abc proteins || the multidrug resistance proteins 3–7

INTRODUCTION

Following the discovery of the multidrug resist-ance protein 1 (MRP1) in 1992 (Cole et al., 1992)(Chapter 19) and the subsequent demonstrationthat the well-known liver canalicular multi-specific organic anion transporter, cMOAT (nowknown as MRP2), was a transporter closelyrelated to MRP1 (Chapter 20), several otherrelated sequences were uncovered by Allikmetset al. (1996) and Kool et al. (1997) in a databasesearch. The existence of a family of MRP-relatedtransporters has been confirmed in subsequentwork and these transporters are now assembledin the C group of ABC transporters (see http://www.nutrigene.4t.com/humanabc.htm) togetherwith CFTR (Chapter 29) and the sulfonylureareceptors (Chapter 27). The present count ofMRPs stands at nine and it is unlikely that thereare more to come. MRP1 and MRP2 are dis-cussed in separate chapters of this book andvery little is known yet about MRP8 and MRP9(Tammur et al., 2001). Here we focus onMRP3–7. Other recent reviews of the MRP fam-ily can be found in (Borst et al., 1999, 2000; Borstand Oude Elferink, 2002; Ishikawa et al., 1994;Keppler, 1999; Renes et al., 1999).

MRPs come in two types of structures, asillustrated in Figure 21.1: the MRP1 type,shared by MRP2, 3, 6 and 7; and the MRP4 type,shared by MRP5 (and probably MRP8 andMRP9). The MRP1 type has an additional NH2-terminal domain, which is thought to have fivetransmembrane segments and is not present in

the MRP4 type. In MRP1, this domain is dispensable for transport function, explainingwhy proteins that differ substantially in sizeand putative structure, like MRP1 and MRP4,can still have similar functions. The sequencedifferences between the known MRPs are sum-marized in Table 21.1.

All MRPs studied thus far are organic anionpumps, but they differ widely in their preferredsubstrate or tissue location, as illustrated byTables 21.2 and 21.3. MRP1–5 are inhibited bysulfinpyrazone, a classical inhibitor of organicanion transport. Note, however, that sulfinpyra-zone is also a substrate of MRP2 (Evers et al.,2000) and that it can actually stimulate transportof S-(2,4-dinitrophenyl) glutathione (GS-DNP)(Evers et al., 1998) or glutathione (GSH) (Evers et al., 2000) at lower concentrations.

MRP3Human MRP3, also known as MOAT-D orcMOAT-2, and formally designated ABCC3,was first spotted by Allikmets and co-workers(1996) in their initial inventory of the humanABC superfamily. Kool et al. (1997) recruitedthis putative transporter to the MRP family andshowed that MRP3 RNA has a rather restrictedtissue distribution, with high concentrations inliver, intestine and adrenal gland. The MRP3gene is located at human chromosome 17q21.3and the corresponding protein is 1527 aminoacids long (Belinsky et al., 1998; Kiuchi et al.,

445

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21THE MULTIDRUG RESISTANCE

PROTEINS 3–7PIET BORST, GLEN REID,

TOHRU SAEKI, PETER WIELINGAAND NOAM ZELCER

CHAPTER

chap-21.qxd 21/Nov/02 12:45 PM Page 445

1998; König et al., 1999; Kool et al., 1999b).Within the MRP family, MRP3 is the MRP mostclosely related to MRP1 (Table 21.1).

Studies on rat Mrp3 have contributed sub-stantially to our present understanding of thesubstrate specificity of human MRP3. An MRP-like protein, called MLP-2, was first identifiedby the group of Suzuki and Sugiyama in theliver of Mrp2 (�/�) rats, in which it is highly

upregulated (Hirohashi et al., 1998). This protein proved to be the rat homologue ofhuman MRP3, and its substrate specificity andinducbility have been studied in some detail(Hirohashi et al., 1999, 2000; Ogawa et al., 2000).A gene knockout (KO) of the mouse Mrp3 genewas recently produced (our unpublishedresults). The mice are healthy and fertile, butremain to be characterized.

ABC PROTEINS: FROM BACTERIA TO MAN446

NH2CHO CHO

CHO

out

inCOOH

out

in COOH

CORE (Pgp-like)TMD0 L0

MRP1

MRP4

NBD1 NBD2

Figure 21.1. Predicted membrane (secondary) structure of the two types of MRP-related proteins representedby their prototypic members, MRP1 (MRP1, 2, 3, 6 and 7) and MRP4 (MRP4 and MRP 5). Shown are theTMD0 (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 SEQUENCEDHUMAN MULTIDRUG RESISTANCE PROTEINS (MRPS)a

MRP1 MRP2 MRP3 MRP4 MRP5 MRP6 MRP7

1531 aa 1545 aa 1527 aa 1325 aa 1437 aa 1503 aa 1492 aa

MRP1 (ABCC1) –MRP2 (ABCC2) 49 –MRP3 (ABCC3) 58 48 –MRP4 (ABCC4) 39 37 36 –MRP5 (ABCC5) 34 35 33 36 –MRP6 (ABCC6) 45 38 43 34 31 –MRP7 (ABCC10) 34 34 36 36 36 34 –

aHomology between human MRPs, expressed as % amino acid (aa) identity; Borst (1999). For the multiple sequencealignment 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.


