Functional characterization of common protein variants in the efflux transporter ABCC11 and identification of T546M as functionally damaging variant.

Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%).

Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors.

Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.

Stratified medicine for the use of antidiabetic medication in treatment of type II diabetes and cancer: where do we go from here?

At present, the global diabetes epidemic is affecting 347 million individuals, 90% of whom are diagnosed with type II diabetes mellitus (T2DM). T2DM is commonly treated with more than one type of therapy, including oral antidiabetic drugs (OADs) and agents used in the treatment of diabetic complications. Several pharmacological classes of OADs are currently available for the treatment of T2DM, of which insulin secretagogues (i.e. sulphonylureas and meglitinides), insulin sensitizers [thiazolidinediones (TZDs)] and biguanides are the most commonly prescribed. Although many of these OADs have been used for more than half a century in the treatment of T2DM, the pharmacogenomic characteristics of these compounds have only recently been investigated, primarily in retrospective studies. Recent advances in pharmacogenomics have led to the identification of polymorphisms that affect the expression and function of drug-metabolizing enzymes and drug transporters, as well as drug targets and receptors. These polymorphisms have been shown to affect the therapeutic response to and side effects associated with OADs. The aim of this review was to provide an up-to-date summary of some of the pharmacogenomic data obtained from studies of T2DM treatment, with a focus on polymorphisms in genes affecting pharmacokinetics, pharmacodynamics and treatment outcome of the most commonly prescribed OADs. In addition, the implications of pharmacogenomics in the use of the OAD metformin in cancer will be briefly discussed. Finally, we will focus on recent advances in novel 'omics' technologies and discuss how these might aid in the personalized management of T2DM.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance.

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.

Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain.

Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.

Human pregnane X receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation.

Delayed graft function (DGF) is an important complication in renal transplantation, contributing significantly to decrease in long-term allograft survival. In addition to donor- and recipient-related risk factors such as immunosuppression, altered renal excretion of xenobiotics by membrane transporters may influence DGF. Using DNA samples from recipients and donors, we assessed the impact on DGF of genetic variants in P-glycoprotein (ABCB1), multidrug resistance protein 2 (ABCC2), and the nuclear pregnane X receptor (PXR/NR1I2), which regulates the transcription of enzymes and transporters. In our local cohort of renal transplant recipients (n = 178), DGF occurred in 27.5%. The PXR 8055TT genotype of the donor only (not of the recipient) was significantly associated with an increased risk for DGF. This finding emerged from univariate as well as multivariate logistic regression analysis including 16 nongenetic factors and held true after correction for multiple testing. Our findings provide the first evidence that PXR may be associated with risk of DGF, independent of previously identified risk factors.

Regulation and translocation of ATP-dependent apical membrane proteins in rat liver.

The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.

ATP-dependent phosphatidylcholine translocation in rat liver canalicular plasma membrane vesicles.

Phosphatidylcholine (PC) translocation was studied in rat liver canalicular plasma membrane vesicles using a fluorescent PC analogue that permitted the quantitation of asymmetric PC distribution in the outer and inner leaflet of the vesicles. PC translocation to the outer leaflet of the canalicular membrane was stimulated by ATP and an ATP-regenerating system in a time and temperature-dependent manner resulting in 200 pmol PC translocated/mg protein per 30 min. A non-hydrolyzable ATP analogue did not support translocation. Translocating activity was observed with PC but not with phosphatidylethanolamine and was specific for inside-out oriented canalicular membrane vesicles. Addition of taurocholate (10 microM), a micelle-forming bile acid, enhanced ATP-dependent PC translocation 1.5 +/- 0.1-fold, whereas addition of taurodehydrocholate (10 microM), a non-micelle-forming bile acid, did not. These results indicate the presence of an ATP-dependent transporter that "flips" phosphatidylcholine from the inner to the outer leaflet of the rat bile canalicular plasma membrane from where it can become associated with bile acids in the canalicular lumen, thereby enhancing ATP-dependent flipping activity. Several lines of evidence suggest that the transporter is Mdr2 P-glycoprotein.

Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide.

Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with a K(m) of 3.3 microm, and 17beta-glucuronosyl estradiol, with a K(m) of 5.4 microm. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain.

A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane.

We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17beta-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17beta-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 microM; its uptake was Na(+) independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver.

Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.

The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin. WIF-B9 cells were transiently and stably transfected with a mdr1-GFP construct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basolateral plasma membrane. At 15 degrees C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocytes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters. Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html

Characterization of the 5'-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene.

The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.

