Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells

Background: Multidrug resistance-associated protein (MRP) 1 and canalicular multispecific organic anion transporter (cMOAT or MRP2) are adenosine trip hosphate-binding cassette transporters that confer resistance to anticancer agents. In addition to these two transporters, there are at least four oth er human MRP subfamily members (MRP3 through MRP6). me and others reported previously that MRP3 is capable of conferring resistance to certain antican cer agents, In this study, me investigated whether MRP4 (MOAT-B), whose tra nscript accumulates to the highest levels in prostate tissue, has the capac ity to confer drug resistance. Methods: MRP4-transfected NIH3T3 cells were generated, and their drug sensitivity was analyzed. The subcellular localiz ation of MRP4 was assessed by immunohistochemical analysis in transfected c ells and in prostate tissue. Statistical tests were two-sided. Results: MRP 4 was detected as a 170-kd protein that was localized in the plasma membran e and cytoplasm of transfected cells, The MRP4 transfectants displayed 5.5- fold increased resistance to methotrexate in short-term drug-exposure assay s (P = .022) and exhibited decreased cellular accumulation of this agent at 4 hours (P = .006) and tl hours (P<.001). In continuous-exposure assays, h owever, the MRP4 transfectants did not display increased resistance for eit her methotrexate or natural product cytotoxic agents (anthracyclines, etopo side, vinca alkaloids, and paclitaxel [Taxol]). However, the transfectants did show increased resistance (2.3-fold) for the anti-acquired immunodefici ency syndrome nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PR-I EA) (P = .022) in continuous-exposure assays. Consistent with MRP4's plasma membrane localization in transfected cells, analysis of prostate tissue sh owed that MRP4 protein was localized primarily in the basolateral plasma me mbranes of tubuloacinar cells. Conclusions: These results indicate that MRP 4 confers resistance to short-term methotrexate and continuous PMEA treatme nt. Given its structure, drug resistance profile and subcellular localizati on, MRP4 probably functions as an amphipathic anion efflux pump whose subst rate range includes glutamate and phosphate conjugates.