Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death
Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death
复制标题
DOI：
10.1074/jbc.m208752200
复制
发表时间：
2003-01-17
影响因子：
4.8
通讯作者：
Van Houten, B
中科院分区：
文献类型：
--
作者：
Santos, JH;Hunakova, L;Van Houten, B
为了了解氧化应激后导致线粒体DNA损伤持续的分子事件，我们在表达人端粒酶逆转录酶(HTERT)的正常人成纤维细胞(NHF)上进行了实验。用定量聚合酶链式反应检测过氧化氢诱导的DNA损伤的形成和修复。结果发现，NHF hTERT暴露于200um H_2O_2后表现出广泛的线粒体DNA损伤(类似于4个损伤/10kb)，在6h的恢复期内可部分修复，同时nDNA似乎完全抵抗损伤。细胞分选实验显示，线粒体DNA持续损伤仅见于线粒体膜电位较低的部分细胞(DeltaPsim)。进一步的分析还显示，这些细胞产生了更多的过氧化氢，随后发生了凋亡。这项工作支持了一种关于活性氧生成和线粒体DNA损伤的前馈级联假说，并提出了线粒体DNA损伤持续存在的可能机制，包括DeltaPsim下降、蛋白质输入受损、二次活性氧生成和修复能力丧失。
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 H2O2-induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage (similar to4 lesions/10 kb) after exposure to 200 muM H2O2, 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 (DeltaPsim). Further analysis also showed increased production of H2O2 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 DeltaPsim, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.