Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint.
Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint.
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发表时间：
1995-09
期刊：
影响因子：
11.2
通讯作者：
M. Hawn;A. Umar;J. Carethers;G. Marra;T. Kunkel;C. Boland;M. Koi
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文献类型：
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作者：
M. Hawn;A. Umar;J. Carethers;G. Marra;T. Kunkel;C. Boland;M. Koi
人结肠肿瘤细胞系HCT 116缺乏野生型hMLH 1，在错配修复（MMR）中有缺陷，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）有耐受性。将3号染色体上的正常hMLH 1拷贝转移到细胞系中，可恢复MMR活性，稳定微卫星位点，并增加细胞对MNNG的敏感性。先前在耐受烷化剂（如MNNG或N-甲基亚硝基脲）的其他细胞系中的研究显示了对6-硫代鸟嘌呤（6 TG）的交叉耐受性，导致对MNNG或6 TG的耐受性可能是MMR缺陷的结果的假设。为了验证这一假设，我们研究了6 TG对MNNG耐受、MMR缺陷的HCT 116细胞系及其MNNG敏感、MMR熟练、MNNG耐受和MMR缺陷衍生物的影响。连续暴露于低剂量的6 TG（0.31-1.25微克/毫升）对MNNG耐受的、MMR缺陷的细胞的集落形成能力（CFA）没有明显影响，而MNNG敏感的、MMR熟练的细胞表现出CFA的剂量依赖性降低。生长动力学和细胞周期分析显示，低剂量6 TG处理后，HCT 116 + chr 3细胞的生长被阻滞在G2期。相比之下，相同的暴露于6 TG没有诱导G2停滞，而是在HCT 116和HCT 116 + chr 2中诱导G1延迟。为了获得MMR对6 TG和MNNG毒性作用的进一步证据，我们从MNNG敏感、MMR熟练的HCT 116 + chr 3细胞系中分离出一个MNNG抗性回复突变体克隆M2，并表征了MMR活性、hMLH 1状态和6 TG应答。结果表明，M2细胞失去了MMR活性以及先前引入的正常hMLH 1基因。用低剂量的6 TG处理后，观察到M2的CFA恢复和G2期阻滞的消失。这些结果表明，错配修复系统与G2检查点相互作用，以响应6 TG或MNNG诱导的DNA损伤。结果进一步表明，任何诱导DNA错配的药物都会导致MMR阳性细胞的G2期阻滞，但不会导致MMR缺陷细胞的G2期阻滞。
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.