesa Fuhrmann, Dipl. BioIng.Diana KreitzMarion KurthBeata SzalayGabriele WarneckeDoris Weidemann

Christian Bauer, Dr. rer. nat.Camino De Juan RomeroJochen Heukeshoven, Dr. rer. nat.Olaf Kühle

Issaaka GigimaSvetland DragicevicMonika MüllerSemsa Reus

Martina Hintz-Malchow


Gäste

Spülküche

Sekretariat

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Abteilung Zellbiologie und Virologie

Joachim Hauber, Prof. Dr. rer. nat.

Jan Chemnitz, Dr. rer. nat.Ilona Hauber, Dr. rer. nat.Kristijana Milovic, Dr. rer. nat.Klaus-Jochen Wiegers, Dr. med.

Nina Albrecht, Dipl.Biol.Christina Ehlers, Dipl.Chem.Barbara Fries, Dipl.Biol.Alexander Prechtel, Dipl.Biol.Birgit Schäfer, Dipl.Biol.Doreen Schwarck, Hum.Biol.

Stephanie FrankenbergerBritta KeyserSusann Schirmer

Andreas ScherzlerKatharina Henkenius

Bettina AbelAndrea BunkCordula Grüttner

Dr. Axel ChoidasDr. Richard GöllesProf. Dr. Heiner Schaal

Margarete HouillonMaria-Christina Moreno

Ute Neumann

Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten


Gäste

Spülküche

Sekretariat

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Abteilung Molekulare Zellbiologie

Frank O. Fackelmayer, Dr. rer. nat.

Ercan Akgün, Dr. rer. nat.

Roger Helbig, Dipl.Biol.Frank Herrmann, Dipl.Biol.Giulia Mearini, Dipl.Biol.Peter Pably, Dipl.Biol.Andrea Schwander, Dipl.Biol.

Amrei HilzPeter KupferKatja ReuschleinGregor Sachse

Oliver Mauermann

Maike Bossert

Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten


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103


Arbeitsgruppe Elektronenmikroskopie und Mikro-Technologie

Heinrich Hohenberg, Dr. rer. nat., Dipl.Ing.

Tina Meins, Dipl.BioIng.

Julia Röglin, cand.med.Martin Ritter, Dipl.Biochem.Norbert Franz, Dipl.Phys.

Vanessa Mutschler

Robert Getzieh

Ulla BergholzBarbara HolstermannCarola Schneider (beurlaubt)

Dr. Mathias Hemmleb

Gruppenleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten


Gäste

Arbeitsgruppe Molekulare Pathologie

Klaus Harbers, Prof. Dr. rer. nat.Jürgen Löhler, Dr. med.

Jörg Cammenga, Dr. med.Stefan Horn, Dr. rer. nat.Maike Täger, Dr. rer. nat.Carol Stocking, Dr. phil.

Afra Engelmann, Dipl.Biol.Meike Fischer, Dipl.Biol.Martin Forster, Dipl.Chem.Heidi Gevesleben, cand.med.Sibyll Hein, Dipl.Biol.Tina Hochgartz, cand.med.Gabriele Iwanski, cand.med.Birte Niebuhr, Dipl.Biol.Michaela Rodenburg, Dipl.Biol.

Birte Niebuhr

Hélène Djuidje KamdemMartina Hurlebaus

Ursula Müller

Karin HeiglSusanne RoscherMarion ZieglerSilvia Wegerich

Dr. Boris FehseDr. Claudia LangeDr. Bernd SchiedlmeierProf. Dr. James McCubreyProf. Dr. Vladimir PrassolovDipl. Biol. Matthias HamdorfDr. Bjorn Steffan

Radmilla KielRadica Karalic

Gruppenleiter

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten

Diplom-Ingenieur


Gäste

Spülküche

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Arbeitsgruppe Somatische Stammzellengenetik

Heinz Lother, PD Dr. rer. nat.

Yasser el Faramawy, Dr. med.Andreas Langer, Dr. rer. nat.Said Abdalla, Dr. rer. nat.

Gökan Arman-Kalcek

Gruppenleiter

Wissenschaftler


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Veröffentlichungen

2003

Bruns, K., Fossen, T., Wray, V., Henklein, P., Tessmer, U., and Schubert, U. (2003).Structural characterization of the HIV-1 Vpr N-terminus: evidence of cis/transproline isomerism. J. Biol. Chem. 278, 43188-43201.

Cantz, T., Zuckerman, D.M., Burda, M.R., Dandri, M., Goricke, B., Thalhammer,S., Heckl, W.M., Manns, M.P., Petersen, J., and Ott, M. (2003). Quantitative geneexpression analysis reveals transition of fetal liver progenitor cells to maturehepatocytes after transplantation in uPA/RAG-2 mice. Am. J. Pathol. 162, 37-45.

Franz, N., Timm, U., and Hohenberg, H. (2003). Monitoring and analysis of theenvironment for biomaterials in the ESEM under „wet-mode“ conditions.Microscopy and Microanalysis 9, pp. 482-484.

Gostissa, M., Hofmann, T.G., Will, H., and Del Sal,G. (2003). Regulation of p53functions: let’s meet at the nuclear bodies. Curr. Opin. Cell Biol. 15, 351-357.

Hauber, J. (2003). Retroviridae. In: Die Infektiologie, D.Adam, H.W.Doerr,H.Link, and H.Lode, eds. (Berlin, Germany: Springer Verlag), pp. 860-870.

Hein, S., Prassolov, V., Zhang, Y., Ivanov, D., Löhler, J., Ross, S.R., and Stocking,C.(2003). Sodium-dependent myo-inositol transporter 1 is a cellular receptorfor Mus cervicolor M813 murine leukemia virus. J.Virol. 77, 5926-5932.

Helbig, R., and Fackelmayer, F.O. (2003). Scaffold attachment factor A (SAF-A) isconcentrated in inactive X chromosome territories through its RGG domain.Chromosoma 112, 173–182.

Hofmann, T.G., and Will, H. (2003). Body language: the function of PML nuclear bodies in apoptosis regulation. Cell Death Differ. 10, 1290-1299.

Hofmann, T.G., Stollberg, N., Schmitz, M.L., and Will, H. (2003). HIPK2 regu-lates TGF-ß-induced JNK activation and apoptosis in human hepatoma cells.Cancer Res. 63, 8271-8277.

Hohenberg, H., Dimmeler, E., and Wepf, R. (2003). Functional states in cellularnano-morphology: from life-like sampling to 3D reconstruction and interpre-tation. Microscopy and Microanalysis 9, 498-500.

Horn, S., Meyer, J., Stocking, C., Ostertag, W., and Jucker, M. (2003). An increasein the expression and total activity of endogenous p60(c-Src) in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1). Oncogene 22, 7170-7180.

Janz, C., Susse, S., and Wiesmuller, L. (2003). p53 and recombination intermedia-tes: Role of tetramerization at DNA-junctions in complex formation and exo-nucleolytic degradation. Oncogene 21, 2130-2140. Erratum in 2003: 22 (18), 2856.

Kalinina, T., Iwanski, A., Will, H., and Sterneck, M. (2003). Deficiency in virionsecretion and decreased stability of the hepatitis B virus immune escape mutantG145R. Hepatology 38, 1274-1281.

Kalinina, T., Riu, A., Fischer, L., Santantonio, T., Will, H., and Sterneck, M. (2003).Selection of a secretion incompetent mutant in the serum of a patient with severehepatitis B. Gastroenterology 125, 1077-1084.

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Kerner, M., Hohenberg, H., Reckermann, M., Ertl, S., and Spitzy, A. (2003). Self-organization of dissolved organic matter producing micelle-like micro-particlesin river water. Nature 422, 150-154.

Kim, E., and Deppert, W. (2003). The complex interactions of p53 with targetDNA: We learn as we go. Biochem. Cell Biol. 81, 141-150.

Kim, E., Günther, W., Yoshizato, K., Meissner, H., Zapf, S., Nüsing, R.M.,Yamamoto, H., VanMeir, E., Deppert, W., and Giese, A. (2003). TumorSuppressor p53 Inhibits Transcriptional Activation of the Invasion GeneThromboxane Synthase Mediated by the Proto-Oncogenic Factor Ets-1.Oncogene 22, 7716-7727.

Koskela, K., Kohonen, P., Nieminen, P., Buerstedde, J.-M., and Lassil O. (2003)Insight into lymphoid development by gene expression profiling of avian B cells.Immunogenetics 55, 412-422.

Li, Z., Schwieger, M., Lange,C., Kraunus, J., Sun, H., van den Akker, E., Modlich,U., Serinsoz, E., Will, E., von Laer, D., Stocking, C., Fehse, B., Schiedlmeier, B.,and Baum, C. (2003). Predictable and efficient retroviral gene transfer intomurine bone marrow repopulating cells using a defined vector dose. Exp.Hematol. 31, 1206-1214.

Maritzen, T., Löhler, J., Deppert, W., and Knippschild, U. (2003). Casein Kinase Idelta (CK1delta) is involved in lymphocyte physiology. Eur. J. Cell. Biol. 82, 1-10.

McIvor, Z., Hein, S., Fiegler, H., Schroeder, T., Stocking, C., Just, U., and Cross, M.(2003). Transient expression of PU.1 commits multipotent progenitors to a myeloid fate whereas continued expression favors macrophage over granulocytedifferentiation. Exp. Hematol. 31, 39-47.

Meier, P., Scougall, C.A., Will, H., Burrell, C.J., and Jilbert, A.R. (2003). A duckHepatitis B Virus strain with a knockout mutation in the putative X ORF showssimilar infectivity and in vivo growth characteristics to wild-type-virus.Virology 371, 291-298.

