DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells:: Bolus addition versus continuous generation of H2O2
DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells:: Bolus addition versus continuous generation of H2O2
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DOI：
10.1016/s0891-5849(02)00967-x
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发表时间：
2002-09-01
影响因子：
7.4
通讯作者：
Galaris, D
中科院分区：
文献类型：
--
作者：
Barbouti, A;Doulias, PT;Galaris, D
本文对过氧化氢诱导DNA损伤和细胞死亡的分子机制进行了研究。将培养物中的Jurkat T细胞暴露于通过葡萄糖氧化酶的作用连续产生的H2 O2的低速率或暴露于相同试剂的推注添加。在第一种情况下，稳态条件占主导地位，而在后者中，H2 O2被去除的细胞防御系统遵循一级动力学。通过使用单细胞凝胶电泳（也称为彗星试验），在暴露于150 μ M H2 O2的团注的细胞中观察到DNA单链断裂形成的初始增加。当H2 O2耗尽时，DNA损伤明显逐渐减少，表明存在有效的单链断裂修复。在100穆尔生长培养基（含1.5 × 10（5）个细胞）中加入10 ng葡萄糖氧化酶，每分钟产生2.0 +/- 0.2 μ M H2 O2。这种处理诱导单链断裂水平增加，达到所用方法的检测上限，并在随后的6小时内继续保持高水平。然而，当检查各种凋亡细胞死亡的标志物（DNA细胞含量，DNA梯状，半胱天冬酶的激活，PARP裂解）时，只有团注添加H2 O2能够诱导凋亡，而该试剂的持续存在抑制了凋亡过程的执行，无论诱导剂是H2 O2本身还是抗Fas抗体。这些观察结果强调，除了明显的遗传毒性和促凋亡作用的H2 O2，它也可以发挥抗凋亡作用时，即使在低浓度，在执行细胞凋亡。(C)2002年爱思唯尔科技有限公司
Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H2O2 by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H2O2 was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 muM H2O2. As the H2O2 was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 mul growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 muM H2O2 per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H2O2 were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H2O2 itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H2O2, it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis. (C) 2002 Elsevier Science Inc.