BIOCHEM DISSECTION OF P53-DEPENDENT APOPTOSIS PATHWAY

P53介导的DNA损伤或癌基因过表达细胞的凋亡是其发挥肿瘤抑制作用的主要机制。许多抗肿瘤治疗，如电离辐射和DNA损伤的化疗药物，通过依赖于P53的细胞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。这项提案中的具体目标代表了我们努力详细定义P53依赖的细胞凋亡的生化途径的组成部分以及通过使用一个概括P53依赖的caspase激活的无细胞系统来调控它的努力的开始。首先，将克隆最近纯化的caspase激活蛋白CAP110的编码基因，并将在无细胞和基于细胞的系统中研究其在P53依赖的细胞凋亡中的作用。其次，基于亲和力的层析方案将用于在无细胞提取物中纯化对P53依赖的caspase激活至关重要的候选蛋白酪氨酸磷酸酶。它的基因将被克隆，并将在细胞中检测其在P53依赖的细胞凋亡中的功能。第三，系统的分级方案将被用来确定无细胞提取物中的其他成分，这些成分是依赖于p53的caspase激活所必需的。第四，将从转化的P53-/-小鼠胚胎成纤维细胞中纯化和克隆抗凋亡活性。Northern印迹分析将确定其表达是否受到P53的负调控。了解p53依赖的细胞凋亡及其在生化过程水平上的调控，将有助于改进目前野生型p53肿瘤的抗癌治疗，并为开发恢复p53缺失癌细胞的凋亡反应的药物提供靶点。大卫·E·费舍尔博士将监督这一项目，并在我向独立调查员过渡的过程中提供指导。

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.