Möller, A., Sirma, H., Hofmann, T.G., Rueffer, S., Klimczak, E., Dröge, W., Will,H., and Schmitz, M.L. (2003). PML is required for HiPK2-mediated p53 phos-phorylation and cell cycle arrest but dispensible for the formation of HiPK2domains. Cancer Res. 63, 4310-4314.

Möller, A., Sirma, H., Hofmann, T.G., Staege, H., Gresko, E., Klimczak, E., Dröge,W., Will, H., and Schmitz, M.L. (2003). Sp100 is important for the stimulatoryeffect of homeodomain-interacting protein kinase-2 on p53-dependent geneexpression. Oncogene 22, 8731-8737.

Müller-Reichert, T., Hohenberg, H., O´Toole, E.T., and McDonald, K. (2003).Cryoimmobilization and three-dimensional visualization of C. elegans ultra-structure. J. Microsc. 212, 71-80.

Münk, C., Prassolov, V., Rodenburg, M., Kalinin, V., Löhler, J., and Stocking, C.(2003). 10-A1-MuLV but not the related amphotropic 4070A MuLV is highlyneurovirolent: importance of sequences upstream of the structural Gag codingregion. Virology 313, 44-55.

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Neidhardt, J., Fehr, S., Kutsche, M., Löhler, J., and Schachner, M. (2003). Tenascin-N:characterization of a novel member of the tenascin family that mediates neuri-te repulsion from hippocampal explants. Mol. Cell. Neuroscience 23, 193-209.

Ott, D.E., Coren, L.V., Sowder II, R.C., Adams, J., and Schubert, U. (2003).Retroviruses have differing requirements for proteasome function in the bud-ding process. J. Virol. 77, 3384-3393.

Prassolov, A., Hohenberg, H., Kalinina, T., Cova, L. Krone, O. Frölich, K., Will, H.,and Sirma, H. (2003). A new hepatitis B virus in cranes with an unexpectedbroad host range. J. Virol. 77, 1964-1976.

Psarras, S., Karagianni, N., Kellendonk, C., Tronche, F., Cosset, F.L., Stocking, C.,Schirrmacher, V., Boehmer, Hv.H., and Khazaie, K. (2004). Gene transfer andgenetic modification of embryonic stem cells by Cre- and Cre-PR-expressingMESV-based retroviral vectors. J. Gene Med. 6, 32-42.

Ritter, M., Sinram, O., Albertz, J., and Hohenberg, H. (2003). Quantitative 3-Dreconstruction of biological surfaces. Microscopy and Microanalysis 9, 476-478.

Sänger, P., Meins, T., Neuber, K., Moodicliff, A., Michel, M., and Hohenberg, H.(2003). Environmental investigation of clinical material using closed prepara-tion cycles with integrated LM control steps. Microscopy and Microanalysis 9,492-494.

Sänger, P., Ritter, M., and Hohenberg, H. (2003). Water reservoirs prevent rapiddehydration of native samples in the ESEM. Microscopy and Microanalysis 9,494-496.

Schubert, U. (2003). Function(s) of the ubiquitin-proteasome-system in retro-virus budding. In Proteasome inhibitors in cancer therapy, Ed. JulianAdams,Humana Press.

Schubert, U. (2003). Neue Wege in der antiretroviralen Therapie. RetrovirusBulletin 4, 2-3.

Sirma, H., Meins, T., Will, H., and Hohenberg, H. (2003). Cryotechniques and theprocessing of vital tissues: The necessity of using micro-methods for micro-samples. Microscopy and Microanalysis 9, 388-389.

Utermöhlen, O., Karow, U., Löhler, J., and Krönke,M.(2003). Severe impairmentin early host defense against Listeria monocytogenes in mice deficient in acidsphingomyelinase. J. Immunol. 170, 2621 - 2628.

Woelcke, J., Reimann, M., Klumpp, M., Goehler, T., Kim, E., and Deppert, W.(2003). Analysis of p53 “latency and “activation” by fluorescence correlationspectroscopy: Evidence for two different modes of high affinity DNA binding. J.Biol. Chem. 278, 32587-32595.

Zander, K., Sherman, M.P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A.I.,Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W.C., and Schubert,U. (2003). Cyclophilin A interacts with HIV-1 Vpr and is required for its func-tional expression. J. Biol. Chem. 278, 43202-43213.

109

Zhang, B., Georgiev, O., Hagmann, M., Günes, Ç., Cramer, M., Faller, P., Vasak,M., and Schaffner, W. (2003). Activity of metal-responsive transcription factor 1by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol.Cell Biol. 23, 8471-85.

2004

AbdAlla, S., Lother, H., Abdel-Baset, A., el Massiery, A., and Quitterer, U. (2004).Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyper-responsiveness in experimental hypertension. J. Mol. Neurosci. In press.

AbdAlla, S., Lother, H., Langer, A., el Faramawy, Y., and Quitterer, U. (2004).Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes atthe onset of atherosclerosis. Cell 119, 343-354.

Alroy, I., Tuvia, S., Greener, T., Gordon, D., Barr, H.M., Taglicht, D., Mandil-Levin,R., Ben-Avraham, D., Konforty, D., Nir, A., Levius, O., Bicoviski, V., Dori, M.,Cohen, S., Yaar, L., Erez, O., Propheta-Meiran, O., Koskas, M., Caspi-Bachar, E.,Alchanati, I., Sela-Brown, A., Moskowitz, H., Tessmer, U., Schubert, U., and Reiss,Y. (2004). The trans-Golgi network-associated human ubiquitin-protein ligasePOSH is essential for HIV type 1 production. Proc. Natl. Acad. Sci. U S A. In press.

Boehden, G. S., Restle, A., Marschalek, R., Stocking, C., and Wiesmuller, L. (2004).Recombination at chromosomal sequences involved in leukaemogenic rearrange-ments is differentially regulated by p53. Carcinogenesis 25, 1305-1313

Bäsecke, J., Schwieger, M., Griesinger, F., Schiedlmeier, B., Wulf, G., Trumper, L.,and Stocking, C. (2004). AML1/ETO promotes the maintenance of early hema-topoietic progenitors in NOD/SCID mice but does not abrogate their lineagespecific differentiation. Leuk. Lymphoma. In press.

Bridgera, J.M., Kallaa, C., Wodrich, H., Weitz, S., King, J.A., Khazai, K.,Kräusslich, H.G., Lichter, P. (2004). Nuclear RNAs confined to a reticular com-partment between chromosome territories. Exp. Cell Research. In press.

Chang, S-F., Chang, S-H., Li, B-C., Will, H., and Netter, H.J. (2004).Characterization of non-conventional hepatitis B viruses lacking the core pro-moter. Virology 330, 437-46.

Chanturiya, A.N., Basanez, G., Schubert, U., Henklein, P., Yewdell, J.W., andZimmerberg, J. (2004). PB1-F2, an influenza A virus-encoded proapoptotic mito-chondrial protein, creates variably sized pores in planar lipid membranes. J.Virol. 78, 6304-6312.

Ehlers, I., Horke, S., Reumann, K., Rang, A., Grosse, F., Will, H., and Heise, T.(2004). Functional Characterization of the Interaction between Human La andHepatitis B Virus RNA in vivo and in vitro. J. Biol. Chem. 279, 43437-47.

Endter, C., Hartl, B., Spruss, T., Hauber, J., and Dobner, T. (2004). Blockage ofCRM1 dependent nuclear export of the adenovirus type 5 early region 1B 55-kDa protein augments oncogenic transformation of primary rat cells. Oncogene[advanced E-pub]

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Veröffentlichungen

Engelhardt, O.G., Sirma, H., Pandolfi, P.P., and Haller, O. (2004). Mx1 GTPaseaccumulates in distinct nuclear domains and inhibits influenza A virus in cellsthat lack PML nuclear bodies. J. Gen. Virol. 85, 2315-26.

Ergün, S., Buschmann, C., Heukeshoven, J., Dammann, K., Schnieders, F., Lauke,H., Chalajour, F., Kilic, N., Strätling, W.H., and Schumann,G. (2004). Cell type-specific expression of LINE-1 open reading frames 1 and 2 in fetal and adulthuman tissues. J. Biol. Chem. 279, 27753-27763.

Fackelmayer, F.O. (2004). A stable proteinaceous structure in the territory of in-active X chromosomes. J. Biol. Chem. (Epub Nov 24)

Fackelmayer, F.O. (2004). Nuclear architecture and transcription factors in thequest for new therapeutics. Curr. Pharma. Design 10, 2851-2860.

Florin, L., Becker, K.A., Sapp, C., Lambert, C., Sirma, H., Müller, M., Streeck, R.E.,and Sapp, M. (2004). Nuclear translocation of papillomavirus minor capsid pro-tein L2 requires Hsc70. J. Virol. 78, 5546-5553.

Fojta, M., Pivonkava, H., Brazdova, M., Nemkova, K., Palecek, J., and Vojtesek, B.(2004). Investigations of the supercoil-selective DNA binding of wild type p53.Eur. J. Biochem. 271, 3865–3876.

Frank, O., Rudolph, C., Heberlein, C., Von Neuhoff, N., Schroeck, E., Schambach,A., Schlegelberger, B., Fehse, B., Ostertag, W., Stocking, C., and Baum, C. (2004).Tumor cells escape suicide gene therapy by genetic and epigenetic instability.Blood 104, 3543-9.

Funk, A., Hohenberg, H., Mhamdi, M., Will, H., and Sirma, H. (2004). Spread ofhepatitis B virus in vitro requires extracellular progeny and may be codeter-mined by polarized egress. J. Virol. 78, 3977-3983.

Funk, A., Lin, L., Mhamdi, M., Will, H., and Sirma, H. (2004). Determinants ofhepadnaviral species and liver cell tropism. Monogr. Virol. 25, 56-65.

