Beere H M, Chresta C M, Alejo-Herberg A, Skladanowski A, Dive C, Larsen A K, Hickman J A
Cancer Research Campaign Molecular and Cellular Pharmacology Group, School of Biological Sciences, University of Manchester, UK.
Mol Pharmacol. 1995 May;47(5):986-96.
Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.
细胞凋亡的特征是DNA的非随机切割。在用拓扑异构酶II抑制剂依托泊苷（50微摩尔）和非基因毒性剂N-甲基甲酰胺（300毫摩尔）连续处理MOLT-4人T淋巴母细胞样细胞后，通过电子显微镜证实了细胞凋亡。通过传统凝胶电泳分析DNA完整性未能检测到核小体间DNA切割。通过场反转凝胶电泳分离DNA显示出50千碱基（kb）的片段。依托泊苷诱导出现一条大于600 kb的额外DNA条带，其在时间上与DNA-蛋白质复合物形成一致，并且在去除药物后迅速可逆。在N-甲基甲酰胺处理后未观察到这条DNA条带。原位DNA末端标记显示生物素化的dUTP掺入到50-kb DNA片段中，但未掺入依托泊苷诱导的大于600 kb的DNA片段中。因此，用末端脱氧核苷酸转移酶进行的DNA末端标记不依赖于核小体间DNA切割，并且大约50 kb的片段具有未被拓扑异构酶II蛋白封闭的游离3'-OH末端。尽管我们认为拓扑异构酶II可能在细胞凋亡过程中高阶染色质的片段化中发挥积极作用，但结果表明拓扑异构酶II诱导的DNA切割产生了大于600 kb的可逆的、与蛋白质相关的片段，而不是不可逆地切割成50-kb片段。DNA可逆切割成大于600 kb的片段似乎是细胞凋亡启动的信号，而不是染色质顺序解旋的初始步骤。
