Yan Tao, Desai Anand B, Jacobberger James W, Sramkoski R Michael, Loh Tamalette, Kinsella Timothy J
Department of Radiation Oncology, LTR 6068, University Hospitals of Cleveland/Ireland Cancer Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068, USA.
Mol Cancer Ther. 2004 Sep;3(9):1147-57.
The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 micromol/L x 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.
DNA错配修复（MMR）系统在介导多种化疗药物处理后的G2-M期检查点停滞及随后的细胞死亡过程中发挥着重要作用。在本研究中，我们使用6-硫鸟嘌呤（6-TG）作为错配诱导药物，研究共济失调毛细血管扩张症突变基因（ATM）/CHK2和ATM与Rad-3相关蛋白（ATR）/CHK1信号通路在错配修复功能正常的人结肠直肠癌RKO细胞的错配修复介导的细胞周期反应中的作用。我们发现，在6-TG（3 μmol/L×24小时）处理后，在错配修复介导的长达6天的细胞周期停滞延长过程中，CHK1在Ser317位点的激活磷酸化[CHK1（pS317）]和CHK2在Thr68位点的激活磷酸化[CHK2（pT68）]被差异性诱导，CHK1（pS317）在1天内被诱导，而CHK2（pT68）随后被诱导。使用信号激酶的化学抑制剂和小干扰RNA，我们表明错配修复介导的6-TG诱导的G2期停滞依赖于ATR/CHK1，而不依赖于ATM/CHK2，并且ATR/CHK1信号通路负责G2期停滞的起始和维持。然而，CHK2（pT68）似乎参与随后的四倍体G1期停滞，这会阻止6-TG处理后从G2-M检查点逃逸的细胞。此外，我们表明6-TG处理后较晚时间CHK2发生过度磷酸化，并且CHK2的磷酸化似乎不依赖于ATM，但在ATR或CHK1减少时上调。因此，我们的数据表明CHK1（pS317）参与错配修复介导的6-TG诱导的G2期停滞，而CHK2（pT68）似乎参与随后的四倍体G1-S检查点。这两种信号激酶似乎协同作用以确保6-TG损伤的细胞在这些细胞周期检查点停滞。