Funk, A., Mhamdi, M., Lin, L., Will, H., and Sirma, H. (2004). Itinerary of hepa-titis B viruses: delineation of restriction points critical for infectious entry. J.Virol. 78, 8289-8300.

Göhler, T., Jäger, S., Warnecke, G., Yahuda, S., Kim, E., and Deppert, W. (2004).Mutant p53 proteins bind DNA in a DNA structure-selective mode. Nucl. AcidRes. In press.

Hanson, S., Kim, E., and Deppert, W. (2004). Redox factor 1 (Ref-1) enhancesspecific DNA binding of p53 by promoting p53 tetramerization. Oncogene. Inpress.

Hauber, I., Bevec, D., Heukeshoven, J., Krätzer, F., Horn, F., Choidas, A., Harrer,T., and Hauber, J. (2004). Identification of cellular deoxyhypusine synthase as anovel target for antiretroviral therapy. J. Clin. Invest. In press.

Henklein, P., Bruns, K., Nimtz, M., Wray, V., Tessmer, U., and Schubert, U. (2004).Influenza A virus protein PB1-F2: synthesis and characterization of the biologi-cally active full length protein and related peptides. J. Pept. Sci. In press.

111

Herrmann, F., and Fackelmayer, F.O. (2004). Arginine methylation of scaffoldattachment factor A (SAF-A) by hnRNP-particle associated PRMT1. J. Biol.Chem. 279, 48774-48779.

Hofmann, T.G., and Will, H. (2004). The role of the PML bodies and its compo-nents in transcriptional regulation. In Mechanisms of Signal Transduction and Inducible Gene Expression, eds. Schmitz,M.L. and S. Bacher. (ResearchSignpost, Kerala, India), pp. 121-139.

Horke, S., Reumann, K., Schweizer, M., Will, H., and Heise, T. (2004). Nucleartrafficking of La protein depends on a newly identified NoLS and the ability tobind RNA. J. Biol. Chem. 279, 26563-70.

Horke, S., Reumann, K., Schulze, C., Große, F., and Heise, T. (2004). The La-motifand the RRMs of hLa contribute individually to RNA recognition and subcellu-lar localization. J. Biol. Chem. 279, 50302-50309.

Janka, C., Selmi, C., Gershwin, ME., Will, H., and Sternsdorf T. (2004) Small ubiquitin-related modifieres: a novel and independent class of autoantigens inprimary biliary cirrhosis. Hepatology. In press.

Jenke, A.C., Stehle, I.M., Herrmann, F., Eisenberger, T., Baiker, A., Bode, J.,Fackelmayer, F.O., and Lipps, H.J. (2004). Nuclear scaffold/matrix attached regionmodules linked to a transcription unit are sufficient for replication and main-tenance of a mammalian episome. Proc. Natl. Acad. Sci. USA. 101, 11322-11327.

Kim, E., and Deppert, W. (2004). Transcriptional activities of mutant p53: Whenmutations are more than a loss. J. Cell. Biochem. 93, 878-886.

Kim, E., Yoshizato, K., Zapf, S., Kluwe, L., Westphal, M., Deppert, W., and Giese,A. (2004). Functional characterization of the p53 pathway in glioma-derivedcells. Anti-Cancer Res. In press.

Klinger, P.P., and Schubert, U. (2004). The role of the ubiquitin-proteasome-system and other cellular factors in HIV replication: potential targets for anti-retroviral therapy. Future drugs. In press.

Knippschild, U., Gocht, A., Wolff, S., Huber, N., Löhler, J., and Stöter, M. (2004).Review: The casein kinase 1 family: participation in multiple cellular processesin eukaryotes. Cellular Signalling. In press.

Kraunus, J., Schaumann, D.H., Meyer, J., Modlich, U., Fehse, B., Brandenburg, G.,von Laer, D., Klump, H., Schambach, A., Bohne, J., and Baum, C. (2004). Self-inactivating retroviral vectors with improved RNA processing. Gene Ther. 11,1568-1578.

Mearini, G., Nielsen, P.E., and Fackelmayer, F.O. (2004). Localization and dyna-mics of plasmid DNA in live mammalian nuclei. Nucl. Acids Res. 32, 2642-2651.

Meier, P., Scougall, C.A., Will, H., Burrell, C.J., and Jilbert, A.R. (2004). Wild-typeand X-knockout DHBV strains show similar in vivo growth characteristics. InProc. Viral Hepatitis and Liver Disease. In press.

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Miyazono,K., Ishikawa,F., Winterhager,E., Deppert,W., Rajewsky,M.F. (2004).Ninth Japanese-german workshop on molecular and cellular aspects of carcino-genesis, Essen, Germany, 18-20 September, 2003. Cancer Sci. 95(3):276-81.

Mohrmann, G., Hengstler, J.G., Hofmann, T.G., Endele, S.U., Lee, B., Stelzer, C.,Zabel, B., Brieger, J., Hasenclever, D., Tanner, B., Sagemüller, J., Sehouli, J., Will, H.,and Winterpacht, A. (2004). SPOC1, a novel nuclear PHD-finger protein: asso-ciation with residual disease and survival in ovarian cancer. Int. J. Cancer. In press.

Petersen, J., Burda, M.R., Dandri, M., and Rogler, C.E. (2004). Transplantation ofhuman hepatocytes in immunodeficient uPA model for the study of Hepatitis BVirus. Methods Mol. Med. 96, 253-260.

Quitterer, U., Lother, H., and AbdAlla, S. (2004). AT1 receptor heterodimers andangiotensin II responsiveness in preeclampsia. Semin. Nephrol. 24, 115-119.

Ritter, M., Hemmleb, M., Sinram, O., Albertz, J., and Hohenberg, H. (2004). Aversatile 3D calibration object for various micro-range objects. InternationalArchives of Photogrammetry and Remote Sensing (ISPRS), pp. 385-390.

Ritz, J.M., Kühle, O., Riethdorf, S., Sipos, B., Deppert, W., Englert, C., and Günes,C. (2004). A novel transgenic mouse model reveals human-like regulation of an8 kbp human TERT gene promoter fragment in normal and tumor tissues.Cancer Res. In press.

Schwieger, M., Löhler, J., Fischer, M., Herwig, M., Tenen, D.G., and Stocking, C.(2004). A dominant-negative mutant of C/EBPa,associated with acute myeloidleukemias, inhibits differentiation of myeloid and erythroid progenitors of manbut not mouse. Blood 103, 2744 - 2752.

Sirma, H., Funk, A., Hohenberg, H., Petrimpol, M., Mhamdi, M., Lin, L.,Schubert, U., and Will, H. (2004). Functional modulation of virus-cell inter-actions by drugs: New concepts for treatment of viral hepatitis and Hepato-cellular carcinoma. In Proc. Viral Hepatitis and Liver Disease. In press.

Sommer, M.N., Bevec, D., Klebl, B., Flicke, B., Holscher, K., Freudenreich, T.,Hauber, I., Hauber, J., and Mett, H. (2004). Screening assay for the identificationof deoxyhypusine synthase inhibitors. J. Biomol. Screen. 9, 434-438.

Sternsdorf, T., Gostissa, M., Sirma, H., Del Sal, G., Ruthardt, M., Schmitz, M.L.,Will, H., and Hofmann, T.G. (2004). PML nuclear bodies: cellular function anddisease association. In: Visions Of The Cell Nucleus, P.Hemmerich andS.Diekmann, eds. (American Scientific Publishers). In press.

Utku, N., Boerner, A., Tomschegg, A., Bennai-Sanfourche, F., Bulwin, G.-C.,Heinemann, T., Löhler, J., Blumberg, R.S., and Volk, H.-D. (2004). TIRC7 defi-ciency causes in vitro and in vivo augmentation of T and B cell activation andcytokine responses. J. Immunol. 173, 2342 -2352.

Vazquez, N., Greenwell-Wild, T., Marions, N.J., Swaim, W.D., Nares, S., Ott, E.D.,Schubert, U., Henklein, P., Orenstein, J.M., Sporn, M.B., and Wahl, S.M. (2004).HIV-1 induced macrophage gene expressions includes p21, a target for viralregulation. J. Virol. In press.

113

2003

Diplomarbeiten

Hermannstädter, A.: „Epigenetische Regulation der Expression von Mutantenp53“. Diplomarbeit, Fachbereich Naturwissenschaftliche Technik, Hochschulefür Angewandte Wissenschaften Hamburg (2003).

Kolster, T.: „Expression und zelluläre Lokalisation von zwei Proteinen des Ubi-quitin-Systems nach Fusion mit fluoreszierenden Proteinen“. Diplomarbeit,Fachbereich Naturwissenschaftliche Technik, Hochschule für AngewandteWissenschaften Hamburg (2003).

Kupfer, P.: „Untersuchungen zur Mobilität von Proteinen des Zellkerns“.Bachelor-Arbeit, Freie Universität Berlin (2003).

Mhamdi, M.: „Ultrastrukturelle und biochemische Analyse der Morphogeneseund Ausschleusung von Hepatitis B Viren“. Diplomarbeit, Fachbereich Biologie,Universität Hamburg (2003).

Piesche, M.: „Charakterisierung der Wechselwirkungen der Stress-induziertenProteinkinase Casein Kinase 1 delta (CK1?) mit zellulären Proteinen“. Diplom-arbeit, Studiengang Biologie der Rheinischen Friedrich-Wilhelms-UniversitätBonn (2003).

Dissertationen

Boehden, G.: „Analyse der Rolle von DNA-cis-Elementen bei der homologenRekombination“. Dissertation, Fachbereich Biologie, Universität Hamburg, 2003.

