Zhao Hong, Traganos Frank, Darzynkiewicz Zbigniew
Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA.
Cell Cycle. 2008 Oct;7(19):3048-55. doi: 10.4161/cc.7.19.6750. Epub 2008 Oct 6.
The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15(P)) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT-induced p53-Ser15(P) with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15(P) had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15(P) cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15(P) appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15(P) was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15(P) expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4-6 h. This maximum expression of p53-Ser15(P) coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1-2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation.
DNA拓扑异构酶I（topo1）抑制剂拓扑替康（TPT）和拓扑异构酶2抑制剂米托蒽醌（MXT）可损伤DNA，诱导DNA双链断裂（DSB）的形成。我们最近研究了用这些药物处理的单个人类肺腺癌A549细胞中，DNA损伤报告分子ATM和Chk2的激活动力学以及组蛋白H2AX的磷酸化情况。使用针对在Ser15位点磷酸化的肿瘤抑制蛋白p53（p53-Ser15(P)）的磷酸化特异性抗体，结合一种能检测无论磷酸化状态的p53的抗体以及多参数细胞术，我们将TPT和MXT诱导的p53-Ser15(P)与ATM和Chk2的激活以及与细胞周期阶段相关的H2AX磷酸化进行了关联。在未处理的间期细胞中，p53-Ser15(P)在整个核质中呈“斑驳状”定位，而有丝分裂细胞显示出强烈的p53-Ser15(P)细胞质免疫荧光（IF）。在未处理的有丝分裂细胞中看到的p53-Ser15的强烈磷酸化，与ATM和Chk2的激活（涉及中心粒）以及H2AX的磷酸化相结合，表明DNA损伤检测/修复机制在控制胞质分裂中发挥作用。在用TPT或MXT处理的细胞的细胞核中，p53-Ser15(P)的表达表现为紧密堆积的强烈IF焦点。TPT处理后，p53-Ser15(P)的诱导在S期细胞中最为明显，而在用MXT处理的细胞中未观察到明显的细胞周期阶段差异。在用TPT或MXT处理4-6小时的细胞中，观察到p53-Ser15(P)表达的最大增加，比其组成型表达水平高出2.5倍。p53-Ser15(P)的这种最大表达在时间上与Chk2激活的峰值一致，但与ATM激活和H2AX磷酸化不一致，后两者在TPT或MXT处理后1-2小时达到峰值。p53-Ser15磷酸化与ATM和Chk2激活的各自动力学表明，响应TPT或MXT引起的DNA损伤时，Chk2而非ATM介导p53磷酸化。
