Role of ROS-Induced DNA Damage in Mitochondria-Regulated Apoptosis

描述(申请人提供)：人类细胞经常暴露在氧化应激下。活性氧物种(ROS)是由于部分还原的O2-泄漏而内源产生的。 线粒体呼吸复合体中的阴离子自由基物种。环境因素，如重金属和其他遗传毒物，也会由于线粒体功能障碍和各种氧化酶而产生ROS。在酶的作用下或通过金属催化的反应，O2-被转化为其他ROS，即H_2O_2和OH自由基。除了具有氧化损伤作用的蛋白质和脂质外，ROS还能引起DNA损伤，如核和线粒体DNA中的突变碱性位点(APS)及其氧化衍生物(OAS)，这与癌症的诱导、衰老过程和其他病理生理有关。由于缺乏保护性的组蛋白和线粒体基因组接近ROS产生的位置，它积累了比核基因组更多的氧化损伤。APE1可能是线粒体DNA碱基切除修复途径中的中心角色，并在其N末端的核定位序列被切割后移位到这个位置来修复线粒体基因组中的APS和OAS。APE1还发挥氧化还原因子1的作用，因为它能够还原激活转录因子，如c-jun和p53。我的赞助人的实验室已经建立了一个条件APE1缺失的小鼠胚胎成纤维细胞(MEF)系统，并将其与人APE1基因连接，以检测APE1在防御细胞凋亡中的作用。在他的实验室所做的研究表明，在这些MEF中，Cre重组酶的表达可以诱导细胞凋亡。Cre重组酶在loxP位点裂解，删除APE1。本项目还提供了一种新的四环素调控的表达HeLa细胞株的APE1siRNA。我们的基本假设是APS和OAS主要积聚在线粒体基因组中，由于APE1‘S修复活性的缺失而导致APS和OAS的积累，从而激活ROS启动的细胞凋亡。我们将通过追求以下三个特定目标来验证这一假说：目的1，我们将构建修复缺陷的和线粒体靶向的APE1MEF和HeLa细胞突变体，以便特异性地检测线粒体中APE1‘S修复活性在调控细胞凋亡中的作用；目标2，我们将通过检测仅缺乏APE1修复活性的MEF中ROS、APS和OAS的产生动力学，来检测APS和OAS作为凋亡信号发起者的作用；目的3，我们将研究外源调控氧化状态对细胞凋亡的影响。因此，这项拟议的研究将揭开从氧化损伤到线粒体介导的细胞凋亡的进展机制。由于这一机制的改变似乎有助于癌细胞逃避凋亡，我们的发现可能有助于设计新的治疗方法来预防和治疗癌症。

DESCRIPTION (provided by applicant): Human cells are constantly exposed to oxidative stress. Reactive oxygen species (ROS) are endogenously produced due to leakage of partially reduced O2- anion radical species from the mitochondrial respiration complexes. Environmental agents, such as heavy metals, and other genotoxicants also generate ROS due to mitochondrial dysfunction and various oxidases. Enzymatically or via metal catalyzed reactions, O2- is converted to other ROS, namely H2O2 and OH radical. In addition to oxidatively damaging proteins and lipids, ROS also cause DNA damage, such as mutagenic abasic sites (APS) and their oxidized derivatives (OAS), in nuclear and mitochondrial DNA, which have implications for the induction of cancers, the aging processes, and other pathophysiologies. Due to the lack of protective histones and mitochondrial genome's proximity to sites of ROS generation, it accumulates more oxidative damage than the nuclear genome. APE1 could be the central player in the DNA base excision repair pathway in the mitochondria and translocates to this site to repair APS and OAS in the mitochondrial genome after cleavage of its nuclear localization sequence present at the N-terminus. APE1 also functions as redox factor 1 due to its ability to reductively activate transcription factors, such as c-Jun and p53. My sponsor's laboratory has established a conditional APE1 null mouse embryonic fibroblast (MEF) system with 'floxed' human APE1 gene to examine the role of APE1 in the defense against apoptosis. Studies done in his laboratory have shown that expression of Cre recombinase, which cleaves at loxP sites to delete APE1 in these MEFs, elicits apoptosis. Also available for this project is a new tetracycline-regulated APE1 siRNA expressing HeLa cell line. Our fundamental hypothesis is that accumulation of APS and OAS mainly in the mitochondrial genome, caused by the absence of APE1's repair activity, would activate ROS-initiated apoptosis. We will test this hypothesis by pursuing the following three specific aims: Aim 1, we will generate repair-deficient and mitochondrial-targeted transductants of the conditional APE1 MEF and HeLa cell mutants in order to specifically examine the role of APE1's repair activity in the mitochondria in modulating apoptosis; Aim 2, we will examine the role of APS and OAS as initiators of apoptotic signaling by measuring kinetics of ROS, APS, and OAS production in MEFs lacking only the repair activity of APE1; Aim 3, we will investigate how exogenous modulation of oxidative state affects apoptosis. Thus, the proposed research should unravel the mechanism of progression from oxidative damage to mitochondria-mediated apoptosis. Since alterations of this mechanism seem to help cancer cells to evade apoptosis, our findings could help design new therapeutic approaches for preventing and treating cancer.