Chemnitz, J.: „Regulation des Intra-S-Phasen Kontrollpunktes in UV-geschädigtenPrimatenzellen“. Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Göhler, T.: „Einfluss der DNA-Konformation auf die Bindungsaffinität von wt undmutiertem p53“. Dissertation, Fachbereich Chemie, Universität Hamburg (2003).

Hanson, S.: „Der Einfluss des Redox-Faktors 1 auf den humanen Tumorsuppressorp53“. Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Hein, S.: „Charakterisierung und Klonierung des Rezeptors für das murineLeukämievirus M813 von Mus cervicolor (Linneaus, 1758)“. Dissertation,Fachbereich Biologie, Universität Hamburg (2003).

Horke, S.: „Identifikation und Charakterisierung funktioneller Domänen des huma-nen La Proteins“. Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Janz, C.: „Molekulare Interaktion des Tumorsuppressors p53 bei der Überwa-chung von Rad51-vermittelten homologen Rekombinationsprozessen“.Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Krepulat, F.: „Mammaspezifische Expression von p53 Hot-Spot-Mutationen desMenschen im transgenen Mausmodell“. Dissertation, Fachbereich Biologie,Universität Hamburg (2003).

Diplomarbeiten,Dissertationen, Habilitationen

114

Diplomarbeiten, Dissertationen, Habilitationen

Mohrmann, G.: „Systematische Analyse von cDNAs aus humaner fetalerWachstumsfuge– Klonierung und Charakterisierung von Kandidatengenen derZelldifferenzierung“. Dissertation, Fachbereich Biologie, Universität Hamburg(2003).

Prechtel, A.: „Untersuchungen zur posttranskriptionellen Prozessierung derMessenger RNA von CD83, dem Reifungsmarker Dendritischer Zellen“. Disser-tation, Naturwissenschaftliche Fakultät II (Biologie), Universität Erlangen-Nürnberg (2003).

Rodenburg, M.: „ Die Bedeutung viraler Determinanten für die Pathogenitätmuriner Leukämieviren“. Dissertation, Universität Hamburg (2003).

Röglin, J.: „Kryokonservierung von Einzelspermatozoen in Zellulosekapillar-röhrchen: Eine neue Methodik im Rahmen der assistierten Reproduktion.“Dissertation, Fachbereich Medizin, Universität Hamburg (2003).

Süße, S.: „Funktionelle Interaktionen zwischen Wildtyp-p53, Rad51 und Poly-ADP-Phosphoribosyl-Transferase bei der Kontrolle von DNA-Rekombinations-prozessen“. Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Wähler, R.: „Adenovirale Immuntherapie solider Tumore am HCC-Modell derRatte“. Dissertation, Fachbereich Biologie, Universität Hamburg (2003).

Habilitationen

Pollok, J.-M.: „Tissue Engineering von Lebergewebe in einem dynamischenFlusskultur-Bioreaktor zur Transplantation“. Fachbereich Medizin, UniversitätHamburg (2003).

Günther, S.: „Natürlich vorkommende Varianten des Hepatitis-B-Virus und ihreklinische Bedeutung“. Fachbereich Medizin, Universität Hamburg (2003).

2004

Diplomarbeiten

Crone, J.: „Charakterisierung eines neuen zellulären HIPK2-Interaktions-partners“. Diplomarbeit, Fachbereich Biologie, Universität Hamburg (2004).

Frankenberger, S.: „Untersuchungen zur Genexpression des Fetuinrezeptors“.Diplomarbeit, Naturwissenschaftliche Fakultät II (Biologie), UniversitätErlangen-Nürnberg (2004).

Keyser, B.: „Untersuchungen zur subzellulären Lokalisierung und zum intrazellu-lären Transport von ICP0 und ICP4 des Herpes Simplex-Virus 2 (HSV-2)“.Diplomarbeit, Fachbereich Biologie, Universität Hamburg (2004).

Mutschler, V.: „Entwicklung neuer Techniken zur immunzytochemischen undfeinmorphologischen Analyse klinischer Zellproben.“ Diplomarbeit, FHHamburg (2004).

115

Niebuhr, B.: „Untersuchungen zur Rolle von Mutanten des TranskriptionsfaktorsC/EBPalpha bei der Leukämogenese“. Diplomarbeit, Fakultät Agrarwissen-schaften, Universität Hohenheim (2004).

Nunez, A.M.: Molekulare Antwort auf DNA Schäden zu definierten Zeiten imZellzyklus: Rolle von p53 und ∆p53“. Diplomarbeit, Universität Konstanz (2004).

Reuschlein, K.: „Untersuchungen zur Mobilität des Kernmatrixproteins SAF-A inlebenden Zellen“. Diplomarbeit, Hochschule für Angewandte WissenschaftenHamburg (2004).

Sachse, G.: „Untersuchungen zur Dynamik des Kerngerüstes in lebenden Zellen“.Diplomarbeit, Fachbereich Biochemie/Molekularbiologie, Universität Hamburg(2004).

Schirmer, S.: „Funktionelle Analysen zur posttranskriptionellen Prozessierungder CD83 mRNA“. Diplomarbeit, Fachbereich Biologie, Technische UniversitätDarmstadt (2004).

Dissertationen

Dittmann, J.: „Charakterisierung des humanen La-Proteins im zellulären RNA-Metabolismus: Modulation des Zellzyklus“. Dissertation, Fachbereich Biologie,Universität Hamburg (2004).

Fleischer, S.: „ Identifizierung neuer Kerndomänen assoziierter Proteine“. Disser-tation, Fachbereich Biologie der Phillips-Universität Marburg (2004).

Funk, A: „Identifizierung und funktionelle Modulation essentieller zellulärerKomponenten für die Propagation von Hepatitis B-Viren“. Dissertation, Fach-bereich Biologie, Technische Universität Darmstadt (2004).

Härle, G.: „Hinweise auf eine Beteiligung von p53 an der DNA-Replikation auf-grund von experimentellen Untersuchungen“. Dissertation, FachbereichMedizin, Universität Hamburg (2004).

Horn, F.: „Hemmung der HIV-1-Rev- und HTLV-I-Rex-Funktion mit Hilfe vonInhibitoren der Desoxyhypusin-Synthase“. Dissertation, Medizinische Fakultät,Universität Erlangen-Nürnberg (2004).

Kraunus, J.: „Kontrolle der RNA-Prozessierung in Retroviren und abgeleitetenGentransfervektoren über regulatorische Motive des untranslatierten erstenExons“. Dissertation, Fachbereich Biologie, Universität Hamburg (2004).

Mearini, G.: „Sequence-Specific Visualization of DNA in live mammalian cells“.Dissertation, Universität Hamburg (2004).

Milovic, K.: „Flash: eine neue PML-Kerndomänenkomponente mit derBindegliedfunktion bei der CD95-vermittelten Apoptose“. Dissertation, Fach-bereich Biologie, Universität Hamburg (2004).

Mohrmann, M.: „Molecular characterization of a chromosome 1;22 translocationin a patient with a phenotype resembling Costello syndrome“. Dissertation,Fachbereich Chemie, Universität Hamburg (2004).


116


Müller, S.: „Genexpressionsanalyse von humanen Gehirntumoren mittels cDNAArray-Analyse“. Dissertation, Fachbereich Chemie, Universität Hamburg(2004).

Reimer, R.: „Cytoplasmatische Sequestrierung von p53 in der Gliomzelllinie C6aus Rattus norvegicus (Berkenhout): Interaktion mit dem Glucocorticoidrezeptorund Verankerung am Cytoskelett“. Dissertation, Fachbereich Biologie, Univer-sität Hamburg (2004).

Schwander, A.: „Die funktionelle Domänenstruktur des humanen KernproteinsScaffold Attachment Factor A“. Dissertation, Fachbereich Biologie, UniversitätHamburg (2004).

Sembritzki-Weß, O.: „Die subzelluläre Lokalisation von p53 in humanen Glio-blastomen in Korrelation zur Expression von Intermediärfilamentproteinen“.Dissertation, Fachbereich Medizin, Universität Hamburg (2004).

Speidel, D.: „Selektivität der p53-Antwort auf genotoxischen Stress“. Dissertation,Fachbereich Chemie, Universität Hannover (2004).

Tettweiler, G.: „Identifizierung und Charkterisierung von Interaktionspartnerndes RNA-bindenden Proteins La“. Dissertation, Fachbereich Biologie,Universität Hamburg (2004).

Tacke, F.: Influence of mutations in the Hepatitis B Virus genome on virus repli-cation and drug resistance. Dissertation, Universität Hannover (2004).

Zingler, N.: „Mechanisms Involved in Target Sequence Recognition andIntegration of Human LINE-1 Retrotransposons“. Dissertation, FachbereichChemie, Universität Hamburg (2004).

Habilitationen

Fischer, L.: „Hepatitis B Virus Infektionen bei Patienten nach Lebertrans-plantationen“. Fachbereich Medizin, Universität Hamburg (2004).

117

Lehrtätigkeit

■ Seminar: Modern biology in disease for Ph.D. students■ Practical introduction into scientific research

■ Seminar: Methoden der Molekularen Zellbiologie und Biochemie■ Seminar: Einführung in wissenschaftliche Originalarbeiten/Journal Club

■ Seminar und Praktikum für Mediziner: Introduction into Molecular Medicine

■ Praktikum für Naturwissenschaftler: Determination of telomerase activity in human cells

■ Seminar: Research Highlights in Molecular Virology and Immunology■ Seminar: Experimental Approaches in Virology

■ Vorlesung: Molekulare Zellbiologie und Virologie■ EMBO-Kurs 2003 an der Universität Prag: „New Electron Microscopy

Techniques in Cell- and Molecular Biology“ ■ EMBO-Kurs 2004 im Institut Pasteur:

„Cryo Electron Microscopy Techniques in Cell- and Molecular Biology“

■ Seminar:Neue Ergebnisse in der Leukämieforschung

■ Seminar: Modern Virus and Tumor Research■ Seminar: Advances in Molecular and Cellular Biology

WolfgangDeppert

Frank O.Fackelmayer

CagatayGünes

JoachimHauber

HeinrichHohenberg

CarolStocking

Hans Will

■ Alexander von Humboldt-Stiftung (W.Deppert, J.Hauber)■ BioPark Regensburg (J.Hauber)■ Deutsche Forschungsgemeinschaft

(W.Deppert, J.Hauber, C.Stocking, H.Will)■ Deutsche Krebshilfe e.V.

