Davis T W, Wilson-Van Patten C, Meyers M, Kunugi K A, Cuthill S, Reznikoff C, Garces C, Boland C R, Kinsella T J, Fishel R, Boothman D A
Department of Human Oncology, University of Wisconsin-Madison, 53792, USA.
Cancer Res. 1998 Feb 15;58(4):767-78.
A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G1 arrest and/or G1-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1+) compared with HCT116 (MLH1-) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G2-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G1, after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazole-induced G2-M block, which corrected the MLH1-mediated G2-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G1 arrest or G1-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G1 cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.
先前有研究表明，Mut L同源蛋白1（MLH1）作为DNA错配修复（MMR）的必要组成部分，在6-硫鸟嘌呤（6-TG）暴露后的G2-M细胞周期检查点停滞中发挥作用。然而，MLH1在损伤后的G1期停滞和/或G1-S期转换中的潜在作用并未被排除。我们报告称，与基因匹配的、MMR校正的人结肠癌（HCT116 3-6）细胞相比，MLH1缺陷的人结肠癌细胞（HCT116）在电离辐射（IR）后存活率降低，同时在G2-M细胞周期检查点停滞方面存在缺陷。与野生型原代胚胎成纤维细胞相比，小鼠MLH1基因敲除细胞也有类似反应。MMR缺陷的HCT116细胞或来自MLH1基因敲除小鼠的胚胎成纤维细胞在6-TG暴露后也表现出典型的DNA损伤耐受反应。有趣的是，与HCT116（MLH1-）细胞相比，在IR或6-TG处理后，HCT116 3-6（MLH1+）细胞中观察到p53蛋白诱导反应增强。然而，逆转录病毒载体介导的E6蛋白表达并未影响与MLH1缺陷的HCT116细胞相比，HCT116 3-6细胞中观察到的增强的G2-M细胞周期停滞。照射后的MLH1缺陷和MLH1正常的人或小鼠细胞之间相似的S期进程也表明，IR后MLH1在G2-M细胞周期检查点控制中发挥作用，而不改变G1期。引入诺考达唑诱导的G2-M阻滞可纠正HCT116细胞中MLH1介导的G2-M停滞缺陷，这清楚地表明，IR后HCT116和HCT116 3-6细胞在G1期停滞或G1-S细胞周期转换方面没有差异。因此，我们的数据表明，MLH1在G1细胞周期转换或停滞中不发挥主要作用。我们还表明，人MLH1和MSH2的稳态蛋白水平不会因IR或6-TG引起的损伤或细胞周期变化而改变。MLH1介导的G2-M细胞周期延迟（由DNA损伤的MMR校对或MLH1蛋白在细胞周期停滞中的直接功能引起）可能对染色体分离前的DNA损伤检测和修复很重要，以消除子细胞中的致癌性损伤（可能由错配修复引起）。
