ICANCER RESEARCH 58, 2741-2747, July I. 1998] Advances in Brief Isolation of MOAT-B, a Widely Expressed Multidrug Resistance-associated Protein/ Canalicular Multispecific Organic Anión Transporter-related Transporter1 Kun Lee, Martin G. Belinsky, Daphne W. Bell, Joseph R. Testa, and Gary D. Krulr Division of Medical Sciences, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 IK. L., M. G. B., J. R. T., G. D. K.ÃOE. and Massachusetts General Hospital Cancer Center, Charleslown, Massachusetts 02129 [D. W. B.I Abstract Multidrug resistance-associated protein (MRP) and canalicular nmil i- specific organic anión transporter (cMOAT) are closely related mamma lian ATP-binding cassette transporters that export organic anions from cells. Transfection studies have established that MRP confers resistance to natural product cytotoxic agents, and recent evidence suggests the possi bility that cMOAT may contribute to cytotoxic drug resistance as well. Based upon the potential importance of these transporters in clinical drug resistance and their important physiological roles in the export of the amphiphilic products of phase I and phase II metabolism, we sought to identify other MRP-related transporters. Using a degenerate PCR ap proach, we isolated a cDNA that encodes a novel ATP-binding cassette transporter, which we designated MOAT-B. The MOAT-B gene was mapped using fluorescence in situ hybridization to chromosome band 13q32. Comparison of the MOAT-B predicted protein with other trans porters revealed that it is most closely related to MRP, cMOAT, and the yeast organic anión transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a —¿200 amino acid NH2-terminal hydrophobic extension that is present in MRP and cMOAT and which is predicted to encode several transmembrane spanning segments. In addition, the MOAT-B tissue distribution is distinct from MRP and cMOAT. In contrast to MRP, which is widely expressed in tissues, including liver, and cMOAT, the expression of which is largely restricted to liver, the MOAT-B transcript is widely expressed, with particularly high levels in prostate, but is barely detectable in liver. These data indicate that MOAT-B is a ubiquitously expressed transporter that is closely related to MRP and cMOAT and raise the possibility that it may be an organic anión pump relevant to cellular detoxification. Introduction Pgp3, the product of the MDR1 gene, was the first ABC transporter shown to confer resistance to cytotoxic agents. Pgp functions as an ATP-dependent efflux pump that reduces the intracellular concentra tion of a variety of chemotherapeutic agents by transporting them across the plasma membrane ( 1). The multidrug resistance phenotype associated with overexpression of Pgp is of considerable clinical interest because natural product drugs are second only to alkylating agents in clinical utility, and many effective chemotherapeutic regi mens contain more than one natural product agent. More recently, we Received 3/3/98; accepted 5/15/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grant CA63173 and American Cancer Society Grant DHP-131 (to G. D. K.l. NIH Grant CA06927 lo Fox Chase Cancer Center, and by an appropriation from the Commonwealth of Pennsylvania. 2 To whom requests for reprints should be addressed, at Fox Chase Cancer Center, 7701 Burholme Ave.. Philadelphia. PA 19111. Phone: (215)728-5317; Fax: (215)728- 3603. 1 The abbreviations used are: Pgp. P-glycoprotein; ABC. ATP-binding cassette; cMOAT, canalicular multispecific organic anióntransporter; MRP, multidrug resistance- associated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SUR, sulfonyl urea receptor; CFTR, cystic fibrosis transmembrane conductance regulator; EST. ex pressed sequence tag; FISH, fluorescence in situ hybridization; YCFI, yeast cadmium factor I. and others have reported transfection studies indicating that MRP, another ABC family transporter (2), confers a multidrug resistance phenotype that includes many natural product drugs, but is distinct from the resistance phenotype associated with Pgp (3-7). MRP shares only limited amino acid identity with Pgp, and this is reflected in the different substrate specificities of the two transporters. In contrast to Pgp, MRP can transport a wide range of anionic organic conjugates, including glutathione 5-conjugates (8). In addition to Pgp and MRP, there may be other transporters that are involved in cytotoxic drug resistance. In the case of natural product drugs, resistant cell lines that display a multidrug-resistant phenotype associated with a drug accu mulation deficit, but do not overexpress Pgp or MRP, have been described (9). ABC transporters have also been linked to cisplatin resistance, and several lines of evidence suggest the possibility that pumps specific for organic anions may be involved: (a) decreased cisplatin accumulation is observed consistently in cisplatin-resistant cell lines (10); (b) cisplatin is conjugated to glutathione in the cell, and this anionic conjugate is toxic in an in vitro biochemical assay (11); and (c) biochemical studies using membrane vesicle preparations have shown that cisplatin-resistant cells lines have enhanced expression of an ATP-dependent transporter of cisplatin-glutathione and other glu tathione 5-conjugates, such as the cystinyl leukotriene C4 (12, 13). Whereas MRP is an organic anión transporter, the reported drug resistance profile of MRP-transfected cells does not extend to this agent (6, 7), and to date only one cisplatin-resistant cell line has been reported to overexpress MRP (14). These observations suggest the possibility that organic anión transporters other than MRP may con tribute to cisplatin resistance. Consistent with this possibility, cMOAT, an MRP-related transporter that functions as a major organic anión transporter in liver, has been reported to be overexpressed in cisplatin-resistant cell lines (15, 16). A more direct link between cMOAT and cytotoxic drug resistance is suggested by a recent report in which transfection of a cMOAT antisense construct into a liver cancer cell line resulted in sensitization to cisplatin, daunorubicin, and other cytotoxic agents (17). In view of the possibility that other MRP-related transporters may be involved in cytotoxic drug resistance, we sought to identify novel, related transporters. In this study, we report the isolation of MOAT-B using a degenerative PCR approach. Analysis of the MOAT-B struc ture indicates that it is closely related to MRP and cMOAT, suggest ing that it may transport similar substrates. In addition, MOAT-B is widely expressed in human tissues, with highest levels in the prostate. These results indicate that MOAT-B is an MRP/cMOAT-related transporter and raise the possibility that it may be an organic anión pump relevant to cellular detoxification in many human tissues. Materials and Methods Isolation of MOAT-B cDNA. Forward (CT(A/G/T) GT(A/G/T) GC(A/ G/T) GT(A/G/T) GT(A/G/T) GG(A/G/OT)] and reverse [(G/A)CT (A/G/C/ T)A(A/G/C) (A/G/C/T)GC (A/G/C/T)(G/C)(T/A) (A/G/C/T)A(A/G) (A/G/C/ T)GG (A/G/C/T)TC <A/G)TC] degenerate oligonucleotide primers were 2741 on May 19, 2016. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from