Santos Janine Hertzog, Hunakova L'uba, Chen Yiming, Bortner Carl, Van Houten Bennett
Laboratory of Molecular Genetics and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2003 Jan 17;278(3):1728-34. doi: 10.1074/jbc.M208752200. Epub 2002 Nov 6.
In order to understand the molecular events following oxidative stress, which lead to persistence of lesions in the mtDNA, experiments were performed on normal human fibroblast (NHF) expressing human telomerase reverse transcriptase (hTERT). The formation and repair of H(2)O(2)-induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage ( approximately 4 lesions/10 kb) after exposure to 200 microm H(2)O(2), which is partially repaired during a recovery period of 6 h. At the same time, the nDNA seemed to be completely resistant to damage. Cell sorting experiments revealed persistent mtDNA damage at 24 h only in the fraction of cells with low mitochondrial membrane potential (Delta Psi m). Further analysis also showed increased production of H(2)O(2) by these cells, which subsequently undergo apoptosis. This work supports a hypothesis for a feed-forward cascade of reactive oxygen species generation and mtDNA damage and also suggested a possible mechanism for persistence of lesions in the mtDNA involving a drop in Delta Psi m, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.
为了了解氧化应激后导致线粒体DNA（mtDNA）损伤持续存在的分子事件，我们对表达人端粒酶逆转录酶（hTERT）的正常人成纤维细胞（NHF）进行了实验。使用定量PCR检测了过氧化氢（H₂O₂）诱导的DNA损伤的形成和修复。结果发现，NHF hTERT在暴露于200微摩尔H₂O₂后显示出广泛的mtDNA损伤（约4个损伤/10 kb），在6小时的恢复期内部分损伤得到修复。与此同时，核DNA（nDNA）似乎对损伤完全具有抗性。细胞分选实验显示，仅在低线粒体膜电位（ΔΨm）细胞部分中，24小时时mtDNA损伤持续存在。进一步分析还表明，这些细胞中H₂O₂的产生增加，随后这些细胞发生凋亡。这项工作支持了活性氧生成和mtDNA损伤的前馈级联假说，还提出了mtDNA损伤持续存在的一种可能机制，涉及ΔΨm下降、蛋白质导入受损、次级活性氧生成以及修复能力丧失。
