[CANCER RESEARCH 54, 5788-5792, November 15, 1994]

Advances in Brief

Detection of the Mr 190,000 MilitidrUg Resistance Protein, MRP, with Monoclonal

Antibodies'

David R. Hipfner, Stephan D Gauldie, Roger G. Deeley, and Susan P@C. Cole2

Cancer Research Laboratories ID. R. H., S. D. G., R. G. D.. S. P. C. C.J and Deportment ofPathology ID. R. H., R. G. D., S. P. C. C.]. Queen's University, Kingston, Ontario,Canada K7L 3N6

proteins as P-gp (6, 7)•4Like P-gp, transfection of drug-sensitive cellswith a full-length MRP cDNA is sufficient to confer multidrug resistance (7).5 However, despite the fact that both P-gp and MRPconfer resistance to a similar spectrum of anticancer drugs, these twoproteins share less than 15% amino acid identity (6).

MAbs against P-gp have played a critical role in determining therelevance of this protein in clinical drug resistance (8). Some antiP-gp MAbs and their derivatives are being investigated for theirtherapeutic potential, particularly those that are able to reverse drugresistance (9—12)or participate in antibody-dependent cell-mediatedcytolysis (13). P-gp-specific MAbs have also been useful in immunolocalization studies of normal tissues and have provided importantclues as to the physiological role of this protein (14, 15). The epitopesof several MAbs have been mapped, providing information aboutP-gp secondary structure and membrane topology (16, 17).

To date, polyclonal antiseraraised against MRP-derivedsyntheticpeptides have been used to quantify MRP protein levels in drugselected and transfected multidrug resistant cell lines and to examineMRP structure, biosynthesis, and subcellular distribution (7, 18,19).@-@However, these studies have been hampered by the unsuitability of polyclonal antisera for many experimental applications and bythe limited availability of these immunoreagents. To obtain betterprobes for immunodetection of MRP, we have produced MAbs frommice immunized with membranes from multidrug resistant H69ARcells which express high levels of this M1 190,000 protein. TheseMRP-specific MAbs should greatly facilitate ongoing investigationsof the biology and clinical relevance of this novel drug resistanceprotein.

Materials and Methods

Cell Culture. The parentalH69; doxorubicin-selected,multidrug-resistantH69AR;andrevertantH69PRsmallcell lungcancercell linesweremaintainedas describedpreviously(4, 20).T5 cellsare HeLacells that havebeenmademultidrug resistant by transfection with a full-length MRP cDNA expressionvector, pRc/CMV-MRP1, and Cl cells are HeLa cells that have been trans

fected with pRc/CMVvector alone (7). These cells were maintainedin thesame medium as the lung cancer cells, supplemented with 400 pg/mi 0-418(Sigma Chemical Co, St. Louis, MO). SP2/0-Ag14 myeloma cells weremaintained in Dulbecco's modified Eagle's medium (Hybri-Max, Sigma)supplementedwith 4 mML-glutamineand 5% heat-inactivatedbovine calfserum. Approximately 1 week prior to fusion, Sp2/0 cells were challenged with0.132 m@i8-azaguanine(Sigma) for one passage.

Generation of Hybridomas. Membrane-enriched fractions were preparedas describedpreviouslyand resuspendedin Tris-sucrosebuffer(10 mMTrisHO, pH 7.5—0.25M sucrose) containing protease inhibitors (7). FemaleBALB/c mice (6—8weeks old) receivedthreei.p. injectionsof 150 g@gH69AR

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

Deeley. Characterization of the 190 kDa multidrug resistance protein (MRP) in drugselected and transfected human tumor cells. Cancer Res., in press, 1994.

5 S. P. C. Cole, K@ E. Sparks, K. Fraser, D. W. Los, C. E. Grant, 0. M. Wilson, and

R. 0. Deeley. Phannacological characterization of multidrug-resistant MRP-transfectedhuman tumor cells. Cancer Res., 54: 5902—5910,1994.

