Winters Z E, Ongkeko W M, Harris A L, Norbury C J
Imperial Cancer Research Fund, Molecular Oncology Laboratory, University of Oxford Institute of Molecular Medicine, John Radcliffe Hospital, UK.
Oncogene. 1998 Aug 13;17(6):673-84. doi: 10.1038/sj.onc.1201991.
We have investigated the influence of p53 on radiation-induced G2 cell cycle arrest using human H1299 cells expressing temperature-sensitive p53. Gamma-irradiated cells lacking p53 arrested transiently in G2 with Cdc2 extensively phosphorylated at the inhibitory sites Thr14 and Tyr15, and with both Cdc2 and cyclin B1 restricted to the cytoplasm. Activation of p53 by temperature shift resulted in a more protracted G2 arrest that could not be overridden by checkpoint-abrogating drugs. Surprisingly, this enhancement of G2 arrest was associated with a marked lack of inhibitory phosphorylation of Cdc2 and with the nuclear localization of both Cdc2 and cyclin B1. While transient expression of an A14F15 mutant form of Cdc2 that is not subject to inhibitory phosphorylation induced mitotic catastrophe in cells lacking p53, the p53-expressing cells were relatively refractory to this effect. Enforced expression of p21WAF1/CJP1 was sufficient to confer nuclear localization on Cdc2 in the p53 null cells, though immunodepletion experiments demonstrated that only a small proportion of Cdc2 was stably associated with p21WAF1/CJP1 in the p53-expressing cells. We conclude that a p53-dependent pathway can operate after exposure of human cells to ionising radiation to promote G2 arrest accompanied by nuclear translocation rather than inhibitory phosphorylation of Cdc2.
我们利用表达温度敏感型p53的人H1299细胞，研究了p53对辐射诱导的G2期细胞周期阻滞的影响。缺乏p53的γ射线照射细胞在G2期短暂停滞，Cdc2在抑制位点Thr14和Tyr15处广泛磷酸化，且Cdc2和细胞周期蛋白B1均局限于细胞质中。通过温度转换激活p53会导致更长时间的G2期阻滞，这种阻滞不能被消除检查点的药物所克服。令人惊讶的是，这种G2期阻滞的增强与Cdc2抑制性磷酸化的显著缺乏以及Cdc2和细胞周期蛋白B1的核定位有关。虽然在缺乏p53的细胞中，不发生抑制性磷酸化的Cdc2的A14F15突变体形式的瞬时表达会诱导有丝分裂灾难，但表达p53的细胞对这种效应相对不敏感。在p53缺失的细胞中，强制表达p21WAF1/CJP1足以使Cdc2定位于细胞核，尽管免疫去除实验表明，在表达p53的细胞中，只有一小部分Cdc2与p21WAF1/CJP1稳定结合。我们得出结论，在人类细胞暴露于电离辐射后，一条p53依赖的途径可以发挥作用，促进G2期阻滞，同时伴有Cdc2的核转位而非抑制性磷酸化。
