Path. Res. Pract. 190,226-307 (1994)

Symposium I Molekularpathologie-Mode? Notwendigkeit? Molecular Pathology - Fashion or Necessity?

1 Abstract not received

2 Abstract not received

3 RELEVANCE OF ACTIVE CELL DEATH (APOPTOSIS) IN TUMOR FORMATION R. Schulte-Hermann , Bettina Grasl-Kraupp, Franziska Oberhammer, W.Bursch Institut fur Tumorbiologie-Krebsforschung, Universitat Wien

Active (or programmed) cell death seems to occur in at least two different morphological types characterized by cell shrinkage, chroma­tin condensation at the nuclear membrane, and cell fragmentation (i.e. apoptosis), or by lysosomal activation and autophagic cell degradation. Both types of cell death are under the control of the growth regulatory system in the organism. Thus, transforming growth factor 81 has been found to be a trigger for apoptosis in the liver.

Active cell death seems to be important in various stages of carcinogenesis. Mathematical and biological findings suggest that initiated cells can be eliminated by active cell death. Thus initiation, at the cellular level, may not always be completely irreversible. In the pro­motion stage of hepatocarcinogenesis, the rate of cell death in putative preneoplastic foci is reduced by tumor promoters which thereby accelerate foci growth and tumor formation. In hormone-dependent tumor cells induction of apoptosis can be triggered by hormone with­drawal or anti-hormones. Fasting seems to induce apoptosis preferentially in putative preneoplastic cells and thereby may protect from cancer development.

4 EPITHELIAL CELL PLASTICITY IN

CARCINOMA. THIERY, J.P., BELLUSCI, S., BOYER, B., DELOUVEE, A., JOUANNEAU, J., RADVANYI, F., SAVAGNER, P., MOENS, G., and VALLES, A.-M. Laboratoire de Physiopathologie du Developpement, CNRS and Ecole Normale Superieure 46, rue d'Ulm, 75230 Paris Cedex 05 France.

A rat bladder carcinoma has been used as a model system to study the conversion of an epithelial to a migratory fibroblast­like state. This morphological transformation is triggered by collagens but not by fibronectin or laminin. A similar conversion is induced by acidic Fibroblast Growth Factor (aFGF) in subconfluent cultures while this multifunctional growth factor acts as a mitogen on high density cultures. In low density cultures, aFGF and several other growth factors acting through tyrosine kinase recept6rs induce a rapid internalization of desmosomes, a major adhesive structure of epithelia. The newly

0344-0338/9410 190-0226$3.5010

Abstracts

formed fibroblasts progressively lose their cytokeratins which are replaced by vimentin intermediate filaments. The transformation is fully reversible upon removal of the growth factor. Acidic FGF also triggers cell motility and production of gelatinases. On collagen substrates, the speed of locomotion is enhanced in the presence of aFGF and under these conditions the bladder carcinoma cells readily invade 3D collagen gels. The bladder carcinoma line can also become fibroblastic after transfection with an expression vector coding for aFGF, most likely through an autocrine mechanism. Clones producing the growth factor can efficiently invade rat bladders maintained in organotypic cultures. These clones grow much more rapidly in nude mice than the original cell line and micrometastases are detected in less than two weeks following subcutaneous injection. Thus, this model system may offer a unique opportunity to evaluate the role of growth factors in the triggering of carcinoma invasion and metastasis.

5 Abstract not received

6 Abstract not received

7 MOLECULAR PATHOLOGY OF HUMAN NEUROBLASTOMA Manfred Schwab Abteilung Zytogenetik - 0130, Deutsches Krebsforschungs­zentrum, D-69120 Heidelberg

Today it is widely accepted that alterations of cellular genes playa pivotal role in the development of cancer, both in animals and humans. The analysis of cancer genes, is currently pursued under three perspectives. First, the spectrum of genetic alterations in different tumor types has to be identified, and it should be examined which of the alterations are non-random for specific tumor types; in extension, oncogenes or tumor suppressor genes as well as the proteins they encode and their biochemical functions in normal and malignant cells have to be defined. Second, it is important to find out if individual non-random alterations are associated with particular tumor phenotypes and if they can be employed in clinical context as parameters in presymptomatic early detection of cancer diseases or if they might be of prognostic value. And third, as a future perspective new strategies of somatic gene therapy might be developed that are based on the knowledge of functions of oncogenes and tumor suppressor genes.

The development of neuroblastoma, a pediatric cancer, is often associated with two non-random genetic alterations, amplification of the oncogene N-myc, and loss of a putative tumor suppressor gene from chromosome 1. N-myc amplification is significantly correlated with poor prognosis. Amplification is associated with elevated expression, both of mRNA and protein. N-myc encodes two nuclear polypeptides of relative masses of 62 and 64 kilodaltons, which act most likely at transcription factors. - The most frequent genetic alteration in neuroblastomas involves rearrangement or deletion of the short arm of chromosome I in band 36.1-2. The specificity and frequency of this alteration, along with the fact that it has been observed in otherwise near dipolid settings suggest an important role in the development of neuroblastomas.

© 1994 by Gustav Fischer Verlag, Stuttgart

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.