(W.Deppert, K.Harbers, J.Löhler, H.Will)■ Eberhard Karls Universität Tübingen,

Medizinische Fakultät (W.Deppert)■ Fonds der Chemischen Industrie (H.Will)■ Forschungszentrum Karlsruhe (W.Deppert)■ Friedrich-Alexander-Universität Erlangen-Nürnberg,

Naturwissenschaftliche Fakultät II (J.Hauber)■ Friedrich-Alexander-Universität Erlangen-Nürnberg,

Medizinische Fakultät (W.Deppert, J.Hauber)■ Heinrich-Heine-Universität Düsseldorf,

Medizinische Fakultät (J.Hauber, H.Will)■ Institut für Molekulare Biotechnologie (IMB), Jena (H.Will)■ Johann Wolfgang Goethe-Universität Frankfurt am Main,

Medizinische Fakultät (H. Will)■ Johannes Gutenberg-Universität Mainz,

Medizinische Fakultät (H.Will)■ Justus-Liebig-Universität Gießen,

Medizinische Fakultät (H.Will)■ Kultusministerium Sachsen-Anhalt (W.Deppert)■ Ludwig-Maximilians-Universität München,

Medizinische Fakultät (H.Will)■ Medizinische Hochschule Hannover (H.Will)■ Nationales Kompetenznetz HepNet (H.Will)■ Nationales Kompetenznetz HIV/AIDS (J.Hauber)■ Philipps-Universität Marburg,

Medizinische Fakultät (W.Deppert)■ Ruprecht-Karls-Universität Heidelberg,

Medizinische Fakultät (J.Hauber, H.Will)■ Studienstiftung des Deutschen Volkes (W.Deppert)■ TU Darmstadt, Fachbereich Biologie, (J.Hauber, H.Will)■ Universität Bremen, Fachbereich Biologie (H.Will)■ Universität Leipzig, Medizinische Fakultät (W.Deppert, H.Will)■ Universität Regensburg, Medizinische Fakultät (H.Will)■ Universität Stuttgart, Medizinische Fakultät (W.Deppert)■ Werner-Otto-Stiftung (W.Deppert, J.Hauber)■ Westfälische Wilhelms-Universität Münster,

Medizinische Fakultät (W.Deppert)■ WGL - Leibniz Gemeinschaft (W.Deppert, J.Hauber)■ Wilhelm Sander-Stiftung (W.Deppert, J.Hauber, H.Will)■ Wissenschaftsrat (J.Hauber)

Gutachtertätigkeitfür nationale

Vereinigungen

118

Gutachtertätigkeit

■ Association for International Cancer Research (W.Deppert)■ Czech Science Foundation (W.Deppert)■ Dutch Cancer Society, Amsterdam (C.Stocking)■ European Inst. of Oncology, Milan, Italy (H.Will)■ German Israeli Foundation (W.Deppert)■ Göteborg Universität, Faculty of Medicine, Sweden (H.Will)■ Institut Pasteur, Paris, France (H.Will)■ Italian Association for Cancer Research (AIRC), Italy (H.Will)■ Leukemia Research Foundation, London (C.Stocking)■ Minerva-Weizmann-Stiftung, Israel (H.Will)■ MIUR – Grant Review Committee.

Italienisches Ministerium für Erziehung,Universitäten und Forschung (F.O.Fackelmayer)

■ Monash-University, Australia (H.Will)■ National Institutes of Health, Bethesda (C.Stocking)■ National Research Foundation, Pretoria, South Africa (H.Will)■ University of Adelaide, Adelaide, Australia (H.Will)■ University of Bratislava, Tschechien (H.Will)■ University of theWitwatersrand, Johannesburg,

South Africa (H.Will)

Gutachtertätigkeitfür internationale

Vereinigungen

119

Gutachtertätigkeit

■ Biochemistry and Cell Biology (W.Deppert)■ Journal of General Virology (H.Will)■ Journal of Microscopy – Special Issue (H.Hohenberg) ■ Journal of Viral Hepatitis (H.Will)■ Nucleic Acids Research (W.Deppert)■ Oncogene (W.Deppert)■ Oncology Reports (W.Deppert)■ Virology (J.Hauber)

120

Mitherausgabe wissenschaftlicher Zeitschriften

■ „Hinterzartener Gesprächskreis“ in Cadenabbia/Comer See,Italien 15.-18.5.2003 (Koorganisation)

■ Japanese-German Workshop in Essen, 18.-20. September 2003(Koorganisation)

■ EMBO Workshop - 3rd Elmau Conference on Nuclear Organization,Schloss Elmau, 6.-10. Oktober 2004 (Koorganisation)

■ Telomerase Symposium at the 26th annuary meeting of the German Cancer Association, Berlin 2004 (Organisation)

■ AIDS 2003. 9. Deutscher und 14. Österreichischer AIDS-Kongress,Hamburg 2003 (Koorganisation)

■ International Workshop „Is everything predetermined? Impact of host genetics on HIV infection“, Regensburg 2004 (Koorganisation)

■ International Microscopy Conference, Dresden 2003 (Koorganisation)■ Symposium „Nano-Organisation of complex biological systems“.

International Microscopy Conference, Dresden 2003 (Chairperson)■ Symposium „Scanning electron microscopy at ambient conditions“.

International Microscopy Conference, Dresden 2003 (Chairperson)■ Symposium “Advanced Methods in Biomedical Electron Microscopy“.

Innsbruck, 2004 (Koorganisation und Chairperson)

■ Fifth International Meeting on Myeloid Stem Cell Development and Leukemia, Annapolis, MD, USA 2003 (Chairperson)

■ XV. Wilsede Meeting „Modern Trends in Human Leukemia“,Wilsede 2003 (Koorganisation)

■ Hepnet-Meeting Tiermodelle, Elmau, 22. Februar 2003 (Koorganisation)

■ Triennial International Symposium on Viral Hepatitis and Liver Disease, Sydney, Australia, 7.-10. April 2003 (Koorganisation)

■ Molecular Biology of Hepatitis B Viruses, Bergamo, Italy,7.-10. September 2003 (Koorganisation)

WolfgangDeppert

CagatayGünes

JoachimHauber

HeinrichHohenberg

CarolStocking

Hans Will

121

Organisation von wissenschaftlichenVeranstaltungen, Vorsitzfunktionen

European patent 03 007 000.7 pending:„Novel human p53 splice variant displaying differential transcriptional activity”

„β-L-5-Methylcytosin-Nucleoside zur Behandlung von Hepatitis B-Infektionen“ (Deutsche und internationale Patentanmeldung gemeinsam mit dem Max-Delbrück-Center, Berlin)

„K203 (SPOC1) – Ein neuer Transkriptionsregulator als „drug target“ und molekularer Marker für Prognose,Therapieresistenz und Residualtumor bei Ovarialkarzinomen und anderen Tumorerkrankungen“ (Deutsche und internationale Patentanmeldung gemeinsam mit der Universität Erlangen-Nürnberg)

„β-L-N4-Hydroxycytosin-Nucleoside zur Behandlung von Hepatitis B- und HIV-Infektionen“(Deutsche Patentanmeldung gemeinsam mit dem Max-Delbrück-Zentrum, Berlin)

2003

W. DeppertI. Dornreiter

H. SirmaL. Li

A. FunkH. Will

T. HofmannG. Mohrmann

H. Will

2004

H. SirmaH. Will

122

Erfindungsmeldungenund Patente

123

03.02.2003 Prof. Dr. Emil Palecek, Laboratory of Biophysical Chemistry andMolecular Oncology, Institute of Biophysics, Academy of Sciences of the CzechRepublic, Brno, Czech Republic: „Electrochemistry of nucleic acids and proteinsin the development of DNA sensors“

14.03.2004 Prof. Dr. Ronald T. Hay, University of St. Andrews, Scotland: „Role ofubiquitin-like proteins in transcriptional regulation“

20.03.2003 Dr. Beate Sodeick, Abteilung für Virologie, Medizinische HochschuleHannover: „Microtubule transport during herpes simplex virus infection“

09.04.2003 Dr. Genrich Tolstonog, Max-Planck-Institut für Zellbiologie,Rosenhof/Ladenburg: „Involvement of the intermediate filament proteins in theactivities of the nuclear and mitochondrial genome“

30.04.2003 Dr. Joachim Kühn, Institut für Mikrobiologie, Universität Münster:„Reciprocal transmission of HSV-1 between epithelium and trigeminal neuronsin an organ model based on embryonic chicken tissue“

30.04.2003 Dr. Felix Unckell, Sigma-Aldrich Chemie GmbH: „Bioinformatik undSynthesetechnik der Oligolibraries für die differentielle Genexpressions-ananlyse“

24.06.2003 Dr. Heike Bantel, Abteilung für Gastroenterologie, Hepatologie undEndokrinologie, Medizinische Hochschule Hannover: „Role of apoptosis ininfectious diseases“

30.06.2003 Dr. Richard Maraia, National Institute of Child Health, Bethesda,U.S.A.: „Multifunctionality of the human La RNA binding protein for precursortRNA in the nucleus and mRNA in the cytoplasm is mediated by differentialpartitioning of its Ser-366 phosphoisoforms“

