ICANCER RESEARCH56, 4124-4129. September 15. 19961

Advances in Brief

AHumanCanalicularMultispecificOrganicAnionTransporter(cMOAT)GeneIsOverexpressed in Cisplatin-resistant Human Cancer Cell Lines with DecreasedDrug Accumulation'

Ken Taniguchi, Morimasa Wada, Kimitoshi Kohno, Takanori Nakamura, Takeshi Kawabe, Mina Kawakami,Kazuhiro Kagotani, Katsuzumi Okumura, Shin-ichi Akiyama, and Michihiko Kuwano2

Department of Biochemistry. Kyushu University School of Medicine, Maidashi, FUkUOICa8i2-82 [K. T., M. W., T. N., T. K., M. Ka., M. Ku]; Department of Molecular Biology,University of Occupational and Environmental Health, Kita-Kyushu 807 1K. Ko.J; Faculty of Bioresources, Mie University, Tsu, Mie 514 [K. Ka, K. 0.]; and Department ofCancer Chemotherapy. Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuraga-oka, Kagoshima 890 (S. Al, Japan

Abstract

By targeting the AlP binding conserved domain in three AlP bindingcassette superfamily proteins (P-glycoprotein, multidrug resistance pro

tein, and cystic fibrosis transmembrane regulator), we isolated the cDNAof a new AlP binding cassette superfamily that was specifically enhancedin a cisplatin-resistant human head and neck cancer KB cell line. A humanclone homologous to rat cnnalicular multispecific organic anion trans.porter (cMOAT) was found and designated human cMOAT. Fluorescencein situ hybridization demonstrated the chromosomal locus of the gene onchromosome 10q24. The human cMOAT eDNA hybridized a 6.5-kbmRNA that was expressed 4- to 6-fold higher by three cisplatin-resistantcell lines derived from various human tumors exhibiting decreased drugaccumulation. Human cMOAT may function as a cellular cisplatin transporter.

Introduction

Acquirement of drug resistance by tumor cells is frequently observed in cancer patients receiving chemotherapeutic treatment. TwoABC3 proteins, the Mr 170,000 Pgp and the Mr 190,000 MRP, areknown to confer multidrug resistant phenotypes to cancer cells (1—3).Although the primary structures of Pgp and MRP share only 15%amino acid homology (4), both Pgp and MRP are involved in drugresistance to a similar profile of chemotherapeutic agents that include

anthracyclines, vinka alkaloids, and epipodophyllotoxins (1—3). By

contrast, cancer cells that overexpress Pgp or MRP do not showcross-resistance to platinum-containing compounds, alkylating agents,and antimetabolites (1—3).MRP-transfectant cells show resistance toanthracyclines, vinka alkaloids, epipodophyllotoxins, and heavy metalanions but are sensitive to platinum-containing compounds (5, 6).However, MRP can transport the cysteinyl leukotriene C4 and otherglutathione conjugates (7—9),suggesting that MRP may be a glutathione-conjugate transporter.

Of the platinum-containing compounds, cisplatin is a potent anticancer agent that has been widely used to treat various malignanttumors, including testicular, head and neck, esophageal, lung, ovarian,

Received 6/4/96; accepted 7/31/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by a grant-in-aid for cancer research from the Ministry of Education,

Science, Sports and Culture, Japan; the Fukuoka Anticancer Research Fund, Fukuoka 21stCentury Medical Fund; and the Yasuda Memorial Medical Grant for Cancer Research.The nucleotide sequence reported in this paper has been deposited in the Genbank database (Accession No. U6397O).

2 To whom requests for reprints should be addressed, at Department of Biochemistry,

Kyushu University School of Medicine, Maidashi, Fukuoka 812-82, Japan. Fax: 81-92-632-4198; E-mail: kuwano@biocheml.med.kyushu-u.ac.jp.

