Achieving high intracellular concentrations of antiretroviral drugs, essential for effective therapy, may be prevented by the specific efflux of nucleoside monophosphates (not di- or triphosphates), however, no specific efflux transporter for these substances has been identified. Our preliminary studies have revealed that in cells resistant to azidothymidine (AZT) and (9-(2-phosphonylmethoxyethyl)adenine) (PMEA), a structural analog of dAMP and AMP, increased energy-dependent efflux of PMEA and AZT monophosphate occurs. Further studies utilizing gene specific reagents and antisera demonstrate only amplification and overexpression of MRP4 (multidrug resistance associated protein 4), a gene currently not linked to chemotherapeutic resistance. Somatic cell fusion studies demonstrated that the resistance could be conferred in a dominant fashion. Further studies, in patients have shown that high levels of MRP4 may contribute to therapeutic failure. To fully understand MRP4s' substrates, inhibitors and resistance properties, we propose to stably express both human and murine MRP4 in cells. . We will also use membrane vesicles prepared from these cells to evaluate transport in cases where anionic substrates are impermeant. We propose to characterize the murine mrp4 gene to facilitate development of the mrp4 nullizygous mouse. Both the mip4 nullizygous mouse and MRP4im~4 cell models will allow evaluation of the therapeutic efficacy and role of MRP4 in transport of antiretroviral drugs and other candidate anionic substrates. Further, the potential biological significance of the MRP4 gene has not escaped us. For a number of years an """"""""efflux system"""""""" has been described for cAMP. The efflux is energy-dependent and a reported inhibitor of cAMP efflux is also an inhibitor of PMEA efflux. Our reagents will allow us to test the novel hypothesis that MRP4 transports cyclic nucleotides, a finding that would profoundly impact our understanding of cyclic nucleotide signaling. Thus, this proposal will elucidate not only the biological role ofMRP4 in cellular transport of organic substrates, but also facilitate the development of more effective therapeutic regimens by revealing MRP4 as a major determinant of intracellular concentration of antiretroviral drugs.
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