03.07.2003 Dr. Peter Hemmerich, Institut für Molekulare Biotechnologie,Abteilung Molekulare Biologie, Jena: „Evolution of multiprotein structures:Lessons from eukaryotic centromere complex“

10.07.2003 Dr. Thomas Dobner, Institut für Medizinische Mikrobiologie undHygiene, Universität Regensburg: „The adenovirus E1B-55-kDa protein: A multi-functional regulator of virus and cell growth“

24.07.2003 Prof. Dr. Rolf Bartenschlager, Abteilung für Molekulare Virologie,Universität Heidelberg: „Studies on HCV replication in cell culture: What did welearn with the replicon system?“

12.08.2003 Dr. Jörg Cammenga, Albert Einstein College of Medicine, New York,U.S.A.: „Transcription factors and their target genes in hematopoietic differen-tiation and leukemia“

16.09.2003 Prof. Gernot Walter, Department of Molecular Pathology, Universityof California, LaJolla, U.S.A.: „Structure and function of protein phosphatase2A, role in cell cycle arrest and cancer“

25.09.2003 Dr. Petra Boukamp, Deutsches Krebsforschungszentrum, Heidelberg:„Telomere length and telomerase regulation in skin cells“

Seminare

124

Seminare

09.10.2003 Dr. Falko Falkner, Baxter BioScience, Biomedical Research Center,Orth/Donau, Österreich: „Vaccinia virus: a tool in research and vaccine develop-ment“

17.11.2003 Dr. Sacha Gnjatic, Ludwig Institute for Cancer Research, New YorkBranch at Memorial Sloan-Kettering Cancer Center, New York, U.S.A.:„Immune responses to tumor antigen NY-ESO-1: Monitoring spontaneousimmunity and vaccination studies“

22.01.2004 Dr. Stefan Busch, Bio-Med Central, London, England: „Open access:The future of science publishing“

05.02.2004 Prof. Dr. Frank Rösl, Forschungsschwerpunkt Angewandte Tumor-virologie, Abteilung Virale Transformationsmechanismen, Deutsches Krebs-forschungszentrum Heidelberg: „Bypassing viral oncogene expression by HDACinhibition: Induction of cell cycle arrest and apoptosis in HPV-positive cells“

19.02.2004 Dr. Mengji Lu, Institut für Virologie, Universitätsklinikum Essen:„Immune modulation and immune therapy of hepatitis B virus infection“

26.02.2004 Prof. Dr. Klaus Schulze-Osthoff, Institut für Molekulare Medizin,Universität Düsseldorf: „Apoptosis: The biochemistry of caspases“

25.03.2004 Dr. Lukas Pelkmans, Max-Planck-Institut für Molekulare Zellbiologieund Genetik, Dresden: „Caveolae in virus entry and endocytic membrane traffic“

07.04.2004 Dr. Sven-Erik Behrens, Institute for Cancer Research, Fox ChaseCancer Center, Philadelphia, U.S.A.: „Host factors and RNA elements regulatethe replication of Flaviviridae“

22.04.2004 Dr. Stefan Pöhlmann, Institut für Klinische und MolekulareVirologie, Universität Erlangen-Nürnberg: „Functional analysis of the spike pro-tein of the SARS associated coronavirus“

27.04.2004 Dr. Holger Kissel, Laboratory of Apoptosis and Cancer Biology, TheRockefeller University, New York, U.S.A.: „Apoptosis without death: Insightsfrom the male germ line“

12.05.2004 Prof. Dr. Ursula Just, Abteilung für Biochemie, Christian-Albrechts-Universität Kiel: „Molecular mechanisms of lineage decisions mediated byNotch signalling in adult and embryonic hematopoietic development“

10.06.2004 Prof. Dr. Udo Schuhmacher, Institut für Anatomie, Universitäts-Krankenhaus Hamburg-Eppendorf: „Clinical relevant model systems for tumormetastasis by use of lectin-defined human tumor cells and immunodeficient mice“

07.07.2004 Dr. C.T. Bock, Abteilung für Molekulare Pathologie, Institut für Patho-logie, Universitätsklinikum Tübingen: „Molecular analysis of hepatitis C virus“

08.07.2004 Dr. Ralph Kehlenbach, Universität Heidelberg, Abteilung fürVirologie: „Analysis of nucleocytoplasmic protein and RNA transport“

15.07.2004 Prof Alexander Steinkasserer, Dermatologische Klinik und Poliklinik,Universitätsklinikum Erlangen: „Role of CD83 in the immune modulation ofdendritic cells“

125

10.08.2004 Prof. J. McCubrey, Department of Microbiology and Immunology,Brody School of Medicine at East Carolina University, Greenville NC, U.S.A.:„On the role of PI3/PTEN/Akt and Raf/MEK/ERK pathways in cancer“

19.08.2004 Prof. Dr. Jan Hengstler, Rudolf Böhm-Institut, Universität Leipzig:„Conditional expression of oncogenes: Are tumors reversible?“

01.09.2004 Dr. Frank Buchholz, Max-Planck-Institut, Dresden: „Large scale genefunction studies in mammalian cells using endoribonuclease prepared siRNAs“

23.09.2004 Dr. Gerald Bacher, Thera-Peptides GmbH, München: „CNI 1493:Activity of an anti-HIV compound in cancer“

20.10.2004 Prof. Dr. Clemens Schmitt, Charité, Berlin: „Cellular senescence inlymphoma development and therapy“

21.10.2004 PD Dr. Ingeborg Zehbe, Medizinische Mikrobiologie, JohannesGutenberg-Universität Mainz: „HPV genome variants in cervical tumorigene-sis“

12.11.2004 Dr. Jörg Baumann, HIV Drug Resistance Program, National CancerInstitute, Frederick MD, U.S.A.: „Characterization of cellular factors interferingwith HIV transmission and infection“

30.11.2004 Prof. Dr. Ourania Andrisani, Purdue University, Department of BasicMedical Sciences, West Lafayette, U.S.A.: „Mechanism(s) of hepatocarcinogene-sis by the hepatitis B virus X-protein“

02.12.2004 PD Dr. Annette Kaiser, Institut für Medizinische Parasitologie,Friedrich-Wilhelms-Universität Bonn: „Spermidine metabolism in Plasmodia“

07.12.2004 Dr. Ourania Andrisani, Purdue University, Department of BasicMedical Sciences, West Lafayette, U.S.A.: „Signal transduction mechanisms inthe differentiation model system of neural crest cells“

13.12.2004 Dr. Eberhard Hildt, Arbeitsgruppe Virusforschung, Robert-Koch-Institut Berlin: „Cell permeable proteins as novel tools for investigation of theviral life cycle“

Seminare

126

Drittmittelprojekte2003 – 2004

Internationale Förderung

„Risk-assessment of hemato- Carol Stockingpoitic stem cell (HSC) genes for leukemia induction“

„Molecular basis of HIV Ulrich Schubertlipodystrophy: Role of Vpr“.In Kooperation mit Baylor College

Europäische Förderung

„Manipulating tumor suppression: Wolfgang Depperta key to improve cancer treatment“

„Mutant p53 as target for improved Wolfgang Deppertcancer treatment“

„Optimizing the oxygen tension Hans Willfor in vitro cell work“

Koordination des EU-Projektes: Wolfgang Deppert„Mutant p53 gain of function activities as determinants for tumor prognosis and therapy“

Bundesförderung

„Entwicklung und Testung von neu- Hans Willartigen Medikamenten mit einem breiten Wirkspektrum (Breitbandvirostatika) zur Vorbeugung und Behandlung hochpathogener Virusinfektionen und zur Abwehr von Bioterror“

„Integrated genome wide approach Hans Willcombining population-based and experimental studies to identify pathways relevant to pathogenesis and drug development in most relevant human infections“. Standort innerhalb des Genomnetzes zu „Infektion/Entzündung“ (NGFN 1)

The Tokyo Metropolitan

Institute

National Institute of Health

Commission of the European Communities




Bundesministeriumfür Gesundheit

und soziale Sicherung

Bundesministeriumfür Bildung

und Forschung

127

Drittmittelprojekte

Bundesförderung

„Integrated genome wide approach Ulrich Schubert combining population-based and experimental studies to identify pathways relevant to pathogenesis and drug development in most relevant human infections“. Standort innerhalb des Genomnetzes zu „Infektion/Entzündung“ (NGFN 1)

„Analysis of hepatitis B virus cccDNA: Hans WillFormation, stability, role in pathogenesis and modulation by antiviral drugs“.Im Rahmen des Kompetenznetz Infektionskrankheiten Hepatitis (Hep-Net)

„Analyse von p53 abhängiger u. p53 Irene Dornreiterunabhängiger Reparatur strahlen-induzierter DNA Brüche während verschiedener Stadien des Zellzyklus“

„Der Zusammenhang von strahlen- Irene Dornreiterinduziertem Zelltod (Apoptose) und dem protektiven G2/M Arrest in menschlichen Zellen“

Bundesministeriumfür Bildung

und Forschung

Medizinische Hochschule Hannover

Bundesamtfür Strahlenschutz

Bundesamtfür Strahlenschutz

128

Drittmittelprojekte

Sonderforschungsbereich DFG

„Einfluss von mutiertem p53 auf die Wolfgang DeppertEntstehung und Progression von indu-zierbarem Mammakarzinom im Modell der transgenen Maus“. Sonderforschungs-bereich „Molekulare Mechanismen gene-tisch bedingter Erkrankungen“ (Hamburg)

Deutsche Forschungsgemeinschaft

„Interferon-induzierte Hans Willposttranskriptionelle Regulations-mechanismen“

„Combined CpG oligodeoxynucleotide/ Hans Willlipopeptide liposomes as a novel immunomodulatory vaccine against hepatitis C virus infection“