3 The abbreviations used are: ABC, AlP binding cassette; Pgp, P-glycoprotein; MRP,

multidrug resistant protein; CFFR, cystic fibrosis transmembrane regulator; cMOAT,canalicular multispecific organic anion transporter; cisplatin, cis-diamminedichloroplatinum(1I); FISH, fluorescence in situ hybridization.

and bladder cancers. However, clinical application of long duration isoften limited because of the development of cisplatin-resistant tumors.To further improve the chemotherapeutic efficacy of this potentanticancer agent, we must understand the mechanisms that controlsensitivity to cisplatin (10, 11). Cisplatin accumulation is decreased inmany specific cisplatin-resistant cells (12—14),and the AlP-dependent active outward efflux of cisplatin is enhanced in both cisplatinresistant human epidermoid cancer KB and human prostatic cancerPC-3 cell lines (12, 14).

Because the overexpression of two ABC-superfamily genes,MDR1/mdrl (15) and MRP (4, 5), is closely associated with Pgp- andnon-Pgp-mediated multidrug resistance, we initially isolated othergenes belonging to the ABC superfamily to explain the decreasingcellular accumulation of cisplatin by tumor cells. Specifically, wetargeted the AlP binding domain conserved in MDRJ, MRP, andCFTR genes in the present study and compared the difference inmRNA from both cisplatin-sensitive and -resistant cells. In this study,we isolated full-length human cMOAT cDNA that is highly homologous to rat cMOAT (16), a homologue of the human MRP gene. Wewill also discuss a possible correlation between human cMOAT andcellular cisplatin accumulation.

Materials and Methods

Cell Lines. Cisplatin-resistantcell lines were derived from variousdrugsensitive parental counterparts: T24IDDP1Ofrom human bladder cancer T24cells (13); P/CDP5 from human prostatic cancer PC-3 cells (12); and KB/KCP4 from human head and neck cancer KB cells (14, 17). Etoposide/teniposide-resistant KB/VP4 cells were derived from KB cells (18). These celllines were cultured at 37°Cunder a humidified atmosphere of 5% CO2 inEagle's MEM (Nissui Seiyaku, Tokyo, Japan) containing 10% fetal bovineserum (Sera-lab, Sussex, United Kingdom), Bactopeptone (1 mg/mI; DIFCOLaboratories, Detroit, MI), glutamine (0.292 mg/ml), kanamycin (100 mg/mi),and penicillin (100 units/mi).

eDNA Cloning. Total RNA was isolated from KB/KCP4 cells and 1-2 @g

were then reverse-transcribed using molony muline leukemia virus reversetranscriptase (Life Technologies, Inc., Gaithersburg, MD). The resultingcDNA was used as a template for PCR using various primer combinations. Inboth nested PCR and heminested PCR, cDNA was reacted with the outerprimer pairs (Cl; Fig. 1A), and an aliquot of the reaction product was thenreamplified with the inner primers (C2, C3, and C4). The primer sequencesused for C-series PCR are shown in Fig. lB. and the expected sizes of PCRproducts are shown in Fig. 1A.PCR cycle temperatures and times were 94°Cfor 30 s, 50°Cfor 30 5, and 72°Cfor 30 s for a total of 35 cycles for allreactions. The PCR products were subcloned in pMOSBlue vector (Amersham, Buckinghamshire, United Kingdom) and sequenced. The overlappingcDNA clones were obtained by screening a Agtll HeLa cell cDNA library(Clontech, Palo Alto, CA), a pCMVSPORT human kidney cell cDNA library(Life Technologies, Inc.), and a AzapIl human liver cDNA library with thecDNA probe. All overlapping cDNA clones were subcloned into the pUCl8

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LIL.@

CISPLATIN RESISTANCE AND HUMAN cMOAT

GRTGAGK—38 aa—IIPQDP 56 aa QRQ@C@—13 aa—D@@4

@ @I

5'—l 5—2 3—2 3—1

Cl(393 bp)

C2(342 bp)

C3(264 bp)

C4(204 bp)

5'-l 5'-(AG)(AC)G(AG)AC(GT)GGA(GT)(GC)(AT)GGGAAG-3'

5'-2 5'-A(AG)T(GC)AT(AC)CC(AC)CAG(AG)A(AC)(GC)-3'

3'-2 5'-.AAGCACA(TC)(AC)A(AG)CTG(GC)(TC)(GT)(Gc)TG-3'

3'—l 5'—(GT)(GC)(AT)GC(AC)(GC)TGG(GC)(TC)TCATC-3'

.r

Fig. 1.A, schematic representation of primer positions and the nested PCR strategy. Top, the conserved amino acid sequence of the AlP binding region. C1-C4, primer pairs forPCR and the expected product sizes. B, the sequence of primers used for nested PCR. C, gel electrophoresis of PCR and nested PCR reaction products. Arrow, 2O4-bp C4 primer pairproducts.

