The contractor plans to develop a panel of well-characterized human tumor sublines that express the MDR (multidrug resistance) phenotype for use in large-scale, disease-oriented anticancer drug screening programs. Initially, the contractor will focus on deriving MDR sublines from established human breast, ovarian and lung lines. The sublines will be obtained using two different strategies: by selection in media containing adriamycin or vinblastine or by gene transfer techniques. For the gene transfer experiments, mdr expression vectors cloned from a human fetal liver cDNA library made in phage lambda gtll will be stably integrated into the genomes of human tumor cell lines by calcium phosphate precipitation or electroporation. Expression plasmids will be constructed using different promoters to develop a system with high efficiency for expression. Sublines will be characterized for multiple drug resistance, mdr gene copy number and mRNA level, as well as reversion of the MDR phenotype after treatment with calcium channel blockers, such as verapamil. The use of these sublines in drug screening program should facilitate the identification and development of new anticancer agents which are effective for the management of tumors that are resistant to current therapies. This study also will contribute to increased understanding of the molecular mechanisms contributing to the MDR phenotype.