5788

Abstract

MRP Is a Mr @°@°®integral membrane phosphoglycoprotein that isoverexpressed in some drug-selected resistant cell lines and has been

shown to cause multidrug resistance in transfected celIs Five marinehybridoma cell lines (QCRL-1, QCRL-2, QCRL-3, QCRL-4, andQCRL-6) have been generated which secrete monoclonal antibodies(MAbs) that react specifically with membrane proteins of MRP-overexpressing, multidrug-resistant, drag-selected H69AR cells and MRP-trans

fected HeLa cells (T5) but not the respective parental (1169) and vectortransfected (Cl) cells. The ability of three of these MAbs (QCRL-1,

QCRL-2, and QCRL-3) to selectively immunoprecipitate a Mr 190@000protein from @S-labeIodH69AR and T5 membranes indicates that theseMAin are specific for MRP. MAb QCRL-1 is also capable ofdetectlng thelow levels of MRP present in revertant H69PR cells by immunoblotanalysis. Indirect immunofluorescence analyses show that MAbs QCRL-l,

QCRL-2, and QCRL-3 strongly and differentially react with fixed T5 andH69AR cells but not with unfixed cells, suggesting that these MAbSrecognizeintracellular MRP epitopes.The availabifityof reagents for thespecific and sensitive immunodetection of MRP should greatly facilitatebiological and clinical studies of this novel drug resistance protein.

Introduction

The inherent or acquired resistance of tumor cells to a spectrum ofstructurally and functionally diverse drugs, termed multidrug resistance, is a serious obstacle to the successful chemotherapeutic treatment of many human cancers. Numerous cell lines have been selectedin vitro for resistance to a variety of cytotoxic drugs, providingexperimental models to study the phenomenon of multidrug resistance(1).ManyoftheseresistantcelllineshavebeenfoundtooverexpressP-gp,3 a M1 170,000 integral membrane protein encoded by the MDR1gene, which is believed to confer resistance by acting as a drug efflux

pump (2, 3). However, a number of cell lines have been described,such as the human small cell lung cancer cell line H69AR, whichdisplay multidrug resistance but do not overexpress this protein (1, 4,5). Recently, cDNA clones corresponding to an mRNA markedlyoverexpressed in H69AR cells were isolated and characterized (6).The previously unidentified M@190,000 integral membrane phosphoglycoprotein encoded by this 65-kilobase mRNA, designated MRP,belongs to the same superfamily of ATP-binding cassette transporter

Received 9/8/94; accepted 10/6/94.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 indicatethis fact.

1 Supported by grants from the Medical Research Council of Canada (to S. P. C. C. and

R. 0. D.) and the CancerResearchSociety (to S. P. C. C. and R. 0. D.). D. R. H. is therecipient of a studentship from the Medical Research Council of Canada and wassupported in part by a Queen's University Graduate Award. R. 0. D. is the StaufferResearchProfessorof Queen's University,and S. P. C. C. is a CareerScientist of theOntario Cancer Foundation.

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

Queen's University, 3rd Floor, Botterell Hall, Kingston, Ontario, Canada K7L 3N6.3 The abbreviations used are: P-gp, P-glycoprotein; eDNA, complementary DNA,

MRP, multidrug resistance protein; MAb, monoclonal antibody; PBS, phosphate-bufferedsaline; TBS, Tris-buffered saline (10 m@.iTris-0.15 MNaCI, pH 7.5); TBS-T, TBS-0.05%Tween-20; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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MRP-5PECIFIC MONOCLONAL ANTIBODIES

membraneprotein(withoutdetergent)in PBS and mixed 1:1 with RIB!MPL+TDM+CWS emulsion (Cedarlane Laboratories, Hornby, Ontario, Canada) at approximately 3-week intervals. Three days before fusion, 100 @gH69AR membrane protein was injected i.v. into a tail vein. Spleen cells werefused with SP2/0 myeloma cells with polyethylene glycol 4000 (Sigma)according to standard methods (21, 22). Cultures were fed with Dulbecco'smodified Eagle's medium containing 100 g@Mhypoxanthine, 04 @Maminopterm, 16 @ZMthymidine, 20% heat-inactivated fetal bovine serum, and 25 @.@g/m1gentamicin (1C14Biomedicals, St. Laurent, Quebec, Canada). After initialscreenin& amino@n was omitted from the growth medium.

