ICANCER RESEARCH54. 5902-5910, November 15, 19941

ABSTRACT

We have previously identified and characterized a novel member of theATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitivecells (Cole et a!., Science (Washington DC), 258: 1650—1654,1992; Grantet aL, Cancer Res. 54: 357—361,1994). In the present study, we havetransfected two different eukaryotic expressionvectors containing MRPcomplementary DNA into HeLa cells to study the pharmacological phe

notype produced exclusively by overexpression of human MRP. The drugresistance patterns of the two MRP-transfected cell populations weresimilar. They were characterized by a moderate (5- to 15-fold) level ofresistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (3-fold) level of resistance to taxol, vinblastine, andcolchicine. The transfectants were not resistant to 9-alkyl anthracydines,mitoxantrone, or cisplatin. The MRP-transfected cells were also resistantto someheavymetal anions includingarsenite, arsenate, and trivalent andpentavalent antimonials but were not resistant to cadmium chloride.Accumulation of radiolabeled vincristine was reduced by 45% in theMRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differgreatly in the sensitive and resistant cells. The cytotoxic effects of yincristineand doxorubicincouldbe enhancedin a dose-dependentfashionby coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree ofchemosensitization by verapamil and cyclosponn A was similar in MRPtransfected cells and in cells transfected with the vector alone, suggestingthat sensitization involved mechanisms independent of MRP expression.Verapamil and cyclosporin A caused a modest increase In vincrlstlneaccumulation in the resistant cells but did not restore levels to those of thesensitive cells. Taken together, these data indicate that drug-resistant celllines generated by transfection with MRP complementaryDNA displaysome but not all of the characteristics of MRP-overexpressing cell linesproduced by drug selection in vitro. They further demonstrate that themultidrug resistance phenotype conferred by MRP is similar but not

identical to that conferred by P-glycoprotein and includes resistance toarsenical and antimonial oxyanlons.

INTRODUCTION

Resistance to multiple drugs is a major impediment to the successful treatment of many human cancers. Drug resistance observed in aclinical setting is very broad and encompasses all classes of chemotherapeutic agents including alkylating agents, antimetabolites, hormones, platinum-containing drugs, and natural products. In sometypes of tumors, this resistance is inherent, while in others, such assmall cell lung cancer, it is usually acquired. Experimental multidrug

Received 6/21/94; accepted 9/19/94.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 grants from the Medical Research Council of Canada (to S. P. C. C. and

R. G. D.) and the National Cancer Institute of Canada with funds from the CanadianCancer Society (to S. P. C. C.). S. P. C. C. is a Career Scientist of the Ontario CancerFoundation. R. G. D. is the Stauffer Research Professor of Queen's University.

2 To whom requests for reprints should be addressed, at Cancer Research Laboratories,

Queen's University,ThirdFloor, BotterellHall, Kingston,Ontario,CanadaK7L 3N6.

resistance involves the simultaneous development of cross-resistanceto structurally diverse compounds with multiple targets within thecell, following selection for resistance to a single drug. The spectrumof drugs included in experimental multidrug resistance is frequentlynarrower than that observed clinically but includes many therapeutically important natural products and their semisynthetic congeners.Such multidrug resistance is known to be conferred by at least twoproteins, the M1 170,000 P-glycoprotein (encoded by the MDR1 gene;Refs. 1 and 2) and the more recently identified 190 kDa MRP@(3—6).Although both P-glycoprotein and MRP belong to the AlP-bindingcassette superfamily of transport proteins, they are structurally verydifferent molecules and share less than 15% amino acid identity (3).At present, they are the only known human members of the Al?binding cassette transporter superfamily that can confer resistance tonatural product anticancer drugs.

MRP is a 1531-amino acid Mr 190,000 N-glycosylated integralmembrane phosphoprotein that is encoded by a 6.5-kilobase mRNA(3, 7, 8). MRP was cloned originally from a multidrug-resistant smallcell lung cancer cell line (3, 9) and has subsequently been found to beoverexpressed in a number of drug-selected cell lines derived from avariety of different tumor types (3, 6, 8, 10—12).The cross-resistancepatterns and pharmacokinetic properties of these in vitro drug-selectedcell lines which overexpress MRP are quite variable. However, inmost cases, there is evidence of coexpression of multiple resistancemechanisms (e.g., alterations in topoisomerase II; Refs. 12—16)so thatthe contribution of MRP to the observed resistance phenotype cannotbe unequivocally identified.

We have recently transfected a full-length MRP cDNA into drugsensitive human tumor cells and demonstrated that overexpression ofMRP is sufficient to confer multidrug resistance (5). In the presentstudy, we have examined the pharmacological characteristics of twoHeLa cell populations transfected with different expression vectors,each containing the complete MRP coding sequence. These cell linesprovide important models for distinguishing those properties whichare directly attributable to MRP from other resistance mechanismsthat may be present in multidrug-resistant cell lines derived in vitro byrepeated exposure to cytotoxic and mutagenic drugs.

MATERIALS AND METHODS

MRP Expression Vectors and Transfected Cell Lines. The assembly andtransfection of the MRP expression vector pRc/CMV-MRP1, which containsthe complete coding region of MRP mRNA and 86 nucleotides of 5' untranslated sequence and 32 nucleotides of 3' untranslated sequence, has beendescribed previously (5). The HeLa cell line transfected with pRc/CMV-MRP1has been designated T5, while the corresponding control cell line transfectedwith the pRc/CMV vector alone has been designated Cl (5). The Cl and T5

cells were maintained in 400 @iWmlG-418 (Sigma Chemical Co., St. Louis,MO). The same MRP full-length cDNA was also cloned into a modified

3 The abbreviations used are: MRP, multidrug resistance protein; cDNA, complemen

tar), DNA; DOX, doxorubicin; SSC, standard sodium citrate; SDS, sodium dodecylsulfate; IC5@,,50% inhibitory concentration; VCR, vincristine.

