Hsiang Y H, Liu L F
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Cancer Res. 1988 Apr 1;48(7):1722-6.
Camptothecin, a plant alkaloid with antitumor activity, has been shown to be a potent inhibitor of nucleic acid synthesis and a strong inducer of DNA strand breaks in mammalian cells. Previous studies have shown that camptothecin inhibits purified mammalian DNA topoisomerase I by trapping a reversible enzyme-DNA "cleavable complex" (Hsiang et al., J. Biol. Chem., 260: 14873-14878, 1985). Our present studies, using L1210 cells and SV40-infected monkey cells, have shown that camptothecin-induced strand breaks are protein linked. The linked protein is most likely DNA topoisomerase I as revealed by immunoblot analysis, using antibodies against purified mammalian DNA topoisomerase I. Brief heating of camptothecin-treated cells to 65 degrees C resulted in a rapid reduction of the number of protein-linked DNA breaks. Reversal of the camptothecin-induced topoisomerase I-DNA complex by heat was also observed in an in vitro system by using purified mammalian DNA topoisomerase I. Our results suggest that camptothecin interferes with DNA topoisomerase I both in cultured mammalian cells and in the purified system by trapping a reversible enzyme-DNA cleavable complex.
喜树碱是一种具有抗肿瘤活性的植物生物碱，已被证明是核酸合成的有效抑制剂，也是哺乳动物细胞中DNA链断裂的强力诱导剂。先前的研究表明，喜树碱通过捕获一种可逆的酶-DNA“可裂解复合物”来抑制纯化的哺乳动物DNA拓扑异构酶I（Hsiang等人，《生物化学杂志》，260: 14873 - 14878，1985）。我们目前使用L1210细胞和SV40感染的猴细胞进行的研究表明，喜树碱诱导的链断裂是与蛋白质相连的。通过使用针对纯化的哺乳动物DNA拓扑异构酶I的抗体进行免疫印迹分析发现，相连的蛋白质很可能是DNA拓扑异构酶I。将喜树碱处理的细胞短暂加热至65摄氏度导致蛋白质相连的DNA断裂数量迅速减少。在体外系统中，通过使用纯化的哺乳动物DNA拓扑异构酶I，也观察到了热对喜树碱诱导的拓扑异构酶I-DNA复合物的逆转作用。我们的结果表明，喜树碱通过捕获一种可逆的酶-DNA可裂解复合物，在培养的哺乳动物细胞和纯化系统中均干扰DNA拓扑异构酶I。
