CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7

CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7 CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7 CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7 CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7 CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7 CHAPTER 21 – THE MULTIDRUG RESISTANCE PROTEINS 3–7

445 21 CHAPTER THE MULTIDRUG RESISTANCE PROTEINS 3–7 PIET BORST, GLEN REID, TOHRU SAEKI, PETER WIELINGA AND NOAM ZELCER INTRODUCTION Following the discovery of the multidrug resistance protein (MRP1) in 1992 (Cole et al., 1992) (Chapter 19) and the subsequent demonstration that the well-known liver canalicular multispecific organic anion transporter, cMOAT (now known as MRP2), was a transporter closely related to MRP1 (Chapter 20), several other related sequences were uncovered by Allikmets et al (1996) and Kool et al (1997) in a database search The existence of a family of MRP-related transporters has been confirmed in subsequent work and these transporters are now assembled in the C group of ABC transporters (see http:// www.nutrigene.4t.com/humanabc.htm) together with CFTR (Chapter 29) and the sulfonylurea receptors (Chapter 27) The present count of MRPs stands at nine and it is unlikely that there are more to come MRP1 and MRP2 are discussed in separate chapters of this book and very little is known yet about MRP8 and MRP9 (Tammur et al., 2001) Here we focus on MRP3–7 Other recent reviews of the MRP family can be found in (Borst et al., 1999, 2000; Borst and Oude Elferink, 2002; Ishikawa et al., 1994; Keppler, 1999; Renes et al., 1999) MRPs come in two types of structures, as illustrated in Figure 21.1: the MRP1 type, shared by MRP2, 3, and 7; and the MRP4 type, shared by MRP5 (and probably MRP8 and MRP9) The MRP1 type has an additional NH2terminal domain, which is thought to have five transmembrane segments and is not present in ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 the MRP4 type In MRP1, this domain is dispensable for transport function, explaining why proteins that differ substantially in size and putative structure, like MRP1 and MRP4, can still have similar functions The sequence differences between the known MRPs are summarized in Table 21.1 All MRPs studied thus far are organic anion pumps, but they differ widely in their preferred substrate or tissue location, as illustrated by Tables 21.2 and 21.3 MRP1–5 are inhibited by sulfinpyrazone, a classical inhibitor of organic anion transport Note, however, that sulfinpyrazone is also a substrate of MRP2 (Evers et al., 2000) and that it can actually stimulate transport of S-(2,4-dinitrophenyl) glutathione (GS-DNP) (Evers et al., 1998) or glutathione (GSH) (Evers et al., 2000) at lower concentrations MRP3 Human MRP3, also known as MOAT-D or cMOAT-2, and formally designated ABCC3, was first spotted by Allikmets and co-workers (1996) in their initial inventory of the human ABC superfamily Kool et al (1997) recruited this putative transporter to the MRP family and showed that MRP3 RNA has a rather restricted tissue distribution, with high concentrations in liver, intestine and adrenal gland The MRP3 gene is located at human chromosome 17q21.3 and the corresponding protein is 1527 amino acids long (Belinsky et al., 1998; Kiuchi et al., Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 446 ABC PROTEINS: FROM BACTERIA TO MAN NH2 CHO CHO out in COOH MRP1 TMD0 L0 CORE (Pgp-like) CHO out in COOH MRP4 NBD1 NBD2 Figure 21.1 Predicted membrane (secondary) structure of the two types of MRP-related proteins represented by their prototypic members, MRP1 (MRP1, 2, 3, and 7) and MRP4 (MRP4 and MRP 5) Shown are the TMD0 (extra transmembrane domain), the linker (L0), and the P-glycoprotein-like MDR core; NBD, Nucleotide-binding domain; CHO, glycosylation site (Adapted from Borst et al., 2000.) TABLE 21.1 PERCENTAGE AMINO ACID IDENTITY BETWEEN FULLY SEQUENCED HUMAN MULTIDRUG RESISTANCE PROTEINS (MRPS)a MRP1 (ABCC1) MRP2 (ABCC2) MRP3 (ABCC3) MRP4 (ABCC4) MRP5 (ABCC5) MRP6 (ABCC6) MRP7 (ABCC10) MRP1 1531 aa MRP2 1545 aa MRP3 1527 aa MRP4 1325 aa MRP5 1437 aa MRP6 1503 aa MRP7 1492 aa – 49 58 39 34 45 34 – 48 37 35 38 34 – 36 33 43 36 – 36 34 36 – 31 36 – 34 – a Homology between human MRPs, expressed as % amino acid (aa) identity; Borst (1999) For the multiple sequence alignment the GAP program from the University of Wisconsin Genetics Group (GCG) package (version 9.1) was used The following accession numbers were used: MRP1, L05628; MRP2, U49248; MRP3, AF009670; MRP4, AF071202; MRP5, AF104942; MRP6, AF076622; MRP7, BAA92227 1998; König et al., 1999; Kool et al., 1999b) Within the MRP family, MRP3 is the MRP most closely related to MRP1 (Table 21.1) Studies on rat Mrp3 have contributed substantially to our present understanding of the substrate specificity of human