The MRP family is comprised of nine related ABC transporters that are able to transport structurally diverse lipophilic anions and function as drug efflux pumps. Investigations of this family have provided insights not only into cellular resistance mechanisms associated with natural product chemotherapeutic agents, antifolates and nucleotide analogs, but also into factors that influence drug distribution in the body, membrane systems that are involved in the extrusion of reduced folates, cysteinyl leukotrienes and bile acids, and the molecular basis of two hereditary conditions in humans. The review will describe the biochemical properties, drug resistance activities and potential in vivo functions of these unusual pumps.

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ost␣-Ost␤ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ost␣-Ost␤ have not been investigated. To determine the role of Ost␣-Ost␤ in intestinal bile acid absorption, the Ost␣ gene was disrupted by homologous recombination in mice. Ost␣ ؊/؊ mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ost␣ ؊/؊ vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ost␣ ؊/؊ Mrp3 ؊/؊ mice. The bile acid pool size was significantly reduced (>65%) in Ost␣ ؊/؊ mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ost␣ ؊/؊ mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ost␣-Ost␤ is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.cholesterol ͉ liver disease ͉ mouse model ͉ nuclear receptor

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5-fluorouracil (ϳ3-fold), 5-fluoro-2-deoxyuridine (ϳ5-fold), and 5-fluoro-5-deoxyuridine (ϳ3-fold), the anti-human immunodeficiency virus agent 2,3-dideoxycytidine (ϳ6-fold) and the anti-hepatitis B agent 9-(2-phosphonylmethoxynyl)adenine (PMEA) (ϳ5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5-fluoro-2-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5-fluorouracil or 5-fluoro-2-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.Cellular extrusion of cyclic nucleotides has been described in prokaryotic and eukaryotic cells (1-4). This process provides extracellular cAMP involved in intercellular signaling, as determined for Dictyostelium discoideum, in which cAMP effluxed by solitary amoebae under low nutrient conditions mediates cellular aggregation and differentiation, and has also been proposed as a potential mechanism that may contribute to the attenuation of intracellular signaling mediated by these second messengers (5). Investigations employing cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux is energy-dependent, and the susceptibility of this process to inhibition by antagonists of organic anion pumps indicates that it is mediated by amphipathic anion transporters (2, 3, 6 -16). Recently, insights into the identities of the cellular components that mediate cyclic nucleotide efflux have come from studies of the MRP 1 family of ABC transporters. MRP4 and MRP5, two members of this extended family of amphipathic anion transporters (17), have been determined to be competent in the transport of cyclic nucleotides (18 -20). By contrast, other characterized MRP family members are able to transport a variety of lipophilic anions, ...

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH 2 -terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17␤-estradiol 17-(␤-D-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9 -13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.

No abstract

NAD(P)H:Quinone Oxidoreductase1 (NQO1) also known as DT-diaphorase is a flavoprotein that catalyzes the two-electron reduction of quinones, quinone imines and azo-dyes and thereby protects cells against mutagenicity and carcinogenicity resulting from free radicals and toxic oxygen metabolites generated by the one-electron reductions catalyzed by cytochromes P450 and other enzymes. High levels of NQO1 gene expression have been observed in liver, lung, colon and breast tumors as compared to normal tissues of the same origin. The transcription of the NQO1 gene is activated in response to exposure to bifunctional (e.g. beta-naphthoflavone (beta-NF), 2, 3, 7, 8 tetrachorodibenzo-p-dioxin (TCDD)) and monofunctional (phenolic antioxidants/chemoprotectors e.g. 2(3)-tert-butyl-4-hydroxy-anisole (BHA)) inducers. The high level of expression of the NQO1 gene and its induction by beta-NF and BHA require the presence of an AP1 binding site contained within the human Antioxidant Response Element (hARE) and are mediated by products of proto-oncogenes, Jun and Fos. Induction of NQO1 gene expression involves transfer of a redox signal from xenobiotics to unknown 'redox protein(s)' which in turn, modify the Jun and Fos proteins for greater affinity towards the AP1 site of the NQO1 gene and activates transcription. The expression and regulation of the NQO1 gene is complex as many additional cis-elements have been identified in the promoter region and is a subject of great future interest. In addition to established tumors, NQO1 gene expression is also increased in developing tumors, indicating a role in cellular defense during tumorigenesis. It has been proposed that low molecular weight substance(s) can diffuse from tumor cells into surrounding normal cells and activate the expression of the NQO1 gene. Purification and characterization of such substance(s) may provide important information in regard to the mechanism of activation of NQO1 gene expression and the role of increased NQO1 expression in tumor development. In view of the general consensus that NQO1 is over-expressed in tumor cells and the realization that NQO1 may either activate or detoxify xenobiotics, it is important to establish the role of NQO1 in the activation, and the detoxification of xenobiotics and drugs and in the intrinsic sensitivity of tumors to bioreductive alkylating aziridinyl benzoquinones such as diaziquone (AZQ), mitomycin C (MMC), and indoloquinone EO9, as well as to the dinitrophenyl aziridine, CB1954, and the benzotriazine-di-N-oxide, SR 4233.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3Ϫ/Ϫ mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide (ϳ2.0-to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3 Ϫ/Ϫ mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3 Ϫ/Ϫ and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATPdependent transport of 17␤-estradiol-(17-␤-D-glucuronide) (E 2 17␤G). E 2 17␤G transport was saturable, with K m and V max values of 57.8 Ϯ 15 M and 53.1 Ϯ 20 pmol/mg/min. By contrast, with E 2 17␤G, only modest enhancement of LTC 4 transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E 2 17␤G was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C 4 (K i(app) , 1.5 M), glycolithocholate 3-sulfate (K i(app) , 34.2 M) and MK571 (K i(app) , 28.5 M), and lipophilic agents such as cyclosporine A (K i(app) , 14.4 M). Of the inhibitors tested, LTC 4 was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E 2 17␤G indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.