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

描述（由申请人提供）：人体细胞持续暴露于氧化应激。 活性氧（ROS）是由于部分还原的O2- 线粒体呼吸复合物中的阴离子自由基。 环境因素，如重金属，和其他遗传毒物也产生ROS由于线粒体功能障碍和各种氧化酶。 酶促或通过金属催化反应，O2-被转化为其他ROS，即H2 O2和OH自由基。 除了氧化损伤蛋白质和脂质外，ROS还引起DNA损伤，例如核和线粒体DNA中的诱变脱碱基位点（APS）及其氧化衍生物（OAS），其与癌症的诱导、衰老过程和其他病理生理学有关。 由于缺乏保护性组蛋白和线粒体基因组接近ROS产生的位点，它比核基因组积累更多的氧化损伤。 APE 1可能是线粒体中DNA碱基切除修复途径的中心参与者，并在N端存在的核定位序列裂解后易位到该位点以修复线粒体基因组中的APS和OAS。 APE 1也作为氧化还原因子1发挥作用，因为它能够还原性激活转录因子，如c-Jun和p53。 我的实验室建立了一个条件性APE 1无效小鼠胚胎成纤维细胞（MEF）系统与'floxed'人APE 1基因，以检查APE 1在防御细胞凋亡的作用。 在他的实验室进行的研究表明，Cre重组酶的表达，在loxP位点切割删除这些MEFs中的APE 1，促进细胞凋亡。 该项目还可获得一种新的四环素调节的APE 1 siRNA表达HeLa细胞系。 我们的基本假设是，APS和OAS的积累主要是在线粒体基因组中，由APE 1的修复活性的缺乏引起的，将激活ROS启动的细胞凋亡。 我们将通过追求以下三个具体目标来测试这一假设：目标1，我们将产生条件APE 1 MEF和HeLa细胞突变体的修复缺陷和细胞靶向转导子，以特异性地检测APE 1在线粒体中的修复活性在调节细胞凋亡中的作用;目的2，我们将通过测量仅缺乏APE 1修复活性的MEFs中ROS、APS和OAS产生的动力学来研究APS和OAS作为凋亡信号传导起始剂的作用;目的3：探讨外源性氧化调节对细胞凋亡的影响。 因此，拟议中的研究应该解开从氧化损伤的进展到细胞凋亡介导的细胞凋亡的机制。 由于这种机制的改变似乎有助于癌细胞逃避凋亡，我们的研究结果可能有助于设计预防和治疗癌症的新治疗方法。

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.