A

B

4

plasmid. Chain elongation and termination were performed with a DyeDeoxyTerminator Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan), andnucleotide sequencing was performed with a DNA sequencing system (model373S; Applied Biosystems). Data were analyzed using GeneWorks software(IntelliGenetics, Mountain View, CA).

Genome DNA Cloning. The EMBL3 human placenta genome DNA library (19) was screened by the previously described method for cDNA libraryscreening. The clones were used for FISH analysis.

Northern Blots. A human multiple tissue Northern blot was commerciallyobtained (Clontech), and total RNA was extracted from exponentially growingcells by the acid guanidinium thiocyanate-phenol-chloroform method (20).RNA (20 i.tg) was subjected to electrophoresis in a 1% agarose gel containing2.2 M formaldehyde and then transferred to a nylon membrane (Hybond N@;Amersham). The filters were hybridized with a 32P-labeled cDNA probe in asolution containing 50% deionized formamide, 10 X Denhardt's buffer,5 X SSC [1 X SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0)], and0.1% SDS for 24 h at 42°Cand then washed twice in 42°C2 X SSC containing0.1% SDS. The amount of human cMOAT encoding mRNA was quantifiedwith a Fujix BAS 2000 image analyzer (Fuji, Tokyo, Japan).

FISH Analysis. Probe labeling and in situ hybridization were performed asdescribed previously (21). Briefly, chromosome spreads were obtained fromphytohemaggulutinin-stimulated blood lymphocytes of a healthy donor afterthymidine synchronization and bromodeoxyuridine incorporation. Genomic

DNA fragments were labeled with biotin-l6-dUTP (Boehringer Mannheim,Mannheim, Germany) by nick translation. In situ hybridization was performedin the presence of the COT-l DNA (Life Technologies, Inc.) competitor.Hybridized probes were detected with FITC-conjugated avidin (Boehringer

Mannheim). Chromosomes were counterstained with 0.2 mg/ml propidium

iodide for R-banding. Fluorescence signals were analyzed using a Zeiss Axioskop epifluorescence microscope equipped with a cooled Charge CoupledDevice camera (model PXL 1400; Photometrics, Tucson, AZ). Image acquisition was performed on a Macintosh Quadra 840AV computer using theWLab@ (Signal Analytics, Vienna, VA) software. The images were thenpseudocolored and merged using Adobe Photoshop'@ 2.5J (Adobe SystemsJapan, Tokyo, Japan). FITC and propidium iodide images were colored whiteand gray, respectively. The merged FITC and propidium iodide images weredirectly printed using a Fuji Pictrography 3000 output device.

Results and Discussion

Human MDR1, MRP, and CFTR have been identified as membersof the ABC transporter superfamily (4, 22). The similarity of thesethree genes resides predominantly in two AlP binding domains.Based on their homology, we designed highly degenerate C-seriesprimers (Fig. 1A). We also designed PCR primer mixtures ranging incomplexity from 64 to 128. We used the C series of primer sequences(Fig. 1B) because we could not produce PCR products of expectedsize when another series of primer pairs were used. The resultsobtained for C-series primer pairs show that nested PCR amplificationof a unique band from primary amplification products contains severalbands (Fig. lC). The PCR products were subcloned, and 43 transformants were subsequently sequenced. Among these clones, only 10had 5' and 3' primer sequences at opposite ends. The sequences of

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CISPLATIN RESISTANCE AND HUMAN cMOAT

