P-glycoprotein is a plasma membrane
protein believed to mediate resistance to
natural product drugs such as
vincristine,
Adriamycin, and
actinomycin D. To facilitate the study of human
P-glycoprotein,
monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against
vincristine-resistant human
neuroblastoma (SH-SY5Y/VCR) cells. The
antibodies recognize a Mr 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in
vincristine-resistant human neuroepithelioma (MC-IXC/VCR),
vinblastine-resistant human
leukemia (CEM/VLB100), and
actinomycin D- or
vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of
proteins in cells metabolically labeled with [35S]
methionine, 32Pi, or [3H]
glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241
antigen by destructive degradation produced a pattern of results typical of a conformation-dependent
protein epitope. HYB-612 recognizes complexes of the Mr 180,000
antigen with an iodinated photoaffinity analogue of
vinblastine or with tritiated
azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of
tritium-labeled
actinomycin D or
vincristine uptake increases the amount of
drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the Mr 180,000
protein is expressed in cells which also contain a Mr 170,000
P-glycoprotein. The relative amounts of the Mr 180,000 and 170,000 species vary from one
drug-resistant cell line to another. Evidence that the Mr 180,000
protein is a
P-glycoprotein and that there is a conserved complex pattern of resistance-related
surface proteins in multidrug-resistant cells is presented in this report.