Dipartimento di Scienze, Università Roma Tre, Roma, Italy; INFN-Sezione di Roma Tre, Roma, Italy.
Mutat Res. 2013 Aug 30;756(1-2):86-94. doi: 10.1016/j.mrgentox.2013.05.010. Epub 2013 May 27.
Mitochondria are the main cellular source of Reactive Oxygen Species (ROS). Alterations of mitochondrial metabolism and consequent loss of mitochondrial membrane potential may lead to redox imbalance and in turn to DNA damage, chromosomal instability and apoptosis. On the other hand, impaired mitochondrial functions may either exacerbate the detrimental effects of geno- and cytotoxic agents or may bring beneficial cellular responses. To study the role of mitochondria within this framework, AG01522 human primary fibroblasts were incubated with the mitochondrial polymerase γ inhibitor 2',3'-dideoxycytidine (ddC), leading to mitochondrial DNA (mtDNA) depletion and to mitochondrial dysfunctions. The successful treatment toward mtDNA depletion was confirmed by Complex-IV subunit I (COX-I) immunofluorescence and western blot assays. mtDNA-depleted cells and their counterparts were ultrastructurally characterized by transmission electron microscopy. mtDNA-depleted cells showed dramatic mitochondrial alterations such as fragmentation and cristae disruption along with a reduction of the mitochondrial membrane potential and elevated levels of ROS. Despite increased ROS levels, we did not find any difference in telomere length between ddC-treated and untreated cells. The spontaneous rate of DNA double-strand breaks (DSBs) and chromosome aberrations was significantly enhanced in mtDNA-depleted cells whereas the induction of DSBs by low-Linear Energy Transfer (LET) (X-rays; 7.7keV/μm protons) and high-LET radiations (28.5keV/μm protons) did not differ when compared with normal cells. However, in irradiated cells impaired mitochondrial functions seemed to bring beneficial cellular responses to the detrimental effect of radiations. In fact, after X-irradiation mtDNA-depleted cells show less remaining unrejoined DSBs than normal cells and furthermore a lower induction of cytogenetic damage. Overall, these data show that active mitochondrial functions are required for the proper maintenance of cellular genome stability in primary fibroblasts.
线粒体是活性氧（ROS）的主要细胞来源。线粒体代谢的改变和随之而来的线粒体膜电位的丧失可能导致氧化还原失衡，进而导致 DNA 损伤、染色体不稳定和细胞凋亡。另一方面，受损的线粒体功能可能加剧遗传毒性和细胞毒性药物的有害作用，也可能带来有益的细胞反应。为了在这个框架内研究线粒体的作用，用线粒体聚合酶γ抑制剂 2'，3'-双脱氧胞苷（ddC）孵育 AG01522 人原代成纤维细胞，导致线粒体 DNA（mtDNA）耗竭和线粒体功能障碍。通过细胞色素 c 氧化酶亚基 I（COX-I）免疫荧光和 Western blot 检测证实了 mtDNA 耗竭治疗的成功。用透射电子显微镜对 mtDNA 耗竭细胞及其对照物进行超微结构特征分析。mtDNA 耗竭细胞显示出明显的线粒体改变，如片段化和嵴破坏，同时线粒体膜电位降低，ROS 水平升高。尽管 ROS 水平升高，但我们没有发现 ddC 处理和未处理细胞之间端粒长度有任何差异。mtDNA 耗竭细胞的 DNA 双链断裂（DSB）和染色体畸变自发率显著增加，而低线性能量传递（LET）（X 射线；7.7keV/μm 质子）和高线性能量传递（28.5keV/μm 质子）诱导的 DSB 没有差异与正常细胞相比。然而，在受照射的细胞中，受损的线粒体功能似乎对辐射的有害作用带来了有益的细胞反应。事实上，与正常细胞相比，X 射线照射后 mtDNA 耗竭细胞中未修复的双链断裂（DSB）残留较少，而且细胞遗传学损伤的诱导也较低。总的来说，这些数据表明，在原代成纤维细胞中，活跃的线粒体功能是维持细胞基因组稳定性的必要条件。
Radiat Prot Dosimetry. 2006
Cell Death Differ. 2013-8-30
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