Tanaka Toshiki, Halicka H Dorota, Huang Xuan, Traganos Frank, Darzynkiewicz Zbigniew
Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA.
Cell Cycle. 2006 Sep;5(17):1940-5. doi: 10.4161/cc.5.17.3191. Epub 2006 Sep 1.
DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named gammaH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
活细胞中的DNA会受到代谢产生的内源性以及外源性氧化剂和氧化剂诱导剂所造成的持续氧化损伤。累积的氧化性DNA损伤被认为是衰老和细胞衰老的关键因素，而抗衰老剂的有效性通常通过其减少此类损伤的能力来评估。氧化性DNA损伤还使细胞易于发生肿瘤转化。DNA损伤的敏感报告分子，特别是DNA双链断裂（DSB）的诱导，是通过ATM在Ser 1981位点的磷酸化以及组蛋白H2AX在Ser 139位点的磷酸化来实现的；H2AX的磷酸化形式被命名为γH2AX。我们回顾了以下观察结果：在正常细胞以及未经过外源性基因毒性剂处理的肿瘤细胞系中，都会发生组成型ATM激活（CAA）和H2AX磷酸化（CHP）。我们推测，通过多参数细胞术相对于细胞周期阶段测量的CAA和CHP是由氧化性DNA损伤引发的。本综述还介绍了各种细胞系中CAA和CHP的差异，以及几种调节这些组蛋白和ATM修饰程度的试剂和生长条件的影响。具体而言，描述了活性氧（ROS）清除剂N-乙酰-L-半胱氨酸（NAC）、谷胱甘肽合成酶抑制剂丁硫氨酸亚砜胺（BSO）的作用，以及在较高细胞密度下生长或在葡萄糖抗代谢物2-脱氧-D-葡萄糖存在下抑制细胞代谢的影响。总体而言，所综述的数据表明，对CHP和/或CAA水平进行多参数细胞术测量可以使人估计正在进行的氧化性DNA损伤的程度，并测量抗氧化剂或减少或放大内源性ROS产生的试剂的DNA保护作用。
