Clifford Brad, Beljin Milos, Stark George R, Taylor William R
Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Cancer Res. 2003 Jul 15;63(14):4074-81.
We have previously found that the overexpression of p53 causes G(2) arrest and represses the synthesis of cyclin-dependent kinase 1 and cyclin B1, two proteins required for cells to traverse from G(2) into M. G(2) arrest occurs in response to DNA damage caused by a variety of agents and treatments. Here, we investigate the role of p53 in the G(2) arrest that occurs in response to the topoisomerase inhibitors etoposide and merbarone. In HT1080 cells expressing a dominant-negative form of p53, treatment with etoposide still caused G(2) arrest, but the arrest could be overcome by additional treatment with caffeine, which inhibits the damage-responsive kinases ataxia telangiectasia mutated (ATM) and atm and rad3-related (ATR). However, caffeine could not overcome etoposide-induced G(2) arrest in HT1080 cells with functional p53. We conclude that etoposide activates two pathways, one of which depends on p53 and the other of which is sensitive to caffeine, and that either pathway is sufficient to activate G(2) arrest. Etoposide inhibits topoisomerase II by trapping the enzyme in a complex with cleaved DNA. Inhibition of topoisomerase II with merbarone, which does not stabilize a cleavage complex, causes G(2) arrest by a checkpoint that monitors the decatenation of chromatin. We find that caffeine can abrogate merbarone-induced G(2) arrest even in cells with functional p53, indicating that p53 does not contribute to the decatenation-sensitive response. Thus, p53 has a differential role in effecting G(2) arrest in response to topoisomerase II inhibitors, depending upon the mechanisms of action of the inhibitors tested.
我们之前发现，p53的过表达会导致G2期阻滞，并抑制细胞周期蛋白依赖性激酶1和细胞周期蛋白B1的合成，这两种蛋白是细胞从G2期进入M期所必需的。G2期阻滞是对多种因素和处理导致的DNA损伤作出的反应。在此，我们研究p53在拓扑异构酶抑制剂依托泊苷和美巴龙引起的G2期阻滞中的作用。在表达p53显性负性形式的HT1080细胞中，用依托泊苷处理仍会导致G2期阻滞，但额外用咖啡因处理可克服这种阻滞，咖啡因可抑制损伤反应激酶共济失调毛细血管扩张症突变基因（ATM）和ATM及Rad3相关蛋白（ATR）。然而，咖啡因不能克服依托泊苷在具有功能性p53的HT1080细胞中诱导的G2期阻滞。我们得出结论，依托泊苷激活两条途径，其中一条依赖p53，另一条对咖啡因敏感，且任何一条途径都足以激活G2期阻滞。依托泊苷通过将拓扑异构酶II捕获在与切割DNA的复合物中而抑制该酶。用美巴龙抑制拓扑异构酶II（美巴龙不会使切割复合物稳定），通过监测染色质解连环的检查点导致G2期阻滞。我们发现，即使在具有功能性p53的细胞中，咖啡因也能消除美巴龙诱导的G2期阻滞，这表明p53对解连环敏感反应没有作用。因此，根据所测试抑制剂的作用机制，p53在拓扑异构酶II抑制剂引起的G2期阻滞中发挥不同作用。