5902

Pharmacological Characterization of Multidrug Resistant MRP-transfectedHuman Tumor Cells'

Susan P. C. Cole,2 Kathryn E. Sparks, Karen Fraser, Douglas W. Loe, Caroline E. Grant, Gerald M Wilson,and Roger G. Deeley

Cancer Research Laboratories (S. P. C. C., K. E. S.. K. F., D. W. L, C. E. G., G. M. W.. R. G. D.J and Department ofBiochemistry [G. M. W., R. G. DI, Queen's University,Kingston, Ontario, Canada K7L 3N6

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MRP-MEDIATEDRESISTANCEIN TRANSFECFEDCELLS

pR.EP7expression vector (Invitrogen, San Diego, CA) designated pCEBV7.4This vector replicates episomally from the Epstein-Barr viral origin of replication (oriP element) and requires the EBNA-1gene product as a trans-actingfactor(17, 18). In addition, pREP7 contains the hp/i gene from Escherichia colithat confessresistanceto hygromycinB and allowsstablemaintenanceof thevector in transfected cells. pCEBV7 was derived by replacing the Roussarcoma viral promoter of pREP7 with a cytomegalovirus promoter. ThepCEBV7-MRP1 construct generates a mRNA transcript of 5.0 to 5.1 kiobases,which includes approximately 170 nucleotides of 5' untranslated sequence and230 nucleotides of 3' untranslated sequence.

HeLa cells were transfected with the pCEBV7 vector or the vector containing the MRP coding sequence (pCEBV7-MRP1) using lipofectin (GIBCO,Burlington, Ontario, Canada) according to the manufacturer's directions.Briefly, approximately 20,000 cells in 2 ml of serum-free minimal essentialmedium were overlaid with 200 @lof serum-free minimal essential mediumcontaining 2 p.g supercoiled DNA and 10 @tlof lipofectin. After 4 h, themedium was replaced with 2 ml RPMI 1640 supplemented with 5% defined/supplemented bovine calf serum (Hyclone Laboratories, Logan, UT) and 4 ms@L-glUtanhinC,and the cells were incubated for a further 48 h. At this time,hygromycin B (Sigma) was added to the cells at 50 @.tWml.After 4 weeks, sixindependently transfected populations of cells were tested for resistance toDOX. A population exhibiting increased resistance, designated T14, and apopulation of cells transfected with the pCEBV7 vector alone, designated C6,were expanded for further analysis. At this time, the hygromycin B concentration in the growth medium was increased to 100 @g/mlwithout anynoticeable effect on the growth rate of the transfected populations. There wereno differences in the cell volumes of the Cl, C6, T5, and T14 cells, asdetermined by measuring cell diameters using a microscope eyepiece micrometer andby analysisof forwardscatteron a CoulterEliteflowcytometer.Allcell lines were free of Mycoplasmacontamination.

RNA Analysis. Polyadenylated RNA was prepared from C6 and T14 cellsusing a micro-FastTrackkit (Invitrogen,San Diego, CA). Approximately 1 pgRNA from each cell line was run on a 1% agarose gel containing 0.66 Mformaldehyde and blotted onto a Zeta-Probe membrane (Bio-Rad, Mississauga,Ontario,Canada)by pressureblottingwith lox SSC(1X = 150nm@NaO-15 mMsodium citrate, pH 7.0@l0XSSPE (lx = somM NaC1,10 mMNaH2PO4,and1 mMEDTA,pH7.4).Theblotwas hybridizedwitha humanMRP1.8-kbcDNA(MRP 10.2) correspondingto nucleotides2185 to 4079 (3, 4) and a glyceraldehydc-3-pbosphate dehydrogenase cDNA (ATCC) which had been labeled with[a-@P]dAlP [specificactivity,3000 CVmmol(NEN-Dupont@Markham,Ontario,Canada; and BRL Random Primers DNA labeling system; GIBCO-BRL LifeTechnOlOgies, Burlington, Ontario, Canada)]. The blot was washed for 15 miss atroom temperature in 1X SSC-0.l% SDS, followed by two times for 30 mm at52°Cin 0.1X SSC-O.l%SDS, oncefor60 miiiat52°Cin o.osx SSC-0.1%SDS,and then exposed to Kodak x-O@at AR film.

Protein Immunoblot Analysis. The relative amounts of the Mr 190,000MRP protein in Cl, C6, T5 and T14 cells were assessed by immunoblotanalysis ofmembrane-enriched fractions prepared as described previously (5).Sampleswere subjectedto electrophoresison 7% polyacrylamidegels andelectroblotted in 50 mM 3-(cyclohexylamino)-l-propanesulfonate (Sigma; pH11) onto Immobion-P membrane(Millipore,Mississauga,Ontario,Canada).Blots were incubated with an affinity purified MRP-specific rabbit polyclonalantibody, MRP-L (3 pg/mI), raised against a unique peptide corresponding toamino acids 932 to 943 in the predicted linker region of MRP. Binding of therabbit antibody was visualized with affinity purified horseradish peroxidaseconjugated goat anti-rabbit IgG (F(ab')@fragment; Jackson Immunoresearch,West Grove, PA) and enhanced chemiluminescence detection (Amersham,Oakville, Ontario, Canada).