SUBSTRATE SPECIFICITY OFMRP3 AND RMRP3

The substrate specificity of MRP3 is summa-rized in Tables 21.4 and 21.5. Drug resistance ofcells transfected with MRP3 cDNA constructsis limited, as shown by the results presented in

Table 21.4. Substantial resistance of the trans-fected cells is found only against etoposide andteniposide, and against methotrexate (MTX) inshort-time (4 h) exposures with high MTX con-centrations. Low vincristine resistance was onlyfound in MRP3-transfected HEK293 cells (Zenget al., 2000), but not in transfected 2008 humanovarian cells (Kool et al., 1999b), pig kidneycells (Haga et al., 2001), or mouse fibroblastcells isolated from triple KO (TKO) mice lackingP-glycoprotein and Mrp1 (Allen et al., 2000). As the transfected TKO cells have the highestetoposide resistance of any MRP3-transfectedcell analyzed (Table 21.4), we think that morework is required to establish unambiguouslytransport of Vinca alkaloids by MRP3. A problemin all these experiments is that expression ofMRP3 is relatively low in most transfected cells(Kool et al., 1999b), complicating the analysis ofthe resistance spectrum associated with the pres-ence of MRP3. Polarized MDCKII cell trans-fectants were used to show that MRP3 cantransport a classical substrate of organic anionpumps, GS-DNP (Kool et al., 1999b).

We have shown that etoposide resistance inTKO cells is associated with diminished drugaccumulation and increased drug extrusion(Zelcer et al., 2001), but the mechanism ofetoposide transport is not yet clear. UnlikeMRP1 and MRP2, MRP3 does not detectablytransport reduced glutathione (GSH) (Kool et al., 1999b) and does not co-transport etopo-side with GSH (Zelcer et al., 2001). Resistance

THE MULTIDRUG RESISTANCE PROTEINS 3–7 447

TABLE 21.2. SUBSTRATE SPECIFICITY OF MRPS

Transport of

MRP GS-X Preferred substrates MDR MTX GSH Sulfinpyrazonepump drugs inhibition

MRP1 � GS-X � � � �

Gluc-XMRP2 � GS-X � � � �

Gluc-XMRP3 � Gluc-X � � � �

Sulf-XMRP4 ? cGMP, cAMP, NMP � � ? �

analogues, Gluc-XMRP5 � cGMP, NMP analogues �? � � �

MRP6 � Peptides? � � ? �

MRP7 ? ? ? ? ? ?

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 drugsbelonging to the multidrug resistance (MDR) spectrum; MTX, methotrexate; NMP, nucleosidemonophosphate. See text for details and references.

TABLE 21.3. TISSUE DISTRIBUTIONAND LOCATION IN THE PLASMA

MEMBRANE OF POLARIZED EPITHELIAOF HUMAN MULTIDRUG RESISTANCE

PROTEINS (MRPS)Tissue distribution Plasma membrane

location

MRP1 Ubiquitous Basolateral(low in liver)

MRP2 Liver, kidney, gut ApicalMRP3 Liver, adrenals, Basolateral

pancreas, kidney, gut, gallbladder

MRP4 Prostate, lung, Apical?muscle, pancreas, testis, ovary, bladder, gallbladder

MRP5 Ubiquitous BasolateralMRP6 Liver, kidney Basolateral?MRP7 Ubiquitous (low) ?

Adapted from Borst et al. (2000).



TABLE 21.4. DRUG RESISTANCE INDUCED BY MRP3 EXPRESSIONIN TRANSFECTED CELLS (EXPRESSED AS RESISTANCE RELATIVE TO

THE UNTRANSFECTED PARENTAL CELL)Drug Resistance of MRP3 cells relative to wild-type

Human ovarian HEK293 cellsb TKO mouse cellsc

2008 carcinomaa

Etoposide 3 4 8Teniposide 3 2 5Podophyllotoxin 1 1Methotrexate (high) 50Methotrexate (low) 1 2Vincristine 1 2 1Daunorubicin 1 1 1Paclitaxel 1 1Mitoxantrone 1 1SN-38 1 1Cisplatin 1 1 1Arsenite 1aKool et al. (1999b).bZeng et al. (2000).cZelcer et al. (2001).

