ROLE OF P53 GENE IN CELLULAR SENSITIVITY TO DNA DAMAGE

这个项目关注的是确定调节 正常人和肿瘤细胞对DNA的反应和敏感性 破坏性物质，对致癌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 致力于癌症研究的事业，并专注于 了解决定癌细胞抗药性的因素 细胞毒剂。在威廉博士的三年实验室经验中 在耶鲁大学，这位候选人发现并描述了 一类逆转多药耐药(MDR)的新型药物的作用， 出版相关出版物8篇，并在以下网站获得国际认可 通过一篇被广泛引用的评论文章，作为一名关于 多药耐药的药物规避。在内科接受临床培训后 在斯坦福大学的医学和内科肿瘤学，这位候选人有一个 在菲利普·哈纳瓦特教授的指导下增加了一年的基础科学培训 斯坦福大学生物科学专业学习分子技术 在他的实验室开发，用来测量特定基因内的DNA修复。其基础是 因为目前的提案是候选人发现人类皮肤 Li-Fraumeni综合征患者成纤维细胞基因突变 的抑癌基因对紫外线的抵抗力显著增强。 辐射效果优于杂合子突变型p53或正常细胞。的目标是 该项目是为了确定p53突变改变的机制 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的损伤剂将使用cell进行评估 生存分析。损伤诱导的细胞程序性死亡的贡献 (细胞凋亡)对突变型和wt p53表达的细胞的敏感性 通过评估细胞在形态上的特征变化来确定 和活力，并用琼脂糖凝胶测定DNA片段化 电泳法。其效果 P53基因突变对DNA修复速度和效率的影响 基因组和特定DNA序列将使用以下方法确定 定量Southern杂交和基因或链特异性探针。 将使用荧光激活的细胞分选来测量损伤和 在细胞周期的特定阶段进行修复，以及转移DNA 合成将被测量以确定存在的DNA损伤程度 在表达突变体和wt p53的细胞中复制。这项工作将是 在哈纳瓦特博士的指导下进行，并拥有广泛的知识 并在集团内部、部门和附近提供技术支持 医疗中心。设施齐全，可进行拟议的工作。由 完成这个项目后，候选人计划获得一名教员。 在大型医疗中心工作，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.