Chemosensitivity Testing. The cross-resistance patterns of the transfectedHeLa cell lines were determinedusing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microtiter plate assay as described previously(19, 20). The resistancefactoris expressedas the ratioof the IC@of the cellline transfected with the MRP expression vector relative to that of the cell linetransfected with vector alone. The effects of VCR and DO@were also testedin the presence of verapamil and cyclosporin A. Dose-response cwves werefirstobtainedforverapamilandcyclosporinA alone,andthesedrugswerethen

4 G. M. Wilson and R. G. Deeley. A eukaryotic episomal expression vector system formonitoringsequencespecificeffectson mRNAstability,submittedforpublication.

examined at concentrations that produced less than 15% cytotoxicity in combination with VCR and DOx. Each drug or drug combination was tested in atleast two independent experiments, and within each experiment, determinations were carried out in quadruplicate. DOX, VCR, colchicine, VP-16, yinblastine, and verapamil were from Sigma; mitoxantrone was from CyanamidCanada, Inc. (Montréal,Québec,Canada); taxol was from Bristol-MyersSquibb (Candiac, Québec,Québec,Canada); cyclosporin A was from Sandoz(Dorval, Québec,Quebec, Canada); and epirubicin and daunorubicin werefrom the pharmacy at the Kingston Regional Cancer Centre. Aclacinomycin Awas a gift from Dr. Peter Twentyman (Cambridge, United Kingdom) and3'-deamino-3'-morpholino-13-deoxo-lO-hydroxycarminomycin (MX2) was a

gift from Dr. N. Saijo (National Cancer Center, Tokyo, Japan). Sodiumarsenite, sodium arsenate, potassium antimony tartrate, potassium antimonate,and cadmium chloride were from J. T. Baker Chemical Co. (Phillipsburg, NJ).

Drug Accumulation and Effiux. Accumulation and efflux of radiolabeledVCR were measuredas describedpreviously(14). To measureaccumulation,cells (5 X 106/ml)were incubated with 1 pM [3HJVCR(specific activity, 4.5Ci/mmol; Amersham; 1 pCi/mI) at 37°Cin RPM! 1640 supplemented with 5%bovineserumand 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid(accumulation medium). Aliquots (0.5 ml) of cells were removed at theappropriate times and added to ice-cold phosphate-buffered saline to stopaccumulation. After centrifugation and two washes at 4°C,cell pellets weresolubilized with 1% SDS; cell-associated radioactivity was determined, andresults were expressed as pmol VCR/l0'@ cells. Zero time points contained less

than 5% of the total radioactivity accumulated at 60 mm. To measure drugefflux, cells were incubated with 1 p@ [3HJVCR for 60 min at 37°Casdescribed above. Cells were centrifuged at 4°C,washed twice in ice-coldserum-free medium, and resuspended in the same volume of drug-free accumulation medium at 37°C.Aliquots (0.5 ml) of cells were removed at varioustimes over the next 120 mm, and the cell-associated radioactivity was determined. Results are expressed as the percentage of radioactivity remainingrelative to that present immediately after washing the cells. Within eachexperiment, determinations were performed in triplicate, and each experimentwas repeated at least two additional times.

To examine the effect of Al? depletionon drugaccumulation,cells werepreincubated for 20 mm in glucose-free RPM! 1640 (GIBCO-BRL LifeTechnologies) containing 10 mMdeoxyglucose (Sigma) and 100 nMrotenone(Sigma).Thecellswerethenincubatedinglucose-freeaccumulationmediumcontaining [3HJVCRin the continued presence of deoxyglucose and rotenonefor an additional 60 mm, and cell-associated radioactivity was measured asabove. These conditions reduced intracellular Al? levels more than 95%, asmeasured by a luciferase-based bioluminescent somatic cell assay kit (Sigma).

To examinethe effect of verapamilandcyclosporinA on VCR accumulation, cells were preincubated for 20 mm in the presence of these agents beforemeasuringthe accumulationof [3HJVCRover 60 mm.

RESULTS

Levels of MRP mRNA and Mr 190,000 ProteIn in TransfectedHeLa Cells. In a previous report, we described the construction andtransfection of a full-length MRP cDNA into HeLa cells using theintegrating expression vector pRc/CMV (5). This vector contains thebacterial aminoglycoside 3'-phosphotransferase gene, which allowsselection of transfected cells in geneticin (G-418). This procedureavoids the use of carcinostatic drugs and diminishes the possibility ofselecting for mutations that might affect the resistance of the transfected cells. The abundance of vector-encoded MRP mRNA in thetransfected T5 cells is similar to that of endogenous MRP mRNA inthe DOX-selected H69AR cell line from which MRP was cloned.However, the transfectants express 5- to 8-fold lower levels of the M1190,000 MRP, which is consistent with the relative levels of resistance observed in T5 cells compared to H69AR cells (5).

We have now transfected HeLa cells with the same full-lengthMRP cDNA using the non-integrating pCEBV7 expression vector(j@CEBV7-MRP1). Since the pCEBV7 vector contains the bacterialhph gene, transfectants were selected with hygromycin alone, and

carcinostatic drugs were not used. The levels of MRP mRNA and5903

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MRP-MEDIATEDRESISTANCE IN TRANSFECFED CELLS

protein in pCEBV7-MRP1 transfected cells and pCEBV7 vectortransfected cells were examined. The MRP mRNA encoded by thepCEBV7-MRP1 expression vector has a predicted length approximately 1 kilobase shorter than the endogenous MRP mRNA becauseit contains only a very small portion of the 3' and 5' untranslatedsequences present in the endogenous 6.5-kilobase MRP mRNA. AnmRNA of 5.1 kilobases was readily detected in the pCEBV7-MRP1transfected cells (population T14) but not in control cells (populationC6) transfected with the pCEBV7 vector alone (Fig. IA). The endogenous 6.5-kilobase MRP mRNA was not detectable in either C6 orT14 cells under the conditions used to generate the northern blotshown. Using a MRP-specific polyclonal antibody, the M1 190,000MRP protein was detected in the T14 and T5 cells but not in thecontrol transfected C6 cells (Fig. 1B). Approximately 2-fold moreMRP protein was expressed in T14 cells than in T5 cells.

