A Human Canalicular Multispecific Organic Anion Transporter (cMOAT) Gene Is Overexpressed in Cisplatin-resistant Human Cancer Cell Lines with Decreased Drug Accumulation1

九州大学学術情報リポジトリ

Kyushu University Institutional Repository

ヒトCanalicular Multispecific Organic Anion Transporter(cMOAT)遺伝子は薬剤蓄積の減少したシ スプラチン耐性ヒトがん細胞株で過剰発現している

谷口, 堅

九州大学医学系研究科分子医学系専攻

https://doi.org/10.11501/3122955

出版情報：Kyushu University, 1996, 博士（医学）, 課程博士 バージョン：

権利関係：

IC1\NChR REShARCJI5o. 4124 412Y. Scptcmher 15. 19961

Advances in Brief

A Human Canalicular Multispecific Organic Anion Transporter (cMOAT) Gene Is Overexpressed in Cisplatin-resistant Human Cancer Cell Lines with Decreased Drug Accumulation1

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

Dfllllrllll<'lll of 1/iot hemr1tn. K\111!¹11 ^U11n l'rll/1 School of Meditine, Mllldl.l.ll/1, f ukuoku /'111-H2 / ^{K. T. M.} W., T N, T K /vi. Ka .. M. Ku.j; Depart mel// of Molecular Biolo�.\.

U111\ enifl fl/ Or'C'llfJIIIIOIILII 1111d t111 rmlllll<'llliil 1/ea/th, Kila-Kw1l111 1'107 f K. Ko.j: Facult1· oj Bwre\Ourc^e\, Mie U11irenit\', Tw. M1 5f.l f K. Ka .. K. 0./: und Oeparl/1/ell/ of Cancer Cht•mollll•ml''. lllltitllle j(,. Callter Re1ean h. Faclllt\ tif. Mt•d1c11u Ka�mhima ^Unn enity, Sakurm;a-oka. Kagfllhima 1'190 {S A /. Japan

Ab. tract

By targeting the TP binding conserved domain in three ATP binding ca. sette superfamily proteins ( P-glycoprotein, multidrug resistance pro­

tein, and cystic fibrosis transmemhrane regulator), we isolated the cO:"/A of a neon ATP binding cassette superfamily that "as specificall) enhanced in a cisplatin-resistant human head and neck cancer KB cell line. A human clone homologous to rat canalicular multi..".pecific organic anion trans­

porter (cMOAT) \\as found and de ignated human c 10AT. Fluore cence

in situ h}bridit.ation demom.trated the chromo omal locu of the gene on chromosome 10q24. The human c,\IOAT cO. hybridized a 6.5-kb mRNA that was expres ed -t-to 6-fold higher b� three cisplatin-resi tant cell line-derived from 'ariou human tumor exhibiting deere· ed drug accumulation. Human c�IO T ma) function a a cellular ci platin tran - porter.

Introduction

cquiremcnt or drug resi!>tance by tumor cell· j.., frequently ob­

..,en-ed in ancer patient:-. re eiving chemotherapeutic treatment. Two B 1 protein!-.. the M, 170.000 Pgp and theM, 190,000 MRP. are known to conl··r multidrug re�i�tant phenotype� to cancer ells ( 1-3).

Although the primary structure� of Pgp and tRP �hare only 159'r amino acid homolog (4). both Pgp and MRP are ill\olved in drug resistance to a :-.imilar profile of chemotherap uti agent.., that include anthrac clines, vinl-.a alkaloid�, and epipodophyllotoxins ( 1-3). By contrast, cancer cells thut overcx.prcss Pgp or 1RP d not :-.hO\\

cro:-.s-resistanct: to platinum-containing compounds, alk_ lating agents, and antimctabolites ( 1-3). MRP-transfectant cells show re:i:-.tance to anthracyclincs, inka al"-aloids, cpipodophyllotoxins, and heavy metal anions but are :-.cnsitive to platinum-containing compounds (5. 6).