MRP3 An MRPlike protein, called MLP-2, was first identified by the group of Suzuki and Sugiyama in the liver of Mrp2 (Ϫ/Ϫ) rats, in which it is highly upregulated (Hirohashi et al., 1998) This protein proved to be the rat homologue of human MRP3, and its substrate specificity and inducbility have been studied in some detail (Hirohashi et al., 1999, 2000; Ogawa et al., 2000) A gene knockout (KO) of the mouse Mrp3 gene was recently produced (our unpublished results) The mice are healthy and fertile, but remain to be characterized THE MULTIDRUG RESISTANCE PROTEINS 3–7 TABLE 21.2 SUBSTRATE SPECIFICITY OF MRPS Transport of MRP GS-X pump Preferred substrates MDR drugs MTX GSH Sulfinpyrazone inhibition MRP1 ϩ ϩ ϩ ϩ ϩ MRP2 ϩ ϩ ϩ ϩ ϩ MRP3 ϩ ϩ ϩ Ϫ ϩ MRP4 ? Ϫ ϩ ? ϩ MRP5 MRP6 MRP7 ϩ Ϫ ? GS-X Gluc-X GS-X Gluc-X Gluc-X Sulf-X cGMP, cAMP, NMP analogues, Gluc-X cGMP, NMP analogues Peptides? ? Ϫ? Ϫ ? Ϫ Ϫ ? ϩ ? ? ϩ Ϫ ? Abbreviations: GS-X, Gluc-X, Sulf-X are conjugates of an organic compound (X) with glutathione (GS), glucuronide (Gluc) or sulfate (Sulf), respectively; MDR drugs are drugs belonging to the multidrug resistance (MDR) spectrum; MTX, methotrexate; NMP, nucleoside monophosphate See text for details and references TABLE 21.3 TISSUE DISTRIBUTION AND LOCATION IN THE PLASMA MEMBRANE OF POLARIZED EPITHELIA OF HUMAN MULTIDRUG RESISTANCE PROTEINS (MRPS) MRP1 MRP2 MRP3 MRP4 MRP5 MRP6 MRP7 Tissue distribution Plasma membrane location Ubiquitous (low in liver) Liver, kidney, gut Liver, adrenals, pancreas, kidney, gut, gallbladder Prostate, lung, muscle, pancreas, testis, ovary, bladder, gallbladder Ubiquitous Liver, kidney Ubiquitous (low) Basolateral Apical Basolateral Apical? Basolateral Basolateral? ? Adapted from Borst et al (2000) SUBSTRATE SPECIFICITY OF MRP3 AND RMRP3 The substrate specificity of MRP3 is summarized in Tables 21.4 and 21.5 Drug resistance of cells transfected with MRP3 cDNA constructs is limited, as shown by the results presented in Table 21.4 Substantial resistance of the transfected cells is found only against etoposide and teniposide, and against methotrexate (MTX) in short-time (4 h) exposures with high MTX concentrations Low vincristine resistance was only found in MRP3-transfected HEK293 cells (Zeng et al., 2000), but not in transfected 2008 human ovarian cells (Kool et al., 1999b), pig kidney cells (Haga et al., 2001), or mouse fibroblast cells isolated from triple KO (TKO) mice lacking P-glycoprotein and Mrp1 (Allen et al., 2000) As the transfected TKO cells have the highest etoposide resistance of any MRP3-transfected cell analyzed (Table 21.4), we think that more work is required to establish unambiguously transport of Vinca alkaloids by MRP3 A problem in all these experiments is that expression of MRP3 is relatively low in most transfected cells (Kool et al., 1999b), complicating the analysis of the resistance spectrum associated with the presence of MRP3 Polarized MDCKII cell transfectants were used to show that MRP3 can transport a classical substrate of organic anion pumps, GS-DNP (Kool et al., 1999b) We have shown that etoposide resistance in TKO cells is associated with diminished drug accumulation and increased drug extrusion (Zelcer et al., 2001), but the mechanism of etoposide transport is not yet clear Unlike MRP1 and MRP2, MRP3 does not detectably transport reduced glutathione (GSH) (Kool et al., 1999b) and does not co-transport etoposide with GSH (Zelcer et al., 2001) Resistance 447 448 ABC PROTEINS: FROM BACTERIA TO MAN TABLE 21.4 DRUG RESISTANCE INDUCED BY MRP3 EXPRESSION IN TRANSFECTED CELLS (EXPRESSED AS RESISTANCE RELATIVE TO THE UNTRANSFECTED PARENTAL CELL) Drug Resistance of MRP3 cells relative to wild-type Etoposide Teniposide Podophyllotoxin Methotrexate (high) Methotrexate (low) Vincristine Daunorubicin Paclitaxel Mitoxantrone SN-38 Cisplatin Arsenite Human ovarian 2008 carcinomaa HEK293 cellsb TKO mouse cellsc 3 50 1 1 1 1 2 1 1 1 1 a Kool et al (1999b) Zeng et al (2000) c Zelcer et al (2001) b TABLE 21.5 SUBSTRATE SPECIFICITY OF RAT MRP3 AND HUMAN MRP3 IN VESICULAR TRANSPORT Substrates Glucuronides Estradiol-17␤-glucuronide Etoposide glucuronide E 3040 glucuronide Glutathione conjugates LTC4 GS-DNP Bile salts (and conjugates) Taurocholate Glycocholate TLC-sulfate Other compounds Methotrexate DHEA-sulfate GSH Hirohashi et al., 1999, 2000 Rat Mrp3 Zeng et al., 2000 Human MRP3 Zelcer et al., 2001 Human MRP3 LLC-PK1a or HeLa HEK293a Sf9a Km Vmax Km 67 415 Vmax Km Vmax 26 76 18 11 474 ϳ138 20 ϩ ϩ 248 183 ϩ ϩ 776 288 ϩ Ϯ Ϯ 16 Ϫ 50 ϩ 162 ϩ ϩ Ϫ Ϫ a Transport was measured using membrane vesicles from transfected cells The LLC-PK1 cells are pig kidney cells; the HeLa cells are human cervical tumor cells; the HEK293 cells are immortalized human embryonic kidney cells; and the Sf9 cells are Spodoptera frugiperda insect cells infected with a recombinant baculovirus MRP3 