Cross-Resistance Patterns ofMRP-transfected HeLa Cells. Thecross-resistance patterns of the MRP-transfected T5 and T14 cells tochemotherapeutic drugs were determined, and the results are summarized in Table 1. The T5 and T14 cells displayed moderate (5- to15-fold) levels of resistance to anthracyclines (DOX, daunorubicin,and epirubicin), the Vinca alkaloid, VCR, and etoposide. The T5 andT14 cells displayed no, or only low level resistance to, mitoxantrone,vinblastine, colchicine, and taxol. T5 cells were also not cross-resistant to the 9-alkyl anthracyclines (aclacinomycin A or MX2) and notcross-resistant, as previously reported (5), to cisplatin. The resistanceof T14 cells to these latter drugs was not tested.

Moderate (3- to 5-fold) levels of resistance to the heavy metalanions, arsenite, arsenate, and antimony (Sb III) tartrate, and low(<2-fold) levels of resistance to antimonate (Sb V) were observed inboth T5 and T14 cells. These cells were not resistant to cadmiumchloride. Typical dose-response curves for C6 and T14 cells areshown in Fig. 2, and a statistical analysis of repeat experiments issummarized in Table 2. Comparable levels of resistance were ob

served in T5 cells (data not shown). This pattern of heavy metalresistance is similar to that conferred by ltpgpA in the trypanosomatidprotozoan Leishmania (21, 22).

Drug Accumulation and Efflux. The time courses of VCR accumutation and efflux in sensitive Cl and resistant T5 cells are shown

GAPDH

in Fig. 3. Steady-state concentrations of VCR were achieved by 60min in both cell lines, and the accumulation of VCR in T5 cells wasapproximately 45% less than that found in Cl cells (Fig. 3, upperpanel). Similar differences in VCR accumulation were observed between C6 and T14 cells (data not shown).

Drug efflux was measured after loading the Cl and T5 cells withradiolabeled VCR at 1 @.LMfor 60 mm to achieve steady-state druglevels, followed by incubation of the cells in drug-free medium anddetermination of cell-associated drug levels over time. Only smalldifferences in rates of efflux were observed between the sensitive andresistant cells (Fig. 3, lower panel). After 60 mm incubation indrug-free medium, there was no significant difference in the percentage of drug remaining in the Cl and T5 cells (64 ±6% versus51 ±9%; P > 0.1). However, after 120 mm, 55 ±4% of the VCRremained associated with the Cl cells while, 35 ±9% remained withthe T5 cells (P < 0.05).

VCR Accumulation after Al? Depletion. To determinewhetherthe reduced accumulation of VCR in MRP-transfected cells wasenergy dependent, VCR accumulation was measured in Cl and T5cells after depleting the cells of Al? with rotenone and deoxyglucose.As shown in Fig. 4, depletion of Al? caused a small increase in VCRaccumulation in control Cl cells (from 43 to 49 pmol VCR/106 cells)and a large increase in T5 cells (from 26 to 41 pmol VCR/106 cells).Thus, depletion of Al? restored the levels of VCR accumulation inT5 cells to those found in control Cl cells.

Effect of Verapamil and Cyclosporin A on DOX and VCRResistance. In preliminary experiments, both the sensitive Cl and C6

cells and resistant T5 and T14 cells showed less than 10% cytotoxicityat concentrations of 30 @aMverapamil and 7 pg/mt cyclosporin A. Insubsequent drug combination experiments, verapamil produced a concentration-dependent increase in the sensitivity of the resistant MRPtransfected (T14 and T5) and sensitive vector control-transfected (C6and Cl) HeLa cells to both VCR and DOX. The results from a typicalexperiment with Cl and T5 cells are shown in Fig. 5 and similarresults were found with C6 and T14 cells (data not shown). For bothVCRandDOX,the degreeof sensitizationby verapamilwas cornparable in sensitive and resistant cells so that the relative resistancefactors were not significantly reduced.

A. B.C6 T14

20 40 20 40T5

20 40 lag

, sp@ MRP

kb

- 9.5

- 7.5

- 4.4

- 2.4

Fig. 1. Levels of MRP mRNA and protein in transfected cell lines. A, levels of MRP mRNA were determined by Northern blot analysis. Polyadenylated RNA from C6 and T14cells was separated by electrophoresis on a formaldehyde-agarose gel and transferred onto a nylon membrane. The blot was probed with a 1.8-kilobase MRP cDNA fragment, whichhybridizeswith a mRNA of approximately5.1 kilobasesas expectedfor the MRPtranscriptencodedby the pCEBV7-MRP1expressionvector.Hybridizationwith a glyceraldehyde3-phosphate dehydrogenase (GAPDH) cDNA indicates the relative loading of the gel. The autoradiograph shown is a 2-h exposure at —-70°Cfor MRP and a 1-h exposure forglyceraldehyde-3-phosphate dehydrogenase. B, the levels of protein encoded by the MRP expression vectors were determined by immunoblot analysis. Proteins from crude membranefractions were separated by polyacrylamide gel electrophoresis and electroblotted onto a nylon membrane. The blot was probed with an affinity purified MRP-specilic polyclonalantibody (3 @g/ml),and a protein of M, 190,000 was detected in MRP-transfccted 15 and T14 cells. The amount of protein loaded per well is indicated at the top of the figure.

