Heiger-Bernays W J, Essigmann J M, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1990 Sep 11;29(36):8461-6. doi: 10.1021/bi00488a037.
An SV40-based in vitro replication system has been used to examine the effects of platinum compounds on eukaryotic DNA replication. Plasmid templates containing the SV40 origin of replication were modified with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) or the inactive analogues [Pt(dien)Cl]+ and trans-DDP. The platinated plasmids were used as templates for DNA synthesis by the DNA polymerases present in cytosolic extracts prepared from human cell lines HeLa and 293. Bifunctional adducts formed by cis- and trans-DDP inhibited DNA replication by 95% at a bound drug to nucleotide ratio [(D/N)b] of less than 9 x 10(-4), in contrast to the monofunctional [Pt(dien)Cl]+ analogues, which required a (D/N)b of 3.4 x 10(-3) for 62% inhibition of DNA replication. An average of two platinum adducts per genome was sufficient for inhibition of DNA replication by cisplatin. When trans-DDP-modified, but not cis-DDP-modified, SV40 origin containing plasmids [(D/N)b = 1.7 x 10(-3)] were allowed to incubate in the 293 cytosolic extracts for 1 h prior to addition of T-antigen to initiate replication, DNA synthesis was restored to 30% of control. This result suggested the presence of an activity in the extracts that reactivates trans-DDP-modified DNA templates for replication. This hypothesis was confirmed by an in vitro nucleotide excision repair assay that revealed activity in 293 and HeLa cell extracts selective for trans-DDP-modified plasmid DNAs. Such selective repair of trans-DDP-damaged DNA in human cells would contribute to its lack of antitumor activity.
一种基于猿猴病毒40（SV40）的体外复制系统已被用于研究铂化合物对真核生物DNA复制的影响。含有SV40复制起点的质粒模板用抗癌药物顺二氯二氨铂（II）（顺铂，cis-DDP）或无活性类似物[Pt(dien)Cl]+和反式-DDP进行修饰。这些铂化质粒被用作模板，供从人细胞系HeLa和293制备的胞质提取物中的DNA聚合酶进行DNA合成。顺铂和反式-DDP形成的双功能加合物在结合药物与核苷酸的比例[（D/N）b]小于9×10^(-4)时，可抑制DNA复制95%，相比之下，单功能的[Pt(dien)Cl]+类似物在（D/N）b为3.4×10^(-3)时，可抑制DNA复制62%。每个基因组平均两个铂加合物就足以抑制顺铂的DNA复制。当含有反式-DDP修饰而非顺铂修饰的SV40复制起点的质粒[（D/N）b = 1.7×10^(-3)]在加入T抗原启动复制之前，在293胞质提取物中孵育1小时后，DNA合成恢复到对照的30%。这一结果表明提取物中存在一种活性，可使反式-DDP修饰的DNA模板重新激活用于复制。体外核苷酸切除修复试验证实了这一假设，该试验显示293和HeLa细胞提取物中存在对反式-DDP修饰的质粒DNA具有选择性的活性。人类细胞中对反式-DDP损伤DNA的这种选择性修复将导致其缺乏抗肿瘤活性。