Screening, Cloning, Isotyping, and Ascites Production. After 11 days ofgrowth in selective medium, 459 hybridoma supernatants were tested for thepresence ofMRP-spccific antibody by immunodot blot analysis. H69, H69AR,and H69PRmembraneproteinsin TBS wereblotted(4 ,&gprotein/dot)ontoImmobilon-P polyvinylidene fluoride membrane (Millipore, Mississauga, Ontario, Canada)using a 96-well vacuum manifold, and blots were kept wet at alltimes. Theblotswere cut into strips such that each strip had spots of membraneproteins from each of the three cell lines. After transfer to 24-slot incubationtrays, stripe were blocked for 1 h in blocking solution (5% bovine calfscrum/5% normal goat serum/1% bovine serum albumin in TBS-T). Hybridomasupernatantswere addeddirectlyto the blockingsolutionat a finaldilution of 1:9. After 90 mis, the strips were washed three times for 5 mm inTBS-T, and secondary antibody [horseradish peroxidase-conjugated goat antimouseIgci + 1gM(H+L) F(ab')2fragment;Pierce,Edmonton,Alberta]wasadded (diluted 1:10,000 in blocking buffer). After 1 h, the stripe were washedfive timesfor 5 mm in TBS-T,andantibodybindingwas determinedbyenhanced chemiluminescence detection (Amersham, Oakville, Ontario, Canada) with exposure on KOdakX-OMAT film.

HybridOmaSwhich showed preferential reactivity with H69AR membranedots were subjected to a second immunodot blot, using strips with Cl and T5membrane protein dots in addition to the H69, H69AR, and H69PR dots.Hybridomas which reacted preferentially with H69AR and T5 membrane dotscompared to 1169,H69PR, and Cl dots were cloned twice by limiting dilutionand then expanded.Immunoglobulinsubtypesof the MAin producedby thefive stablehybridomaclonesobtainedweredeterminedusingan isotypingreagent kit (Sigma). To produce ascites, 5 X 106hybridoma cells resuspendedin PBS were injected i.p. into pristane-pretreated BALB/c (nWnu) mice.Ascites fluid was collected over the next 1-2 weeks, andMAbswere purifiedby passageover Econo-PacDEAEBlue cartridges(Bio-Rad,Mississauga,Ontario, Canada) according to the manufacturer's instructions.

Immunoblotting and Immunoprecipitation of MRP Membrane proteinwas solubilized in Laemmli buffer (23) and subjected to SDS-PAGE andelectroblotting as described previously (7). !mmunoblouing was performed asdescribed above for dot blot strips. In some experiments, a polyclonal antiserum (MRP-2) that was raised against a peptide corresponding to amino acids1418 to 1432 of MRP,andwhich is knownto cross-reactwith P-gp,was used

as a positive control for MRP detection.4MAb C219 (Centocor, Malvern, PA)was used at 1 pg/mi for specific detection of P-gp.

