Hawn M T, Umar A, Carethers J M, Marra G, Kunkel T A, Boland C R, Koi M
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0586, USA.
Cancer Res. 1995 Sep 1;55(17):3721-5.
The human colon tumor cell line HCT116 is deficient in wild-type hMLH1, is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Transferring a normal copy of hMLH1 on chromosome 3 into the cell line restores MMR activity, stabilizes microsatellite loci, and increases the sensitivity of the cell to MNNG. Previous studies in other cell lines tolerant to alkylating agents such as MNNG or N-methylnitrosourea have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypothesis that tolerance to MNNG or 6TG may be the result of MMR deficiency. To test this hypothesis, we studied the effects of 6TG on the MNNG-tolerant, MMR-deficient HCT116 cell line and its MNNG-sensitive, MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Continuous exposure to low doses of 6TG (0.31-1.25 micrograms/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dose-dependent decrease in CFA. Growth kinetics and cell cycle analysis revealed that the growth of 6TG-treated HCT116 + chr3 cells was arrested at G2 after exposure to low dose of 6TG. In contrast, the same exposure to 6TG did not induce G2 arrest but rather a G1 delay in HCT116 and HCT116 + chr2. To obtain further evidence for the role of MMR on 6TG and MNNG toxicity, we isolated an MNNG-resistant revertant clone, M2, from the MNNG-sensitive, MMR-proficient HCT116 + chr3 cell line and characterized the MMR activity, hMLH1 status, and 6TG response. The results showed that M2 cells lost MMR activity as well as the previously introduced normal hMLH1 gene. Restoration of the CFA of M2 and an absence of G2 arrest were observed after treatment with low doses of 6TG. These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The results further suggest that any agent that induces DNA mispairs will cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells.
人结肠肿瘤细胞系HCT116缺乏野生型hMLH1，错配修复（MMR）存在缺陷，表现出微卫星不稳定性，并且对N-甲基-N'-硝基-N-亚硝基胍（MNNG）具有耐受性。将3号染色体上的hMLH1正常拷贝转入该细胞系可恢复MMR活性，稳定微卫星位点，并增加细胞对MNNG的敏感性。先前在其他对烷化剂如MNNG或N-甲基亚硝基脲具有耐受性的细胞系中的研究表明，对6-硫鸟嘌呤（6TG）存在交叉耐受性，从而产生了一种假说，即对MNNG或6TG的耐受性可能是MMR缺陷的结果。为了验证这一假说，我们研究了6TG对MNNG耐受、MMR缺陷的HCT116细胞系及其MNNG敏感、MMR功能正常、MNNG耐受和MMR缺陷的衍生物的影响。持续暴露于低剂量的6TG（0.31 - 1.25微克/毫升）对MNNG耐受、MMR缺陷的细胞的集落形成能力（CFA）没有明显影响，而MNNG敏感、MMR功能正常的细胞则表现出CFA呈剂量依赖性下降。生长动力学和细胞周期分析表明，低剂量6TG处理后的HCT116 + chr3细胞在暴露于6TG后其生长停滞在G2期。相反，相同的6TG暴露并未诱导HCT116和HCT116 + chr2细胞出现G2期停滞，而是导致G1期延迟。为了获得关于MMR在6TG和MNNG毒性作用中作用的进一步证据，我们从MNNG敏感、MMR功能正常的HCT116 + chr3细胞系中分离出一个MNNG抗性回复克隆M2，并对其MMR活性、hMLH1状态和6TG反应进行了表征。结果表明，M2细胞失去了MMR活性以及先前导入的正常hMLH1基因。用低剂量6TG处理后，观察到M2细胞的CFA恢复且未出现G2期停滞。这些结果表明，错配修复系统在响应6TG或MNNG诱导的DNA损伤时与G2检查点相互作用。结果还进一步表明，任何诱导DNA错配的试剂都会在MMR功能正常的细胞中导致G2期停滞，但在MMR缺陷的细胞中则不会。
