APOPTOSIS OF HUMAN SEMINOMA CELLS UPON DISRUPTION OF THEIR MICROENVIRONMENT

One of the main obstacles encountered when trying to culture human sem inoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain sing le-cell suspensions for in vitro culture, results in the onset of apop tosis. Using morphological analysis and ill situ end labelling, less t han 4% of apoptotic tumour cells were detected in intact tissue from 1 1 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not de tected on electrophoresis gels. In contrast, three SEs with over 12% o f apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions. obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders. Flow cytometric analysis of end lab elled SE suspensions showed DNA breaks in up to 85% of the tumour cell s. As indicated by cell morphology and DNA degradation, SE cells appea r to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inh ibitor bcl-2 was detectable in intact SE tissue or cell suspensions. O ur data suggest that abrogation of apoptosis might be crucial to succe ed in culturing human SE cells in vitro.