Olie R A, Boersma A W, Dekker M C, Nooter K, Looijenga L H, Oosterhuis J W
Laboratory of Experimental Patho-Oncology, Dr Daniel den Hoed Cancer Center (Academic Hospital), Rotterdam, The Netherlands.
Br J Cancer. 1996 May;73(9):1031-6. doi: 10.1038/bjc.1996.200.
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.
在体外培养人精原细胞瘤（SE）细胞时遇到的主要障碍之一是肿瘤细胞的大量退化。我们研究了将肿瘤组织解离以获得用于体外培养的单细胞悬液是否会导致细胞凋亡的发生。通过形态学分析和原位末端标记，在14例SE中的11例完整组织中检测到不到4%的凋亡肿瘤细胞。在这11个肿瘤中，电泳凝胶上未检测到指示核小体间双链DNA断裂的凋亡特异性DNA梯带。相反，在完整组织中凋亡肿瘤细胞超过12%的3例SE以及所有经分析的（纯）SE细胞悬液（通过完整肿瘤组织机械解离获得）中均显示出DNA梯带。对末端标记的SE悬液进行流式细胞术分析显示，高达85%的肿瘤细胞存在DNA断裂。从细胞形态和DNA降解情况来看，SE细胞在其微环境受到机械破坏后似乎会迅速进入凋亡途径。在完整的SE组织或细胞悬液中未检测到p53和凋亡抑制因子bcl-2的表达。我们的数据表明，消除凋亡可能是成功在体外培养人SE细胞的关键。