However. MRP ·an transport the c steinyl leukotriene C4 and other glutathione conjugates (7 9), suggesting that RP may be a gluta­

thione-conjugate transporter.

or the rlatinum-containing compounds, ci�platin is a potent anti­

cancer agent that has been \vide! used to treat \arious malignant tumors, including testicular, head and neck. esophageal. lung, O\'arian,

Rtort:ll't:d 6/4/96: :.cn:plcd 7/31/96.

The cos I' of puhlicaiHlll of thb arlicll' ^\I ere ddnt) ed in pan h) I he pa) ment of page charge' Th" an1cle mu'1 Ihercfore be hen:h) mar�cd mlnTit\1'1111'11/ 111 a -cor fann: "ilh

I X U.S.C'. Seclion 173-l 'olelv 10 TtH.li..:alc thi' facl.

1 Suppntled by a gtanl-tll··aid for cancu re,carch from Ihc lini,lr) of Fducminn.

Science. Sporb and Cullurc. Japan; I he t'uJ..uoJ..n nlicanccr Rc;,earch Fund. Fu�uoJ..a 21,1 Cenlury Medical Fund: and Ihe Yasuda t\1entotial 1\kdical ()rani lot Cancer Research.

The nucleotide ;,cquencc reported in I hi;, papc1 ha;, been dcp""l 'U in Ilw .cnhanJ.. daia ba:-.e ( cce,,iun o. U6.l<J70).

�To \1 hom requcsls for rcprinls ,hould be addtcssed. at Depanmt·nt of Biochcmi>tt').

K ushu Univcr;,iJy School of Medicine, l\1aidashi. l'ukuo�a 812-82. Japan. 1-';n: lll-92- 632-4 19K: 1�-mai 1: kuwuno0�hioch�ntl.metl.l.,yushu-u.:tc.jp

'The abbn:viation' "''-'d an:: AHC. TP binding c:t-.st:llc: Pg.p. P-gl)n>prnlcin: 1\tRP.

muliidrug tc;,i;,lanl proit·in: CI·TR. cy>tic lihro'i' Ir;uhll11.'11lhranc rcgula10r: ci\IOAT.

canalicular mullispccilic organic anion Iran.,pnner: cbplutin. Cl.l-diamminedichlll!oplal­

inum(ll): 1'1.11, nuorc,ccncc in siru hyhridinHion.

and bladder cancers. Howe\·er. clinical application of long duration is often limited because of the development of cisplatin-resistant tumors.

To further improve the chemotherapeutic efficacy of this potent anticancer agent. we must understand the mechani In'> that control

. ensitivity to ci platin ( 10. l l ). Cisplatin accumulation i · decrea ed in

many pecific cisplatin-resi rant cell!> (12-14 ), and the A TP-depend­

ent active outward efflux of ci, platin is enhanced in both ci. platin­

re istant human epidermoid cancer KB and human pro tatic cancer PC-3 cell line. ( 12. 14).

Becau e the overexpression of two ABC- uperfamily genes, MDRI/mdrf ( 15) and MRP (4, 5). is closely associated \-\ith Pgp- and non-Pgp-mediated multidrug re i ranee. we initially i olated other genes belonging to the ABC superfamily to explain the decreasing cellular accumulation of ci. platin by tumor cells. Specifically. we targeted the ATP binding domain con·erved in MDRJ, MRP, and CFTR genes in the present study and compared the difference in mR A from both cisplatin-. ensiti\ e and -resi tant cells. ln thi . tudy . we isolated full-length human cMOAT eDNA that i. highly homol­

ogous to rat cMOAT (16). a homologue of the human MRP gene. We will al:o discu a po ·sible correlation between human cMOAT and cellular cisplatin accumulation.

