DESCRIPTION: (Applicant's Abstract) Ovarian cancer is a disease characterized by high initial response rates to aggressive platinum- based combination chemotherapy, however patients with advanced disease are rarely cured by this modality. Therapy fails because of the emergence of tumor cells resistant to drugs and irradiation. The applicant's long term goal is to identify the genetic basis for resistance to platinum-containing anticancer drugs so that better strategies for treatment can be developed. In order to identify these mechanisms, the applicant has used in vitro selection with cisplatin to produce ovarian cancer cell lines with various levels of primary cisplatin resistance and cross-resistance to diverse drugs. His study of these cell lines has identified DNA repair as an important mechanism of resistance, but he has also learned that resistance is multifactorial and is accompanied by diverse genetic changes. Some of these alterations in gene expression likely contribute to the phenotype whereas others could well be irrelevant. These findings have directed the development of this application which includes as a critical component, strategies to identify the clinical significance of his findings. The importance of DNA repair to cisplatin resistance in his model and those of others directed the development of his Specific Aim 1: Establish the presence of increased DNA repair activity in extracts from cisplatin resistant cell lines, purify the rate-limiting components and determine their clinical relevance. Identification of the critical rate-limiting component(s) to platinum-DNA adduct repair as they relate specifically to cisplatin resistance will provide the tools to ascertain clinical relevance and identify potential new drug targets. The applicant has found that resistance is accompanied by both causal and casual genetic changes and is multifactorial with components, in addition to increased DNA repair, which include (i) decreased cell- associated cisplatin; (ii) increased potential for drug inactivation; (iii) alterations in the types of platinum-DNA adducts formed; and (iv) increased amounts of platinum-DNA damage required for cytotoxicity. This has directed the development of Specific Aim 2: Identify cisplatin resistance genes using a functional assay for resistance. This expression cloning strategy substantially eliminates the identification of genetic changes associated with cisplatin resistance that are not causally involved in the process. The multifactorial nature of cisplatin resistance has also directed the development of Specific Aim 3: Determine the relative contribution of an individual mechanism to the total cisplatin resistance phenotype. This aim will use gene knockout to silence a potential resistance gene or pathway in resistant cells. Using this approach on a clinically relevant gene in appropriate models will establish the magnitude of a specific gene's contribution to resistance.
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