Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis
Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis
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DOI：
10.1002/cyto.a.20241
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发表时间：
2006-04-01
期刊：
影响因子：
3.7
通讯作者：
Darzynkiewicz, Z
中科院分区：
文献类型：
--
作者：
Kurose, A;Tanaka, T;Darzynkiewicz, Z
背景：DNA复制应激常导致DNA损伤。抗肿瘤药物羟基脲（HU）是一种有效的核糖核苷酸还原酶抑制剂，通过对细胞脱氧核苷酸池的影响来阻止DNA复制，已被证明可损伤DNA诱导双链断裂（DSB）。阿非迪霉素（APH），一种α-样DNA聚合酶的抑制剂，也被报道会引起DNA损伤，但APH诱导DSB的证据并不简单。组蛋白H2 AX在Ser139上磷酸化以响应DSB，磷酸化H2 AX的蛋白激酶之一是共济失调毛细血管扩张突变（ATM）; ATM的激活是通过其Ser1981的磷酸化。本研究进行揭示是否H2AX是磷酸化的细胞暴露于HU或APH，其磷酸化是否是由ATM介导的。材料和方法：HL-60细胞在培养物中处理0.1 - 5.0 mM HU或1 - 4 μ M APH长达5小时。ATM和H2AX磷酸化的激活分别使用对Ser1981-ATM或Ser139-H2AX表位特异的Ab进行免疫细胞化学检测，同时测量细胞DNA含量。而暴露于HU导致细胞在S期选择性地磷酸化H2AX，并且细胞进展通过S期的早期部分。（DI = 1.1 - 1.4）的细胞比晚S期（DI = 1.6 - 1.9）的细胞受影响更大，而HU不激活ATM。事实上，在细胞周期的所有阶段，在0.1 - 5.0 mM HU时，组成性（"程序性"）ATM磷酸化水平明显受到抑制。细胞暴露于APH也导致H2AX在ser139处磷酸化，没有ATM活化的证据，并且在HU的情况下，早期S期细胞比晚期S期细胞受到更多的影响。在暴露于HU或APH 2小时后，凋亡细胞频率的上升变得明显，并且所有凋亡细胞的H2AX-Ser139和ATM-Ser1981磷酸化水平均显著升高。H2AX磷酸化和ATM活化之间缺乏相关性表明ATM以外的蛋白激酶（ATR和/或DNA依赖性蛋白激酶）被复制应激诱导的DSB激活。有趣的是，HU抑制未处理细胞中ATM磷酸化的组成性（"程序化"）水平。然而，凋亡过程中的DNA片段化激活ATM并显著增加H2AX磷酸化水平。(c)2006年国际分析细胞学学会。
Background: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM.Materials and Methods: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 mu M APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser 1981-ATM or Ser139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content.Results: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic, cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation.Conclusions: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. (c) 2006 International Society for Analytical Cytology.