Characterization of Monoclonal Antibodies Recognizing a M_r 180,000 P-Glycoprotein: Differential Expression of the M_r 180,000 and M_r 170,000 P-Glycoproteins in Multidrug-resistant Human Tumor Cells

Abstract

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 M_r 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, ³²P₁, 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 M_r 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 M_r 180,000 protein is expressed in cells which also contain a M_r 170,000 P-glycoprotein. The relative amounts of the M_r 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the M_r 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.