Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.
Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.
复制标题
研究药物诱导细胞凋亡中观察到的高阶染色质断裂机制。
作者：
H. Beere;C. Chresta;A. Alejo;A. Składanowski;C. Dive;A. Larsen;J. Hickman
细胞凋亡的特征是DNA的非随机裂解。在用拓扑异构酶II抑制剂依托泊苷（50 μ M）和非遗传毒性剂N-甲基甲酰胺（300 mM）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。用常规凝胶电泳分析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的DNA解析显示50个酶（kb）的片段。依托泊苷诱导了一条> 600 kb的额外DNA条带的短暂出现，这与DNA-蛋白质复合物的形成在时间上一致，并且在药物去除后迅速可逆。在N-甲基甲酰胺处理后未观察到该DNA条带。原位DNA末端标记显示生物素化的dUTP掺入到50-kb的DNA片段中，但没有依托泊苷诱导的> 600 kb的DNA片段。因此，用末端脱氧核苷酸转移酶标记的DNA末端不依赖于核小体间DNA切割，并且约50 kb的片段的特征在于未被拓扑异构酶II蛋白封闭的游离3 '-OH末端。虽然我们认为拓扑异构酶II在细胞凋亡过程中对高级染色质的断裂可能起积极作用，但结果表明，拓扑异构酶II的DNA切割诱导了> 600 kb的可逆的蛋白质相关片段，而不是不可逆的50 kb片段切割。DNA可逆切割成> 600 kb的片段似乎是参与细胞凋亡的信号，而不是染色质顺序解旋的初始步骤。
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.