PHARMACOLOGICAL CHARACTERIZATION OF RAT MULTIDRUG RESISTANCE PROTEIN 1 ( MRP 1 )

Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH). The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents. Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents. Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation. Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1). Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17 -estradiol-17-( -Dglucuronide) (E217 G) and estrone 3-sulfate, relatively poorly. Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity. Despite the fact that the rodent proteins transport E217 G poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported. Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins. Human multidrug resistance protein 1 (hMRP1) was originally cloned from a multidrug-resistant human small cell lung cancer cell line, H69AR, that was selected in the presence of doxorubicin (Cole et al., 1992). When overexpressed in tumor cells, hMRP1 confers resistance to anticancer drugs that include Vinca alkaloids, epipodophyllotoxins and anthracyclines, as well as some heavy metal oxyanions (Cole et al., 1994; Grant et al., 1994; Zaman et al., 1994). MRP1 is now known to be a primary active transporter of conjugated organic anions (Leslie et al., 2001a; Borst and Oude Elferink, 2002). The list of substrates transported by MRP1 includes GSH conjugates such as the leukotriene LTC4, as well as a number of glucuronide and sulfateconjugates (Leier et al., 1994; Muller et al., 1994; Jedlitschky et al., 1996; Loe et al., 1996a, 1997; Leslie et al., 2001b; Qian et al., 2001). Transport of certain anionic conjugates, as well as some unmodified chemotherapeutic drugs to which MRP1 confers resistance is augmented in the presence of GSH (Loe et al., 1996b, 1998; Ding et al., 1999; Renes et al., 1999; Leslie et al., 2001b; Qian et al., 2001). In some cases, cotransport of GSH can be demonstrated but not in others (Rappa et al., 1997; Loe et al., 1998, 2000; Leslie et al., 2001b; Qian et al., 2001). GSH alone is inefficiently transported by MRP1, although transport can be stimulated by compounds such as verapamil and a variety of different flavonoids (Loe et al., 2000; Qian et al., 2001; Leslie et al., 2001b, 2003). Despite a high level of sequence conservation, the murine ortholog of MRP1, mMRP1, differs from the human protein with respect to its ability to confer resistance to anthracyclines and to transport the conjugated estrogen E217 G (Stride et al., 1996, 1997). Thus, the mouse protein fails to confer resistance to several commonly used anthracyclines and transports E217 G very poorly. These differences place some limits on the usefulness of the mouse as a preclinical model for the development of agents that might be used to selectively reverse drug resistance mediated by MRP1. To date, the substrate specificity of other mammalian MRP1 orthologs has not been characterized in any detail. However, bovine MRP1 (bMRP1) has been cloned recently, and the drug resistance phenotype of transfected cell This work was supported by Ono Pharmaceutical Co. in Japan and a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run. 1 Canada Research Chair in Cancer Biology and Senior Scientist of Cancer Care Ontario. 2 Stauffer Research Professor of Queen’s University. 3 Abbreviations used are: hMRP1, human multidrug resistance protein 1; bMRP1, bovine MRP1; rMRP1, rat MRP1; GSH, glutathione; LTC4, leukotriene C4; VP-16, etoposide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, Sprague-Dawley; HEK, human embryonic kidney; TMs, transmembrane helices; E217 G, 17 -estradiol-17-( -D-glucuronide); PC7, episomal expression vector pCEBV7; PCR, polymerase chain reaction; kb, kilobase(s); S, sense; AS, antisense; mAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NBD, nucleotide binding domain; MSD, membrane-span-