Dynamic monitoring of oxidative DNA double-strand break and repair in cardiomyocytes.
Dynamic monitoring of oxidative DNA double-strand break and repair in cardiomyocytes.
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DOI：
10.1016/j.carpath.2015.10.010
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发表时间：
2016-03
期刊：
影响因子：
--
通讯作者：
Li F
中科院分区：
文献类型：
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作者：
Ye B;Hou N;Xiao L;Xu Y;Xu H;Li F
DNA双链断裂（DSBs）是最危险的病变。为了确定氧化应激是否可以诱导DSB及其如何在心肌细胞（CMs）中修复，我们用不同剂量的H2O2处理培养的新生大鼠CMs，并随访长达72小时，以监测DSB病灶DNA修复蛋白组装/拆卸的时空动态。139丝氨酸磷酸化的组蛋白H2AX （γ-H2AX）在处理30min后，在50、100和200µmol/L H2O2条件下，蛋白水平和病灶数量成比例增加。当H2O2≥400µmol/L时，γ-H2AX以泛核为主；在200µmol/L H2O2处理30 min后，γ-H2AX水平在第1 h内最高，随后在恢复过程中逐渐下降，并在48 h时恢复到基础水平。DNA损伤换能器激酶中，共济失调毛细血管扩张突变（ATM）被H2O2显著激活，而共济失调毛细血管扩张突变和rad3相关（ATR）被H2O2轻度激活。DSB结合蛋白、p53结合蛋白1形成不同的核灶，与γ-H2AX灶共定位，磷酸化ATM。我们的研究结果表明H2O2可以诱导DNA双链断裂，而ATM是介导心肌细胞DSB修复的主要激酶。因此，监测DSB修复可以评估心肌细胞的氧化损伤和反应。
DNA double strand breaks (DSBs) are most dangerous lesions. To determine whether oxidative stress can induce DSBs and how they are repaired in cardiomyocytes (CMs), cultured neonatal rat CMs were treated with different doses of H2O2 and followed for up to 72 hours for monitoring the spatiotemporal dynamics of DNA repair protein assembly/disassembly at DSB foci. The protein levels and foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) increased proportionally to 50, 100 and 200 µmol/L H2O2 after 30 min treatment. When H2O2 was at or above 400 µmol/L, γ-H2AX became predominantly pan-nuclear. After 30 min, 200 µmol/L of H2O2 treatment, γ-H2AX levels were highest within the first hour and then gradually declined during the recovery and returned to basal levels at 48 hours. Among DNA damage transducer kinases, ataxia telangiectasia mutated (ATM) was significantly activated by H2O2 in contrast to mild activation of ataxia telangiectasia mutated and Rad3-related (ATR). DSB binding protein, p53 binding protein 1 formed distinct nuclear foci that colocalized with γ-H2AX foci and phosphorylated ATM. Our findings indicate that DNA double strand breaks can be induced by H2O2 and ATM is the main kinase to mediate DSB repair in cardiomyocytes. Therefore, monitoring DSB repair can assess oxidative injury and response in cardiomyocytes.