CYCLIC ADP RIBOSE METABOLISM IN OXIDATIVE CELL DEATH

氧化性细胞死亡中的循环 ADP 核糖代谢

• 批准号: 6351880
• 负责人: MYRON K JACOBSON
• 金额: $ 29.27万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351880
• 关键词: ADP ribosylation; BCL2 gene /protein; CD38 molecule; N methyl N' nitro N nitrosoguanidine; NAD nucleosidase; PC12 cells; antisense nucleic acid; apoptosis; calcium flux; calcium indicator; enzyme induction /repression; oxidative stress; peroxides; second messengers; transfection; tumor necrosis factor alpha

DESCRIPTION (Adapted from applicant's abstract): Oxidative stress, defined as an abnormal accumulation of reactive oxygen species (ROS), disrupts normal cell signaling pathways often resulting in cell death by apoptosis or necrosis. Calcium signaling is involved in pathways leading to oxidant induced cell death. In preliminary experiments, the investigator and his associates show that oxidant induced apoptosis results in a marked elevation of cyclic adenosine diphosphoribose (cADPR), which is a metabolite of NAD that elicits calcium mobilization. The elevation of cADPR is due to activation of a membrane cADPR synthase, linked to the activation of the mitochondrial permeability transition (PT), and causally related to increases in levels of intracellular free calcium. These data have led to the investigator's hypothesis, that cADPR functions in a signaling pathway that is activated by oxidant stress and leads to disruption of calcium homeostasis and to oxidant induced cell death. He will test this hypothesis in PC12 cells, which are widely used as a model system of neuronal cell death relating to oxidant toxicity. Studies will be conducted in PC12 cells overexpressing Bcl-2, a potent inhibitor of the mitochondrial PT and apoptosis, and in a control cell line containing only vector, to allow segregation of events upstream and downstream from the effector phase of apoptosis. The first specific aim is to identify the cADPR synthase(s) activated by oxidant stress. A molecular genetic approach will be used to determine if the target enzyme is one of two known cADPR synthases (CD38 or BST-l) or a novel enzyme. According to the investigator, identification of the synthase is a prerequisite to studies of the mechanism of oxidant induced activation of cADPR and evaluation of the therapeutic potential of this metabolism. The second specific aim will establish whether cADPR formation is obligatory in oxidant induced apoptosis and will identify specific functions of cADPR in cell death. PC12 cells will be stably transfected with inducible sense and antisense cDNA constructs encoding the cADPR synthase activated by oxidant stress. Enzyme depletion and overexpression will then be used to assess the role of cADPR synthase in the cell death pathway, as monitored by examination of specific biochemical markers of apoptosis. The third specific aim will determine if cADPR synthase activation is involved in initiating pathways of apoptosis specific to ROS, or whether it results from ROS generated in the effector phase of apoptosis. Initiators will include tumor necrosis factor alpha (TNF-(), anti-Fas antibodies, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Staurosporine, and serum withdrawal. The studies are designed to advance our understanding of mechanisms of cell death and provide potentially new targets for reduction of oxidant induced cell death that results from re-perfusion injury following vascular or cerebral ischemia.

描述(改编自申请人的摘要)：氧化应激，定义为 活性氧物种(ROS)的异常积累，扰乱正常细胞 信号通路通常通过细胞凋亡或坏死而导致细胞死亡。 钙信号参与氧化诱导细胞的通路 死亡。在初步实验中，调查员和他的同事们表明 氧化剂诱导的细胞凋亡导致细胞周期显著升高 腺苷二磷酸核糖(CADPR)，是NAD的代谢物，可引起 钙动员。CADPR的升高是由于膜的激活 CADPR合酶，与激活线粒体通透性有关 转变(PT)，并与细胞内水平的增加有关 游离钙。这些数据导致了研究人员的假设，即cADPR 在氧化应激和铅激活的信号通路中发挥作用 破坏钙稳态和氧化剂诱导的细胞死亡。他会的 在PC12细胞中验证这一假设，PC12细胞被广泛用作 与氧化剂毒性有关的神经细胞死亡。研究将在#年进行 PC12细胞过表达Bcl-2，一种有效的线粒体PT和 在只含有载体的对照细胞系中，允许 从效应器阶段的上游和下游分离事件 细胞凋亡。第一个特定目的是鉴定cADPR合成酶(S) 被氧化应激激活。分子遗传学方法将被用来 确定靶酶是否是两种已知的cADPR合成酶(CD38或 Bst-L)或一种新的酶。根据调查人员的说法，对 合酶是研究氧化剂诱导氧化机制的前提 CADPR的激活及其治疗潜力的评价 新陈代谢。 第二个具体目标将确定成立cadpr是否在#年是强制性的。 氧化剂诱导细胞凋亡并鉴定cADPR在细胞中的特定功能 死亡。可诱导正义和反义基因稳定导入PC12细胞 编码氧化应激激活的cADPR合成酶的cdna构建。酶 耗竭和过度表达将被用来评估cADPR的作用 合酶在细胞死亡途径中的作用，如通过检测特异性 细胞凋亡的生化标志物。第三个具体目标将决定是否 CADPR合酶激活参与启动细胞凋亡途径 特定于ROS，或者它是否是在效应器阶段产生的ROS的结果 对细胞凋亡的影响。启动者将包括肿瘤坏死因子α(TNF-())， 抗Fas抗体，N-甲基-N‘-硝基-N-亚硝基(MNNG)， 星状孢子素和停用血清。这些研究旨在推动我们的 了解细胞死亡的机制并提供潜在的新靶点 用于减少再灌流引起的氧化剂诱导的细胞死亡 血管或脑缺血后的损伤。