Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies

Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR ) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR 3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably m inor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can tran sport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibod ies are required for wide-scale studies on the putative contribution of the se closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of mo noclonal antibodies (Mabs) detecting several MDR-related transporter protei ns in both human and animal tissues. The panel consists of P3II-1 and P3II- 26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M 2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5I I-54 for MRP5. All Mabs in the panel appeared to be fully specific for thei r cognate antigens, both in Western blots and cytospin preparations, as rev ealed by lark of crossreactivity with any of the other family members. Inde ed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunoh istochemical analyses, distinct differences in reactivity and suitability w ere noted. These Mabs should become valuable tools in studying the physiolo gical functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.