Materials and Methods

Cell Line . Cisplatin-resi-.tant ell lines \\'ere deri1ed from \'ariou. drug­

sen�iti\'e parental counterpart\: T2-l/DDP I 0 from human bladder cancer T2-l cells (13): P/CDPS from human pro,tatic cancer PC-3 elb (12): and KB/

K P-l from human head and neck cane r KB cell-, il-l, 17). Etopo,ide/

tenipo�ide-re�i,tant KB P-l cells were deri\'ed from KB cells (It). These cell lines 1\ere cultured at 3rC under a humiditi d atmo,phere of 50[ C02 in Eagle·s 1EM ( 1issui eiyaku. Tok�o. Japan) containing 10c-c fetal bo\'ine

-;erum ( era-lab. usse'\. nited Kingdom). Bactopeptone (I mg/ml: DlFCO

Laboratories. Detroit. .\111). glutamine (0.292 mg/ml), J...anamycin (I 00 rng/ml ).

and penicillin ( 100 unit,/rnl).

eDNA Cloning. Total R was isolated fr m KB/KCP-l celb and 1-2 1-lg were then re\'erse-transcribed using molon) muline leukemia \'irus reverse tran$criptase (Life Technologies, Inc .. Gaithersburg. MDJ. The re. ulting cDN ll'as used as a t mplat for PCR using variou · primer ·omb1nations. In both nest d P R and heminested PCR. cDJ A \\a' reacted with the outer primer pairs (C I: Fig. lrl ), and an aliquot of the rea ·tion product wa-, then reamplifi d with the inner primers (C2. C3. and C-l). The primer 'equence' used for C -series PCR are shown in Fig. I B. and the expected 'it: s of PCR products arc shown in Fig. lA. PCR cycle temperature� and times were 94°C for 30 �- 50°C for 30 s, and 72°C for 30 s for a total of 35 C)CI " for all reaction�. The PCR produ ·ts were subcloned in p 10 Blue vector (Amer­ sham. Buckinghamshire. nited Kingdom) and �equenced. The overlapping eDNA clones were obtained b) screening a Agtl l HeLa cell cD A library (Ciontech. Palo Alto, . ). a pCMV PORT human kidnc) cell cD A library (Life Technologies. Inc.). and a Azapll human liver eDNA librar) with the cD1 probe. II O\'erlapping cO clones were -.ubcloned into the pUC Ill

CJSPI ATI1 RES!. TA CF \ND IILIJ\1AN r,\IOA I'

A

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:

K-38 aa-IIPQ

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: ^�i

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(393 bp)

C2

(342 bp) .. ..

C3

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(264 bp) ..

C4

_..

(204 bp)

B

5'-1 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'-1 5'-(GT)(GC)(AT)GC(AC)(GC)TGG(GC)(TC)TCATC-3'

Fig. I. A. schematic repre;entation of primer po;ition� and the nested PCR strategy. Top. the cono,erved ammo �cid '>Cquence of the AlP hind1ng region ('J.c-1. pnmer pan' for PCR and the expected product

;

^{izes. B.} the equence of pnmero-. u;ed for nested PCR. C. gel electrophoreo.,1 of PCR and nc,ted PCR reacuon pn>ducl\. Arro11. :!04 hp 4 primer JXIIr products.

plasmid. Chain elongation and termination were performed with a DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosy terns. Tokyo. Japan). and nucleotide sequencing wa, performed with a D A sequencing ystem (model 373S: Applied Biosy�tems). Data were analyzed using GeneWorks software OntelliGenetics. Mountain View. CA).

Genome DNA Cloning. The EMBL3 human placenta genome D A li­

brary ( 19) v. as �creened by the previously described method for cD A library screening. The clones were u�ed for FISH analysis.

Northern Blots. A human multiple tissue orthem blot wa commercially obtained <Ciontech). and total R A was extracted from exponentially growing cells by the acid guanidinium thiocyanate-phenol-chloroform method (20).

RNA (20 11-g) was subjected to electrophoresis in a I o/c agarose gel containing 2.2 M formaldehyde and then transferred to a nylon membrane (Hybond r\�:

Amersham). The filter<, were hybridized with a 12P-labeled cD A probe in a '>Olution containing 50%- deionized fonnamide. I 0 X Denhardt' s buffer.

