Zhao Hong, Tanaka Toshiki, Halicka H Dorota, Traganos Frank, Zarebski Miroslaw, Dobrucki Jurek, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.
Cytometry A. 2007 Nov;71(11):905-14. doi: 10.1002/cyto.a.20469.
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 microM 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% O2) 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 celecoxib (5 microM) 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.
氧化代谢过程中产生的活性氧物种所导致的持续性DNA损伤，被认为是促成细胞衰老以及使细胞易于发生肿瘤转化的关键因素。我们之前推测，在未经处理的正常细胞和肿瘤细胞中观察到的相当一部分组成型组蛋白H2AX磷酸化（CHP）和ATM的组成型激活（CAA），是对这种DNA损伤的反应。在本研究中，我们提供了进一步的证据来支持这一推测。我们通过免疫细胞化学检测了在经H2O2处理以及在已知或怀疑会影响内源性氧化剂水平的条件下生长的WI-38、A549和TK6细胞中ATM激活和H2AX磷酸化的水平。细胞暴露于100或200 microM H2O2 30至60分钟会导致H2AX磷酸化水平和ATM激活增加，在S期细胞中尤为明显（近五倍）。在缺氧条件（3% O2）下细胞生长24至48小时会明显降低CHP和CAA的水平，而对细胞周期进程影响较小。用0.1或0.3 mM 3-溴丙酮酸（一种糖酵解和线粒体氧化磷酸化的抑制剂）处理（4小时）可降低CHP水平（高达四倍），同时也降低CAA水平。WI-38细胞在2%血清浓度下生长48小时，与在10%血清中生长的细胞相比，CHP和CHA分别降低了25%和30%。抗氧化剂维生素C（2 mM）处理24小时后可使CHP和CAA降低20 - 30%，而COX-2抑制剂塞来昔布（5 microM）对CHP和CAA影响较小，尽管它降低了H2O2诱导的H2AX磷酸化和ATM激活水平。相反，已知通过其对丙酮酸脱氢酶激酶的作用使代谢从无氧糖酵解转变为有氧糖酵解的二氯乙酸，增强了CHP和CAA的水平。我们目前的数据和早期观察结果有力地支持了这样一种推测，即很大一部分CHP和CAA是对代谢产生的氧化剂所导致的DNA损伤的反应。对CHP和CAA的细胞分析为测量外源性因素的有效性提供了手段，这些外源性因素要么通过降低有氧代谢要么通过中和自由基来保护DNA免受此类损伤。
