Analysis of the MRP4 Drug Resistance Profile in Transfected NIH3T3 Cells

Abstract

BACKGROUND: Multidrug resistance-associated protein (MRP) 1 and canalicular multispecific organic anion transporter (cMOAT or MRP2) are adenosine triphosphate-binding cassette transporters that confer resistance to anticancer agents. In addition to these two transporters, there are at least four other human MRP subfamily members (MRP3 through MRP6). We and others reported previously that MRP3 is capable of conferring resistance to certain anticancer agents. In this study, we investigated whether MRP4 (MOAT-B), whose transcript accumulates to the highest levels in prostate tissue, has the capacity to confer drug resistance. METHODS: MRP4-transfected NIH3T3 cells were generated, and their drug sensitivity was analyzed. The subcellular localization of MRP4 was assessed by immunohistochemical analysis in transfected cells and in prostate tissue. Statistical tests were two-sided. RESULTS: MRP4 was detected as a 170-kd protein that was localized in the plasma membrane and cytoplasm of transfected cells. The MRP4 transfectants displayed 5.5-fold increased resistance to methotrexate in short-term drug-exposure assays (P=.022) and exhibited decreased cellular accumulation of this agent at 4 hours (P=.006) and 24 hours (P<.001). In continuous-exposure assays, however, the MRP4 transfectants did not display increased resistance for either methotrexate or natural product cytotoxic agents (anthracyclines, etoposide, vinca alkaloids, and paclitaxel [Taxol]). However, the transfectants did show increased resistance (2.3-fold) for the anti-acquired immunodeficiency syndrome nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) (P=.022) in continuous-exposure assays. Consistent with MRP4's plasma membrane localization in transfected cells, analysis of prostate tissue showed that MRP4 protein was localized primarily in the basolateral plasma membranes of tubuloacinar cells. CONCLUSIONS: These results indicate that MRP4 confers resistance to short-term methotrexate and continuous PMEA treatment. Given its structure, drug resistance profile and subcellular localization, MRP4 probably functions as an amphipathic anion efflux pump whose substrate range includes glutamate and phosphate conjugates.