gene construct Values for Km and Vmax are ␮M and pmol mgϪ1 (protein) minϪ1, respectively For some substrates, no Km or Vmax was determined and are presented as follows: (ϩ) ϭ transport, (Ϯ) ϭ marginal transport, (Ϫ) ϭ no transport Abbreviations: DHEA, dihydroepiandrosterone; GSH, glutathione; GS-DNP, S-(2,4-dinitrophenyl)glutathione; LTC4, leukotriene C4 THE MULTIDRUG RESISTANCE PROTEINS 3–7 is not due to intracellular conversion of etoposide to glucuronosyl-etoposide (which is a good substrate for MRP3, see below) and the simplest interpretation of the available results is that MRP3 transports unmodified etoposide by itself Vesicular transport studies, summarized in Table 21.5, confirmed that the substrate specificity of MRP3 differs from that of MRP1 and MRP2 in that glutathione conjugates are relatively poor substrates for MRP3 (Hirohashi et al., 1999) Hirohashi et al (2000) found that rat Mrp3 also transports several bile salts at a high rate and with high affinity (Table 21.5) The best substrates were taurolithocholate3-sulfate and taurochenodeoxycholate-3-sulfate (also substrates of MRP2) (Chapter 20), and taurocholate and glycocholate (also substrates of BSEP, the bile salt export pump) Competition experiments suggest that rat Mrp3 may also interact with other organic sulfate compounds, such as estrone sulfate (Hirohashi et al., 1999), but MRP3 does not transport a prominent human steroid derivative, dehydroepiandrosterone sulfate (DHEA-sulfate) The results of Hirohashi et al (1999) with rat Mrp3 have been reproduced to some extent with human MRP3 (Table 21.5) Differences are that the human MRP3 appears to transport bile salts more sluggishly than rat Mrp3, and glutathione conjugates more briskly Although Zeng et al (2000) found a low Vmax for transport of glutathione conjugates by vesicles derived from MRP3-overexpressing HEK293 cells, much higher rates were observed in the baculovirus system, in which the transport rate for DNP-GS is similar to that of estradiol-17␤glucuronide (E217␤G), in agreement with the substantial transport of DNP-GS observed in MDCKII cells transfected with MRP3 (Kool et al., 1999b) Like MRP1 and MRP2, MRP3 is inhibited by common organic anion transport inhibitors, such as sulfinpyrazone (1 mM), benzbromarone (250 ␮M), and indomethacin (250 ␮M), and less efficiently by probenecid (1 mM) (Zelcer et al., 2001) In summary, MRP3 is a typical organic anion pump, able to transport acidic drugs, such as MTX, and conjugates of organic compounds with GSH, glucuronate or sulfate It is inhibited by sulfinpyrazone and other inhibitors of organic anion transport A major difference compared with MRP1 and MRP2 is the inability of MRP3 to transport free GSH This may limit its ability to transport unconjugated drugs and may explain, at least in part, the very restricted drug resistance spectrum associated with MRP3 (Table 21.4) Another remarkable property of rat Mrp3 is its ability to transport a range of bile salts at a high rate Whether human MRP3 has this same property remains to be verified TISSUE DISTRIBUTION AND REGULATION OF MRP3 EXPRESSION Kool et al (1997) found substantial amounts of MRP3 RNA in adrenal gland, colon, small intestine and liver, and low amounts in kidney, bladder, pancreas, stomach, lung, spleen and tonsil Similar results were obtained in a more limited survey by Belinsky et al (1998), König et al (1999) and Uchiumi et al (1998), and for rat tissues by Kiuchi et al (1998) The only other tissues found to be weakly positive were placenta and prostate Noteworthy is the absence of detectable MRP3 expression in muscle, heart, brain, mammary gland, thyroid, salivary gland, testis and ovary (see overview in Scheffer et al., 2002) The presence of MRP3 has been verified at the protein level in gut, pancreas, gallbladder, liver, spleen and adrenal gland (Scheffer et al., 2002) In the adrenals, MRP3 is only present in the cortex, and staining was restricted to the two innermost zones, the zona fasciculata and the zona reticularis In the kidney, MRP3 is only seen in the distal convoluted tubules and the ascending loops of Henle (Scheffer et al., 2002) In denaturing acrylamide gels, MRP3 from most tissues and cell lines migrates as two separate bands with apparent masses of 170 kDa and 190 kDa (Kool et al., 1999b; Scheffer et al., 2002) The two bands reduce to a single 150 kDa band in cells incubated with tunicamycin, an inhibitor of N-linked glycosylation Why glycosylation results in two distinct MRP3 protein bands rather than a smear is not known In polarized epithelia, MRP3 is located in the basolateral membrane (König et al., 1999; Kool et al., 1999b) Some confusion was created when Ortiz et al (1999) reported that a polyclonal antibody raised against MRP3 stained the canalicular (apical) membrane of the hepatocyte In view of the unambiguous basolateral localization of