5904

C6 T14

MRP

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Table1ResistanceoIMRP-transfected HeLa cellsto chemotherapeuncdrugsDrugI@Resistance

factor1'IC@Resistancefactor1'ClT5C6T14Doxorubicin0.28

±0.10(n = 15)1.58

±0.90@?(n = 15)5.60.26

±0.07(n = 10)2.12

±0.62@?(n =10)8.2Daunorubicin0.09

±0.05(n=3)0.46

±0.32c(n=3)5.10.23

±0.13(n=3)1.70

±0.27'@(n=3)7.4Epirubicin0.103

(0.086,0.120)0.93 (1.17,0.69)9.00.14 (0.21,0.07)1.15 (0.59,1.70)8.2Aclacinomycin

A0.057(0.055,0.059)0.069 (0.077,0.068)1.2ND―@i@dMX23.53±2.44

(n=3)5.83±4.25(n=3)1.7@1@d@1@dMitoxantrone0.51

±0.25(n = 3)0.60

±0.28(n =3)1.20.97 (0.54, 1.4)0.81 (0.82,0.80)0.8Vincristine6.14

±1.21e(n=9)51.33

±20.74@(n=9)8.44.74

±2.66e(n=5)69.60

±42.81@e(n=5)14.7Vinblastine0.76e

(0.90,0.62)33e (5.2,1.4)4@33ff5.Oe1.7VP-164.6

(4, 5.2)54 (75, 32)11.67.03±1.00

(n =3)62.67±22.14c

(n =3)8.9Colchicine20'

(5,34)22e (10,34)1.130±4e

(n=3)93@ 16@e

(n=3)3.1Taxol3.6―

(3.9,3.2)7.1! (8.0,6.1)2.013.5 (5.9, 21.0)18.5 (13,24)1.4Cisplatin1.70

±0.75(n=3)1.60

±0.30(n=3)0.9@d@d

MRP-MEDIATEDRESISTANCEIN TRAN5FECFEDCELLS

a@ IC@ of each drug was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and is expressed as ps@iconcentrations,

unlessotherwiseindicated.Thevaluesshownaremeans±SDfromindependentexperimentswiththenumberof experimentsdoneshowninparentheses.Where<3experimentswerecarriedout,theresultsof individualexperimentsareshownin parentheses.Where 3 independentexperimentswerecarriedout,statisticalsignificancewasdeterminedusingan unpaired: test.

b p@@ factors were calculated as the ratio of the IC@ of HeLa cells transfected with the pRc/CMV-MRP1 (T5) and pCEBV7-MRP1 (T14) expression

vectors to the IC@ of the HeLa cells transfected with the corresponding pRc/CMV (Cl) or pCEBV7 (C6) vectors alone.C Values Obtained with the resistant cell lines, T5 and 114, are significantly different from those obtained with their corresponding sensitive cell lines, Cl and

c@s@@P< 0.05).ND not done.

S as.

‘nWml.

Cyclosporin A also sensitized the transfected HeLa cells to thecytotoxic effects of VCR, and once again, the degree of sensitizationwas comparable in control Cl and resistant T5 cells (Fig. 6, left panel)as it was in control C6 and resistant T14 cells (data not shown).However, cyclosporin A was less effective in sensitizing the HeLacells to DOX. In Cl and T5 cells, cyclosporin A had no effect onDOX resistance (Fig. 6, right panel) and had only a small butsignificant effect in C6 and T14 cells (data not shown).

Effect olVerapamil and Cyclosporin A on VCR Accumulation.To determine whether the sensitization of Cl and T5 cells to VCR byverapamil and cyclosporin A was accompanied by an increase in drugaccumulation, [3H]VCR accumulation was measured after preincubating cells in medium containing the chemosensitizing agents (Fig.7).VerapamilhadnoeffectonVCRaccumulationin Cl cellsat3 @LM.At 30 g.LM,it caused only a small increase to 111 ±4% of controllevels. In comparison, these concentrations of verapamil reduced theIC@IJVCRof Cl cells approximately 3- and 25-fold, respectively (Fig.5, leftpanel). In T5 cells, verapamil at 3 and 30 pM increased levelsof VCR accumulation from 56 ±6% to 72 ±6% and 86 ±10% ofcontrol (Cl) levels, respectively. In comparison, these concentrationsof verapamil reduced the IC@IJVCRof T5 cells approximately 6- and40-fold, respectively (Fig. 5, left panel).

Cyclosporin A at 7 p@g/rnlhad no effect on VCR accumulation inCl cells (Fig. 7), although this concentration of chemosensitizerreduced the IC50VCR in these cells from 6.67 ± 0.67 n@i to1.23 ±038 n@i(Fig. 6, left panel). Cyclosporin A caused a smallincrease in VCR accumulation in T5 cells (from 56 ±6% to 70±3%of control Cl levels; Fig. 7) while reducing the IC50VCR of T5 cells

from 31.33 ±6.66 nMto 7.50 ±3.50 flM,which is comparable to theIC50 of Cl cells in the absence of the reversing agent (Fig. 6, leftpanel). These experiments suggest that increases in drug accumulation cannot account for the chemosensitization of these transfectedHeLa cells by verapamil and cyclosporin A.

