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.

The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C4 (LTC4) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V(max) and K(m) values for LTC4 transport by membrane vesicles from T14 cells were 529 +/- 176 pmol mg(-1) min(-1) and 105 +/- 31 nM, respectively. At 50 nM LTC4, the K(m) (ATP) was 70 micron. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC4 transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC4 and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC4 transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC4 was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC4 transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC4 transport (K(i(app)) 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC4 transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC4 transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC4 transport and prevents photolabeling of MRP by LTC4, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent.

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-

In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

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).