Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine

Two photoactive radiolabeled analogs of colchicine, N-(p-azido[3,5-[3H]benzoyl)aminohexanoyldeacetylcolchicine ([3H]NABC]) and N-(p-azido-[3-125I]salicyl)aminohexanoyldeacetylcolchicine ([125I]NASC) were synthesized and used to identify colchicine-specific acceptor(s) in membrane vesicles from multidrug resistant (MDR) variant DC-3F/VCRd-5L Chinese hamster lung cells. Both [3H]NABC and [125I]NASC specifically photolabeled a prominent 150-180 kDa polypeptide in membrane vesicles from DC-3F/VCRd-5L cells. The photolabeled polypeptide was immunoprecipitated by monoclonal antibody C219 specific for the MDR-related P-glycoprotein (P-gp) indicating the identity of this protein with P-gp. Colchicine at 1000 microM reduced [3H]NABC photolabeling of P-gp by 72%. Furthermore, 100 microM of colchicine, vincristine, vinblastine, doxorubicin and actinomycin D inhibited [125I]NASC photolabeling by 45, 88.8, 91.1, 61.5, and 51% respectively. However, methotrexate did not affect the [125I]NASC photolabeling of P-gp, indicating the multidrug specificity of the P-gp colchicine acceptor for drugs to which these cells are resistant.