TABLE 21.5. SUBSTRATE SPECIFICITY OF RAT MRP3 AND HUMAN MRP3 INVESICULAR TRANSPORT

Hirohashi et al., 1999, Zeng et al., 2000 Zelcer et al., 2001

2000 Rat Mrp3 Human MRP3 Human MRP3

LLC-PK1a or HeLa HEK293a Sf9a

Substrates Km Vmax Km Vmax Km Vmax

GlucuronidesEstradiol-17�-glucuronide 67 415 26 76 18 474Etoposide glucuronide 11 �138E 3040 glucuronide �

Glutathione conjugatesLTC4 � 5 20 �

GS-DNP � 6 4 �

Bile salts (and conjugates)Taurocholate 16 50 � �

Glycocholate � 248 183 �

TLC-sulfate 3 162Other compounds

Methotrexate � 776 288 �

DHEA-sulfate �

GSH �

aTransport 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 mg�1 (protein) min�1, respectively. For some substrates, no Km or Vmax was determined and are presented as follows: (�) � transport, (�) � marginal transport, (�) � no transport.Abbreviations: DHEA, dihydroepiandrosterone; GSH, glutathione; GS-DNP, S-(2,4-dinitrophenyl)glutathione; LTC4, leukotriene C4.

chap-21.qxd 21/Nov/02 12:45 PM 48

is not due to intracellular conversion of etopo-side to glucuronosyl-etoposide (which is a goodsubstrate for MRP3, see below) and the sim-plest interpretation of the available results isthat MRP3 transports unmodified etoposide byitself.

Vesicular transport studies, summarized inTable 21.5, confirmed that the substrate speci-ficity of MRP3 differs from that of MRP1 andMRP2 in that glutathione conjugates are rela-tively poor substrates for MRP3 (Hirohashi et al., 1999). Hirohashi et al. (2000) found that rat Mrp3 also transports several bile salts at ahigh rate and with high affinity (Table 21.5). The best substrates were taurolithocholate-3-sulfate and taurochenodeoxycholate-3-sulfate(also substrates of MRP2) (Chapter 20), and taurocholate and glycocholate (also substrates ofBSEP, the bile salt export pump). Competitionexperiments suggest that rat Mrp3 may alsointeract with other organic sulfate compounds,such as estrone sulfate (Hirohashi et al., 1999), butMRP3 does not transport a prominent humansteroid derivative, dehydroepiandrosteronesulfate (DHEA-sulfate).

The results of Hirohashi et al. (1999) with ratMrp3 have been reproduced to some extentwith human MRP3 (Table 21.5). Differences arethat the human MRP3 appears to transport bilesalts more sluggishly than rat Mrp3, and glu-tathione conjugates more briskly. AlthoughZeng et al. (2000) found a low Vmax for transportof glutathione conjugates by vesicles derivedfrom MRP3-overexpressing HEK293 cells,much higher rates were observed in the bac-ulovirus system, in which the transport rate for DNP-GS is similar to that of estradiol-17�-glucuronide (E217�G), in agreement with thesubstantial transport of DNP-GS observed inMDCKII cells transfected with MRP3 (Kool et al., 1999b). Like MRP1 and MRP2, MRP3 isinhibited by common organic anion transportinhibitors, 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 anionpump, able to transport acidic drugs, such asMTX, and conjugates of organic compounds withGSH, glucuronate or sulfate. It is inhibited bysulfinpyrazone and other inhibitors of organicanion transport. A major difference comparedwith MRP1 and MRP2 is the inability of MRP3to transport free GSH. This may limit its abilityto transport unconjugated drugs and mayexplain, at least in part, the very restricted drug

resistance spectrum associated with MRP3(Table 21.4). Another remarkable property of ratMrp3 is its ability to transport a range of bilesalts at a high rate. Whether human MRP3 hasthis same property remains to be verified.

