ROLE OF P53 GENE IN CELLULAR SENSITIVITY TO DNA DAMAGE

该项目涉及确定管理 正常和肿瘤人类细胞对DNA的反应和敏感性 破坏剂，对致癌作用和癌症都很重要的过程 治疗学候选人的研究和临床培训反映了 致力于癌症研究事业，并专注于研究 了解决定癌细胞抵抗 细胞毒性剂。在威廉博士的3年实验室经验中， 在耶鲁大学，这位候选人发现并描述了 逆转多药耐药（MDR）的一类新药的作用， 导致8个相关出版物，并在 会议，并通过广泛引用的评论文章作为专家， 药物规避MDR。经过内部临床培训后， 医学和医学肿瘤学在斯坦福大学，候选人有一个 在Philip Hanawalt教授的指导下进行了一年的基础科学培训， 在斯坦福大学学习生物科学专业的分子技术 在他的实验室开发的测量特定基因内的DNA修复。基础 候选人发现人类皮肤 Li-Fraumeni综合征患者的成纤维细胞突变纯合子 的p53肿瘤抑制基因，显着更耐紫外线- 与杂合p53突变体或正常细胞相比，的目标 该项目旨在确定p53突变改变 细胞对辐射和化疗DNA损伤剂的敏感性。 含有野生型的肿瘤细胞和非肿瘤细胞的敏感性 (wt)或突变型p53对DNA损伤剂的作用将使用细胞 存活测定。损伤诱导的程序性细胞死亡 （细胞凋亡）对表达突变型和野生型p53的细胞的敏感性将 通过评价细胞的形态学特征变化来确定 和活力，并通过琼脂糖凝胶测量DNA片段化 电泳效果 p53突变对DNA修复的速率和效率的影响 基因组和特定的DNA序列内将确定使用 定量Southern杂交和基因或链特异性探针。 将采用活化细胞分选来测量损伤， 在细胞周期的特定阶段内修复，以及跨损伤DNA 将测量合成以确定存在的DNA损伤水平 在表达突变型和野生型p53的细胞中复制期间。这项工作将 在Hanawalt博士的指导下进行，广泛的知识分子 在集团、部门和附近提供技术支持 医学中心为拟议的工作配备了齐全的设施。由 完成这个项目后，候选人计划获得一个教师 在一家大型医疗中心工作，继续临床和实验室工作， 癌症抗药性的研究。

This project is concerned with determining the mechanisms that regulate the response and sensitivity of normal and neoplastic human cells to DNA damaging agents, processes important to both carcinogenesis and cancer therapeutics. The candidate's research and clinical training reflect a commitment to a career in cancer research, and an investigative focus on understanding factors that determine resistance of cancer cells to cytotoxic agents. During a 3 year laboratory experience with Dr. William Hait at Yale, the candidate discovered and characterized the mechanism of action of a new class of drugs which reverse multidrug resistance (MDR), leading to 8 related publications, and international recognition at conferences and through a widely cited review article as an expert on the pharmacologic circumvention of MDR. Following clinical training in internal medicine and medical oncology at Stanford, the candidate has had an additional year of basic science training under Prof. Philip Hanawalt in Biological Sciences at Stanford learning specialized molecular techniques developed in his lab to measure DNA repair within specific genes. The basis for the current proposal is the candidate's finding that human skin fibroblasts from patients with Li-Fraumeni syndrome homozygous for mutation of the p53 tumor suppressor gene are significantly more resistant to UV- irradiation than heterozygous p53 mutants or normal cells. The objective of the project is to determine the mechanism by which p53 mutations alter cellular sensitivity to radiation and chemotherapeutic DNA damaging agents. The sensitivity of neoplastic and non-neoplastic cells containing wild-type (wt) or mutant p53 to DNA damaging agents will be assessed using cell survival assays. The contribution of damage induced programmed cell death (apoptosis) to the sensitivity of mutant and wt p53 expressing cells will be determined by evaluating cells for characteristic changes in morphology and viability, and measuring DNA fragmentation by agarose gel electrophoresis. The effect of p53 mutations on the rate and efficiency of DNA repair in the overall genome and within specific DNA sequences will be determined using quantitative Southern hybridization and gene or strand specific probes. Fluorescence-activated cell sorting will be employed to measure damage and repair within specific phases of the cell cycle, and translesional DNA synthesis will be measured to determine the level of DNA damage present during replication in cells expressing mutant and wt p53. The work will be performed under the guidance of Dr. Hanawalt, and extensive intellectual and technical support is available within the group, department and nearby medical center. Facilities are fully equipped for the work proposed. By the completion of this project, the candidate plans to secure a faculty position at a major medical center and continue clinical and laboratory research into cancer drug resistance.