MRP3 with several independent monoclonal antibodies in hepatocytes and in other epithelia (König et al., 1999; Kool et al., 1999b; Scheffer et al., 2002), the result of Ortiz et al (1999) must be an artifact The inducibility of MRP3 in the liver has generated intense interest Hirohashi et al (1998) discovered that Mrp3 RNA is very low 449 450 ABC PROTEINS: FROM BACTERIA TO MAN in normal rat liver and upregulated in Mrp2 (Ϫ/Ϫ) rats or after bile duct ligation Less dramatic induction was obtained by feeding rats phenobarbital or ␣-naphthylisothiocyanate, a compound that induces cholestasis in rats (Ogawa et al., 2000) Initially, these results seemed in contradiction with results reported for human liver RNA Kool et al (1997) found high levels of MRP3 RNA in liver samples and this was also observed in some other laboratories (Belinsky et al., 1998; König et al., 1999; Uchiumi et al., 1998) Immunohistochemistry of normal human liver showed, however, only prominent staining of the intra-hepatic bile duct epithelial cells (cholangiocytes) and weak staining of a small subset of hepatocytes surrounding the portal tracts This resembles the staining seen in normal rat liver (Soroka et al., 2001) Presumably, some of the initial human liver RNA samples with high MRP3 RNA came from damaged livers Absence of MRP2 also leads to strong induction of MRP3 in humans (König et al., 1999; Scheffer et al., 2002), and increased MRP3 in hepatocytes was also seen in patients with hepatitis, biliary atresia, and especially patients lacking the MDR3 P-glycoprotein (Scheffer et al., 2002) However, not all cholestatic patients upregulate MRP3 in their hepatocytes, as a patient with obstructive cholestasis and patients lacking BSEP (ABCB2) had no increase in MRP3 in their hepatocytes, even though they were highly cholestatic and had high concentrations of MRP3 in their proliferating bile ducts (Scheffer et al., 2002) Induction of MRP3 in the liver is therefore not a simple consequence of cholestasis, but requires a more specific signal generated in some, but not all, cholestatic conditions The nature of this signal is not known PHYSIOLOGICAL FUNCTION OF MRP3 AND ITS POSSIBLE ROLE IN DRUG RESISTANCE OF TUMOR CELLS The physiological function of MRP3 is not known, but on the basis of its substrate distribution and location in the body, several functions have been proposed At the top of the list is a function in the cholehepatic and enterohepatic circulation of bile salts (Hirohashi et al., 2000; König et al., 1999; Kool et al., 1999b; Ogawa et al., 2000) Bile salts are secreted in the liver On their way to the gut they may enter epithelial cells lining the bile ducts, either actively through the apical bile salt transporter (ASBT) or passively MRP3 may help the bile salts to leave the epithelial cells at the basolateral side The presence of MRP3 in the ductules of the pancreas and induction of MRP3 in the hepatocyte may serve an analogous function In the gut, bile salts are taken up, passively or through ASBT, and MRP3 may be the basolateral transporter allowing exit of the bile salts from the enterocyte A second possibility is that MRP3 has a defense function and contributes to the elimination of toxic organic anions, notably glucuronosyl derivatives Humans produce at least 15 UDP-glucuronosyltransferases and these enzymes can glucuronidate a wide range of endogenous and exogenous toxic compounds, not only in the liver and gastrointestinal tract, but also in many other tissues in the body (reviewed by Tukey and Strassburg, 2000) MRP3 and MRP1 may allow cells to export the glucuronosyl derivatives produced intracellularly The relatively high affinity of MRP3 for E217␤G (Table 21.5), and the high concentration of MRP3 in the adrenal cortex, suggest a role in steroid metabolism, but the physiological substrates transported are not yet known The Mrp3 (Ϫ/Ϫ) mouse, recently generated, should provide a test model for these speculations Whether MRP3 can contribute to clinical drug resistance is also still unclear Kool et al (1997) found no association between drug resistance and MRP3 in a diverse panel of cell lines Young et al (1999, 2001) studied a series of 30 lung cancer cell lines and observed that MRP3 was increased in many of the non-small cell lung cancers, but not in the small cell lung cancers They found a significant correlation between MRP3 levels and doxorubicin resistance, and a weaker association with resistance to vincristine, etoposide and cisplatin This does not fit the resistance spectrum of the MRP3-transfected cells described in Table 21.4 A further complication is the positive correlation between overexpression of MRP3 and MRP1 in these cell lines (Young et al., 2001) It is therefore difficult to assess whether the association between MRP3 levels and resistance is not due to the association between MRP3 and MRP1 