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.