A@ ATP 2-28 PCR fragment —

28—9

AK1—8

1—1

cs-i86

4—1

15—9

L3

Bhianan cMOAT @LEKr@S----TF@1lSSF -LDSPEA@P@ PPIQLLHVYKSRTKRSSTFLLYLAfl@VFVGFI1fLAAIEEI LVLTEbSGQA 94

I4IQLYSVYRS RTKRSSIfrfr YL*@VFWF UI[LAAIDL@ LAL*bTGQA 93rat cMOAT -DK@C@S- ---TFIVbLSL -LESPEAbtP F LVW GF 8 LAhuman MRP LRG@fAD GSDPL@IWNV TWNTSNP@,4T F N LVW F 8 CF @YFLYLSRHDRGYIQI*J'L NK*J'ALGFL @p4JJICWAD@JYSFViJISRGI 100

human cP4OAT@ LVIQYSRQWCVQKN@FLSL @i@LSILflT FQFQTLflTL fl@GDNSNLAYSCL-flISYG FQILILIFSA @NNESSNN 193rat cMOAT TVPPII@YTN LC L LAVQHSRQWC VRKI@IFLSL f$P1SVLL@V FQFQTLk@AL @4(DSKSNMAY SYL-i4VSYG FQIVLLILTA @SIPSDSTQT 192

TFLIQLERRK GVQS@IMLT @@VALV@LAILRSK@ITA @j(EDAQVDLF RDIT@J'VYFS LLLI1@.JLSC @@1RSPLFSE 200human t@P FLAP@JLVS GI L

hianan cMOAT PSSIASFLSS ITY@t4YDSII LKGYKRPF@L EDVWEVD@M KTKTLVSnr11'HMKRELQKA RRALQRRQEflSQQNSGARL PGLNKN@QS QOALVLEDVE 293rat cP4OAT PSVTASFLSS ITF@PYDRTV LKGYKHPLIL EDVWDID F KTRSVTSIt(@ @AMTKDLQKA RQAFQRRLQ@ @QR-KPEATL HGLNKK@@QS QDVLVLEEAK 291human @P TIHDPNPCPE SSA@JLSRIT FW-WITG@jV RGYRQPL S DLWSL-N@flJ@TSEQVVPVL VKNWKKECA@JIRKQPVKVVY SSKDPA@KE SSKVDANEEV 297

r—wri@ DTYLPIL@VLCAII1.flAALI@ Lf@kLflIKVR393htunan cMOAT KKKKKSGTK@@PKSW1I6

human MRP EALIVKSPQ -NP@1J@@ KLLI @JfNDTKAPOkJl@Y@Y1*J.@J'TACL L F F S 1K 396KLLI Ei'KSS NSYVPIUYtC A4@.ff@VTLI FC S F @L@VR 389rat cMOAT KKSEK--TT YPKS

human cMOAT@@ F@VlVWE! @ILST@@KTI@VKNW1 493TNYMQLV$@S@VIP@TL$IF 8 GPSrat cMOAT ITIrVNSSII'RALILSI4@@J NL MSVD L@J GPS@ VMAM@J@KTYPVJH@$J496RN4@V@NkI 489kAGVt@IIMVL1 VLAT

human P4W @VIGAV@@@JIITNJRK E NL MSVD F

human cP4OAT@ @NL@Krr9IM1AFSQf@CVV@JLI@1(V@VVfl LV@N@ TkFNILRFPLI 589@ @@ll1TLFNILRFPL1593L KI&LRFGQ@tSLL LQI@@@@VVF IVYVLVb@Arat cMOAT (IN EIL@IKILK I'4WE@'S E GI K L

human t.@P@ D Al E@@J4..J(KSAYU AVG C V LC F @I@N@j 4FNILRFPLI 596

hianan cMOAT St4:@R4Ir@Il L@V@E@@KYflGDDfl TSAVI--flIDC NFD--KAMQFS@WTW@HDSE4flfRDVNL @@4brvzv@-@@ 689rat cMOAT SP4.Pt4'T@St L@ASVSVDkL @R'rUGDDLb TS4j.-It@VS NFD--KAVKF s&@s@rwbpo LEAITIQDVNL VAV G@ L M 685human P@P NIkPPI@J @ASVS@.K@JIFL.JHEEU PDS@JR@VK DGGGTNSITV RP4AJ@TW@RSDPPITJ.NGITF VAV G L A 696