overexpression MRP4 MRP4 first appeared in the literature as one of the 21 new ABC genes found by Allikmets et al (1996) by screening the EST database Kool et al (1997) then showed that a cDNA THE MULTIDRUG RESISTANCE PROTEINS 3–7 S NH2 N N N O OϪ P OϪ N O O N N N H N H 2N O HO N HN O HO O OϪ P OϪ N N O HO O O P O PMEA Thio-IMP OϪ cGMP Figure 21.2 Chemical structures of the MRP4 and MRP5 substrates PMEA, thio-IMP and cGMP corresponding to the 3Ј-half of MRP4 is expressed at low levels in several organs, and that the MRP4 gene is located on chromosome 13, a location that has since been refined to 13q32 (Schuetz et al., 1999) The MRP4 cDNA, first reported by Lee et al (1998), has a reading frame encoding a protein of 1325 amino acids and a predicted secondary structure most closely resembling that of MRP5 (Figure 21.1) The first substrates of MRP4 were deduced with the human T-lymphoid cell line CEM-r1 This cell line, generated by continual selection on the antiviral agent 9-(2-phosphonylmethoxyethyl) adenine (PMEA) (Figure 21.2), an analogue of AMP, is highly resistant to PMEA and related compounds, as well as other nucleoside analogues, but shows no cross-resistance to typical MRP1 substrates such as vinblastine (Robbins et al., 1995) This cell line was shown to rapidly efflux PMEA and other nucleoside monophosphates, such as AZTMP The finding that the CEM-r1 cells have an amplification of the MRP4 gene led to the conclusion that MRP4 can transport nucleoside monophosphate analogues (Schuetz et al., 1999) SUBSTRATE SPECIFICITY OF MRP4 Initial studies with MRP4 using the PMEAselected CEM-r1 line suggested a relatively broad spectrum of transportable substrates However, as was apparent at the time, the other genetic changes present in this cell line have a considerable influence on the drug resistance phenotype This is especially true for the downregulation of adenylate kinase activity in these cells, which increases the pool of transportable nucleoside monophosphates (Robbins et al., 1995; Schuetz et al., 1999) Lee et al (2000) transfected NIH3T3 cells with MRP4 cDNA and, somewhat surprisingly, found these cells exhibited resistance only against PMEA and short-term MTX exposure In contrast to the cross-resistance of CEM-r1 cells to a variety of nucleoside analogues, the NIH3T3/MRP4 cells showed no resistance against AZT, 3TC, ddC or d4T Further work by Schuetz and co-workers (presented at the FEBS 2001 ABC Meeting in Gosau), however, broadened the substrate specificity to include thiopurine derivatives Resistance to 6-mercaptopurine (6-MP) and thioguanine (TG) was recently found in the MRP4-transfected NIH3T3 cells studied by the group of Kruh (Chen et al., 2001) We have transfected a variety of cell lines with the MRP4 cDNA (kindly provided by J Schuetz), and found the highest MRP4 expression in HEK293 cells Initial experiments with these HEK293/MRP4 cells showed that they efflux PMEA when loaded with bis-POM-PMEA (a membrane permeable form of PMEA), and show resistance under continuous exposure to PMEA, to 6-MP and to the antiviral nucleoside analogue abacavir (our unpublished results) In common with the exogenous expression of other MRPs, it appears relatively difficult to obtain substantial levels of the protein after transduction A further complication is the endogenous expression of MRP4 and MRP5 in many of the cell lines used for transfection studies For example, HEK293 cells contain levels of MRP4 mRNA comparable to those found in the most MRP4rich tissues (our unpublished results) A recently published study suggests that expression of MRP4 in insect cells could be the best way to overcome problems of endogenous transporter background Chen et al (2001) used MRP4-containing baculovirus to infect insect cells from which they made inside-out membrane vesicles Taking into account the structural and substrate similarities between MRP4 and MRP5, and a previous study on MRP5 (Jedlitschky et al., 2000) (see later section on substrate specificity of human MRP5 for 451 452 ABC PROTEINS: FROM BACTERIA TO MAN details), they demonstrated that cyclic nucleotides are substrates for this transporter Low rates of transport were observed for 3Ј,5Ј-cyclic GMP (cGMP) (Figure 21.2) (Km and Vmax values of 10 ␮M and pmol mgϪ1 minϪ1) and 3Ј,5Ј-cyclic AMP (cAMP) (Km and Vmax values of 45 ␮M and pmol mgϪ1 minϪ1) Remarkably, estradiol E217␤G was a relatively good substrate (Km and Vmax values of 30 ␮M and 102 pmol mgϪ1 minϪ1) TISSUE DISTRIBUTION OF MRP4 EXPRESSION Initial reports concerning the expression of MRP4 described a gene with a restricted tissue distribution, as determined by polymerase chain reaction (PCR) mapping (Allikmets et al., 1996) and RNase protection assays (Kool et al., 1997) More recent data suggest that the gene is more widely expressed Lee et al (1998) detected MRP4 protein in most of the tissues