DISCUSSION

Overexpression of the Mr 170,000 kDa drug efflux pump P-glycoprotein has been observed in many multidrug-resistant cell lines andsome human tumors (1, 2, 23—25).However, it has been clear forsome time that alternate mechanisms of multidrug resistance mustexist both in vivo and in vitro. A number of multidrug-resistant cell lineshave been described that do not overexpress P-glycoprotein but displaydrug resistance patterns very similar to cell lines that do (9, 15, 26—29).The procedures used to derive these cell lines appear no different fromthose which result in P-glycoprotein overexpression, and it is presentlyunknown what determines whether a cell line will acquire resistance viaa P-glycoproteinmechanism or a non-P-glycoproteinmechanism.

Until recently, non-P-glycoprotein-mediated multidrug resistancehas been generally attributed to a variety of mechanisms, includingenhanced expression of drug detoxification enzymes and alterations intopoisomerase II (30—33).In addition, elevated expression of a number of uncharacterized proteins has been reported in some non-Pglycoprotein multidrug-resistant cell lines. The cloning of MRP (3)permitted the identification of one of these, P190, as a member of theAlP-binding cassette transporter superfamily (6, 34, 35), and wesubsequently demonstrated that its overexpression was sufficient to

5905

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Table2 ResistanceoJMRP-transfectedHeLa cells to heavymetalsCompoundlC@

(@g/mI)°Resistance

factor1'C6T14Sodium

arsenite (As III)0.622.253.6Sodium

arsenate (As V)(0.58,0.65) (2.30,2.20)

50 ±9 181 ±47'(n=4)(n=3)3.6Potassium

antimony tartrate (Sb III)2.310.54.6Potassium

antimonate (Sb V)(2.8,1.7)(15,6)

162 ±32 252 ±18'(n=3)(n=3)1.6Cadmium

chloride (Cd II)44 ±2 45 ±3(n=4) (n=4)1

MRP-MEDIATED RESISTANCE IN TRANSFECrED CELLS

C0

0

0

0

100

75

50

25

00

Fig. 2. Resistance of HeLa cells transfectedwith the pCEBV7 vector (C6, 0) or with thepCEBV7-MRP1 vector (T14, •)to heavy metalanions. The compounds tested included sodium

arsenite (As III), sodium arsenate (As V), potassium antimony tartrate (Sb III), potassium antimonate (Sb V), and cadmium chloride (CdC1,).The results shown are those of a typical experimentin whicheachdatapointrepresentsthe meanof quadruplicatedeterminations.Errorbars havebeen omitted for clarity but were usually less than7% of the mean values.

100!\ 7525

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overexpression cannot be identified with certainty. For this reason, theT5 and T14 MRP-transfected HeLa cells described in the presentstudy provide important models for characterizing the MRP-mediatedmultidrug resistance phenotype.

The levels of pRc/CMV-MRP1 vector-encoded MRP mRNA in T5cells and pCEBV7-MRP1 vector-encoded MRP mRNA in T14 cellsare comparable to those of endogenous MRP mRNA in drug-selectedH69AR cells. However, the levels of MRP protein in H69AR cells areapproximately 8- and 4-fold higher than in T5 and T14 cells, respectively (Fig. 1; Ref. 5). These relative amounts of protein are generallyconsistent with the relative resistance of the three different cell lines.The reason for the low levels of MRP protein compared to mRNA inthe transfected cells is presently unknown. It may be the result of alower translational efficiency of vector-encoded MRP mRNA, sincethe construct used lacks part of the 5' untranslated region of theendogenous mRNA. An alternative explanation, i.e., that the stability ofthe mature protein is lower in the transfected cells, has been ruled outrecently by pulse chase experiments.5 However, we cannot exclude thepossibility that a significant fraction of MRP is degraded very rapidly inthe transfectants. Whatever the explanation, these data suggest that caution should be exercised in extrapolating from mRNA levels to protein.

The MRP-transfected T5 and T14 cells display drug resistancepatterns that are similar in many respects to those observed in Pglycoprotein overexpressing cells, but there are some potentiallyimportant differences (Table 1). For example, both T14 and T5 cellswere not resistant to mitoxantrone and displayed either no, or verylow, resistance to colchicine and taxol. These three highly lipophilicdrugs are normally included in the resistance spectrum of P-glycoprotein (29). Whether this apparent preference for more hydrophilic xenobiotics is a fundamental property of MRP or is influenced by the host cell in which MRP is expressed awaits furtherinvestigation.

Of the more than 50 members of the AlP-binding cassette superfamily that have been identified to date, MRP shares the greatest se

5 K. C. Almquist, D. W. Loe, D. R. Hipfner, J. E. Mackie, R. 0. Deeley, and S. P. C.

Cole. Characterization of the 190 kDa multidrug resistance protein (MRP) in drugselected and transfected human tumor cells, submitted for publication.

a The ICso of each compound was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the results shown are mean values from 2—4independentexperiments.Withineach experiment,determinationswere done in quadruplicate. Where :3 independentexperimentswere conducted,statisticalsignificancewasdeterminedusing an unpaired: test.

b The resistance factor was calculated as the ratio of the IC50 of T14 cells to the IC50

of C6 cells.C Values obtained with the resistant T14 cells are significantly different from those

obtained with the sensitive C6 cells (P < 0.05).

confer multidrug resistance on previously sensitive cells (5). Thesestudies provided the first unequivocal evidence of the existence of analternate multidrug resistance gene that is not a member of theP-glycoprotein family. Since the cloning of MRP cDNA from themultidrug-resistant H69AR cell line, overexpression of MRP mRNAor protein has been reported in many other previously describednon-P-glycoprotein multidrug-resistant cell lines (3, 6, 8, 10, 11, 36).These observations suggest that MRP may be a relatively commonmediator of resistance, at least in vitro.