@@ $LSGGQKQRI @@@VbNL@ 789human I@P@ CAL LPDLE P IGEK @ILSGGQKQR$ LARA @DA P@IY@LbDPLSI 785htanan cMOAT

Ii LFGkEYN@K K CAL LPDLE P IGEKGrat cP4OAT@ILSGGQKQRt LARA @NA@ 796

SY AII―kKK@WikNL KWLRHTGPf@TVHDGSE EEDDDY@LIS 889human cMOAT @VDAHVGKHI @@@Lfr11@MHFPQVD

human @P @VDAHVGKHIF TR@ 14TJ'---AST(JIJ@DAEENGVTGVSGP@J.-K 891rat cMOAT @VDAHVGKHI@ TR F%9T HIIHF PQVD KKhWAIINWK@1JMKHSGP@@fITVNNDSEAEDDDI44IP 885

human cMOAT SVEEIPflIAA SITMRRENSF@ NGRHLKSLRN PLKTRNVNSL @DEELVKGQ @E@KKVFIDf'@f1@}flflE @t@)@FSI FFI@.AFVP41 989rat cMOAT TMEEIP@1AA SLAMRRENSL ltfl.SItS S SSRRGKSLKN SLKIKNVNVL P(@KEKEVEGQ I(L@KKEFVEJ G@4iK@IYtK @4.t@4@lWSI LFIIII.FYGLIII 985human P@P EAKQM-@jlGM LVTDSAGKQL c@JLSJ S Y SG---DISRH HNSTAELQ-- @EAKKEEN @J4EADKAQtG@K@J%@D @*J@J.FIS FLS@JLFMC@J 985

h@anancMOAT@ F@W1@YPASQ@ IAHFWSAFGFV1g@llI@Q 1WFI@bGDIS 1089

human @P ALA N LS I -I ---EH 1K S A LG S GI VF GYSMAVSIGGI S C H D@ IIINRF@GDIS 1085rat cMOAT FIG N LS I ON @@@NSSSHK G @,,@@APGIJLL ISTLWSIYAC R@@A H Q

LWNR.F@KELD 1081

htsnan cMOAT IiV5bTLRSL GSWITCFLGI@@@@ 1189rat cMOAT @TVDIILLP@TLRSWP4ICFFGI AGTLVI.EtP41P@FA I I L IL S Qhuman t@P y@t4I@VI KMFMGSLFNVIGACIV@J.L T AA I P L LI Q ‘(VS RQL L V R SHF T L S RAFE@ IStFI.AWNEKQ 1185

YVTRQL L VKP SHF P RAFE@,@FJHQSDLK 1181

human cMOAT 1flII@k'tVFS@@@@ 1288rat cP4OAT I@*I4QKtVFS i4t@s RWLA kELI1GNtV1@ jC L L I ItkILtGDYkG @V A @J1'Y1.NWI.VRP.@@ Y ET@JtkAPW@IQET 12811@@13NWLVRM@@SE@TNIVAVERRSY NVE P&APWI-VTDK i2a*@ SL.JAGL@JSSVhuman l4tP @@ft@rrP @Ji RWLA J@@jI@J.F L A I

M@W@Y@@SLTCF @DI@ GLHDLR@KLF IIPQDPr1@fl 1388rat cMOAT P P H I V V YR L@ IIDG DVKII GLHDLRLRL@ IIPQDPIIFSI 1384

human cMOAT Kfl@flVD@E@fl ______ ___hOG N4J4__GLHDLRIKI@__IIPQDP@j@J 1381humanI@P P PV YL YR L

