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: 6479399
• 负责人: MYRON K JACOBSON
• 金额: $ 16.58万
• 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/6479399
• 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 (ROS) 的异常积累会破坏正常细胞 信号通路通常导致细胞凋亡或坏死。 钙信号传导参与导致氧化诱导细胞的途径 死亡。在初步实验中，研究人员和他的同事表明 氧化剂诱导的细胞凋亡导致循环周期显着升高 腺苷二磷酸核糖 (cADPR)，它是 NAD 的代谢产物，可引发 钙动员。 cADPR 的升高是由于膜的激活 cADPR 合酶，与线粒体通透性的激活有关 转变（PT），并且与细胞内水平的增加有因果关系 游离钙。这些数据导致了研究者的假设，即 cADPR 在氧化应激激活的信号通路中发挥作用并导致 钙稳态的破坏和氧化剂诱导的细胞死亡。他会 在 PC12 细胞中检验这一假设，PC12 细胞被广泛用作 与氧化剂毒性相关的神经元细胞死亡。研究将在 PC12 细胞过表达 Bcl-2（线粒体 PT 的有效抑制剂） 细胞凋亡，并且在仅含有载体的对照细胞系中，允许 效应器阶段上游和下游事件的分离 细胞凋亡。第一个具体目标是鉴定 cADPR 合酶 被氧化应激激活。分子遗传学方法将用于 确定目标酶是否是两种已知的 cADPR 合酶（CD38 或 BST-1) 或一种新型酶。据调查人员称，经鉴定， 合酶是研究氧化诱导机制的先决条件 cADPR 的激活并评估其治疗潜力 代谢。 第二个具体目标将确定 cADPR 的形成是否是强制性的 氧化剂诱导细胞凋亡，并鉴定细胞中 cADPR 的特定功能 死亡。 PC12细胞将被诱导有义和反义稳定转染 编码由氧化应激激活的 cADPR 合酶的 cDNA 构建体。酶 然后，耗尽和过度表达将用于评估 cADPR 的作用 细胞死亡途径中的合酶，通过检查特定的 细胞凋亡的生化标志物。第三个具体目标将决定是否 cADPR 合酶激活参与细胞凋亡的启动途径 特定于 ROS，或者是否是效应器阶段产生的 ROS 的结果 细胞凋亡。引发剂包括肿瘤坏死因子α（TNF-()、 抗 Fas 抗体、N-甲基-N'-硝基-N-亚硝基胍 (MNNG)、 星形孢菌素和血清戒断。这些研究旨在推进我们的 了解细胞死亡机制并提供潜在的新靶标 减少再灌注导致的氧化剂诱导的细胞死亡 血管或脑缺血后的损伤。