Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes
Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes
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DOI：
10.1002/cyto.a.20469
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发表时间：
2007-11-01
期刊：
影响因子：
3.7
通讯作者：
Darzynkiewicz, Zbigniew
中科院分区：
文献类型：
--
作者：
Zhao, Hong;Tanaka, Toshiki;Darzynkiewicz, Zbigniew
氧化代谢过程中产生的活性氧引起的DNA损伤被认为是导致细胞衰老的关键因素，也是细胞向肿瘤转化的预适应因素。我们以前推测，在未经处理的正常和肿瘤细胞中，有很大一部分组蛋白H_2AX磷酸化(CHP)和ATM的结构性激活(CAA)发生在这种DNA损伤的反应中。在本研究中，我们提供了进一步的证据来支持这一假设。免疫细胞化学检测到，在用过氧化氢处理的WI-38、A549和TK6细胞中，以及在已知或怀疑影响内源性氧化剂水平的条件下生长的细胞中，已经监测到ATM激活和H2AX磷酸化的水平。细胞暴露于100或200 mU的H_2O_2 30~60分钟后，H_2AX的磷酸化水平和ATM的激活水平均增加，尤以S期细胞最为明显(近5倍)。细胞在低氧(3%02)条件下生长24~48h，CHP和CAA水平明显降低，而对细胞周期进程的影响较小。用糖酵解和线粒体氧化磷酸化抑制剂3-溴丙酮酸(0.1或0.3 mM)处理4h后，CHP水平降低4倍，CAA水平也降低。与在10%血清中生长的细胞相比，在2%血清浓度下培养48h，细胞的CHP和CHA分别减少了25%和30%。抗氧化剂维生素C(2 MM)处理24 h后，CHP和CAA减少20-30%，而COX-2抑制剂Cclecoxib(5 MM)对CHP和CAA影响不大，但可降低H_2O_2诱导的H_2O_2诱导的H_2AX磷酸化和ATM激活水平。相比之下，已知的二氯乙酸酯通过对丙酮酸脱氢酶激酶的作用而将代谢从厌氧糖酵解转变为氧化糖酵解，从而提高了CHP和CAA的水平。我们目前的数据和早期的观察有力地支持了这一假设，即很大一部分CHP和CAA发生在代谢生成的氧化剂引起的DNA损伤的反应中。CHP和CAA的细胞学分析提供了测量外源因素有效性的手段，外源因素可以通过降低有氧代谢或中和自由基来保护DNA免受这种损害。(C)2007国际分析细胞学学会。
The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty-to 60-min exposure of cells to 100 or 200 mu M H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% 02) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor cclecoxib (5 mu M) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.(c) 2007 International Society for Analytical Cytology.