____________@@ @ILE@V@rILVLDEATAAVD@ 1488rat cMOAT kSLRMNLDPF SDEE A1.ELAHL@S LG S@ LS QRQL L ItAI&R4KIL VLDEATAAVD@ 1484human @P @SLRP44LDPF@ @E1.AHL@D OK H 1.: QRQL L @@.LRK@j(ILVLDEATAAVD LETD IQ I TVD@@J 1481

htanan cMOAT @SLRP*LDPF

rat cMOAT IITIAHRL@ .1@@ NVNSTKF 1545h@auancMOAT I1TIAHRLIj@@DKV LD

@ G E NVNHTEL 1541human @P L@TIAHRL@I TRV_____ Q GV 1531

Fig. 2. A, schematic representation of overlapping cDNA clones. These cDNA clones encompass the coding region for human cMOAT. B, comparison of amino acid sequences ofhuman cMOAT, human MRP (HUMMRPX), and rat cMOAT. Boxes, amino acid identity. Amino acid differences are shown in their corresponding positions. Dashes, gaps that areintroduced to maximize identity.

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five clones were identical to those of MRP, and four clones hadunrecognized sequences with no homology to sequences in the Gen

bank database. One clone, pATP2-28, showed homology with MRP(4) and, more significantly, with rat cMOAT (16). Therefore, we

tentatively designated this product human cMOAT. Screening of thehuman cervical cancer HeLa cell cDNA library, the human kidney cellcDNA library, and the human liver cDNA library with a 207-bpATP2-28 and walking probe allowed the isolation of overlappingcDNA clones (Fig. 2A). Sequencing each of these clones revealed anopen reading frame coding for 1545 amino acids that showed 46%similarity to that of human MRP (Fig. 2B). The EMBL3 humanplacenta genomic DNA library was also screened, and several positiveclones were identified and then used for FISH analysis, revealingpositive signals at 10q24 (Fig. 3). Because human MDRJ, CFTR, andMRP genes are localized on 7q21.l, 7q3l, and l6pl3.l2-l3, respectively (4, 23, 24), the human cMOAT gene seems to be different fromthese other genes.

Multiple-tissue Northern blot analyses with the CS-i cDNA proberevealed that human cMOAT mRNA was expressed in liver butundetectable in other tissues (Fig. 4A). The tissue profile of humancMOAT expression was identical to that of rat cMOAT expression.TR@ rats, an animal model of the Dubin-Johnson syndrome, aredefective in cMOAT, which mediates the hepatobiliary excretion ofnumerous organic anions including glutathione S-conjugates. We havepreviously reported that cellular cisplatin accumulation was markedlydecreased in the cisplatin-resistant human head and neck cancer KBcell line KBIKCP4 (14), the cisplatin-resistant human prostatic cancerPC-3 cell line PICDP5 (12), and the cisplatin-resistant human bladdercancer T24 cell line T24IDDP1O (13). The transport of leukotriene C4in membrane vesicles prepared from cisplatin-resistant KB/KCP4cells was facilitated by an AlP-dependent pump that seemed similarto the GS-X pump (17). Therefore, we examined whether expressionof the human cMOAT gene was enhanced in these drug-resistant celllines exhibiting decreased cisplatin accumulation. Human cMOATmRNA was overexpressed 4.0- to 6.0-fold in all three cisplatinresistant cell lines as compared to their parental drug-sensitive coun

CISPLATIN RESISTANCE AND HUMAN cMOAT

I..

Fig. 3. Mapping of the gene region encodinghuman cMOAT by FISH. Four genomic cloneswere isolated using a cDNA probe and subjected toFISH analysis. Arrowheads, fluorescence signalson the R-banded metaphase chromosomes. Basedon observations of more than 10 metaphase chromosomes, a band is identified at 10q24.

I

I.'

A@ F@ c'-. !•1

4-

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terparts (Fig. 4B). In contrast, human cMOATgene expression was notenhanced in KB/VP4 cells that overexpress the MRP gene (Fig. 4, Cand D). The KB/VP4 cell line, derived from KB cells resistant to theepipodophyllotoxin anticancer agent etoposide, had a multidrug resistance phenotype and exhibited decreased drug accumulation (18).

The size of the human cMOAT mRNA was estimated to be about 6.5kb (Fig. 4B), similar to that of MRP mRNA (Ref. 4; see Fig. 4D) butlarger than the 4.5-kb human MDR1 mRNA.

