Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry
Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry
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DOI：
10.1007/978-1-61779-182-6_6
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Podhorecka, Monika
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作者：
Darzynkiewicz, Zbigniew;Halicka, H. Dorota;Podhorecka, Monika
细胞同步通常是通过抑制DNA复制来实现的。在羟基脲、阿菲霉素或胸苷等抑制剂存在下培养的细胞在进入S期时被阻滞，当从阻滞中释放出来时，它们同步通过S， G(2)。我们最近报道，将细胞暴露于这些通常用于同步细胞群的抑制剂浓度下，通过激活共济失调毛细血管扩张突变和rad3相关蛋白激酶（ATR），导致Ser139上的组蛋白H2AX磷酸化（诱导γ H2AX）。这些发现表明，这些抑制剂诱导的DNA复制应激激活了DNA损伤反应信号通路，并且在解释使用细胞同步（如代表未受干扰的细胞）获得的数据时要谨慎。本章提出的方案描述了在DNA复制抑制剂处理的细胞中评估组蛋白H2AX-Ser139、SQ/TQ簇结构域Ser/Thr上的ATM/ATR底物以及共济失调毛细血管扩张突变（ATM）蛋白激酶磷酸化的方法。这些蛋白的磷酸化在单个细胞中用磷酸化特异性抗体（Ab）免疫细胞化学检测，并通过流式细胞术测量。同时测量细胞DNA含量和磷酸化蛋白，然后进行多参数细胞分析，可以将它们的磷酸化程度与细胞周期相关联。
Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G(2), and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of gamma H2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.