TISSUE DISTRIBUTION AND REGULATIONOF MRP3 EXPRESSION

Kool et al. (1997) found substantial amounts ofMRP3 RNA in adrenal gland, colon, small intes-tine and liver, and low amounts in kidney, blad-der, pancreas, stomach, lung, spleen and tonsil.Similar results were obtained in a more limitedsurvey by Belinsky et al. (1998), König et al.(1999) and Uchiumi et al. (1998), and for rat tis-sues by Kiuchi et al. (1998). The only other tis-sues found to be weakly positive were placenta and prostate. Noteworthy is the absence ofdetectable 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 verifiedat the protein level in gut, pancreas, gallbladder,liver, spleen and adrenal gland (Scheffer et al.,2002). In the adrenals, MRP3 is only present inthe cortex, and staining was restricted to the twoinnermost zones, the zona fasciculata and thezona reticularis. In the kidney, MRP3 is onlyseen in the distal convoluted tubules and theascending loops of Henle (Scheffer et al., 2002).In denaturing acrylamide gels, MRP3 from mosttissues and cell lines migrates as two separatebands with apparent masses of 170 kDa and190 kDa (Kool et al., 1999b; Scheffer et al., 2002).The two bands reduce to a single 150 kDa bandin cells incubated with tunicamycin, an inhibitorof N-linked glycosylation. Why glycosylationresults in two distinct MRP3 protein bandsrather 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 createdwhen Ortiz et al. (1999) reported that a poly-clonal antibody raised against MRP3 stainedthe canalicular (apical) membrane of the hepa-tocyte. In view of the unambiguous basolaterallocalization of MRP3 with several independentmonoclonal antibodies in hepatocytes and inother 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 hasgenerated intense interest. Hirohashi et al.(1998) discovered that Mrp3 RNA is very low



in normal rat liver and upregulated in Mrp2(�/�) rats or after bile duct ligation. Less dramatic induction was obtained by feedingrats phenobarbital or �-naphthylisothiocyanate,a compound that induces cholestasis in rats(Ogawa et al., 2000). Initially, these results seemedin contradiction with results reported forhuman liver RNA. Kool et al. (1997) found highlevels of MRP3 RNA in liver samples and thiswas also observed in some other laboratories(Belinsky et al., 1998; König et al., 1999; Uchiumi et al., 1998). Immunohistochemistry ofnormal human liver showed, however, onlyprominent staining of the intra-hepatic bile ductepithelial cells (cholangiocytes) and weak stain-ing of a small subset of hepatocytes surround-ing the portal tracts. This resembles the stainingseen in normal rat liver (Soroka et al., 2001).Presumably, some of the initial human liverRNA samples with high MRP3 RNA came fromdamaged livers.

Absence of MRP2 also leads to strong induc-tion of MRP3 in humans (König et al., 1999;Scheffer et al., 2002), and increased MRP3 inhepatocytes was also seen in patients with hepatitis, biliary atresia, and especially patientslacking the MDR3 P-glycoprotein (Scheffer et al., 2002). However, not all cholestatic patientsupregulate MRP3 in their hepatocytes, as apatient with obstructive cholestasis and patientslacking BSEP (ABCB2) had no increase in MRP3in their hepatocytes, even though they werehighly cholestatic and had high concentrationsof MRP3 in their proliferating bile ducts(Scheffer et al., 2002). Induction of MRP3 in theliver is therefore not a simple consequence ofcholestasis, but requires a more specific signalgenerated in some, but not all, cholestatic condi-tions. The nature of this signal is not known.

PHYSIOLOGICAL FUNCTION OF MRP3AND ITS POSSIBLE ROLE IN DRUGRESISTANCE OF TUMOR CELLS

The physiological function of MRP3 is notknown, but on the basis of its substrate distri-bution and location in the body, several func-tions have been proposed. At the top of the listis a function in the cholehepatic and enterohe-patic 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 theliver. On their way to the gut they may enterepithelial cells lining the bile ducts, eitheractively through the apical bile salt transporter

(ASBT) or passively. MRP3 may help the bilesalts to leave the epithelial cells at the basolat-eral side. The presence of MRP3 in the ductulesof the pancreas and induction of MRP3 in thehepatocyte may serve an analogous function.In the gut, bile salts are taken up, passively orthrough ASBT, and MRP3 may be the basolat-eral transporter allowing exit of the bile saltsfrom the enterocyte.

A second possibility is that MRP3 has adefense function and contributes to the elimi-nation of toxic organic anions, notably glu-curonosyl derivatives. Humans produce at least 15 UDP-glucuronosyltransferases and theseenzymes can glucuronidate a wide range ofendogenous 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 theglucuronosyl derivatives produced intracellu-larly. The relatively high affinity of MRP3 forE217�G (Table 21.5), and the high concentrationof MRP3 in the adrenal cortex, suggest a role insteroid metabolism, but the physiological sub-strates transported are not yet known. The Mrp3(�/�) mouse, recently generated, should pro-vide a test model for these speculations.