examined, with levels ranging from very high in the prostate to barely detectable in the liver Using a semi-quantitative reverse transcriptase PCR (RT-PCR) method to standardize MRP4 transcript levels to ␤-actin, we find high levels of expression in the kidney, with lower but substantial expression in the gallbladder, testis and prostate We also find MRP4 mRNA in all cell lines tested More recently, we generated a new monoclonal antibody against human MRP4, which we have used to confirm the high expression of MRP4 in the kidney, as well as its presence in cell lines Lee et al (2000) found MRP4 in the basolateral membrane of the acinar cells in the prostate In contrast, Van Aubel reported at the FEBS 2001 ABC Meeting in Gosau that MRP4 is in the apical membrane, not the basolateral membrane, of rat and human kidney cells Whether MRP4 is indeed localized to different membranes in different epithelial tissues needs verification with antibodies that enable more conclusive immunohistochemistry PHYSIOLOGICAL FUNCTION OF MRP4 AND ROLE IN DRUG RESISTANCE There are still very few clues as to the normal physiological function of MRP4, and the role, if any, played by MRP4 in anticancer drug resistance The recent discovery by Chen et al (2001) that MRP4, like MRP5 (Jedlitschky et al., 2000), can serve as an efflux pump for cGMP and cAMP indicates that MRP4 is able to remove physiologically relevant (cyclic) nucleoside monophosphates from the cell Any role MRP4 may have in drug resistance is also under investigation As nucleobase and nucleoside analogues are used extensively in anticancer and antiviral therapies, there is potential for MRP4 to mediate resistance to these compounds As pointed out by Chen et al (2001), 6-MP and MTX are both used in the treatment of childhood leukemias and MRP4 is the only drug transporter known thus far that can transport both drugs In a screen of drug-selected human cancer cell lines by RNase protection assays, Kool et al (1997) found MRP4 to be expressed at low levels in all cell lines, but this did not correlate with resistance However, the cell lines tested were not selected by nucleobase or nucleoside analogues nor tested for resistance against these compounds MRP5 Human MRP5, also known as MOAT-C, and formally called ABCC5 (GenBank: AF146074), was cloned by several groups (Belinsky et al., 1998; Jedlitschky et al., 2000; McAleer et al., 1999; Wijnholds et al., 2000) The mouse homologue of human MRP5, called mrp5 or Mrp5 (GenBank AB019003), turned out to be the same as the previously identified sMRP (Suzuki et al., 1997; Tusnady and Varadi, 1998), a cloning artifact missing the part encoding the first transmembrane domain (Suzuki et al., 2000) Like MRP4, MRP5 is an organic anion pump with a high affinity for nucleotide analogues and cyclic nucleotides (Jedlitschky et al., 2000; Wijnholds et al., 2000) No known human disease is associated with MRP5 defects and the Mrp5 KO mouse has no phenotype thus far (Wijnholds et al., 2000) (our unpublished results) SUBSTRATE SPECIFICITY OF HUMAN MRP5 Cells transfected with MRP5 cDNA constructs were used by McAleer et al (1999) and Wijnholds et al (2000) to study the substrate specificity of MRP5 McAleer et al (1999) found reduced accumulation in MRP5 cells for the acidic organic dyes, 5-chloromethylfluorescein diacetate (CMFDA), 5-fluorescein diacetate (FDA), and 2Ј,7Ј-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), but not for structural analogues, THE MULTIDRUG RESISTANCE PROTEINS 3–7 the anionic calcein and the cationic tetramethylrosamine Further evidence that MRP5 is a typical organic pump, like other MRPs, came from Wijnholds et al (2000), who showed that MRP5 transports DNP-GS and GSH, and that MRP5 is inhibited by nonspecific organic anion transport inhibitors, such as sulfinpyrazone and benzbromarone MRP5 does not seem to transport MTX, in contrast to MRP1-4 There is also no evidence that MRP5 can mediate resistance to any of the anticancer drugs belonging to the MDR spectrum and transported by MRP1 with one exception Wijnholds et al (2000) found a low level of etoposide resistance but this was not found by McAleer et al (1999) Conversely, resistance against cadmium chloride and potassium antimonyl tartrate was found by McAleer et al (1999) but this could not be reproduced by Wijnholds et al (2000) Like MRP4, MRP5 can also cause resistance against the nucleoside monophosphate analogue PMEA (Figure 21.2) When cells are loaded with the membrane-permeable PMEA precursor bis-POM-PMEA, MRP5 mediates excretion of PMEA, but not of the di-(PMEAp) and triphosphate (PMEApp) of PMEA (Wijnholds et al., 2000), which are formed intracellularly (Balzarini et al., 1991) The ability of MRP5 to transport nucleotide analogues may also explain the resistance of MRP5-transfected cells to the thiopurines 6-MP and TG As shown in Figure 21.3, these