BIOCHEMICAL DISSECTION OF P53 DEPENDENT APOPTOSIS PATHW

P53 依赖性细胞凋亡途径的生化剖析

• 批准号: 2885057
• 负责人: HAN-FEI DING
• 金额: $ 11.84万
• 依托单位: DANA-FARBER CANCER INST
• 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/2885057
• 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损伤或癌基因过表达的细胞凋亡是其作为肿瘤抑制剂的主要机制。 许多抗肿瘤治疗，如电离辐射和DNA损伤化疗剂通过p53依赖性凋亡途径杀死癌细胞。 这些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。 在这个建议的具体目标代表了我们的努力，以详细定义的生化途径的p53依赖性细胞凋亡和它的监管采用无细胞系统，recapitulates p53依赖性激活半胱天冬酶的组件的开始。 首先，将克隆编码最近纯化的半胱天冬酶激活蛋白CAP 110的基因，并在无细胞和基于细胞的系统中研究其在p53依赖性细胞凋亡中的作用。其次，将使用基于亲和性的色谱方案来纯化无细胞提取物中p53依赖性半胱天冬酶活化所必需的候选蛋白酪氨酸磷酸酶。 将克隆其基因，并在细胞中检测其在p53依赖性细胞凋亡中的功能。 第三，将采用系统分级方案来鉴定无细胞提取物中p53依赖性半胱天冬酶活化所需的其他组分。 第四，从转化的p53-/-小鼠胚胎成纤维细胞中纯化并克隆抗凋亡活性。将进行北方印迹分析以确定其表达是否受p53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控将有助于改善目前野生型p53肿瘤的抗癌治疗，并为开发恢复p53缺陷癌细胞凋亡反应的药物提供靶点。大卫博士E.费舍尔将监督这个项目，并提供指导，在我过渡到一个独立的调查员。