Whether MRP3 can contribute to clinical drugresistance is also still unclear. Kool et al. (1997)found no association between drug resistanceand MRP3 in a diverse panel of cell lines. Younget al. (1999, 2001) studied a series of 30 lung cancer cell lines and observed that MRP3 wasincreased in many of the non-small cell lungcancers, but not in the small cell lung cancers.They found a significant correlation betweenMRP3 levels and doxorubicin resistance, and aweaker association with resistance to vincristine,etoposide and cisplatin. This does not fit theresistance spectrum of the MRP3-transfectedcells described in Table 21.4. A further complica-tion is the positive correlation between over-expression of MRP3 and MRP1 in these cell lines(Young et al., 2001). It is therefore difficult toassess whether the association between MRP3levels and resistance is not due to the associationbetween MRP3 and MRP1 overexpression.

MRP4MRP4 first appeared in the literature as one ofthe 21 new ABC genes found by Allikmets et al. (1996) by screening the EST database. Kool et al. (1997) then showed that a cDNA



corresponding to the 3�-half of MRP4 isexpressed at low levels in several organs, andthat the MRP4 gene is located on chromosome13, a location that has since been refined to13q32 (Schuetz et al., 1999). The MRP4 cDNA,first reported by Lee et al. (1998), has a readingframe encoding a protein of 1325 amino acidsand a predicted secondary structure most closelyresembling that of MRP5 (Figure 21.1).

The first substrates of MRP4 were deducedwith the human T-lymphoid cell line CEM-r1.This cell line, generated by continual selec-tion on the antiviral agent 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) (Figure 21.2),an analogue of AMP, is highly resistant to PMEAand related compounds, as well as other nucleo-side analogues, but shows no cross-resistance totypical MRP1 substrates such as vinblastine(Robbins et al., 1995). This cell line was shown to rapidly efflux PMEA and other nucleosidemonophosphates, such as AZTMP. The findingthat the CEM-r1 cells have an amplification ofthe MRP4 gene led to the conclusion that MRP4can transport nucleoside monophosphate ana-logues (Schuetz et al., 1999).

SUBSTRATE SPECIFICITY OF MRP4

Initial studies with MRP4 using the PMEA-selected CEM-r1 line suggested a relativelybroad spectrum of transportable substrates.However, as was apparent at the time, the othergenetic changes present in this cell line have aconsiderable influence on the drug resistancephenotype. This is especially true for thedownregulation of adenylate kinase activity inthese cells, which increases the pool of trans-portable nucleoside monophosphates (Robbinset al., 1995; Schuetz et al., 1999).

Lee et al. (2000) transfected NIH3T3 cellswith 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-r1cells to a variety of nucleoside analogues, theNIH3T3/MRP4 cells showed no resistanceagainst AZT, 3TC, ddC or d4T. Further work by Schuetz and co-workers (presented at the FEBS 2001 ABC Meeting in Gosau), how-ever, broadened the substrate specificity toinclude thiopurine derivatives. Resistance to 6-mercaptopurine (6-MP) and thioguanine(TG) was recently found in the MRP4-trans-fected NIH3T3 cells studied by the group ofKruh (Chen et al., 2001).

We have transfected a variety of cell lines withthe MRP4 cDNA (kindly provided by J. Schuetz),and found the highest MRP4 expression inHEK293 cells. Initial experiments with theseHEK293/MRP4 cells showed that they effluxPMEA when loaded with bis-POM-PMEA(a membrane permeable form of PMEA), andshow resistance under continuous exposure toPMEA, to 6-MP and to the antiviral nucleosideanalogue abacavir (our unpublished results). Incommon with the exogenous expression of otherMRPs, it appears relatively difficult to obtainsubstantial levels of the protein after transduc-tion. A further complication is the endogenousexpression of MRP4 and MRP5 in many of thecell lines used for transfection studies. For exam-ple, HEK293 cells contain levels of MRP4 mRNAcomparable to those found in the most MRP4-rich tissues (our unpublished results).

A recently published study suggests thatexpression of MRP4 in insect cells could be thebest way to overcome problems of endogenoustransporter background. Chen et al. (2001) used MRP4-containing baculovirus to infectinsect cells from which they made inside-out membrane vesicles. Taking into account thestructural and substrate similarities betweenMRP4 and MRP5, and a previous study onMRP5 (Jedlitschky et al., 2000) (see later sectionon substrate specificity of human MRP5 for


P O O

O

O

O

O

O

O

O

O�

O�

O�

O

O�

O�

N

N N

NNH

HO

HO

N

N

N

NH2S

P

P

HO

N

N

N

HN

H2N

PMEA Thio-IMP cGMP

Figure 21.2. Chemical structures of the MRP4 and MRP5 substrates PMEA, thio-IMP and cGMP.


details), they demonstrated that cyclic nucleo-tides are substrates for this transporter. Lowrates of transport were observed for 3�,5�-cyclicGMP (cGMP) (Figure 21.2) (Km and Vmax val-ues of 10 �M and 2 pmol mg�1 min�1) and3�,5�-cyclic AMP (cAMP) (Km and Vmax valuesof 45 �M and 4 pmol mg�1 min�1). Remarkably,estradiol E217�G was a relatively good sub-strate (Km and Vmax values of 30 �M and102 pmol mg�1 min�1).