thiopurines are converted into the corresponding nucleoside monophosphates (e.g thio-IMP) (Figure 21.2) and these are excreted via MRP5 (Wijnholds et al., 2000) (our unpublished results) Whether 6-Me-MP TPMT MRP5 can mediate excretion of methylated thiopurine derivatives, as reported for MRP4 by J Schuetz at the 2001 FEBS 2001 ABC Meeting in Gosau, remains to be tested Using vesicular transport by plasma membrane vesicles made from hamster V79 cells overexpressing MRP5, Jedlitschky et al (2000) identified cGMP as a MRP5 substrate with a micromolar affinity for the pump cAMP was also transported, but with a lower affinity Interestingly, they also found that the phosphodiesterase (PDE) inhibitors sildenafil (better known as Viagra), trequinsin and zaprinast, which prevent intracellular breakdown of cGMP, inhibited the MRP5-mediated cGMP transport as well MRP5 does not only transport purine-based compounds In unpublished experiments (J Wijnholds, P.W and P.B.), we have also found transport of a pro-drug of 3Ј-deoxy 2Ј,3Ј-didehydrothymidine 5Јmonophosphate (d4TMP), alaninyl-d4TMP, an antiviral agent (Balzarini et al., 1996) MRP5 can therefore also transport nucleotide analogues with a normal pyrimidine (thymine) ring EXPRESSION OF CELL LINES MRP5 IN TISSUES AND Analysis of tissue RNA suggested that MRP5 is ubiquitously expressed (Table 21.3) The highest levels are found in skeletal muscle and brain (Belinsky et al., 1998; Kool et al., 1997; McAleer et al., 1999; Zhang et al., 2000) All attempts to generate antibodies that allow the localization of MRP5 in tissues have failed thus 6-MP PRPP HGPRT PPi 6-Me-thio-IMP TPMT Thio-IMP IMPDH Higher phosphorylation Thio-XMP GMPS Thio-GMP Higher phosphorylation Figure 21.3 A simplified schematic diagram of 6-mercaptopurine metabolism With the metabolites: 6-MP, 6-mercaptopurine; thio-IMP, 6-thio-inosine monophosphate; thio-XMP, 6-thio-xanthidine monophosphate; thio-GMP, thio-guanosine monophosphate; 6-Me-MP, 6-methyl-mercaptopurine; 6-Me-thioIMP, 6-methyl-thio-inosine monophosphate; PRPP, phosphoribosyl pyrophosphate; PPi, pyrophosphate; and the enzymes: HGPRT, hypoxanthine-guanosine phosphoribosyltransferase, TPMT, thiopurine methyltransferase; IMPDH, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphate synthetase (Adapted from Zimm et al., 1985.) 453 454 ABC PROTEINS: FROM BACTERIA TO MAN far This is presumably because the expression levels are too low, as these antibodies readily detect MRP5 in transfected cells (Wijnholds et al., 2000) On Western blots, MRP5 can be detected in human and murine erythrocytes (Jedlitschky et al., 2000) (our unpublished results) and MRP5 might be the cGMP pump detected in erythrocytes (Schultz et al., 1998), although this needs to be shown using red cells from Mrp5 (Ϫ/Ϫ) mice Levels of MRP5 protein were also high in brain extracts, but not in skeletal muscle, in contrast to the MRP5 RNA levels in this tissue The negative results obtained on intact tissues using antibodies against human or murine MRP5 make it impossible to decide whether MRP5 is present in many cell types or only in some cell types present in all tissues, for example, endothelial cells In a recent survey using Western blots, we found MRP5 in all human tumor cell lines analyzed, including colon, breast, ovarian, lung carcinoma lines, leukemia and embryonic kidney cell lines This suggests, but does not prove, that MRP5 is present in many different normal cell types THE POSSIBLE INVOLVEMENT OF MRP5 IN DRUG RESISTANCE OR DISEASE Base, nucleoside and nucleotide analogues are used in antiviral and in anticancer therapy Potentially, elevated levels of MRP5 could therefore contribute to clinical resistance to these agents In MRP5-transfected cells, unambiguous resistance has only been found thus far against 6MP, TG, PMEA, 5-hydroxypyrimidine-2-carboxaldehyde thiosemicarbazone (an experimental anticancer drug), and an aryloxyphosphoramidate derivative of 2Ј,3Ј-dideoxyadenosine (Cf 1093) Whether clinical resistance against these compounds is ever associated with elevated MRP5 levels remains to be studied No resistance was observed thus far in MRP5 cells for other base or nucleoside analogues used in cancer or antiviral chemotherapy, such as ara-C, 5-fluorouracil, cidofovir and fludarabine (our unpublished results) No human disease has been associated with alterations in MRP5, and the Mrp5 KO mouse, generated by Wijnholds et al (2000), has no obvious phenotype It is possible, however, that the overlapping substrate specificities of MRP5 and MRP4 (and possibly MRP8 and MRP9) may hide the physiological function of Mrp5 (e.g in cyclic nucleotide transport), and that the generation of mice lacking all these transporters may lead to an understanding of the physiological function of each of them MRP6 MRP6 sprung into prominence when defects in the MRP6 gene were identified