BIOCHEM DISSECTION OF P53-DEPENDENT APOPTOSIS PATHWAY

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因子功能的主要机制。许多抗肿瘤治疗如电离辐射和dna损伤化疗药物通过p53依赖性凋亡途径杀死癌细胞。所提出的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。本提案的具体目标代表了我们努力详细定义p53依赖性凋亡生化途径的组成部分及其调控的开始，通过采用无细胞系统来概括p53依赖性caspases的激活。首先，将克隆最近纯化的caspase激活蛋白CAP110的基因编码，并在无细胞和基于细胞的系统中检测其在p53依赖性凋亡中的作用。其次，基于亲和的色谱方案将用于纯化候选蛋白酪氨酸磷酸酶，该候选蛋白酪氨酸磷酸酶是无细胞提取物中p53依赖性半胱天冬酶激活所必需的。我们将克隆其基因，并在细胞中研究其在p53依赖性凋亡中的作用。第三，将采用系统的分离方案来鉴定无细胞提取物中p53依赖性caspases激活所需的其他成分。第四，纯化和克隆转化的p53-/-小鼠胚胎成纤维细胞的抗凋亡活性。Northern blot分析确定其表达是否受p53负调控。了解p53依赖的细胞凋亡及其在生化过程水平上的调控，将有助于改善目前对野生型p53肿瘤的抗癌治疗，并为开发恢复p53缺失癌细胞凋亡反应的药物提供靶点。David E. Fisher博士将监督这个项目，并在我过渡到独立调查员的过程中提供指导。

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.