TISSUE DISTRIBUTION OF MRP4EXPRESSION

Initial reports concerning the expression ofMRP4 described a gene with a restricted tissuedistribution, as determined by polymerase chainreaction (PCR) mapping (Allikmets et al., 1996)and RNase protection assays (Kool et al., 1997).More recent data suggest that the gene is morewidely expressed. Lee et al. (1998) detectedMRP4 protein in most of the tissues examined,with levels ranging from very high in theprostate to barely detectable in the liver. Using a semi-quantitative reverse transcriptase PCR(RT-PCR) method to standardize MRP4 tran-script levels to �-actin, we find high levels ofexpression in the kidney, with lower but sub-stantial expression in the gallbladder, testis andprostate. We also find MRP4 mRNA in all celllines tested. More recently, we generated a newmonoclonal antibody against human MRP4,which we have used to confirm the high expres-sion of MRP4 in the kidney, as well as its pres-ence in cell lines. Lee et al. (2000) found MRP4 in the basolateral membrane of the acinar cells in the prostate. In contrast, Van Aubel reportedat the FEBS 2001 ABC Meeting in Gosau thatMRP4 is in the apical membrane, not the baso-lateral membrane, of rat and human kidneycells. Whether MRP4 is indeed localized to dif-ferent membranes in different epithelial tissuesneeds verification with antibodies that enablemore conclusive immunohistochemistry.

PHYSIOLOGICAL FUNCTION OF MRP4AND ROLE IN DRUG RESISTANCE

There are still very few clues as to the normalphysiological function of MRP4, and the role, ifany, played by MRP4 in anticancer drug resist-ance. The recent discovery by Chen et al. (2001)that MRP4, like MRP5 (Jedlitschky et al., 2000),can serve as an efflux pump for cGMP andcAMP indicates that MRP4 is able to remove

physiologically relevant (cyclic) nucleosidemonophosphates from the cell. Any role MRP4may have in drug resistance is also under inves-tigation. As nucleobase and nucleoside ana-logues are used extensively in anticancer andantiviral therapies, there is potential for MRP4to mediate resistance to these compounds. Aspointed out by Chen et al. (2001), 6-MP and MTXare both used in the treatment of childhoodleukemias and MRP4 is the only drug trans-porter known thus far that can transport bothdrugs. In a screen of drug-selected human can-cer cell lines by RNase protection assays, Kool et al. (1997) found MRP4 to be expressed at lowlevels in all cell lines, but this did not correlatewith resistance. However, the cell lines testedwere not selected by nucleobase or nucleosideanalogues nor tested for resistance against thesecompounds.

MRP5Human MRP5, also known as MOAT-C, and for-mally called ABCC5 (GenBank: AF146074), wascloned by several groups (Belinsky et al., 1998;Jedlitschky et al., 2000; McAleer et al., 1999;Wijnholds et al., 2000). The mouse homologue ofhuman MRP5, called mrp5 or Mrp5 (GenBankAB019003), turned out to be the same as the pre-viously identified sMRP (Suzuki et al., 1997;Tusnady and Varadi, 1998), a cloning artifactmissing the part encoding the first transmem-brane domain (Suzuki et al., 2000). Like MRP4,MRP5 is an organic anion pump with a highaffinity for nucleotide analogues and cyclicnucleotides (Jedlitschky et al., 2000; Wijnholds et al., 2000). No known human disease is associ-ated with MRP5 defects and the Mrp5 KOmouse has no phenotype thus far (Wijnholds et al., 2000) (our unpublished results).

SUBSTRATE SPECIFICITY OFHUMAN MRP5

Cells transfected with MRP5 cDNA constructswere used by McAleer et al. (1999) andWijnholds et al. (2000) to study the substratespecificity of MRP5. McAleer et al. (1999) found reduced accumulation in MRP5 cells forthe acidic organic dyes, 5-chloromethylfluores-cein diacetate (CMFDA), 5-fluorescein diac-etate (FDA), and 2�,7�-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein acetoxymethyl ester(BCECF-AM), but not for structural analogues,



the anionic calcein and the cationic tetra-methylrosamine. Further evidence that MRP5 isa typical organic pump, like other MRPs, camefrom Wijnholds et al. (2000), who showed thatMRP5 transports DNP-GS and GSH, and thatMRP5 is inhibited by nonspecific organic aniontransport inhibitors, such as sulfinpyrazoneand benzbromarone. MRP5 does not seem totransport MTX, in contrast to MRP1-4. There isalso no evidence that MRP5 can mediate resist-ance to any of the anticancer drugs belonging tothe MDR spectrum and transported by MRP1with one exception. Wijnholds et al. (2000)found a low level of etoposide resistance butthis was not found by McAleer et al. (1999).Conversely, resistance against cadmium chlo-ride and potassium antimonyl tartrate wasfound by McAleer et al. (1999) but this could notbe reproduced by Wijnholds et al. (2000).