as the cause of pseudoxanthoma elasticum (Bergen et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000), a connective tissue disease affecting multiple organs MRP6 is a protein of 1503 amino acids (Belinsky and Kruh, 1999; Kool et al., 1999a), 45% identical to MRP1, and its gene is located next to MRP1 on chromosome 16 in a tail-to-tail configuration (Kool et al., 1999a) Human MRP6 is mainly expressed in liver and kidney (Belinsky and Kruh, 1999; Kool et al., 1997, 1999a), like Mrp6 (MLP-1), its rat homologue (Hirohashi et al., 1998, 1999; Madon et al., 2000), but low RNA levels have also been detected in other tissues In initial immunofluorescence studies, Madon et al (2000) localized rat Mrp6 in the basolateral and apical membranes of hepatocytes More recent work reported at the FEBS 2001 ABC Meeting at Gosau strongly indicates, however, that MRP6 is in the basolateral membrane of polarized cells In contrast to some other MRPs, expression of Mrp6 appears stable, whatever damage is inflicted on the liver (Madon et al., 2000) The substrate specificity of MRP6 is still a mystery Madon et al (2000) tested a series of typical MRP substrates in vesicular transport studies and found only transport of BQ-123, an anionic cyclopentapeptide and endothelin A receptor antagonist Endothelin-1 itself was transported by Mrp2, but not by Mrp6 These results suggest that MRP6 could be a highly selective organic anion pump It should be noted, however, that Madon et al (2000) only tested radioactive substrates at relatively low concentrations No competition experiments were done with high competitor concentrations, substrates such as MTX were not tested, and standard inhibitors of MRPs were not tested either MRP6 AND DRUG RESISTANCE Amplification of the 3Ј-part of the MRP6 gene was found in leukemia cells selected for anthracycline (epirubicin) resistance (Kuss et al., 1998; Longhurst et al., 1996; O’Neill et al., 1998) The THE MULTIDRUG RESISTANCE PROTEINS 3–7 anthracycline resistance was initially thought to be due to a new resistance determinant, called the anthracycline resistance gene ARA Subsequent work has shown, however, that the epirubicin resistance of cell lines with ARA gene amplification can be explained by co-amplification of the MRP1 gene with the 3Ј half of the adjacent MRP6 gene (Belinsky and Kruh, 1999; Kool et al., 1999a) There is no indication that the MRP6 gene is ever associated with anticancer drug resistance MRP6 is expressed at low or undetectable levels in all cancer cell lines tested (Kool et al., 1997, 1999a; Madon et al., 2000) and no correlation between expression and drug resistance was observed (Kool et al., 1997) MRP6 AND PXE Pseudoxanthoma elasticum is a heritable disorder characterized by calcification of elastic fibers in skin, arteries and retina, resulting in loss of elasticity of the skin, arterial insufficiency and retinal hemorrhage Why loss of a highly specialized pump located in the basolateral membrane of liver and kidney cells would lead to such a generalized connective tissue disease is unclear Speculations include indirect effects on Ca2ϩ metabolism or elastic fiber assembly, through excretion of cytokine-like organic anionic peptides (Bergen et al., 2000; Le Saux et al., 2000; Ringpfeil et al., 2000) (see Chapter 28) MRP7 Hopper et al (2001) identified an MRP homologue designated MRP7 by a database search MRP7 cDNA encodes a protein of 1492 amino acids and, translated in a reticulocyte lysate system, the cDNA produces a protein of approximately 158 kDa MRP7 has a predicted secondary structure and topology similar to that of MRP1, with an extra NH2-terminal transmembrane domain Although clearly an MRP homologue, MRP7 has the lowest overall homology with other members of the MRP family (33–36% sequence identity) MRP7 seems to be ubiquitously expressed as assessed by RT-PCR, but at a low level, since the mRNA transcript could not be detected by RNA blot analysis The substrate specificity and mechanism of transport of MRP7 have not yet been studied Whether or not MRP7 is an organic anion transporter also remains to be tested ACKNOWLEDGMENTS We thank Drs Marcel Kool, Alfred Schinkel and Jan Wijnholds for their helpful comments 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O’Neill et al., 1998) The THE MULTIDRUG RESISTANCE PROTEINS 3–7 anthracycline resistance was initially thought to be due to a new resistance determinant, called the anthracycline resistance gene ARA... structural analogues, THE MULTIDRUG RESISTANCE PROTEINS 3–7 the anionic calcein and the cationic tetramethylrosamine Further evidence that MRP5 is a typical organic pump, like other MRPs, came from... literature as one of the 21 new ABC genes found by Allikmets et al (1996) by screening the EST database Kool et al (1997) then showed that a cDNA THE MULTIDRUG RESISTANCE PROTEINS 3–7 S NH2 N N N O