5 X SSC f I X SSC = 0. 15 M NaCI and 0.015 \1 �odium citrate (pH 7 .O)j, and 0.1 9t SDS for 24 h at 42°C and then wa�hed twice in 42°C 2 X SSC containing 0.1 o/c SDS. The amount of human cMOAT encoding mRNA wa� quantified with a Fujix BAS 2000 image analyzer (fuji. Tokyo. Japan).

FISH Analysis. Probe labeling and in situ hybridization were performed a�

described previou�Jy (21 ). Briefly, chromosome spreads were obtained from phytohemaggulutinin-�timulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation. Genomic D A fragments were labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) by nick tram.lation. In situ hybridization wa� performed in the pre�ence of the COT-I DNA (Life Technologie�. Inc.) competitor.

Hybridized probes were detected with FITC-conjugated avidin (Boehringer

Mannheim). Chromo�ome\ were counter,taJncd � 1th 0.2 mg/ml prop1dJUm iodide for R-banding. Fluorescence signab were anal.rted using a Zt:I\S ioskop epifluorescence micro\cope equipped v.tth a coolt:d harge oupkd Device camera (model PXL 1400: Photometric�. Tucson, AZJ. Image acqui­

sition was performed on a Macintosh Quadra X40A V computet using the IPLab1'1 (Signal Analytics, Vienna. VA) software. The images were then pseudocolored and merged using Adobe Photoshop1 '1 2 SJ (Adobe Syo,temo, Japan, Tokyo. Japan). FITC and propidium iodide images were colored v. h11c and gray. respecti\ely. The merged FITC and prop1dium todide 1mages v..ere directly printed u;ing a Fuji Pictrography 3000 output device.

Results and Discussion

Human MDR I, MRP, and CFTR have been identified as member.., of the ABC tran�porter superfamily (4, 22). The �imilarity of these tbree genes re�ides predominantly in two ATP binding domain�.

Based on their homology, we designed highly degenerate -�-ocrie1-.

primers (Fig. lA). We also designed PCR primer mixtures ranginn in complexity from 64 to 128. We used the �cries of primer sequences (Fig. I B) because we could not produce PCR product" of expected size when another series of primer pairs were used. The rc�-oult�

obtained for C-series primer pairs show that nested PCR amplification of a unique band from primary amplification products contains several band!> (Fig. I C). The PCR products were suhcloncd, and 43 transfor­

mants were sub�equently sequenced. Among these clones, only I 0 had 5' and 3' primer sequences at opposite end�. The sequence" or 4125

A

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<I�PI.ATIN Rr.SJSTAN J;. A D HUMAN cMOAT

ATP 2-28 PCR fragment

28-9---

AKl-8--- 1-1---

CS-1--- H6 ---

4-1--- 15-9---

L3---

TVPA�YTN[lllG�ffi TVPP YTN LC l FLAP LVS GI L PSSIASFLSS ITY�YDSII PSVTASFLSS ITF YDR1V TIHOPNPCPE SSA LSRIT

LVIQYSRQWC LAVQHSRQWC TFLIQLERRK LKGYKRP�l LKGYKHP

^l

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GFmlA �QLLHVYKS RTKRSSml Yl�VFVGF 00AAIE� LVL�SGQA GF W LA QL YSVYRS RTKRSSI F YL VFVVF AAID LAL TGQA FY W CF YFL YLSRH DRGYIQ

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mlSilnT FQFQTL�Tl VRKN F Sl LSVL V FQFQTL Al GVQS IML T VALV l AILRSK TA EDVWEVD�M KTKTLVSBDHMKRELQKA EDVYIDID F KTRSVTS AMTKDLQKA RGYRQPL

⁵

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�GDNSNLAY SCL�ISYG DSKSNMAY SYL- VSYG EDAQVDLF RDI VYFS RRALQRRQEOSQQNSGARL RQAFQRRLQ QR-KPEATL VKNWKKECA RKQPVKVVY

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EALIVKSPQ W-NPS TF PYF F F IH G Q