MRP shares less than 15% amino acid identity with P-glycoprotein,and this resides almost exclusively within the nucleotide-bindingdomains, which are relatively highly conserved throughout the Al?binding cassette superfamily (37). Despite the structural divergencebetween the two proteins, no consistent differences in the resistancepatterns of MRP- and P-glycoprotein-overexpressing cell lines havebeen noted (29). However, resistance in drug-selected cell lines isfrequently multifactorial, and features solely attributable to MRP

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MRP-MEDIATEDRESISTANCE IN TRANSFECFED CELLS

quence identity with Leishmarna 4pgpA (32%) and yeast YCF-1 (43%)(38).However,in contrastto MRP,whichconfersresistanceto naturalproduct anticancer drugs (5), lapgpA confers resistance to arsenical andantimonial oxyanions (21, 22), and YCF-1 confers resistance to Cd2@(38). One feature which MRP, ltpgpA, and YCF-1 all share, and whichdistinguishes them from P-glycoprotein, is the presence of small, conserved deletions in their NH2-proximal nucleotide-binding domains (3,38). The functional significance ofthese deletions arepresently unknown.Although the high degree of amino acid identity between MRP andY-cFl s@g@ts that these two proteins may be true homologues, MRPdearly does not confer QJ2@ @is@ceon human cells (Thble 2; Fig. 2).On the other hand, like the ltpgpA gene (21, 22), MRP does conferresistance to trivalent and pentavalent arsenicals, as well as trivalent and,to a lesser extent, pentavalent antimonials. This cross-resistance wasunanticipated and is a second feature which distinguishes MRP fromP-glycoprotein-mediated resistance. Whether MRP mediates resistance tothese heavy metal oxyanions by a transport or a binding mechanismremains to be established.

Evidence that MRP binds xenobiotics and pumps them out of thecell in a fashion analogous to P-glycoprotein is equivocal. It has beenreported previously that membrane proteins from DOX-selected

Cl T5

50

@3o

@2O

10

0

Fig. 4. Energy dependence of reduced VCR accumulation in MRP-transfected HeLacells. HeLa cells transfected with the pRc/CMV vector (CI) or the MRP expression vectorpRc/CMV-MRP1 (T5) were incubated with radiolabeled VCR for 60 miii at 37°Candcell-associated radioactivity determined. To measure the effect of Al? depletion, drugaccumulation was measured after preincubation of the cells for 20 mm in glucose-freemedium supplemented with 10 mt@ideoxyglucose and 100 as rotenone (@). Controls (0)were preincubatedin medium containing 10 nmi glucose. Data, the mean of triplicatedeterminations in a single experiment; bars, SD. Similar results were obtained in oneadditional experiment.

HL6O/Adr cells, which are now known to overexpress MRP, (6) couldnot be labeled with photoaffinity analogues of vinblastine (34). Similarly, we could find no evidence of MRP labeling in H69AR cellswith a photoaffinity analogue of doxorubicin.6 The association ofreduced drug accumulation and enhanced drug efflux with the overexpression of MRP is less consistent than that reported for P-glycoprotein (12, 14, 39—42).There is considerable variability in the timecourse of drug accumulation and efflux, not only among different celllines, but also with the particular drug and drug concentration exammed. Drug accumulation and efflux were unchanged in the H69ARcell line from which MRP was isolated (14). However, the MRPtransfected HeLa cells described in this study clearly display reducedVCR accumulation which is AlP-dependent (Fig. 3, upperpanel; Fig.4). On the other hand, differences in the rates of VCR efflux fromsensitive and resistant transfected cells were slight (Fig. 3, lowerpanel) and similar to those reported previously for daunomycin effluxfrom drug-selected H69AR cells (14). One interpretation of the reduced accumulation in the transfected cells without a significantincrease in efflux is that MRP acts to remove drug from the plasmamembrane before it enters the cell. Alternatively, if MRP acts both asa plasma membrane pump and promotes drug accumulation in intracellular vesicles, cytoplasmic concentrations of free drug in MRPtransfected cells may be lower than total cellular drug measurementssuggest. This could result in a lower rate of efflux. The methodscommonly used to measure drug accumulation and efflux assume thatthe intracellular distribution of drug in resistant and sensitive cells isidentical with respect to location and exchangeability (23). They alsopresume that active transport is an exclusive property of the plasmamembrane. In the case of MRP-mediated resistance, neither assumption may be valid. Altered intracellular distribution of fluorescentanthracyclines has been reported in the MRP-overexpressing cell linesH69AR, HL6O/Adr, and COR-L23/R (7, 31, 42—44).Furthermore,MRP in HL6O/Adrcells has been reported to be predominantly in theendoplasmic reticulum (35), while preliminary subcellular fraction

6 A. R. Safa, K. C. Ahnquist, R. 0. Deeley, and S. P. C. Cole, unpublished observations.

time (mm)

.1

100

I

0@0 20 40 60 eo 100 120

time (mm)

Fig. 3. Accumulation and efflux of radiolabeled VCR in HeLa cells transfected withthe pRc/@MVvector (Cl, 0) or with the pRc/CMV-MRP1 vector (T5, •).Upper panel,to measuredrugaccumulation,cellswereincubatedin thepresenceof[3HJVCRat 37°C,and cell-associated radioscilvity was determined at the times indicated. The results shownare those of a typicalexperiment.Similarresultswere obtainedin three additionalindependentexperiments.Lowerpane4tomeasuredrugefflux,cellswerepreincubatedinthe presence of VCR for 60 mm at 37°C,washed, and exposed to drug-free medium.Cell-associated radioactivity was determined at the times indicated and expressed as apercentageof the radioactivitypresentimmediatelyafterwashingthe cells.Eachpointrepresentsthe meanof valuesobtainedin threeindependentexperiments;bars, SD.