„Untersuchung Interferon-induzierter Hans WillHemmung von Hepatitis B Virus in vitro“

„Hemmung des Hepatitis B Virus Tilman Heise RNA-Abbaus durch das La-Protein in Hepatocyten“

„Modulation des NFAT-Transkriptions- Michael Brunsfaktors durch Hepatitis B-Viren und dessen Bedeutung für die virale Infektion“

„Molekulare Mechanismen der infektiösen Hüseyin SirmaAufnahme und des zellulären Transports von Hepatitis B-Viren und deren Bedeutung für den viralen Wirts-und Lebertropismus“

„Untersuchungen zur Regulation der Thomas HofmannZellproliferation durch den potenziellen Tumorsuppressor HIPK2 (Homeodomain-interacting protein kinase 2)“

„Spezifische Interaktion von Mutanten Wolfgang Deppertp53 mit MAR-DNA Elementen:Molekulare Basis der dominant-onkogenenWirkung von Mutanten p53“

SFB 545

129

Drittmittelprojekte

Deutsche Forschungsgemeinschaft

„Funktionelle Analyse der HIV-1 Rev Joachim HauberTransaktivierung“

„Molekulare Analyse von Konformations- Klaus Wiegersveränderungen viraler Proteine des HIV-1 Kapsids“

„Biochemische Charakterisierung der Frank FackelmayerSAF-Box, einer evolutionär konservierten SAR-spezifischen DNA-Bindedomäne“

„The role of a scaffold attachment factor Frank Fackelmayerin nuclear assembly and architecture“

„Die funktionelle und strukturelle Frank FackelmayerBedeutung der Arginin-Methylierung von Proteinen, am Beispiel des Scaffold Attachment Factors A (SAF-A)“

„Untersuchung von nativem biologischen Heinrich HohenbergMaterial und hydratisierten organischen Trägerschichten bei höchster Auflösung im Environmental-REM“

„Photogrammetrische Rekonstruktion Heinrich Hohenbergder Oberflächenform biologischer Objekte mittels REM-Bilddaten“

„Identifizierung von Zielproteinen und Klaus HarbersUntersuchungen zur Spezifität des Ubiquitin-konjugierenden Enzyms UbcM4“

„U-Box-Proteine und ihre Funktion im Klaus HarbersUbiquitin/Proteasom-System:Untersuchungen am Beispiel des nukleären U-Box-Proteins UIP5“

„Mechanismus der Entstehung Carol Stockingeiner Stammzell-Leukämie in MuLV-infizierten Mäusen“

„Mechanismen der Desensibilisierung Heinz Lothervon G-Protein-gekoppelten Rezeptoren“

130

Drittmittelprojekte

Deutsche Krebshilfe

„Studien zur Funktion von Hans Willzellulären und viralen Proteinen in ND-10 Kerndomänen bei akuter promyelozytischer Leukämie und anderen malignen Erkrankungen“

„Funktionsanalyse von SPOC1: Hans WillEin neuer mit der Entstehung und Progression von Ovarialkarzinom assoziierter Transkriptionsregulator und mögliches Therapieziel von Tumorerkrankungen“

„Funktionelle Analyse des Wolfgang DeppertTumorsuppressors p53“

„Analyse der Apoptoseresistenz Wolfgang Deppertin Wildtyp p53 exprimierenden Tumoren“

„Epigenetische Inaktivierung von p53 Wolfgang Bohndurch zytoplasmatische Verankerung“

„Regulation der Telomerase während Cagatay Günesder Entstehung und Progression von Mammakarzinomen in transgenen Mausmodellen“

„Kainatrezeptoren normaler Heinz Lotherund maligner hämatopoetischer Zellen“

131

Drittmittelprojekte

Förderung durch weitere Stiftungen

„Untersuchungen zur Rolle des Tilman HeiseOTT/MAL Fusionsproteins in der Entwicklung der jugendlichen akuten Megakaryozytenleukämie.“

„Untersuchungen zur Rolle des Tilman HeiseOTT Proteins in der Entwicklung neuronaler Zellen“

„Untersuchungen zur Rolle Thomas Hofmanndes potenziellen Tumorsuppressors HIPK2 bei der Kanzerogenese von Hirntumoren“

„Analyse von mutiertem p53 als Wolfgang DeppertProgressionsfaktor in einem WAP-mutp53 transgenen, induzierbaren Mausmodell für das Mammakarzinom“

„Identifizierung prognostischer, Wolfgang Deppertmolekularer Marker der Metastasierungdes Mammakarzinoms im Modell der transgenen Maus“

„Verlust der Wachstumskontrolle in Wolfgang Bohnprimären Glioblastomen des Menschen“

„Molekulare Analyse der CD83 Expression Joachim Hauberin Dendritischen Zellen“

„Die Rolle von AML1-ETO bei Carol Stockingder Entstehung akuter myeloischer Leukämie (AML): Untersuchungen mit Hilfe von Mausmodellen“

„Molekulare Determinanten der Hüseyin SirmaWirtsspezifität der Hepatitis B-Viren“

„Morphogenese und intrazellulärer Hüseyin SirmaTransport von Hepatitis B-Viren“

Deutsche José CarrerasLeukämie-Stiftung e.V.

Stiftung zur Bekämpfung neuroviraler

Erkrankungen

Stiftung zur Bekämpfung neuroviraler

Erkrankungen

Erich u. Gertrud Roggenbuck-Stiftung

Hamburger Stiftung zur Förderung derKrebsbekämpfung

Erich u. Gertrud Roggenbuck-Stiftung

Wilhelm Sander-Stiftung

Deutsche José CarrerasLeukämie-Stiftung e.V.

DAAD

Studienstiftung des deutschen Volkes

132

Drittmittelprojekte

Industriekooperationen

„Geometrical Calibration of Heinrich HohenbergDual Beam SEM and Accuracy of FIB Applications“

„Proteologics Phase I“ Ulrich Schubert

„Proteologics Phase II“ Ulrich Schubert

„Untersuchung der subzellulären Joachim HauberLokalisation sehr früher Genprodukte des humanen Herpesvirus Typ 1 und Typ 2 (Herpes Simplex-Virus 1 und 2)“

„Identification of relevant signal Joachim Haubertransduction targets and antiviral

compounds for HIV-1 therapy“

„Investigation of apoptosis in Joachim HauberHIV-1-infected cells and analysis of mode of action of selected antiretroviral compounds“

„Use of fin-needle biopsy and advanced Heinrich Hohenbergcryotechniques for microscopic characterization of skin and the potential use for other soft tissues and food systems“

„Analyse von Varianten Hans Willdes Hepatitis B-Virus“

Fei Electron Optics b.v.

Proteologics Inc.

Proteologics Inc.

BiomedForschungsges.mbH

AxximaPharmaceuticals AG

AxximaPharmaceuticals AG

NesTec

Dade-Behring

133

Finanzielle FörderungEndstand 2003

Einnahmen Personalkosten Sachkosten Investitionen Gesamtwert

Institutionelle Förderung 8.457.050 4.205.787 2.181.983 1.539.278 7.927.048

Summe Drittmittel 2.425.022 1.605.490 1.029.908 0 2.635.398

Gesamtes HPI 10.882.072 5.811.277 3.211.891 1.539.278 10.562.446

davon:

Bereich Grundfinanzierung 7.057.050 1.126.145 706.323 0 1.832.468Bereich Gebäude und Technik 1.400.000 250.759 837.461 787.587 1.875.808Bereich Wissenschaft 0 2.828.882 638.199 751.691 4.218.772

Summe Inst. Förderung 8.457.050 4.205.787 2.181.983 1.539.278 7.927.048

Drittmitteleinnahmen

MiGeb DFG 565.074 463.080 177.230 0 640.309MiGeb Diverse 13.387 13.161 6.497 0 19.658MiGeb EU 88.290 66.767 49.983 0 116.750MiGeb International 0 0 0 0 0MiGeb Krebshilfe 458.425 329.412 109.938 0 439.350MiGeb Öffentliche 725.424 454.788 339.806 0 794.595MiGeb Stiftungen 229.781 144.139 45.601 0 189.740MiGeb Unternehmen 344.640 134.144 300.852 0 434.996

Summe Drittmittel 2.425.022 1.605.490 1.029.907 0 2.635.398

134

Finanzielle FörderungVorläufiger Endstand 2004

Einnahmen Personalkosten Sachkosten Investitionen Gesamtwert

Institutionelle Förderung 9.748.141 4.212.166 2.177.693 2.607.178 8.997.036

Summe Drittmittel 3.042.038 1.673.201 941.091 0 2.614.291

Gesamtes HPI 12.790.179 5.885.367 3.118.783 2.607.178 11.611.328

davon:

BereichGrundfinanzierung 7.139.143 1.135.697 611.858 0 1.747.556Bereich Gebäude und Technik 2.608.998 278.624 782.384 1.857.965 2.918.974Bereich Wissenschaft 0 2.797.844 783.450 749.213 4.330.507

Summe Inst. Förderung 9.748.141 4.212.166 2.177.693 2.607.178 8.997.036

DrittmitteleinnahmenMiGeb DFG 683.098 443.759 189.613 0 633.372MiGeb Diverse 54.070 73.767 117.174 0 190.940MiGeb EU 565.058 93.018 237.018 0 330.036MiGeb International 0 0 0 0 0MiGeb Krebshilfe 494.265 396.123 92.272 0 488.395MiGeb Öffentliche 430.318 253.160 125.638 0 378.798MiGeb Stiftungen 543.846 322.401 74.963 0 397.364MiGeb Unternehmen 271.383 90.973 104.412 0 195.386

Summe Drittmittel 3.042.038 1.673.201 941.091 0 2.614.291

135

Die Verwaltung des Heinrich-Pette-Instituts befindet sich in einem Prozess derRestrukturierung ihrer internen Arbeitsabläufe und der Neubestimmung desVerhältnisses zur Wissenschaft.