Immunoprecipitationswere carriedout as follows. Cells were incubatedfor24 h in L-methionine-deflCient RPM! 1640 (Sigma) supplemented with 10%dialyzed bovine calf senim and 50 @aCi/m1[35Sjmethionine (1110 Ci/mmol,cell labeling grade; Dupont NEN, Markham,Ontario,Canada).Cells werewashed twice with PBS and resuspended at approximately 6 X iO@cells/mi insolubilizingbuffer [1% 3-(3-cholamidopropyl)dimethylamino-1-propanesulfonate, 100 mM KU, and 50 mMTris-HC1(jH 7.5)] containing proteaseinhibitors(7). After 1 h on ice, insolublematterwas removedby ultracentrifugation.Aliquots of the supernatantwere broughtup to 250 g.dwith solubilizing buffer and incubated for at least 2 h at 4°Cwith hybridoma supernatantdiluted1:6.Antibody-MRPcomplexeswererecoveredby incubationwith25%w/v GammaBindPlus Protein G Sepharose (30 pi) or 10% w/v proteinA-SepharoseCL-4B (25 @d)(Pharmacia,Bale D'Urfé,Québec,Canada)insolubiization buffer for at least 2 h at 4°C.The samples were sequentiallywashed (24), and precipitatedproteins were eluted from the beads withLaemmli buffer and analyzed by SDS-PAGE and fluorography.

Indirect Immunofluorescence and Flow Cytometry. Cells were washedtwice with cold PBS and fixed with either 0.5% paraformaldehyde (Sigma) inPBS for 30 mm at 4°Cor with 70% methanol at —20°Cfor 10 mm. Allsubsequent procedures were done at 4°C. Cells were washed once withblockingsolution(1%bovineserumalbumin/5%normalgoatserum/PBS).ForMAbs QCRL-2 and QCRL-3, the cells were incubated in blocking solutionwith 01% Triton X-100 for 30 mm, followed by direct addition of hybridomasupernatant or ascites diluted as required. After incubation for 1 h, the cells

were washedonce in blocking solutionwith 0.1%TritonX-100, followed bya wash in blocking solution alone. The washed cells were incubatedwithfluorescein-conjugatedgoat anti-mouseIgO (H+L) F(ab')2fragment(Pierce)diluted1:50in blockingsolutionfor 30 mm andthenwashedtwice in blockingsolution with 0.1% Triton X-100. For MAb QCRL-1, cells were treatedsimilarlyexcept Tween-20was used at 0.1%insteadof TritonX-100 andwasincludedin all washes andincubations.Finally,cells were resuspendedin 1%paraformaldehydein PBS andeitheranalyzedon a CoulterEpic flow cytometer or cytospinswere preparedfor examinationby fluorescencemicroscopy.

Results and Discussion

Using spleens from mice immunized with MRP-enriched membranes, murine hybridomas were generated and screened for theirability to detect MRP in nondenatured membranes. In the end, fivestable cloned hybridoma cell lines, designated QCRL-1, QCRL-2,QCRL-3, QCRL-4, and QCRL-6, were obtained. MAbs QCRL-1,QCRL-4, and QCRL-6 were determined to be of the Ig0@ subclass;MAb QCRL-2 was an IgO2b; and MAb QCRL-3 was an Ig0@. The

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Fig@1. Immunodot blot analyses with hybridoma supernatants. Membrane-enriched fractionswere prepared from parental H69, multidrug realstent H69AR, and revertant H69PR cells, as well asfromHeLacellstransfectedwithvectoronly(Cl)and MRPcDNA(T5), and 8226/Dox4Ocells whichoverexpressP-gj@.H69,H69AR,H69PR,and8226/Dox4Omcmbraneproteins(2.5 paJdot)and Cl and13 membraneproteins(5 @&g/dot)were spottedonto stripsof polyvinylidenefluoridemembrane.Stripswereincubatedwiththehybridomasupematents,indicatedat thetopof thestrips,or anti-P-gpMAb @19(1 p@/ml).AntibOdybindingwas determinedby enhancedchemiluminescencedetec

..@— H69PR

@T5

.l@— H69

.‘@— H69AR

@8226/Dox4O

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MRP.SPECIFICMONOCLONALANTIBODIES

fected T5 cells but not from Cl control cells (data not shown). Takentogether, these data provide confirmation of the MRP specificity ofMAbs QCRL-1, QCRL-2, and QCRL-3. None of the membranesolubilization conditions tested to date have enabled the precipitationof MRP (or other membrane proteins) with MAbs QCRL-4 andQCRL-6,althoughthe MAbs themselves can be precipitatedby protein 0. Thus, at the present time, the MRP specificity of these MAbscannot be claimed with absolute certainty.

