Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note.
Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note.
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作者：
S. Kaufmann
用拓扑异构酶II抑制剂依托泊苷处理人HL-60或KG 1A白血病细胞导致广泛的DNA降解。当通过琼脂糖凝胶电泳分析DNA完整性时，在向细胞中加入依托泊苷后1.5-2 h，核小体梯状条带变得明显，在6 h内强度增加，并持续到24 h。在加入药物后6小时，94 +/- 4%的细胞排除台盼蓝，即使多达90%的DNA已降解为寡核苷酸片段。将细胞暴露于10 μ g/ml（17 μ M）依托泊苷45分钟就足以在4小时后诱导这种DNA损伤。与二硝基苯酚预孵育消除了依托泊苷的作用，这表明在内切核酸切割之前或期间发生了一个需要能量的步骤。与此相反，依托泊苷的效果没有阻止HL-60细胞与RNA合成抑制剂5，6-二氯-1-β-呋喃核糖基苯并咪唑或蛋白质合成抑制剂放线菌酮或嘌呤霉素预孵育。相反，高浓度的5，6-二氯-1-β-呋喃核糖基苯并咪唑、放线菌酮或嘌呤霉素本身诱导了相同的核酸内切裂解，各种不同的细胞毒性剂，包括喜树碱（0.1 μ M）、秋水仙胺（0.1 μ g/ml）、顺铂（20 μ M）、甲氨蝶呤（1 μ M）和1-β-D-阿拉伯呋喃糖基胞嘧啶（3 μ M）也诱导了相同的核酸内切裂解。这些结果表明，由预先存在的细胞酶的核酸内切DNA损伤后不久发生的HL-60细胞与各种细胞毒性剂的任何治疗。观察到多种核蛋白[包括聚（ADP-核糖）聚合酶、核纤层蛋白B、拓扑异构酶I、拓扑异构酶II和组蛋白H1]与DNA片段化同时降解，这就对DNA降解过程的选择性提出了质疑。这些结果的影响（a）目前的理论，专注于核酸内切损伤的DNA作为一个关键的早期事件在细胞死亡，和（B）使用拓扑异构酶导向的药物来映射拓扑异构酶结合位点的活性染色质进行了讨论。
Treatment of human HL-60 or KG1A leukemia cells with the topoisomerase II inhibitor etoposide resulted in extensive DNA degradation. When DNA integrity was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident 1.5-2 h after addition of etoposide to cells, increased in intensity over 6 h, and persisted at 24 h. Six h after addition of the drug, 94 +/- 4% of the cells excluded trypan blue even though as much as 90% of the DNA had been degraded to oligosomal fragments. Exposure of cells to 10 micrograms/ml (17 microM) etoposide for as little as 45 min was sufficient to induce this DNA damage 4 h later. Preincubation with dinitrophenol abolished the effect of etoposide, suggesting that an energy-requiring step occurred prior to or during the endonucleolytic cleavage. In contrast, the effect of etoposide was not prevented by preincubation of HL-60 cells with the RNA synthesis inhibitor 5,6-dichloro-1-beta-ribofuranosylbenzimidazole or the protein synthesis inhibitors cycloheximide or puromycin. On the contrary, high concentrations of 5,6-dichloro-1-beta-ribofuranosylbenzimidazole, cycloheximide, or puromycin by themselves induced the same endonucleolytic cleavage, as did a variety of diverse cytotoxic agents, including camptothecin (0.1 microM), colcemid (0.1 microgram/ml), cis-platinum (20 microM), methotrexate (1 microM), and 1-beta-D-arabinofuranosylcytosine (3 microM). These results suggest that endonucleolytic DNA damage by a preexisting cellular enzyme occurs soon after treatment of HL-60 cells with any of a variety of cytotoxic agents. The observation that a variety of nuclear proteins [including poly(ADP-ribose) polymerase, lamin B, topoisomerase I, topoisomerase II, and histone H1] are degraded concomitant with the DNA fragmentation calls into question the selectivity of the degradative process for DNA. The implications of these results for (a) current theories which focus upon endonucleolytic damage of DNA as a critical early event during cell death, and (b) use of topoisomerase-directed drugs to map topoisomerase-binding sites in active chromatin are discussed.