Functional Characterization of Human Breast Cancer Resistance Protein ( BCRP , ABCG 2 ) Expressed in the Oocytes of Xenopus laevis

To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity. Cancer drug resistance remains a major obstacle to the success of curative treatment regimens for human malignancies. In experimental systems, resistance of cancer cells to multiple drugs is often associated with reduced intracellular drug accumulation and overexpression of ATP binding-cassette (ABC) transporter proteins such as Pgp (Gottesman et al., 1996) or MRP1 (Loe et al., 1996). Recently, BCRP was isolated from multidrug resistant MCF7/AdrVp human breast cancer cells (Doyle et al., 1998), which were selected with doxorubicin (Adriamycin) in the presence of verapamil (Chen et al., 1990). Human BCRP is the second member of the G subfamily of the ABC transporter proteins (ABCG2), and is closely related to the white gene of Drosophila melanogaster. In normal tissues, BCRP expression is high in placenta, venules, and stem cells (Allikmets et al., 1998; Doyle et al., 1998; Bunting, 2002), and in the apical membrane of hepatocytes and gut epithelium (Jonker et al., 2000), where it may play a role in defense from xenobiotics and drug clearance. In human cancer cells in culture, BCRP expression confers a high level of resistance to a variety of cancer chemotherapeutic drugs, including MX (Doyle et al., 1998; Miyake et al., 1999; Ross et al., 1999), camptothecin-derived topoisomerase I inhibitors (Maliepaard et al., 1999; Nakatomi et al., 2001), methotrexate (Volk et al., 2002), and FLV (Robey et al., 2001). BCRP overexpression is found in drug-selected cancer cells derived from various types of human solid tumors (Doyle et al., 1998; Maliepaard et al., 1999; Miyake et al., 1999; Ross et al., 1999), and seems to be independent of Pgp or MRP expression. BCRP This work was supported in part by a Department of Veterans Affairs Merit Review Grant (to D.D.R.) and by a Leukemia and Lymphoma Society Translational Research Program grant and National Cancer Institute grant R01CA77545 (to D.D.R. and L.A.D). ABBREVIATIONS: ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; MX, mitoxantrone; FLV, flavopiridol; Pgp, P-glycoprotein; MRP1, multidrug resistance-associated protein 1; MDR1, multidrug resistance gene 1; DNR, daunorubicin; ADR, doxorubicin (Adriamycin); TPT, topotecan; Rho123, rhodamine 123; PCR, polymerase chain reaction; UTR, untranslated region; HPLC, high-performance liquid chromatography; FTC, fumitremorgin C. 0026-895X/03/6406-1452–1462 MOLECULAR PHARMACOLOGY Vol. 64, No. 6 U.S. Government work not protected by U.S. copyright 2659/1108697 Mol Pharmacol 64:1452–1462, 2003 Printed in U.S.A. 1452 at A PE T Jornals on O cber 9, 2017 m oharm . aspeurnals.org D ow nladed from expression in clinical cancers is currently under investigation. Some reports suggest that the expression of BCRP mRNA or protein may correlate with resistant disease or immature phenotype, respectively, in acute myeloid leukemia (van den Heuvel-Eibrink et al., 2002; van der Kolk et al., 2002; Nakanishi et al., 2003). Furthermore, high expression of BCRP has been detected immunohistochemically in a variety of human solid tumors (Diestra et al., 2002). Ironically, expression of BCRP in human breast cancer biopsies seems to be low (Faneyte et al.,