Unter der Prämisse einer dienstleistungsorientierten Ausrichtung von Verwal-tungsprozessen werden die Schnittstellen zwischen Verwaltung und Wissenschaftneu definiert. Die Reorganisation der Verwaltung ist dabei einerseits durch einestärkere ökonomische Steuerung durch die Zuwendungsgeber (Programm-budgets) und einer zunehmend dezentralen Ressourcenbewirtschaftung der wis-senschaftlichen Abteilungen geprägt.

Dementsprechend ist in den letzten Jahren durch den Aufbau einer Kosten- undLeistungsrechnung ein zentrales Informationsinstrument aufgebaut worden, dasden Ressourcenverzehr und die internen Leistungsbeziehungen zwischen denAbteilungen und den Dienstleistern des Hauses dokumentiert.

In 2005 und 2006 wird die bestehende Haushalts- und Kostenstellenrechnung inRichtung einer Kostenträgerrechnung ausgebaut, so dass die zukünftigenProgrammbudgets kostenrechnerisch verarbeitet werden können. Durch denAufbau eines Informationsportals der Verwaltung ist hierfür in 2004 einInformationsmanagementsystem geschaffen worden, das die zeitnahe Kommuni-kation von Daten des Finanz- und Rechnungswesens mit den Informations-bedürfnissen der wissenschaftlichen Abteilungen ermöglicht.

Innerhalb der Personalabteilung werden zwei wesentliche Aufgabenschwerpunkteverfolgt. Einerseits wird durch den Aufbau einer komfortableren Personal-abrechnung die Integration der Personalabrechnung mit dem Finanz- undRechnungswesen verbessert und zum anderen wird durch die Schaffung einesPersonalmanagementsystems die Digitalisierung von Routineprozessen in derPersonalbeschaffung, -betreuung und -entwicklung angestrebt. Da sowohl die

VerwaltungLeiter: Dr. Volker Uhl

Die Mitarbeiter der Verwaltung des HPI

136

Verwaltung

Personalabrechnung als auch das Personalmanagementsystem mit den Systemender Finanzbuchhaltung kompatibel sind, ist eine weitgehende Integration desDatenflusses zwischen der Personalabteilung und dem Finanz- und Rechnungs-wesen verwirklicht. Aufwendige Abstimmungsprozesse und Mehrfacheingabenvon Daten werden somit abgebaut, so dass Personalstatistiken und -erhebungenzeitnaher dargestellt werden können. Für die Personalabteilung wird eineEntlastung von Routinetätigkeit angestrebt, so dass arbeitsrechtliche Service-funktionen und die Dienstleitung der Personalbetreuung ein größeres Gewichtgewinnen werden.

Im Bereich des Einkaufs ist für 2005 die Einrichtung einer webbasierten Ein-kaufssoftware geplant, wodurch der Arbeitsprozess im Einkauf neu gestaltet wer-den kann. Während bisher die Bedarfsanforderungen manuell in den wissen-schaftlichen Abteilungen erstellt wurden und erst im Einkauf eine elektronischeDatenerfassung der Bestellanforderung realisiert wurde, soll zukünftig dieBedarfsanforderung in den wissenschaftlichen Abteilungen elektronisch erfasstwerden. Für einkäuferische Tätigkeiten, wie das Aushandeln von Rahmen-verträgen und eine aktive Ausschreibungspolitik, können somit Ressourcen frei-gesetzt werden.

Für alle Abteilungen der Verwaltung bedeutet der Neubau des Ersatz- undErweiterungsbaus II eine besondere Herausforderung. Die Administration undbautechnische Überwachung dieses Sonderprojektes erfordert eine besondersenge Zusammenarbeit in der Verwaltung, die in einem abteilungsübergreifendenProjektteam erfüllt wird.

Mit dem Neubau ergibt sich darüber hinaus die Möglichkeit, das Energiekonzeptdes Heinrich-Pette-Instituts neu zu bestimmen. Die Analyse der bestehendenEnergieverbräuche hat dabei gezeigt, dass Einsparpotenziale in der Energie-versorgung bestehen. Von der Abteilung Haustechnik wird dieses zentrale Projektzur Kosteneinsparung bei den Betriebskosten realisiert.

137

VerwaltungBesondere Ereignisse im Zeitraum2003 / 2004

■ Prüfung des Bundesrechnungshofes zur „Einreihung der Angestellten in die Vergütungsstruktur“

■ Projektstart: „Personaldatenbank“■ Projektabschluss: „Digitales Vergabemanagement zur

elektronischen Bearbeitung von Beschaffungsprozessen“■ Erarbeitung des Raumbedarfs für den Ersatz- und Erweiterungsbau II

anhand von Bestands- und Funktionsanalysen

■ Wirtschaftsplanverhandlungen mit den Zuwendungsgebern für das Rechnungsjahr 2004

■ Begleitung der Erstellung der HU-Bau für den Ersatz- und Erweiterungsbau II

■ Aufstellung des Jahresabschlusses■ Beschluss des Hamburger Senats für den Ersatz-

und Erweiterungsbau II

■ Vertragsunterzeichnung mit der Fördergemeinschaft Kinder-Krebs-Zentrum Hamburg e.V.

■ Erste europaweite Ausschreibung für Investitionen im Gerätebereich

■ Erstellung eines Grobkonzepts über die Durchführung vonVerwaltungstätigkeiten in der Bauphase

■ Sozialversicherungsrechtliche Betriebsprüfung■ Projektstart: Web-Marktplatz zur internetgestützten Abwicklung von

Einkaufsprozessen

■ Wechsel der Abteilungsleitung im Personalbereich

■ Abschluss der Entwurfsplanung mit überarbeiteter Kostenberechnung für den Neubau

■ Einreichung des Antrags auf Baugenehmigung■ Durchführung eines Tages der offenen Tür in der Verwaltung am 20.11.2003

■ Abschluss der technischen Implementation des KLR-Projektes■ Kassenmäßiger Jahresabschluss

2003

Januar

Februar

März

April/Mai

Juni

Juli

August

September

Oktober

November

Dezember

■ Lohnsteuer-Außenprüfung durch die zuständige Finanzbehörde■ Wechsel des Lohn- und Gehaltsprogrammes

■ Wirtschaftsplanverhandlung für die Rechnungsjahre 2005 und 2006(Integration des Projektes „Bioterrorismus“ in den Institutshaushalt)

■ Aufstellung des Jahresabschlusses■ Präsentation der HPI-Verwaltungskomplettlösung auf der CeBit/

Public-Sector

■ Abschlussprüfung mit dem Wirtschaftsprüfer

■ 1. Workshop mit der Wissenschaft im Projekt „Programmbudgets“

■ Konzeption eines Info-Portals der Verwaltung für die Wissenschaft■ Einführung der Voice-Mailboxen ■ Projektstart eines webbasierten Personalmanagementsystems

■ Ausscheiden des Verwaltungsleiters

■ 2. Workshop mit der Wissenschaft im Projekt „Programmbudgets“■ Präsentation der Verwaltung auf dem Kongress „Innovatives Management“■ Projektstart: Energiestudie des Heinrich-Pette-Instituts

■ Einstellung einer Controllerin

■ Implementation der Systemdaten für die Aufbereitung von Programmbudgets■ Nachbesetzungsverfahren für die Verwaltungsleitung■ Grundsteinlegung Ersatz- und Erweiterungsbau II■ Sozialversicherungsrechtliche Prüfung der Versorgungsbezüge■ Reorganisation der Stickstoffversorgung und Erneuerung der Brutschränke

■ Kassenmäßiger Jahresabschluss■ Freigabe des Infoportals der Verwaltung für die Wissenschaft■ Vorläufiger Abschluss des Projektes „Programmbudgets“

2004

Januar

Februar

März

Mai

Juni

Juli

August

September

Oktober

November

Dezember

138

Verwaltung /Besondere Ereignisse

139

Personal der Verwaltungund Allgemeine Dienste

Verwaltung

Dr. Elmar Schultz bis August 2004 Jörn Weidt ab September 2004

Alexa Bäns,Regina Dickschas

Jörn WeidtDetlef Maetzing bis Juni 2004Sylvia BräuAnke WirdemannLilia SpannagelHelena Kuhn

Jörg SchinkelClaudia WienrankStefanie Fink

Bodo BrisséLiane KönigAnke Tepper

Carl-Heinz LohmIngo GregorzewskiOlaf NehlsMichael HabenichtPetra Plarre

Allgemeine Dienste

Dr. Jürgen Löhler

Dr. Norbert Zangenberg

Arne Düsedau

Dr. Angela Homfeld

Hasso MündAnke DorendorfBeate MikscheMatthias Timmermann

Leitung

Sekretariat

Finanz- undRechnungswesen

Personal-Abteilung

Einkauf

Technik undWarenannahme

Bibliothek

EDV

Wissenschaftliche Dienste

Öffentlichkeitsarbeit

Tierhaltung

140

141

Zusammenfassung derKostenträgerstruktur

142

143

144

145

Nach alter Bautradition wird neben anderen Dokumenten auch ein Bild von Edith und Heinrich Pettein den Grundstein der neuesten und größten baulichen Erweiterung des HPI eingelassen. Das neueGebäude wird genau an der Stelle erbaut, an der Heinrich Pette 1952 sein erstes Laboratoriums-gebäude errichtete. Das Heinrich-Pette-Institut steht somit für Tradition, Kontinuität und Erneuerunggleichermaßen.

146

Notizen

Documents

Tätigkeitsbericht - hpi-hamburg.de · A duck hepatitis B virus strain with a knockout ... Diese neue Struktur gewährleistet eine Output-orientierte Förderung auf der Basis einer