Tanaka Toshiki, Kurose Akira, Halicka H Dorota, Traganos Frank, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10532, USA.
Cell Cycle. 2006 Apr;5(8):878-82. doi: 10.4161/cc.5.8.2681. Epub 2006 Apr 17.
Histone H2AX phosphorylated on Ser-139, defined as gammaH2AX, is a reporter of DNA double-strand breaks (DSBs). While H2AX undergoes phosphorylation after induction of DNA damage by genotoxic agents or during physiological events that involve DNA recombination, it also is phosphorylated in untreated normal and tumor cells. We recently reported that this constitutive H2AX phosphorylation (CHP) is markedly reduced by the antioxidant N-acetyl-L-cysteine (NAC), and postulated that it reflects the oxidative DNA damage ("endogenous DSBs") induced by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle. In the present study, we provide evidence that growth of cells from three human lymphoblastoid cell lines TK6, NH32 and WTK1 in the presence of the glucose antimetabolite 2-deoxy-D-glucose (2-DG) led to a distinct reduction in the level of CHP. The reduction of CHP was more pronounced in S and G(2)M than in G(1) phase cells. Constitutive activation of ATM was also reduced. The data suggest that a decrease in a cell's metabolic activity as a result of inhibition of glycolysis by 2-DG reduces generation of ROS which leads to the reduction of oxidative DNA damage. The data also point out that ATM may play a role in CHP induced by oxidative DNA damage. Therefore, the assay of CHP by multiparameter cytometry provides the means to measure effects of antioxidants and metabolic inhibitors on endogenous oxidative DNA damage in relation to cell cycle phase.
在丝氨酸139位点磷酸化的组蛋白H2AX，即γH2AX，是DNA双链断裂（DSB）的一个标志物。虽然H2AX在遗传毒性剂诱导DNA损伤后或在涉及DNA重组的生理事件中会发生磷酸化，但在未经处理的正常细胞和肿瘤细胞中它也会被磷酸化。我们最近报道，抗氧化剂N - 乙酰 - L - 半胱氨酸（NAC）可显著降低这种组成性H2AX磷酸化（CHP），并推测它反映了细胞周期进程中代谢活性产生的活性氧（ROS）诱导的氧化性DNA损伤（“内源性DSB”）。在本研究中，我们提供证据表明，在葡萄糖抗代谢物2 - 脱氧 - D - 葡萄糖（2 - DG）存在的情况下，三种人类淋巴母细胞系TK6、NH32和WTK1的细胞生长导致CHP水平明显降低。CHP的降低在S期和G2M期比在G1期细胞中更明显。ATM的组成性激活也降低了。数据表明，2 - DG抑制糖酵解导致细胞代谢活性降低，从而减少了ROS的产生，进而导致氧化性DNA损伤的减少。数据还指出，ATM可能在氧化性DNA损伤诱导的CHP中起作用。因此，通过多参数细胞术检测CHP提供了一种手段，可用于测量抗氧化剂和代谢抑制剂对与细胞周期阶段相关的内源性氧化性DNA损伤的影响。