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1545 1541 1531

l'ig. 2 :I. 'chc•matic •cprc,c·ntation or m crlapping eDN clone•>. J'he'c cD, '\ clone> cncompas> I he coding region for human c 10AT. B. comparison of amino acid sequences of hiiiiHIII cMOA 1'. human MRI' (Ill 11\IMRP'\). and raJ cMOi\T. Hoi!'.'· amino acid idcn1i1y. Amino acid differences arc 'hO\\n in their corrc�ponding positions. Dashes. gaps that are 111troduccd In ma�imi1c· 1dcnlll)

41�6

Fig. 3. Mapping of the gene region encoding human cMOAT by FISH. Four genomic Iones were i olated using a eDNA probe and ubjected to FISH analy is. Arrowheads. fluore cence ignaL on the R -banded metaphase chromosome . Ba. ed on ob ervalions of more than I 0 metaphase chro­

mo ornes. a band is identified at 10q24.

I 'PLATIJ RE I Tf\ 1 E AND !lUMAN cMOAT

five clone were identical to tho e of MRP. and four clone had ter-part� (Fig. 48). In contrast. human eM

OAT

_{gene expre�'>}ion �a� not unrecognized sequences with no homology to sequences in the Gen- enhanced in KBNP-1- cells that ov re pre � the MRP gene (Fig. -1-. C bank database. One clone, pATP2-28, hawed homology with MRP and

D).

The KBNP4 cell line. derived from KB celb re�i tam to the (4) and, more significantly, with rat cMOAT (16). Therefore, we epipodophyllotoxin anticancer agent etoposide, had a multidrug re- tentatively de ignated thi product human cMOA T. Screening of the si tance phenotype and exhibited decrea�ed drug accumulation ( l ).

human cervical cancer HeLa cell cD A library, the human kidney cell The size of the human eM OAT mR A was estimated to be about 6.5 eDNA ubrary, and the human liver eDNA library with a 207-bp kb (Fig. 48), imilar to that of MRP mR (Ref. 4: �ee Fig. 4D) but ATP2-28 and walking probe allowed the isolation of overlapping larger than the 4.5-kb human MDR I mR ' .

eDNA clones (Fig. 2A). Sequencing each of these clones revealed an We isolated human cMOAT cO A by targeting the ATP binding open reading frame coding for 1545 amino acid that hawed 46o/c domain conserved in three human AB �uperfamily gene'> [MD

R

_I

similarity to that of human MRP (Fig. 2B). The EMBL3 human (P-gp),

MRP,

_and

CFTR]

and found human cMOAT wa-, a homologue placenta genomic DNA library was also screened, and several po itive of human MRP (4) and the recently de. cribed rat cMOAT gene (Ref.

clones were identified and then used for FISH analysis, revealing 16: see Fig. 2). Mayer et a/. (25) have reported that the ab'>encc of positive signals at l0q24 (Fig. 3). Because human

MDRJ, CFTR,

_and MRP or its isoform from the canalicular membrane i� the ba'>i" for the

MRP

genes are localized on 7q21.1, 7q31, and 16p l3.12-13, respec- hereditary defect of the hepatobiliary excretion of anionic conjugate� tively (4, 23, 24), the human

cMOAT

gene seems to be different from in the TR rat model of human Dubin-John!>on �yndrome. P<.JUlu�ma

the e other gene . eta/. ( 16) have reported selective expression of rat cMO T by liver

Multiple-tissue Northern blot analyses with the C5-l eDNA probe hepatocytes as well as reduced mRNA levels in the TR rat. Exprt;�- revealed that human cMOAT mRNA was expressed in liver but sion of human cMOAT was observed in liver but wa� not detectable undetectable in other tis ue (Fig. 4

A

). The tis ue profile of human in other tissues, sugge�ting that human cMOAT may mediate the cMOAT expression was identical to that of rat cMOAT expression. hepatobiliary excretion of numerou'> organic anion'> and that human TR- rat, an animal model of the Dubin-John on syndrome, are cMOAT mutation may result in the Dubin-.John�on �yndromc. Wear defective in cMOAT, which mediates the hepatobiliary excretion of now exploring this possibility in human patient�.

