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Apoptosis of human seminoma cells upon disruption of their microenvironment
R A Olie1, A W Boersma, M C Dekker
1Laboratory of Experimental Patho-Oncology, Dr Daniel den Hoed Cancer Center (Academic Hospital), Rotterdam, The Netherlands.
Abstract:
One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis. Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels. In contrast, three SEs with over 12% of 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 labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells. As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions. Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro.
Insights
Dissociating human seminoma cells for in vitro culture triggers apoptosis, causing massive cell degeneration. Preventing this apoptosis is key to successfully culturing these tumor cells.
Area of Science:
- Oncology
- Cell Biology
Background:
- Human seminoma (SE) cell culture in vitro is hindered by significant tumor cell degeneration.
- Understanding the mechanisms behind this degeneration is crucial for advancing SE research.
Purpose of the Study:
- To investigate if the dissociation of seminoma tumor tissue into single-cell suspensions induces apoptosis.
- To identify factors that may influence apoptosis onset during SE cell preparation for in vitro studies.
Main Methods:
- Morphological analysis and in situ end labeling to detect apoptosis in intact SE tissue and cell suspensions.
- Electrophoresis to identify DNA ladders indicative of apoptosis.
- Flow cytometry to quantify DNA breaks in SE cell suspensions.
Main Results:
- Less than 4% apoptotic cells in intact SE tissue from most samples; DNA ladders were absent.
- Three SEs with >12% apoptotic cells in intact tissue and all dissociated SE cell suspensions showed DNA ladders.
- Flow cytometry revealed DNA breaks in up to 85% of dissociated SE cells, indicating rapid apoptosis induction upon mechanical disruption.
Conclusions:
- Mechanical disruption of the tumor microenvironment rapidly induces apoptosis in human seminoma cells.
- The absence of p53 and bcl-2 expression suggests these pathways are not primary regulators of apoptosis in this context.
- Abrogating apoptosis is likely essential for successful in vitro culturing of human seminoma cells.