We isolated human cMOAT cDNA by targeting the AlP bindingdomain conserved in three human ABC superfamily genes [MDRJ(P-gp),MRP,andCFTR]andfoundhumancMOATwasahomologueof human MRP (4) and the recently described rat cMOAT gene (Ref.16; see Fig. 2). Mayer et a!. (25) have reported that the absence ofMRP or its isoform from the canalicular membrane is the basis for thehereditary defect of the hepatobiliary excretion of anionic conjugatesin the TR rat model of human Dubin-Johnson syndrome. Paulusmaet a!. (16) have reported selective expression of rat cMOAT by liverhepatocytes as well as reduced mRNA levels in the Th rat. Expres

sion of human cMOAT was observed in liver but was not detectablein other tissues, suggesting that human cMOAT may mediate thehepatobiliary excretion of numerous organic anions and that humancMOAT mutation may result in the Dubin-Johnson syndrome. We arenow exploring this possibility in human patients.

Expression of human cMOAT was enhanced in cisplatin-resistant human cancer cell lines with decreased cellular cisplatin accumulation but was not enhanced in lines with normal levels of drugaccumulation. We have previously reported that two cisplatinresistant lines, KB/KCP4 and P/CDP5, that do not overexpress Pgpor MRP show enhanced active ATP-dependent efflux of cisplatin(12, 14). We also demonstrated by a cell-cell hybridization test thatboth the drug resistance and the accumulation defect found inKB/KCP4 cells are dominant traits (14), suggesting the existenceof an active efflux system for cisplatin in KB/KCP4 and P/CDP5cells. Ishikawa et a!. (26) have suggested that an ATP-dependentpump may function to transport glutathione-conjugates (GS-Xpump). Ishikawa and Ali-Osman (27) have additionally reported

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CISPLATIN RESISTANCE AND HUMAN cMOAT

A B

°ohcMOAT'@

28S —

18S—

V °@GAPDHm@

0

4@ ‘,) I

@c'4@4 @/&Z@'

D

MRPN°28S—

18S —IGAPDH@@

Fig. 4. Northern blot analysis. A, human multiple tissue blots wereprobed with human cMOAT CS-I cDNA fragment (see Fig. 2A).B, variousparental and cisplatin-resistant cell lines were analyzed using the sameprobe. Northern blots of KB and KB/VP4 cell lines were probed withhuman cMOAT (0 and MRP (D). C

that the GS-X pump may effuse glutathione-conjugated cisplatinfrom Ll2lO murine leukemia cells. Cisplatin molecules werefound to interact very quickly with two glutathione molecules ingrowing mammalian cells (28). It remains unclear whether humancMOAT is closely coupled with the activity of the glutathioneconjugated cisplatin pump.

On the other hand, MRP functions as an ATP-dependent exportpump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds, including various anticancer agents (29). However, allthree cisplatin-resistant lines derived from KB, PC-3, and T24 cellsdo not overexpress MRP (1 2—14),and cancer cell lines that overexpress MRP, including KBIVP4 cells, are not cross-resistant tocisplatin (5, 6, 18). It is thus unlikely that MRP is involved inintrinsic or acquired cisplatin resistance due to decreased drugaccumulation. Further studies using human cMOAT cDNA transfection will confirm that human cMOAT is directly involved incisplatin resistance and can serve as a multispecific organic aniontransporter. Transfection of the complete human cMOAT cDNA is

Acknowledgments

We thank Yukiko Mine of our laboratory for preparing this manuscript. Wethank Mayumi Ono (Kyushu University) and Hiroshi Taguchi (Mie University) for fruitful discussion.

References

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q)@ @.°Q@ 4@ q

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hcMOAT@'@'

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CI5PLATIN RESISTANCE AND HUMAN cMOAT

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1996;56:4124-4129. Cancer Res   Ken Taniguchi, Morimasa Wada, Kimitoshi Kohno, et al.   Cancer Cell Lines with Decreased Drug Accumulation(cMOAT) Gene Is Overexpressed in Cisplatin-resistant Human A Human Canalicular Multispecific Organic Anion Transporter

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