BIOCHEM DISSECTION OF P53-DEPENDENT APOPTOSIS PATHWAY

P53 依赖性细胞凋亡途径的生物化学剖析

• 批准号: 6522438
• 负责人: HAN-FEI DING
• 金额: $ 15.28万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6522438
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; affinity chromatography; apoptosis; biochemistry; cell free system; computer assisted sequence analysis; cysteine endopeptidases; enzyme activity; gene expression; gene mutation; immunoprecipitation; laboratory mouse; molecular cloning; northern blottings; nucleic acid sequence; p53 gene /protein; polymerase chain reaction; protein structure function; protein tyrosine phosphatase

p53-mediated apoptosis of cells with DNA damage or oncogene overexpression is a major mechanism for its function as a tumor suppressor. Many antitumor treatments such as ionizing radiation and DNA-damaging chemotherapy agents kill cancer cells through the p53-dependent apoptosis pathway. The long-term goal of the proposed studies is to understand the molecular mechanism controlling p53-mediated apoptosis so that better therapeutic strategies can be developed. The specific aims in this proposal represent the beginning of our efforts to define in detail the components of the biochemical pathway of p53-dependent apoptosis and its regulation by employing a cell-free system that recapitulates p53-dependent activation of caspases. First, the gene coding for a recently purified caspase-activating protein CAP110 will be cloned, and its role in p53-dependent apoptosis will be examined in both cell-free and cell-based systems. Second, an affinity-based chromatographic scheme will be used to purify a candidate protein tyrosine phosphatase essential for p53-dependent caspase activation in the cell-free extracts. Its gene will be cloned and its function in p53-dependent apoptosis will be examined in cells. Third, a systematic fractionation scheme will be employed to identify other components in the cell-free extracts that are required for p53-dependent activation of caspases. Forth, an anti-apoptotic activity from transformed p53-/- mouse embryo fibroblasts will be purified and cloned. Northern blot analysis will be performed to determine whether its expression is negatively regulated by p53. Understanding p53-dependent apoptosis and its regulation at the levels of biochemical process will help improve current anticancer therapies for tumors with wild-type p53 and provide targets for the development of drugs which restore the apoptotic response in p53 deficient cancer cells. Dr. David E. Fisher will supervise this project and provide guidance in my transition to an independent investigator.

p53 介导的 DNA 损伤或癌基因过度表达的细胞凋亡是其作为肿瘤抑制因子发挥作用的主要机制。 许多抗肿瘤治疗，例如电离辐射和 DNA 损伤化疗药物，通过 p53 依赖性细胞凋亡途径杀死癌细胞。 拟议研究的长期目标是了解控制 p53 介导的细胞凋亡的分子机制，以便开发更好的治疗策略。 该提案中的具体目标代表了我们努力详细定义 p53 依赖性细胞凋亡的生化途径的组成部分及其通过采用无细胞系统来概括 p53 依赖性半胱天冬酶激活的调节的开始。 首先，将克隆编码最近纯化的 caspase 激活蛋白 CAP110 的基因，并在无细胞和基于细胞的系统中检查其在 p53 依赖性细胞凋亡中的作用。其次，基于亲和力的色谱方案将用于纯化无细胞提取物中 p53 依赖性 caspase 激活所必需的候选蛋白酪氨酸磷酸酶。 其基因将被克隆，并在细胞中检查其在 p53 依赖性细胞凋亡中的功能。 第三，将采用系统分级分离方案来鉴定无细胞提取物中 p53 依赖性半胱天冬酶激活所需的其他成分。 第四，将纯化并克隆来自转化的p53-/-小鼠胚胎成纤维细胞的抗凋亡活性。将进行 Northern blot 分析以确定其表达是否受到 p53 的负调控。 了解p53依赖性细胞凋亡及其在生化过程水平上的调节将有助于改善目前针对野生型p53肿瘤的抗癌疗法，并为开发恢复p53缺陷癌细胞凋亡反应的药物提供靶标。 David E. Fisher 博士将监督这个项目，并为我向独立研究者的过渡提供指导。