MOLECULAR MECHANISM MULTIDRUG RESISTANCE--MAMMALIAN CELL

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Development of drug resistance to cytotoxic agents by malignant cells is a major reason for failur to present chemotherapy in the management of cancer. Development of such resistance to cytotoxic drugs that interact with structural proteins or DNA occurs not only to the selective agents but also to a wide variety of other, seemingly unrelated, compounds. The establishment of multidrug-resistance (MDR) in tissue culture cells represents a complicated process that is associated with gene amplificiation and over-expression of a plasma-membrane glycoprotein, termed P-glycoprotein. The proposed study is designed to analyze the role expression of P-glycoprotein gene(s) plays in the establishment and maintenance of drug resistance in multidrug- resistant sublines of the parent Chinese hamster lung (CHL) cell line, DC-3F. The experimental strategy to characterize these geen transcripts will be to first prepare cDNA libraries in pUC9 vectors from MDR sublines containing overexpressed genes. These libraries wil be constructed under conditions that will generate full-length cDNAs and screended with nick-translated P- glycoprotein cDNA probes that have been prepared from drug resistant lines. cDNAs corresponding to the nonamplified transcripts from the parent line, DC-3F, will be constructed in lambda gt10 vectors and these libraries will be screened with similar probes. Full-length cDNAs isolated after such screening will be compared to each other using fine restriction mapping, and if necessary, sequencing studies to determine the extent to which heterogeneity of multidrug-resistant-associated (MDRA) gene expression exists between the various sublines. Transfection of a functional full-length MDRA cDNA from a resistant subline to the drug-sensitive parent line will be carried out to assess the actual role the MDRA gene expression, other experiments are designed to use anti-sense RNAs to inhibit expression of members of the MDRA multigene family. Effects of such anti-sense inhibition will be assayed by assessing possible changes in drug-resistance as determined by ED50 values during clonogenic assays. Finally, to determine if differential gene expression is controlled at the level of 5' regulatory sequences, experiments will be carried out to isolate and characterize promoter sequences of DC-3F and one of its resistant sublines, DC-3F/ADXC. The personnel and laboratory facilities of Sloan-Ketting Institute are well equipped to undertake this project and such basic research may lead to the eventual development of strategies to overcome multidrug- resistance in the clinical setting.