Martin G. Belinsky

The MRP family of drug efflux pumps.

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.

NAD(P)H:Quinone oxidoreductase1 (DT-diaphorase) expression in normal and tumor tissues

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 oneelectron 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. β-naphthoflavone (β-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 β-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 tumorogenesis. 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.

Analysis of the structure and expression pattern of MRP7 (ABCC10), a new member of the MRP subfamily

The MRP subfamily of ABC transporters currently consists of at least six members, several of which have been demonstrated to transport amphipathic anions and to confer in vitro resistance to chemotherapeutic agents. In searching the data bases we identified the product of a cDNA sequencing project that bears significant similarity to MRP subfamily transporters. In this report the predicted coding sequence, protein product and expression pattern of this cDNA, termed MRP7, are analyzed. The MRP7 cDNA sequence encodes a 1492 amino acid ABC transporter whose structural architecture resembles that of MRP1, MRP2, MRP3, and MRP6, in that its transmembrane helices are arranged in three membrane spanning domains. However, in contrast to the latter transporters, a conserved N-linked glycosylation site is not found at the N-terminus of MRP7. Comparisons of the MRP7 amino acid sequence indicated that while it is most closely related to other MRP subfamily members, its degree of relatedness is the lowest of any of the known MRP-related transporters. The integrity of the predicted MRP7 coding sequence was confirmed by the synthesis of an approximately 158 kDa protein in reticulocyte lysates programmed with the MRP7 cDNA. While MRP7 transcript was detected in a variety of tissues by RT/PCR, it was not readily detectable by RNA blot analysis, suggesting that it is expressed at low levels in these tissues. Fluorescence in situ hybridization indicated that MRP7 maps to chromosome 6p12-21, in proximity to several genes associated with glutathione conjugation and synthesis. On the basis of these findings and evolutionary cluster analysis, we conclude that MRP7 is a member of the MRP subfamily of amphipathic anion transporters.

Analysis of the drug resistance profile of multidrug resistance protein 7 (ABCC10): resistance to docetaxel.

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH2-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 17β-estradiol 17-(β-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.

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.

The organic solute transporter α-β, Ostα-Ostβ, is essential for intestinal bile acid transport and homeostasis

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 Ostα-Ostβ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostα-Ostβ have not been investigated. To determine the role of Ostα-Ostβ in intestinal bile acid absorption, the Ostα gene was disrupted by homologous recombination in mice. Ostα−/− mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostα−/− vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostα−/−Mrp3−/− mice. The bile acid pool size was significantly reduced (>65%) in Ostα−/− mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostα−/− mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostα-Ostβ 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.

ABCC10, ABCC11, and ABCC12

Multidrug resistance protein (MRP)7, MRP8, and MRP9 (gene symbols ABCC10, ABCC11, and ABCC12) are recently identified members of the MRP family that are at relatively early stages of investigation. Of these proteins, a physiological function has only been established for MRP8, for which a single nucleotide polymorphism determines wet vs dry earwax type. MRP7 and MRP8 are lipophilic anion pumps that are able to confer resistance to chemotherapeutic agents. MRP7 is competent in the transport of the glucuronide E217βG, and its resistance profile, which includes several natural product anticancer agents, is distinguished by the taxane docetaxel. MRP8 is able to transport a diverse range of lipophilic anions, including cyclic nucleotides, E217βG, steroid sulfates such as dehydroepiandrosterone (DHEAS) and E1S, glutathione conjugates such as leukotriene C4 and dinitrophenyl-S-glutathione, and monoanionic bile acids. However, the constituent of earwax that is susceptible to transport by MRP8 has not been identified. MRP8 has complex interactions with its substrates, as indicated by the nonreciprocal ability of DHEAS to stimulate E217βG transport. Similar to the case for other MRPs that possess only two membrane spanning domains (MRP4 and MRP5), MRP8 is a cyclic nucleotide efflux pump that is able to confer resistance to nucleoside-based agents, such as PMEA and 5FU. The functional characteristics of MRP9 are currently unknown.

Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3–/– and Abcc4–/– Mice

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3–/– and Abcc4–/– mice using a cassette dosing approach. In the livers of Abcc3–/– and Abcc4–/– mice, the basolateral excretory clearance of acetaminophen sulfate was reduced ∼20 and ∼20%, 4-methylumbelliferyl sulfate was reduced ∼50 and ∼65%, and harmol sulfate was decreased ∼30 and ∼45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by ∼96, ∼85, and ∼40%, respectively, in the livers of Abcc3–/– mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

Physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 as determined from recent studies on gene-disrupted mice

The MRP family is composed of nine transporters, at least eight of which are lipophilic anion transporters that are capable of conferring resistance to various anticancer agents. Recently, mice with gene disruptions in Mrp2, Mrp3 and Mrp4 have been developed. This review will discuss insights into the physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 afforded by investigations of these new mouse models.

Overexpression of insulin‐like growth factor 1 receptor and frequent mutational inactivation of SDHA in wild‐type SDHB‐negative gastrointestinal stromal tumors

Approximately 15% of gastrointestinal stromal tumors (GISTs) in adults and 85% in children lack mutations in KIT and PDGFRA and are known as wild‐type GISTs. Wild‐type GISTs from adults and children express high levels of insulin‐like growth factor 1 receptor (IGF1R) and exhibit stable genomes compared to mutant GISTs. Pediatric wild‐type GISTs, GISTs from the multitumor Carney–Stratakis syndrome, and the Carney triad share other clinicopathological properties (e.g., early‐onset, multifocal GISTs with epitheliod cell morphology), suggesting a common etiology. Carney–Stratakis is an inherited association of GIST and paragangliomas caused by germline mutations in succinate dehydrogenase (SDH) genes. The connection between defective cellular respiration and GIST pathology has been strengthened by the utilization of SDHB immunohistochemistry to identify SDH deficiency in pediatric GISTs, syndromic GISTs, and some adult wild‐type GISTs. SDHB and IGF1R expression was examined in 12 wild‐type and 12 mutant GIST cases. Wild‐type GISTs were screened for coding‐region alterations in SDH genes and for chromosomal aberrations using genome‐wide single‐nucleotide polymorphism and MIP arrays. SDHB‐deficiency, identified in 11/12 wild‐type GIST cases, was tightly associated with overexpression of IGF1R protein and transcript. Biallelic inactivation of the SDHA gene was a surprisingly frequent event, identified in 5 of 11 SDHB‐negative cases, generally due to germline point mutations accompanied by somatic SDHA allelic losses. As a novel finding, inactivation of the SDHC gene from a combination of a heterozygous coding‐region mutation and hypermethylation of the wild‐type allele was found in one SDHB‐negative case.