Like MRP4, MRP5 can also cause resistanceagainst the nucleoside monophosphate ana-logue PMEA (Figure 21.2). When cells areloaded with the membrane-permeable PMEAprecursor bis-POM-PMEA, MRP5 mediatesexcretion of PMEA, but not of the di-(PMEAp)and triphosphate (PMEApp) of PMEA(Wijnholds et al., 2000), which are formed intra-cellularly (Balzarini et al., 1991). The ability ofMRP5 to transport nucleotide analogues mayalso explain the resistance of MRP5-transfectedcells to the thiopurines 6-MP and TG. Asshown in Figure 21.3, these thiopurines areconverted into the corresponding nucleosidemonophosphates (e.g. thio-IMP) (Figure 21.2)and these are excreted via MRP5 (Wijnholds et al., 2000) (our unpublished results). Whether

MRP5 can mediate excretion of methylatedthiopurine derivatives, as reported for MRP4 by J. Schuetz at the 2001 FEBS 2001 ABCMeeting in Gosau, remains to be tested.

Using vesicular transport by plasma mem-brane vesicles made from hamster V79 cellsoverexpressing MRP5, Jedlitschky et al. (2000)identified cGMP as a MRP5 substrate with amicromolar affinity for the pump. cAMP wasalso transported, but with a lower affinity.Interestingly, they also found that the phospho-diesterase (PDE) inhibitors sildenafil (betterknown as Viagra), trequinsin and zaprinast,which prevent intracellular breakdown ofcGMP, inhibited the MRP5-mediated cGMPtransport as well.

MRP5 does not only transport purine-basedcompounds. In unpublished experiments (J. Wijnholds, P.W. and P.B.), we have alsofound transport of a pro-drug of 3�-deoxy 2�,3�-didehydrothymidine 5�monophosphate(d4TMP), alaninyl-d4TMP, an antiviral agent(Balzarini et al., 1996). MRP5 can therefore alsotransport nucleotide analogues with a normalpyrimidine (thymine) ring.

EXPRESSION OF MRP5 IN TISSUES ANDCELL LINES

Analysis of tissue RNA suggested that MRP5 isubiquitously expressed (Table 21.3). The high-est levels are found in skeletal muscle andbrain (Belinsky et al., 1998; Kool et al., 1997;McAleer et al., 1999; Zhang et al., 2000). Allattempts to generate antibodies that allow thelocalization of MRP5 in tissues have failed thus


6-Me-thio-IMP

6-Me-MP

PRPP

HGPRT

PPi

TPMT

TPMT

IMPDH GMPSThio-IMP Thio-XMP Thio-GMP

6-MP

Higher phosphorylation 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, thiopurinemethyltransferase; IMPDH, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphatesynthetase. (Adapted from Zimm et al., 1985.)


far. This is presumably because the expressionlevels are too low, as these antibodies readilydetect MRP5 in transfected cells (Wijnholds et al., 2000). On Western blots, MRP5 can bedetected in human and murine erythrocytes(Jedlitschky et al., 2000) (our unpublishedresults) and MRP5 might be the cGMP pumpdetected in erythrocytes (Schultz et al., 1998),although this needs to be shown using red cellsfrom Mrp5 (�/�) mice. Levels of MRP5 proteinwere also high in brain extracts, but not in skele-tal muscle, in contrast to the MRP5 RNA levelsin this tissue.

The negative results obtained on intact tis-sues using antibodies against human or murineMRP5 make it impossible to decide whetherMRP5 is present in many cell types or only insome cell types present in all tissues, for exam-ple, endothelial cells. In a recent survey usingWestern blots, we found MRP5 in all humantumor cell lines analyzed, including colon,breast, ovarian, lung carcinoma lines, leukemiaand embryonic kidney cell lines. This suggests,but does not prove, that MRP5 is present inmany different normal cell types.

