During the past two years as a hematology-oncology research fellow in Dr. Levy's laboratory, I developed a series of sequentially selected, adriamycin resistant murine erythroleukemia cells. This research was supported by an individual NRSA from the NCI. The project focused on the earliest mutants to arise and their progression to high-level, multidrug resistance. One particular mutant, PC4-40, was found to efflux anthracyclines but did not overexpress mdr or P-glycoprotein. An in-depth study of drug transport and the molecular basis for efflux in this cell line forms the basis for this current proposal. Anthracycline uptake and efflux will be studied using cytoplasts and intracellular drug distribution examined using digitized fluorescence microscopy. The substrate specificity for the efflux system will be determined. Photoaffinity labelling techniques should identify the new efflux protein and confirm whether efflux is related to a newly expressed 45-kd membrane protein. Microsequencing of the protein's N-terminus will be attempted after separation by 2D electrophoresis or after immunoprecipitation. Synthetic oligonucleotides based on protein sequence data will be used to screen a PC4-40 cDNA library. The technique of differential hybridization, subtracted probes, or subtracted libraries will be used as alternative approaches to cloning the gene encoding the PC4-40 efflux protein. After cloning, the gene will be sequenced and transfected to prove its role in acquired drug resistance. This proposal represents a new direction in my research training. It makes use of findings from my previous work, but now introduces the molecular biology discipline in identification and characterization of this efflux protein. New collaboration with Dr. Robert Taub for the drug distribution studies and new molecular biology advisors here at Tufts - New England Medical Center (Drs. T. Krontiris and D. Chikaraishi) will significantly broaden my research experience. This scientific environment here has been extremely beneficial to me with intellectual and technical support from the many members of the departments of Medicine and Molecular Biology and Microbiology. This award will provide in-depth training in molecular biology and protein chemistry which will enhance my development to an independent investigator.