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+

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i::i ci

Fig. 7. Effect of verapamil and cyclosporin A on VCR accumulation in MRP-transfected HeLa cells. Accumulation of [3HJVCRwas determined in HeLa cells transfectedwith the pRc/CMV vector (Cl, 0) or the pRcICMV-MRP1 vector (T5, @)60 mis afterexposure to drug alone (control) or to drug after preincubation of the cells in verapamilor cyclosporin A (CycA). Data, the mean of triplicate determinations in a single experiment; bars, SD. Similar results were obtained in one additional experiment.

MRP-MEDIATED RESISTANCE IN TRANSFECrED CELLS

Fig. 5. Effect of verapamil on VCR and DOXresistance in transfected HeLa cells. The resistanceof HeLacells transfectedwiththepRc/CMVvector(Cl, U@or the MRP expression vector pRc/CMVMRP1(T5,U)toVCRandDOXwasdeterminedinthe absenceor presenceof the indicatedconcentrations of verapamil. These concentrations had beenshown previouslyto cause less than 10%cytotoxicity when tested alone. Data, the mean of quadruplicate determinationswithin a single experiment.Similar results were obtained in at least one additional experiment.

ation studies with COR-L23/R (11), H69AR, and transfected T5 cells5indicate that the majority of MRP in these cells is in the plasmamembrane, although it can also be detected in some intracellularmembrane fractions. Whether these apparent differences in localization are truly cell type specific or simply reflect differences in thekinetics of MRP processing and trafficking to an ultimate, commondestination, such as the plasma membrane, is under investigation.

Resistance in P-glycoprotein-overexpressing cell lines can be reversed by a wide variety of compounds, although the mechanism(s)underlying chemosensitization is poorly understood (45). The mostextensively studied of the reversing agents are the calcium channelblocker, verapamil, and the immunosuppressive cyclopeptide, cyclosporin A. It has been proposed that these compounds reverse resistanceby directly binding to P-glycoprotein and inhibiting its drug effluxactivity. The demonstration that P-glycoprotein can be labeled withphotoaffinity analogues of verapamil and cyclosporin A (46, 47)supports this proposed mechanism of action. However, there is alsoevidence that verapamil and cyclosporin A may enhance cytotoxicitythrough a mechanism(s) unrelated to a direct interaction with Pglycoprotein (48—52).

Chemosensitization by verapamil and cyclosporin A in MRP-overexpressing cell lines is somewhat variable and at most can be de

scribed as modest, particularly with respect to cyclosporin A (20, 30,39, 43, 53—55).Consequently, the marked chemosensitization byverapamil of MRP-transfected T5 and T14 cells with respect to VCRand DOX (Fig. 5) was unexpected. However, a similar degree ofchemosensitization was found in control-transfected Cl and C6 cells

5908

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Fig. 6. Effect of cyclosporin A on VCR andDOX resistance in transfected HeLa cells. Theresistance of HeLa cells transfected with the pRc/CMVvector(CI) or the MRPexpressionvectorpRc/CMV-MRP1 (T5) to VCR and DOX weredetermined in the absence (0) or presence (@) ofcyclosporin A (7 @g/ml).Data, the mean of valuesobtained in three independent experiments; bars,SD.

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MRP-MEDIATEDRESISTANCE IN TRAN5FECItD CELLS

and verapamil only partially restored VCR accumulation in T5 cells(Fig. 7). Like verapamil, cyclosporin A also markedly increased VCRsensitivity in MRP-transfected cells and caused a similar chemosensitizing effect in control cells (Fig. 6). However, cyclosporin A hadless of an effect on DOX resistance (Fig. 6) and only slightly increased VCR accumulation in the transfected cells (Fig. 7). Thus, themechanism(s) by which verapamil and cyclosporin A enhance thedrug sensitivity of these cells appears unlikely to be the result of adirect interaction of these agents with MRP. The observation thatmembranes from drug-selected HL6O/Adr cells, which overexpressMRP (6) could not be labeled with a photoactive analogue ofverapamil, supports the suggestion that verapamil does not interactdirectly with MRP (34). The apparent variability of chemosensitization in different MRP-overexpressing cell lines suggeststhat the mechanisms by which these reversing agents act maydepend on the cell type involved, but this remains to bedetermined.

In conclusion, our data suggest that there are significant differencesin the pharmacological properties of MRP-mediated and P-glycoprotein-mediated multidrug-resistant cells. MRP-mediated resistance intransfected HeLa cells is characterized by low level resistance tolipophilicxenobioticsand by only slightly increasedrates of drugefflux. In addition, MRP-transfected cells are moderately resistant toarsenical and antimonial anions. Whether or not these data are indicative of a physiological role for MRP as an oxyanion or organic aniontransporter is the subject of ongoing investigations.

ACKNOWLEDGMENTS

We thankDr.DennisThiele(Universityof Michigan)forhelpfuldiscussions and for making data available on YCF-1 prior to publication. Theassistance of SandraCaine and ChristineWattersin the preparationof themanuscript is gratefully acknowledged.

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1994;54:5902-5910. Cancer Res   Susan P. C. Cole, Kathryn E. Sparks, Karen Fraser, et al.   MRP-transfected Human Tumor CellsPharmacological Characterization of Multidrug Resistant

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