Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis
Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis
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DOI：
10.1002/cyto.a.20186
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发表时间：
2005-11-01
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：ATM 激酶通过磷酸化多种蛋白质（包括组蛋白 H2AX、CHK1 和 CHK2 激酶以及 p53）来调节细胞周期检查点。 ATM 通过 Ser-1981 的自磷酸化或转磷酸化来响应 DNA 损伤，特别是诱导 DNA 双链断裂 (DSB)。本研究的目的是揭示用 DNA 拓扑异构酶 (topo) I（托泊替康；Tpt）或 topo2（米托蒽醌；Mtx）抑制剂处理的细胞中 ATM 激活与 H2AX 磷酸化、细胞周期阶段和细胞凋亡之间可能的相关性。 材料和方法：用 Tpt 或 Mtx 处理 HL-60 细胞培养物 各种时间间隔。使用分别针对 Ser-1981 (ATMS1981(P)) 上磷酸化的 ATM 或 Ser-139 上磷酸化的 H2AX (γ H2AX) 的特异性抗体，通过免疫细胞化学方法检测 ATM 或 H2AX 磷酸化，同时分析细胞 DNA 含量。通过流式细胞术测量细胞荧光。结果：未处理的细胞在整个细胞周期中显示出适度但可变的 ATM-S1981(P) Ab 标记水平，但有丝分裂细胞被强烈标记。将细胞暴露于 150 nM Tpt，在 10 分钟内诱导 ATM 磷酸化，同时 H2AX 磷酸化；两种蛋白质的磷酸化基本上仅限于 S 期，并被咖啡因和渥曼青霉素（PI-3 样激酶抑制剂）抑制。细胞暴露于 Mtx 也会导致 ATM 和 H2AX 磷酸化，与 Tpt 相比，磷酸化发生较晚，并且不具有细胞周期阶段特异性。 Tpt 或 Mtx 处理的培养物中的 HL-60 细胞分别在 2 或 4 小时后检测到细胞凋亡，并且仅限于 S 期细胞。 结论：这些数据与 ATM 作为 H2AX 磷酸化介质响应 topol (Tpt) 或 topo 2 (Mtx) 抑制剂 DNA 损伤的作用一致。观察到的 Tpt 与 Mtx 响应的细胞周期阶段相关差异表明，虽然 DNA 复制叉与由 topol 抑制剂稳定的“可裂解复合物”的碰撞是 Tpt 诱导 DSB 的主要原因，但 RNA 聚合酶分子与由 topo2 抑制剂稳定的复合物的碰撞在 Mtx 诱导 DSB 中起主要作用。本报告是第一份报告，(i) 描述了 ATM 激活的细胞计数分析，(ii) 证明了酶（激酶）的激活及其后果（底物磷酸化），两者都与细胞周期阶段和同一细胞内细胞凋亡的开始有关。 (c) 2005 年国际分析细胞学学会。
Background: The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-a-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor.Materials and Methods: Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATMS1981(P)) or for H2AX (gamma H2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry.Results: Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells.Conclusions: The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topol (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the "cleavable complexes" stabilized by topol inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells. (c) 2005 International Society for Analytical Cytology.