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（改编自申请人摘要）：氧化应激，定义为 活性氧（ROS）的异常积累，破坏正常细胞 信号通路通常导致细胞凋亡或坏死而死亡。 钙信号参与氧化诱导细胞凋亡的途径 死亡在初步的实验中，研究人员和他的同事表明， 氧化剂诱导的细胞凋亡导致细胞周期的显著升高， 腺苷二磷酸核糖（cADPR），是NAD的代谢产物， 钙动员cADPR的升高是由于细胞膜的激活， cADPR合酶，与线粒体通透性的激活有关 过渡（PT），并与细胞内 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起作用， 钙稳态的破坏和氧化剂诱导的细胞死亡。他将 在PC 12细胞中测试这一假设，PC 12细胞被广泛用作 与氧化剂毒性有关的神经元细胞死亡。研究将在 过表达Bcl-2（一种有效的线粒体PT抑制剂）的PC 12细胞， 在仅含有载体的对照细胞系中， 分离的事件上游和下游的效应阶段， 凋亡第一个具体目标是鉴定cADPR合酶 被氧化应激激活将使用分子遗传学方法来 确定靶酶是否是两种已知的cADPR转移酶（CD 38或 BST-1）或新的酶。据调查人员称， 合成酶是研究氧化剂诱导细胞凋亡机制的前提 cADPR的激活和评估其治疗潜力 新陈代谢. 第二个具体目标是确定cADPR的形成是否是强制性的， 氧化剂诱导细胞凋亡，并将确定cADPR在细胞中的特定功能 死亡PC 12细胞将用诱导型正义和反义寡核苷酸稳定转染。 编码由氧化应激激活的cADPR合酶的cDNA构建体。酶 然后将使用cADPR的缺失和过表达来评估cADPR的作用。 合成酶在细胞死亡途径中的作用，如通过检查特异性 凋亡的生化标志物。第三个具体目标将决定， cADPR合酶激活参与启动细胞凋亡途径 特异于ROS，或者是否由效应期产生的ROS引起 细胞凋亡。启动剂将包括肿瘤坏死因子α（TNF-α）， 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 用于减少由再灌注引起的氧化剂诱导的细胞死亡 血管或脑缺血后的损伤。