8 EXPRESSION OF SMALL HEAT SHOCK PROTEINS IN

RESPONSE TO STRESS, HOMONES AND ONCOGENES AND

IN HUMAN TUMORS

R. Klemenz (a.G.), B. Scheier (a.G.), A. Aoyama (a.G.)

Dept. of Pathology, University Hospital, CH-809l Zurich

Two small heat shock protein have been identified in mammals:

Hsp27 and aBcrystallin (aBc). Their genes are transcriptionally

activated by various fonns of stess and by the steroid hormones ~­

estradiol and glucocorticoides, respectively. We are interested in the

honnonal regulation of aBc expression. This gene is induced by

honnones in a delayed manner. A glucocorticoid honnone response

element has been identified in the promoter region by footprint

analyses. An additional promoter element which does not resemble

any known transcription factor binding site is essential for hormonal

induction. Sustained Ha-ras oncogene expression attenuates aBc

gene activation by glucocoritcoides.

Both augmented and reduced a Bc expression has been found

associated with many diseases. We observe strongly reduced amounts

of aBc in a large portion of analysed human breast carcinomas. In

contrast, the related stess protein Hsp27 accumulated in many of these

tumors.

Symposium II Molekulare Tumorbiologie und Diagnostik Molecular Tumor Biology and Diagnostics

9 Abstract not received

1 0 Abstract not received

11 Abstract not received

12 IN SITU-HYBRIDIZATION IN PATHOLOGY

H. Hofler

Institutes of Pathology TU Munich and GSF Neuherberg

The specific detection and localization of DNA and RNA sequences

in histological sections and cytological preparations employing in

situ hybridization techniques (ISH) represents already a routine

procedure in many laboratories. Its application, however, is still

restricted to few areas in diagnostic pathology: e. g. visualization of

viral DNA (CMV, HSV, HPV typing, etc.), detection of mRNA of

specific hormones in endocrine tumors, and rare situations when

specific antibodies for the immunohistochemical detection of

expression products do not exist.

Technical refinements in probe construction (e. g. ss antisense

DNA, strand specific oligonucleotide cocktails), probe labelling

Abstracts· 227

(digoxigenin), probe detection (autometallography) allow the

application of these techniques on formalin fixed and paraffin

embedded tissue.

The increased sensitivity is often associated with an increasing risk

of non specific reactions and implies the consequent use of time

consuming controls.

Methods merging ISH with other molecular techniques including in

situ PCR or in situ 3SR should currently be used for scientific

applications only.

13 Application of the Polymerase Chain Reaction (PCR) to Diagnostic and Experimental Pathology M. Dietel, Institut f. Pathologie, Charita, Humboldt-Universitat Berlin

peR produces an exponential increase of a 100-2000 bp nucleic acid template which flanking sequences have to be known. After multiple cycles of the enzyme driven reaction PCR allows the am­plification of extremely small amounts of DNA, down to single-copy. To perform PCR a DNA template, synthetic oligonucleotide pri­mers, nuclesoside triphosphates, a compatible buffer system and the thermostable Taq polymerase are required. The options to analyze PCR products are gel electrophoresis, dot blotting, denatu­ring gradient gels, etc. Strategies with coamplification of one tem­plate of known quantity and one of unknown quantity together in one tube opens the way for direct quantification of the PCR pro· duct. In many instances, PCR is one step of more complex analy· ses, like direct sequencing, detection of loss of heterozygosity or single strand conformation polymorphism. For pathologists, the possibility of employing PCR directly on tissue sections or cytological smears is of special importance. This proce­dure avoids cell destruction. It is called in situ PCR. The main applications to the field of pathology include • detection of genetic alterations related to cancer cells, e.g. bcr/ab/ translocation in myelodysplasia syndrom, amplification of the cyto­static drug resistance associated mdr1 gene, point mutations of ras genes and the p53 gene, T-cell receptor rearrangement etc., • detection of infectious non-eukaryontic DNA or RNA, e.g. from virus, bacteria, etc. - this is an upcoming field of in situ PCR- and • disclosure of heriditary diseases caused by genetic abnormalities. There exist several technical problems (contamination, unex­spected ssDNA "bubbles", nonspecific priming/annealing, etc.) which must be considered and the possibility of false negative but even more important of false positive results has always to be kept in mind, e.g. false positivity for HIV. Besides these restrictions PCR is an extremely powerful technique for the solution of many scien­tific and diagnostic questions and will for shure be introduced in the upcomming decade as a routine method of diagnostic pathology.