numerous organic anions including glutathione S-conjugates. We have Expression of human cMOAT was enhanced in cisplatin-resi:-,t- previously reported that cellular cisplatin accumulation was markedly ant human cancer cell lines with decreased cellular ci..,platin accu- decreased in the ci platin-resistant human head and neck cancer KB mulation but wa� not enhanced in lines with normal level� of drug cell line KB/KCP4 (14), the cisplatin-resistant human prostatic cancer accumulation. We have previously reported that two cisplatin- PC-3 cell line P/CDP5 (12), and the cisplatin-resistant human bladder resistant lines, KB/KCP4 and P/ DPS, that do not ovcrexpress Pgp cancer T24 cell line T24/DDPIO (13). The transport of leukotriene C4 or MRP show enhanced active ATP-dependent efrtux of isplatin in membrane vesicles prepared from cisplatin-resistant KB/KCP4 ( J 2, 14 ). We also demonstrated hy a cell-cell hybridization test that cells wa facilitated by an ATP-dependent pump that seemed similar both the drug resistance and the accumulation defect found in to the GS-X pump (17). Therefore, we examined whether expression KB/KCP4 cells are dominant traits ( 14), �uggesling !he existence of the human

cMOAT

gene was enhanced in these drug-resistant cell of an active efflux system for cisplatin in KB/K P4 and P/ DPS Jines exhibiting decreased cisplatin accumulation. Human eM OAT cells. Ishikawa et a/. (26) have suggested that an ATP-dcp ·nd nt mRNA was overexpressed 4.0- to 6.0-fold in all three cisplatin- pump muy function to transport glututhione-conjugates (GS-X re i tant ce!J lines as compared to their parental drug-sensitive coun- pump). Ishikawa and Ali-Osman (27) have additionally rep )rt�d

4127

)

CISPLATfN RESISTA 'CE AND IIUMA cMOAT

I 1g. 4. orthem blot analy"'· A, human multiple u sue blot<; were pmhcd \'lth human cMOAT C5-l eDNA fragment (sec Fig. 2A). B. variou., parental and ci<,plmm-r.:si\tant cell lines were anal)•ted using the >arne prohc Northern hloh ol KB and KBIVP4 cell lines '-'Cre probed '-'lth human cMO T CCI and MRP !D)

that the pump may effuse glutathione-conjugated ci platin from L 1210 murine leukemia cells. Ci. plat in molecule. were round to interact very quickly with two glutathione molecules in growing mammalian celb (2 ). lt remains unci ar whether human cMOAT is closely coupled with the activity of the glutathione­

conjugated cisplatin pump.

On th other hand, MRP functions as an ATP-dep ndent export pump not onl for glutathi ne conjugate. but al o for glucu­ ronidated and sulfated endogcnou� as \ ell as exogenous com­

pounds, including various anti ancer agents (29). However. II three cisplatin-resistant lines derived from KB. PC-3. and T24 cells d not overexpress MRP (12-14), and ancer cell lines that over­ express MRP. including KB/VP4 cells. are n t cross-re 'istant to cisplatin ( , 6, 18). It is thus unlikely that MRP is involved in intrinsic or a quirecl cisplatin r sistan e due to d crea ed drug accumulation. Further studies u ing human cMO T eDNA trans­ f ction will confirm that human cMOAT i. directly involved in cisplatin resistance and can serve as a rnultispecific organic ani n transporter. Transf'ection of the complete human cMOAT cO A is in progres · in our laboratory.

Acknowledgments

B

_.hcMOAT.,..

28S-

D

MRP.,.

ZBS-

18S­

GAPDH.,..

We thank Yukiko Mine of our laboratory for preparing thi manuscript. We thank Mayumi Ono (Kyu ·hu Uni\'ersity) and Hiroshi Taguchi (Mie Uni\'er­

·ity) for fruitful di cussion.

References

I. Gotlesman. 1. M. and Pa:;tan, I. Biochemistry of multidrug re istance mediated b)

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