The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

Multidrug Resistance Protein 1 (MRP1) 1 is a member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that has been shown to confer resistance to a variety of natural product type drugs (1-6). The drug resistance phenotype conferred by MRP1 is similar to that resulting from overexpression of P-glycoprotein (P-gp) (reviewed in Refs. 7-9) and is typically associated with an ATP-dependent decrease in drug accumulation and an increase in drug efflux (4, 6). Although both ABC proteins can function as energy-dependent efflux pumps for a range of natural product type drugs, there is very limited primary structure similarity between them, and phylogenetic analyses suggest that they evolved from different ancestral proteins. There is also considerable evidence that the mechanisms by which MRP1 and P-gp transport drugs are different (reviewed in Ref. 8).In addition to its ability to confer multidrug resistance, MRP1, unlike P-gp, has been shown by in vitro studies using inside-out membrane vesicles to transport a structurally diverse array of organic, anionic conjugates (reviewed in Ref. 9). These include GSH-, glucuronide-, and sulfate-conjugated aliphatic, prostanoid, and heterocyclic compounds. The two highest affinity substrates identified to date are the proinflammatory cysteinyl leukotriene C 4 (LTC 4 ) (10 -12) and the GSH-

Multidrug resistance is frequently characterized by an ATPdependent reduction in cellular drug accumulation. This phenotype can occur in mammalian cells by overexpression of either the multidrug resistance protein (MRP) 1 or P-glycoprotein (MDR1) (1-5). MRP and P-glycoprotein belong to the ATPbinding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity (1). Nevertheless, both proteins confer resistance to a broad range of cytotoxic xenobiotics including doxorubicin, vincristine, and VP-16 (etoposide), drugs that are widely used in the treatment of many human cancers. However, there is growing evidence that the mechanisms by which MRP and P-glycoprotein reduce cellular drug accumulation are not the same, suggesting that there are major differences in the drug-protein interactions of these two molecules (6 -8).Like most eukaryotic ABC proteins, MRP and P-glycoprotein contain hydrophobic membrane spanning domains (MSDs) and cytoplasmic nucleotide binding domains (NBDs) (9). To understand how drugs interact with P-glycoprotein, there has been considerable interest in determining the precise topology of this integral membrane protein. Investigations in most experimental systems support a model in which P-glycoprotein is organized as a symmetrically arranged, tandemly duplicated molecule with each half consisting of six transmembrane segments followed by a NBD (10), but alternate models have also been proposed (11,12).At present, little is known about the membrane topology of MRP. The topological model we proposed when MRP was cloned in 1992 was based on computer-assisted hydropathy analyses of its deduced amino acid sequence and alignment with the predicted structure of ltpgpA (1). LtpgpA is an ABC protein cloned from Leishmania tarentolae which was the most closely related protein to MRP known at that time (13). In the original model, we suggested that MRP consisted of eight transmembrane segments and an NBD in its NH 2 -proximal half and only four transmembrane segments and an NBD in its COOH-proximal half (1). More recently, alignment of the hydropathy profiles of human MRP with those of its murine ortholog (14) and several members of the ABC superfamily (including the related sulfonylurea receptor, SUR (15), and the yeast cadmium resistance factor, YCF1 (16), as well as Pglycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)) suggested to us a different topology for MRP. In this later model, we predicted that MRP contains two MSDs of six transmembrane helices in a "6 ϩ 6" configuration typical of several eukaryotic ABC transporters (9), plus an extremely hydrophobic NH 2 -terminal MSD of approximately 220 amino acids (4,14,17,18). This additional hydrophobic domain is predicted to contain four to six transmembrane segments and is not present in ABC proteins such as P-glycoprotein and CFTR. Thus it is a characteristic feature of members of the MRP branch of the ABC transporter superfamily (4,14).

Overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only distantly related. In contrast to P-glycoprotein, human MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates, some of which may be physiological substrates. At present, the mechanism by which MRP transports these compounds and mediates multidrug resistance is not understood. With the objective of developing an animal model for studies on the normal functions of MRP and its ability to confer multidrug resistance in vivo, we recently cloned the murine ortholog of MRP (mrp). To assess the degree of functional conservation between mrp and MRP, we directly compared the drug cross-resistance profiles they confer when transfected into human embryonic kidney cells, as well as their ability to actively transport leukotriene C4, 17beta-Estradiol 17beta-(D-glucuronide), and vincristine; mrp and MRP conferred similar drug resistance profiles, with the exception that only MRP conferred resistance to the anthracyclines tested. Consistent with these findings, accumulation of [3H]vincristine and [3H]VP-16 was decreased, and efflux of [3H]vincristine was increased in both murine and human MRP-transfected cell populations, whereas only human MRP-transfected cells displayed decreased accumulation and increased efflux of [3H]daunorubicin. Membrane vesicles derived from both transfected cell populations transported leukotriene C4 in an ATP-dependent manner with comparable efficiency, although the efficiency of 17beta-estradiol 17beta-(D-glucuronide) transport was somewhat higher with MRP transfectants. ATP-dependent transport of vincristine was also observed with vesicles from mrp and MRP transfectants but only in the presence of glutathione. These studies reveal intrinsic differences between the murine and human MRP orthologs with respect to their ability to confer resistance to a major class of chemotherapeutic drugs.

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein.