Among different types of drug resistance in cancer, the phenomenon of multidrug resistance constitutes a particularly important clinical problem since it involves resistance to many commonly used anticancer agents, including Vinca alkaloids, anthracyclines and epypodophyllotoxins. Multidrug resistance in human cells results from increased expression of a gene designated mdrl. The mdrl gene is a member of a multigene family, which includes at least one other expressed gene, mdr2. The product of the mdrl gene is a large membrane protein (P-glycoprotein), which most probably functions as an efflux pump, providing for decreased drug accumulation in multidrug-resistant cells. An altered pattern of cross-resistance to different drugs was found in one case to result from point mutations in the mdrl gene leading to a single amino acid substitution in P-glycoprotein. Many aspects of P- glycoprotein function are poorly understood. In particular, it is unknown what determines the specificity of P-glycoprotein interaction with structurally different drugs, and how mdrl gene expression is regulated. In the proposed studies the intron/exon structure of the mdrl gene will be determined by comparison of cDNA and genomic clones. The presence of point mutations in the mdrl gene in different multidrug-resistant cell lines will be investigated by RNase protection assays. The role of the tentatively identified drug-binding, nucleotide-binding and glycosilation sites of P-glycoprotein will be analyzed by oligonucleotide-directed mutagenesis. Localized random mutagenesis will be used to identify other amino acid residues involved in the binding of monoclonal antibodies, drugs and P-glycoprotein inhibitors. Regulation of mdrl gene expression will be studied by identifying the compounds which induce the expression of this gene in tissue culture and by identifying and analyzing cis-regulatory sequences in the mdrl gene. The role of the mdr2 gene will be studied by cloning and sequencing of full-length mdr2 cDNA, analyzing mdr2 expression in different types of cells and by gene transfer and expression of mdr2. The specificity and role of DNA rearrangements observed in the mdr2 gene will also be analyzed. Finally, protocols for detection of mdrl expression in clinical tumor samples, based on in situ RNA hybridization, will be developed and optimized for clinical studies.
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