Urate transport in Homarus americanus hepatopancreas: studies on membrane vesicles and R cells.

[2-14C]urate uptake was studied in hepatopancreatic basolateral membrane vesicles and in R cell suspensions of the American lobster by Millipore filtration techniques. Unspecific binding of urate to the vesicular membrane was 25.5 +/- 3.0% of equilibrium. Vesicular uptake showed a diffusional and a saturable component (Km) 0.37 +/- 0.04 mM and maximal velocity (Vmax) 16.5 +/- 1.2 pmol urate.mg protein-1.s-1). [2-14C]urate uptake was significantly trans-stimulated by urate. Purine analogues, probenecid, p-aminohippuric acid, pyrazinoic, and oxonic acid cis-inhibited urate transport. Urate uptake was not affected by Na+ or K+ transmembrane gradients but stimulated by 1 mM 2-oxoglutarate at the cis-side in Na(+)-containing media. Cellular urate uptake was inhibited by pyrazinoic acid. Uptake was saturable (Km 0.53 +/- 0.11 mM and Vmax 3.7 +/- 0.4 pmol urate.mg protein-1.s-1) and Na(+)-independent. However, 2-oxoglutarate stimulated uptake in Na(+)-containing media. These results suggest that urate uptake across the basolateral membrane occurs via a specific, Na(+)-independent transport system that may operate in the exchange mode accepting 2-oxoglutarate as countertransported substrate. In vivo, urate uptake thereby would be a tertiary active system driven by a 2-oxoglutarate gradient established across the cell membrane by the operation of a Na(+)-2-oxoglutarate cotransport system.

MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.

The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.

Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B.

The polarized rat hepatoma/human fibroblast hybrid cell line, WIF-B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF-B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190-kd membrane glycoprotein mediating adenosine triphosphate (ATP)-dependent transport of glucuronides, glutathione S-conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF-B cells was shown by reverse-transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789-bp fragment. Immunoblotting, using antibodies reacting with the amino-terminal or with the carboxyl-terminal sequence of rat Mrp2, detected the 190-kd glycoprotein in WIF-B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF-B cells with a membrane-permeable ester of the calcium-dependent fluorescent indicator, Fluo-3, was followed by Mrp2-mediated secretion of the amphiphilic anion, Fluo-3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP-dependent transport into Mrp2-containing plasma membrane vesicles in comparison with Mrp2-deficient vesicles established that Fluo-3 is transported by Mrp2 with a Km value of 3.7 micromol/L. Our results indicate that the polarized WIF-B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo-3.

Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome.

The Dubin-Johnson syndrome is an inherited disorder characterized by conjugated hyperbilirubinemia. The deficient hepatobiliary transport of anionic conjugates is caused by the absence of a functional multidrug-resistance protein 2 (MRP2, symbol ABCC2) from the apical (canalicular) membrane of hepatocytes. Mechanisms underlying this deficiency may include rapid degradation of mutated MRP2 messenger RNA (mRNA) or impaired MRP2 protein maturation and trafficking. We investigated the consequences of the mutation MRP2Delta(R,M), which leads to the loss of 2 amino acids from the second ATP-binding domain of MRP2. The MRP2Delta(R,M) mutation is associated with the absence of the MRP2 glycoprotein from the apical membrane of hepatocytes. Transfection of mutated MRP2 complementary DNA (cDNA) led to an MRP2Delta(R,M) protein that was only core glycosylated, sensitive to endoglycosidase H digestion, and located in the endoplasmic reticulum (ER) of transfected HEK293 and HepG2 cells. This indicated that deletion of Arg1392 and Met1393 leads to impaired maturation and trafficking of the protein from the ER to the Golgi complex. Inhibition of proteasome function resulted in a paranuclear accumulation of the MRP2Delta(R,M) protein, suggesting that proteasomes are involved in the degradation of the mutant protein. This is the first mutation in Dubin-Johnson syndrome shown to cause deficient MRP2 maturation and impaired sorting of this glycoprotein to the apical membrane.

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.

Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver. The MDR phenotype can be caused by ATP-dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P-glycoprotein family or of the multidrug resistance protein (MRP) family. To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs. Semiquantitative RT-PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10-fold higher than MRP1 mRNA expression. MRP2 immunostaining was observed in 87% (33/38) of HCC samples. MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement. MRP3 was detected in all samples examined (9/9) by RT-PCR and by immunofluorescence microscopy. MRP3 was localized to the basolateral membrane of carcinoma cells. Double-label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously. When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells. Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of HCC.