THE POSSIBLE INVOLVEMENT OF MRP5IN DRUG RESISTANCE OR DISEASE

Base, nucleoside and nucleotide analogues areused in antiviral and in anticancer therapy.Potentially, elevated levels of MRP5 could there-fore contribute to clinical resistance to theseagents. In MRP5-transfected cells, unambiguousresistance has only been found thus far against 6-MP, TG, PMEA, 5-hydroxypyrimidine-2-carbox-aldehyde thiosemicarbazone (an experimentalanticancer drug), and an aryloxyphosphorami-date derivative of 2�,3�-dideoxyadenosine (Cf1093). Whether clinical resistance against thesecompounds is ever associated with elevatedMRP5 levels remains to be studied. No resist-ance was observed thus far in MRP5 cells forother base or nucleoside analogues used in can-cer or antiviral chemotherapy, such as ara-C, 5-fluorouracil, cidofovir and fludarabine (ourunpublished results).

No human disease has been associated withalterations 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 MRP5and 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 transportersmay lead to an understanding of the physiologi-cal function of each of them.

MRP6MRP6 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 multipleorgans. MRP6 is a protein of 1503 amino acids(Belinsky and Kruh, 1999; Kool et al., 1999a),45% identical to MRP1, and its gene is locatednext to MRP1 on chromosome 16 in a tail-to-tailconfiguration (Kool et al., 1999a). Human MRP6is 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 inother tissues. In initial immunofluorescencestudies, Madon et al. (2000) localized rat Mrp6 inthe basolateral and apical membranes of hepato-cytes. More recent work reported at the FEBS2001 ABC Meeting at Gosau strongly indicates,however, that MRP6 is in the basolateral mem-brane of polarized cells. In contrast to someother MRPs, expression of Mrp6 appears stable,whatever damage is inflicted on the liver(Madon et al., 2000).

The substrate specificity of MRP6 is still amystery. Madon et al. (2000) tested a series oftypical MRP substrates in vesicular transportstudies and found only transport of BQ-123, ananionic cyclopentapeptide and endothelin Areceptor antagonist. Endothelin-1 itself wastransported by Mrp2, but not by Mrp6. Theseresults suggest that MRP6 could be a highlyselective organic anion pump. It should benoted, however, that Madon et al. (2000) onlytested radioactive substrates at relatively lowconcentrations. No competition experimentswere done with high competitor concentrations,substrates such as MTX were not tested, andstandard inhibitors of MRPs were not testedeither.

MRP6 AND DRUG RESISTANCE

Amplification of the 3�-part of the MRP6 genewas found in leukemia cells selected for anthra-cycline (epirubicin) resistance (Kuss et al., 1998;Longhurst et al., 1996; O’Neill et al., 1998). The



anthracycline resistance was initially thoughtto 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 3� halfof the adjacent MRP6 gene (Belinsky and Kruh,1999; Kool et al., 1999a).

There is no indication that the MRP6 gene isever associated with anticancer drug resist-ance. MRP6 is expressed at low or undetectablelevels in all cancer cell lines tested (Kool et al.,1997, 1999a; Madon et al., 2000) and no correla-tion between expression and drug resistancewas observed (Kool et al., 1997).

MRP6 AND PXE

Pseudoxanthoma elasticum is a heritable disor-der characterized by calcification of elastic fibersin skin, arteries and retina, resulting in loss ofelasticity of the skin, arterial insufficiency andretinal hemorrhage. Why loss of a highly spe-cialized pump located in the basolateral mem-brane of liver and kidney cells would lead tosuch a generalized connective tissue disease isunclear. Speculations include indirect effects onCa2� metabolism or elastic fiber assembly,through excretion of cytokine-like organicanionic peptides (Bergen et al., 2000; Le Saux etal., 2000; Ringpfeil et al., 2000) (see Chapter 28).

MRP7Hopper et al. (2001) identified an MRP homo-logue designated MRP7 by a database search.MRP7 cDNA encodes a protein of 1492 aminoacids and, translated in a reticulocyte lysatesystem, the cDNA produces a protein ofapproximately 158 kDa. MRP7 has a predictedsecondary structure and topology similar tothat of MRP1, with an extra NH2-terminaltransmembrane domain. Although clearly anMRP homologue, MRP7 has the lowest overallhomology with other members of the MRPfamily (33–36% sequence identity). MRP7seems to be ubiquitously expressed as assessedby RT-PCR, but at a low level, since the mRNAtranscript could not be detected by RNA blotanalysis. The substrate specificity and mecha-nism of transport of MRP7 have not yet beenstudied. Whether or not MRP7 is an organicanion transporter also remains to be tested.

ACKNOWLEDGMENTS

We thank Drs Marcel Kool, Alfred Schinkel andJan Wijnholds for their helpful comments onthe manuscript. T.S. was supported by a post-doctoral fellowship from the Japanese Societyfor the Promotion of Science and our researchwas supported by grants (NKI 2001-2474 and1998-1794) of the Dutch Cancer Society to P.B.

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abc proteins || the multidrug resistance proteins 3–7

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