Lin X, Ramamurthi K, Mishima M, Kondo A, Christen R D, Howell S B
Department of Medicine and the Cancer Center, University of California, San Diego, La Jolla 92093-0058, USA.
Cancer Res. 2001 Feb 15;61(4):1508-16.
This study examined how the DNA mismatch repair (MMR) system and p53 interact to maintain genomic integrity in the presence of the mutagenic stress induced by cisplatin (DDP). Sensitivity to the cytotoxic and mutagenic effect of DDP was assessed using a panel of sublines of the MMR-deficient HCT116 colon carcinoma cells in which MMR function had been restored by transfer of a copy of MLH1 on chromosome 3 or in which p53 function had been disabled by expression of HPV-16 E6. Loss of p53 function by expression of E6 in MMR-proficient HCT116+ ch3 cells conferred only 1.1-2.0-fold resistance to a panel of commonly used chemotherapeutic agents, whereas disruption of p53 in MMR-deficient HCT116 cells resulted in substantial levels of resistance to some agents (paclitaxel, 1.9-fold; gemcitabine, 2.7-fold; 6-thioguanine, 3.3-fold; and etoposide, 4.4-fold) but sensitization to other agents (topotecan, 2.5-fold; and DDP, 3.3-fold). Loss of MMR or p53 alone had only a minor effect on sensitivity to the mutagenic effect of DDP as measured by the appearance of variants resistant to 6-thioguanine, etoposide, topotecan, gemcitabine, and paclitaxel in the population 10 days later (1.0-2.4-fold), whereas loss of both p53 and MMR had a more profound effect (1.7-6.5-fold). Loss of both p53 and MMR increased the basal frequency insertion/deletion mutations detected by a shuttle vector-based assay to a greater extent than loss of either alone. In association with DDP-induced injury, loss of p53 or MMR alone resulted in 1.2- and 1.7-fold more mutations, whereas loss of both resulted in a 5.1-fold increase in mutant frequency. Examination of the impact of loss of p53 and/or MMR on the DDP-induced cell cycle checkpoint activation, p53 induction, ability of the cell to tolerate adducts in its DNA, and the rate of disappearance of platinum from genomic DNA indicated the effects of the loss of p53 and/or MMR on all of these parameters, suggesting a multifactorial etiology for the changes in sensitivity to the cytotoxic and mutagenic effects of DDP. These results indicate that p53 and MMR can cooperate to control sensitivity to the cytotoxic effect of DDP and to limit its mutagenic potential in the colon cancer cells.
本研究探讨了在顺铂（DDP）诱导的诱变应激存在下，DNA错配修复（MMR）系统与p53如何相互作用以维持基因组完整性。使用一组MMR缺陷的HCT116结肠癌细胞亚系评估对DDP细胞毒性和诱变作用的敏感性，这些亚系中MMR功能已通过在3号染色体上转移MLH1拷贝得以恢复，或者p53功能已通过HPV-16 E6的表达而失活。在MMR功能正常的HCT116+ ch3细胞中通过表达E6使p53功能丧失，对一组常用化疗药物仅产生1.1至2.0倍的抗性，而在MMR缺陷的HCT116细胞中破坏p53导致对某些药物产生显著水平的抗性（紫杉醇，1.9倍；吉西他滨，2.7倍；6-硫鸟嘌呤，3.3倍；依托泊苷，4.4倍），但对其他药物敏感（拓扑替康，2.5倍；DDP，3.3倍）。单独缺失MMR或p53对DDP诱变作用的敏感性影响较小，通过10天后群体中对6-硫鸟嘌呤、依托泊苷、拓扑替康、吉西他滨和紫杉醇耐药变体的出现来衡量（1.0至2.4倍），而同时缺失p53和MMR则有更显著的影响（1.7至6.5倍）。与单独缺失p53或MMR相比，同时缺失p53和MMR使基于穿梭载体测定法检测到的碱基插入/缺失突变的基础频率增加得更多。与DDP诱导的损伤相关，单独缺失p53或MMR分别导致突变增加1.2倍和1.7倍，而同时缺失两者则导致突变频率增加5.1倍。研究p53和/或MMR缺失对DDP诱导的细胞周期检查点激活、p53诱导、细胞耐受其DNA中加合物的能力以及基因组DNA中铂消失速率的影响，表明p53和/或MMR缺失对所有这些参数均有影响，提示对DDP细胞毒性和诱变作用敏感性变化的多因素病因。这些结果表明，p53和MMR可以协同控制对DDP细胞毒性的敏感性，并限制其在结肠癌细胞中的诱变潜力。
