MECHANISMS FOR OXIDATIVE STRESS INDUCED APOPTOSIS

描述：(改编自申请人的摘要)：硫醇一般 想到一种抗氧化剂，它可以防止由 不同的试剂和条件，如电离辐射，一些 化疗药物，神经退行性疾病，自身免疫性疾病， 缺血/再灌注等。然而，在某些情况下，例如，在 存在微量的过渡金属，一些抗氧化剂会变成 促氧化剂，导致细胞死亡，包括细胞凋亡。我们已经开发出 一个详细的、可测试的、逐步的模型来解释 细胞凋亡中的硫醇。根据模型，在一定的细胞条件下， 条件下，硫醇：(I)发生金属催化氧化，产生 活性氧类、H_2O_2、O(2)-和·OH(II)破坏细胞内 细胞内临界硫醇或二硫化物的变化引起的钙稳态 钙调节蛋白，以及(Iii)激活转录因子 核因子-kB，(Iv)导致细胞凋亡。我们进一步假设bcl2可以 在这一途径中的一个或多个步骤起作用，以抑制硫醇诱导的细胞凋亡。 特定的目标旨在单独测试模型中的每个步骤 以及演示将这些步骤集成到从 硫醇对细胞凋亡的影响。此外，我们还将获得有关 硫醇处理后细胞凋亡与克隆性丧失的关系 解决细胞凋亡是否导致所有细胞死亡的问题 是否存在抑制细胞凋亡的药物(例如，钙络合剂或 Bcl2)增加细胞的长期存活率或仅延缓细胞死亡。大部分 这些研究将在人类白血病中使用模型硫醇二硫苏糖醇 HL-60细胞。细胞凋亡将通过形态和DNA片段化表现出来 标准，并用DNA片段化和TUNEL法进行定量。哦，会吗？ 使用敏感的、特定的荧光技术进行测量，钙将 用荧光细胞内钙探针和核因子-kB进行检测 活性将使用凝胶迁移率变化分析进行评估。 对使用硫醇和其他抗氧化剂预防或治疗感兴趣 据信至少部分是由活性氧引起的情况 物种正在迅速增长。因此，重要的是理解可能 这些毒剂的有害影响。此外，由于他们的 已记录的在氧化应激、钙稳态、核因子-kB激活中的作用 和细胞凋亡，硫醇是研究化学，酶和 这些过程中的每一个过程都涉及到遗传机制 导致细胞死亡的相互作用。归根结底，在这个过程中获得的知识 研究可能会导致新的、特定的疗法的开发，基于 针对特定胞内结构域的硫醇的性质 为了“开启”保护通路或“关闭”不想要的细胞过程， 例如，增强对肿瘤细胞的杀伤或防止对肿瘤细胞的杀伤 艾滋病、自身免疫性疾病、神经退行性疾病等 压力可能起到一定作用。

DESCRIPTION: (Adapted from the applicant's abstract): Thiols are generally thought of a antioxidants which protect against oxidative stress imposed by diverse agents and conditions such as ionizing radiation, some chemotherapeutic drugs, neurodegenerative diseases, autoimmune diseases, ischemia/reperfusion, etc. However, in some circumstances, e.g., in the presence of traces of transition metals, some antioxidants become pro-oxidants, causing cell killing, including apoptosis. We have developed a detailed, testable, stepwise model to explain this paradoxical action of thiols in apoptosis. According to the model, under certain cellular conditions, thiols: (i) undergo metal-catalyzed oxidation producing the reactive oxygen species, H2O2, O(2)- and .OH, (ii) disrupt intracellular calcium homeostasis by alteration of critical thiols or disulfides in calcium-regulating proteins, and (iii) activate the transcription factor NF-kB, (iv) resulting in apoptosis. We further hypothesize that bcl-2 can act at one or more steps in this pathway to inhibit thiol-induced apoptosis. The specific aims are designed to test each step in the model individually as well as to demonstrate the integration of the steps into a pathway from thiols to apoptosis. In addition, we will obtain quantitative data on the relationship between apoptosis and loss of clonogenicity in thiol-treated cells to address whether apoptosis accounts for all the cell death and whether agents that appear to inhibit apoptosis (e.g., calcium chelators or bcl-2) increase long-term cell survival or only delay cell death. Most of these studies will use the model thiol dithiothreitol in human leukemia HL-60 cells. Apoptosis will be shown by morphological and DNA fragmentation criteria and quantitated using DNA fragmentation and TUNEL assays. .OH will be measured using sensitive, specific, fluorometric techniques, calcium will be measured using fluorescent intracellular calcium probes, and NF-kB activity will be assessed using the gel mobility shift assay. Interest in using thiols and other antioxidants to prevent or treat conditions believed to be caused, at least in part, by reactive oxygen species is growing rapidly. Thus, it is important to understand possible detrimental effects of these agents. Furthermore, because of their documented roles in oxidative stress, calcium homeostasis, NF-kB activation and apoptosis, thiols are unique probes for study of chemical, enzymatic and genetic mechanisms involved in each of these processes and in their interactions that cause cell death. Ultimately the knowledge gained in this study may lead to development of novel, specific therapeutics, based on the properties of thiols, that are targeted to particular intracellular domains to "turn on" protective pathways or "turn off" undesired cellular processes, e.g., to enhance cell killing in tumor cells or prevent cell killing in AIDS, autoimmune diseases, neurodegenerative diseases, etc., where oxidative stress may play a role.