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Publications (2)17.3 Total impact

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    ABSTRACT: ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.
    Cancer Research 08/2003; 63(14):4048-54. · 8.65 Impact Factor
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    ABSTRACT: Breast cancer resistance protein (BCRP)/MXR/ABCG2 is a new member of the family of ATP-dependent drug efflux proteins. Whereas overexpression of another member of this family, P-glycoprotein, minimally affects the cytotoxicity of camptothecins (CPTs), overexpression of wild-type as well as certain mutant BCRPs confers resistance to CPT analogues that are used clinically, including topotecan and irinotecan. Relatively little is known regarding the effects of BCRP on other CPT analogues. We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). The results indicate that overexpression of either P-glycoprotein, multidrug resistance protein type 1, or multidrug resistance protein type 2 has little effect on the cytotoxicity of 9-NC or 9-AC. By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Furthermore, overexpression of wild-type or mutant BCRP is associated with reduced intracellular accumulation of 9-AC, but not 9-NC. In addition, immunoblotting studies indicate that whereas increased BCRP expression is evident in cells selected for resistance to irinotecan, BCRP expression is not detectable in two different cell lines selected for resistance to 9-NC. Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Furthermore, the results suggest that polar groups at the 9 or 10 position of the CPT A ring facilitate interaction with BCRP and have implications for the clinical development of new CPT analogues.
    Cancer Research 07/2003; 63(12):3228-33. · 8.65 Impact Factor