Activation of DNA damage response signaling in mouse embryonic stem cells
Activation of DNA damage response signaling in mouse embryonic stem cells
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DOI：
10.4161/cc.7.18.6699
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发表时间：
2008-09-15
期刊：
影响因子：
4.3
通讯作者：
Pospelov, Valery A.
中科院分区：
文献类型：
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作者：
Chuykin, Ilya A.;Lianguzova, Maria S.;Pospelov, Valery A.
小鼠胚胎干细胞（mESC）具有高增殖活性。mESC对基因毒性应激高度敏感，并且在dna损伤时不经过G(1)/S检查点。mESC被认为是通过DNA损伤修复或通过凋亡消除缺陷细胞来维持基因组完整性的敏感机制。mESC如何识别损伤并执行DNA修复的问题仍有待研究。我们分析了由磷酸化的ATM激酶和组蛋白H2AX （γ H2AX）抗体标记的DNA修复病灶的动力学。我们发现mESC显示非诱导DNA单链断裂（SSBs），如彗星测定所示，并且具有明显的伽马H2AX染色背景。mESC暴露于γ辐照诱导磷酸化的atm -激酶在细胞核中积累，并形成额外的γ - H2AX灶，此后消失。为了降低未照射细胞的γ - H2AX染色背景，我们用低浓度的诺可达唑在短时间内（6小时）以G(2)/M预同步mESC。然后对细胞进行照射和γ - H2AX染色。辐照诱导G(2)期和有丝分裂细胞中γ - H2AX灶的形成，证明在mESC细胞周期的这些阶段dna损伤信号通路处于活跃状态。由于G(1)/S检查点在mESCs中被破坏，我们检查了野生型p53 （ATM激酶的靶点）是否在γ辐照下被磷酸化。在辐照下，p53几乎没有磷酸化，这与p53靶基因p21/Waf1的表达非常低有关。因此，尽管p53/Waf1通路功能障碍且缺乏细胞周期检查点，但mESC能够激活ATM并诱导γ H2AX病灶形成，这是激活DNA损伤反应所必需的。
Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G(1)/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (gamma H2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of gamma H2AX staining. Exposure of mESC to gamma-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional gamma H2AX foci, which disappeared thereafter. To decrease the background of gamma H2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G(2)/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for gamma H2AX. Irradiation induced the formation of gamma H2AX foci both in G(2)-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G(1)/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to gamma-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing gamma H2AX foci formation, which are necessary for the activation of DNA damage response.