Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.
Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.
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发表时间：
2001-11
期刊：
影响因子：
11.2
通讯作者：
T. Yan;J. Schupp;H. Hwang;M. Wagner;S. E. Berry;S. Strickfaden;M. Veigl;W. D. Sedwick;D. Boothman;T. Kinsella
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文献类型：
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作者：
T. Yan;J. Schupp;H. Hwang;M. Wagner;S. E. Berry;S. Strickfaden;M. Veigl;W. D. Sedwick;D. Boothman;T. Kinsella
我们之前的数据表明，在高剂量率电离辐射（IR）后，缺乏MutL同源物-1 （MLH1）表达的细胞G(2)阻滞减少且时间缩短，这表明错配修复（MMR）系统介导了这种细胞周期检查点。我们使用另外两个等基因匹配的人类MLH1 （hMLH1）缺陷和成熟的人类肿瘤细胞系统证实了这一观察结果：人类卵巢癌细胞A2780/CP70，有或没有异位表达hMLH1，人类结直肠癌细胞RKO，有或没有阿扎胞苷治疗来重新表达hMLH1。我们还检测了匹配的MutS同源物-2 （hMSH2）缺陷和精通的人子宫内膜癌he59细胞系，以确定hMSH2和MMR是否参与ir相关的G(2)阻滞反应。与mlh1缺陷细胞一样，缺乏hMSH2的细胞在IR （6 Gy）下表现出类似的G(2)阻滞改变。无论同步细胞是在G(0)/G(1)期还是S期辐照，MMR熟练细胞和缺陷细胞在红外诱导的G(2)阻滞方面都存在差异，这表明MMR确实显著影响G(2)-M检查点阻滞。然而，与6-硫鸟嘌呤暴露的mmr依赖性损伤耐受性反应不同，在高剂量率IR后，mmr缺陷细胞与mmr正常细胞的克隆存活没有显著差异。为了确定mmr介导的G(2)阻滞的信号转导机制，我们检测了cdc2 （phospho-Tyr15-cdc2）的酪氨酸15磷酸化水平，cdc2是G(2)-M转变的关键调节因子。IR后，在mmr熟练和缺陷细胞系中均观察到磷酸化tyr15 -cdc2水平升高。然而，在MMR （hMLH1或hMSH2）缺陷细胞系中，磷酸化tyr15 -cdc2的水平在从ir诱导的G(2)阻滞到M期的过程中迅速下降。因此，高剂量率IR后磷酸化tyr15 -cdc2水平的差异与观察到的IR诱导的G(2)阻滞的差异在时间上是一致的，这表明MMR蛋白可能通过cdc2信号通路对IR诱导的G(2)阻滞发挥作用。尽管MMR状态对高剂量率IR后的细胞存活没有显著影响，但它似乎调节G(2)-M检查点，并可能影响总体突变率。
Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.