Wesierska-Gadek Józefa, Gueorguieva Marieta, Schloffer Daniela, Uhl Maria, Wojciechowski Jacek
Cell Cycle Regulation Group, Institute of Cancer Research, Faculty of Medicine, University of Vienna, Vienna, Austria.
J Cell Biochem. 2003 Aug 15;89(6):1222-34. doi: 10.1002/jcb.10586.
We examined the action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G(1) cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate.
我们研究了N-甲基-N'-硝基-N-亚硝基胍（MNNG）对HeLa细胞的作用，并将其与顺铂（CP）的作用进行了比较。MNNG在1小时内直接杀死了大量细胞，并导致强烈的DNA损伤，彗星试验证明了这一点。尽管出现了DNA损伤，但p53蛋白并未被激活。通过流式细胞术和Hoechst染色分析用MNNG处理1小时、3小时和6小时的HeLa细胞，分别未发现任何亚G(1)细胞群和凋亡特有的染色质浓缩/片段化。此外，未检测到凋亡典型的生化变化，如caspase-3的激活或细胞色素C从线粒体的释放。PARP-1的失活降低了MNNG施加的直接细胞毒性。我们的结果表明，尽管HeLa细胞短期暴露于MNNG后出现了严重的DNA损伤，但p53反应既未激活也未诱导凋亡，这意味着在HeLa细胞中产生强烈的DNA损伤不足以稳定p53蛋白。我们的数据明确表明，认真确定遗传毒性剂诱导的细胞死亡类型是必要的。需要基于至少几个独立标准来评估变化，以区分凋亡和坏死。由于烷化剂会产生DNA链断裂，因此采用基于DNA切割测定（如DNA梯状条带或TUNEL测定）的方法来评估凋亡是不够的。