Immunoblot analyses with the MAbs were carried out under bothreducing and nonreducing conditions. In both cases, only MAbQCRL-1 detected a proteinof Mr190,000 (Fig. 2B, leftpanel). Thisprotein is easily detectable at high levels in membranes from H69ARand T5 cells using QCRL-1 hybridoma supernatant, and the relativelevels in the two cell types are approximately the same as those wehave reported previously using MRP-specific polyclonal antisera (7)•4The very low levels of the Mr 190,000 protein found in the drugsensitive revertant H69PR cells could also be detected with a veryhigh degree of specificity using MAb QCRL-1 ascites (Fig. 2B,middle panel). The Mr 170,000 P@f),detectable in 8226/Dox4O cellswith MAb C219 (Fig. 2B, right panel), was not detected in immunoblotswith MAb QCRL-1, consistent with the immunodotblotanalyses.

To be widely useful for thedetectionof MRPin manyexperimentalapplications and in the analysis of clinical samples, it is important thatMRP-specific MAbs are able to recognize MRP epitopes in fixed cellsand tissues. For this reason, labeling of H69, H69AR, H69PR, Cl, andT5 cells with MAbs QCRL-1, QCRL-2, and QCRL-3 was examinedby flow cytometry and indirect immunofluorescence microscopy.None of the MAbs reacted with unfixed cells (data not shown),suggesting that the MRP epitopes detected by these MAbs are not

exposed on the cell surface. However, the epitopes recognized bythese three MAbs remained intact after fixation of cells with either

0.5% paraformaldehyde or 70% methanol. MRP reactivity with MAbsQCRL-1 and QCRL-3 also remains intact after fixation with 10%formalin.6 Representative flow cytometry histograms obtained withMAb QCRL-3 and cells fixed with 0.5% paraformaldehyde are shownin Fig. 3. MAb QCRL-3 clearly discriminated between H69AR cells,in which high levels of MRP are detected in essentially all cells, andparental H69 cells, in which the Mr 190,000 protein is not detected

(Fig. 3A). A small differencein immunofluorescentlabelingwas alsoobserved between the parental H69 cells and revertant H69PR cells,which express slightly higher levels of MRP than H69 cells (6). WhenMRP-transfected T5 cells were labeled with MAb QCRL-3, an asym

metric distribution of relative fluorescence intensity was observed(Fig. 3B). These findings were not unexpected since T5 cells are anuncloned population and, therefore, individual cells within this population are likely to express different levels of MRP (7). Similarhistograms were obtained with MAbs QCRL-1 and QCRL-2 (data notshown).

Using indirect immunofluorescence microscopy, all three MAbswere observed to react intensely with resistant T5 and H69AR cellsbut not with Cl and H69 cells. Representative results obtained withMAb QCRL-3 are shown in Fig. 4. Labeling of H69AR cells wasuniform while staining of T5 cells was somewhat heterogeneous,consistent with the flow cytometric analyses. Both MRP-positive T5cells and H69AR cells showed predominantly plasma membranelabeling. These data are consistent with subcellular fractionation studies, which also indicate a predominantly plasma membrane localiza

tion of MRP in these cells.4 However, some granular cytoplasmic

6 H. S. L Chan, G. Haddad, D. R. Hipfner, R. 0. Deeley, and S. P. C. Cole, manuscript

in preparation.

5790

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0cRL@1 0@RL-1 @2iCFig. 2. Immunodetection of M, 190,000 MRP with MAbs QCRL-1, QCRL-2, and

QCRL-3. A, H69AR cells were metabolically labeled with [35S]methionine and thensolubilized in buffer containing 1% 3-(3-cholamidopropyl)dimethylamino-1-propanesul

fonate. Soluble proteins were incubated with polyclonal antiserum MRP-2 (diluted 1:30)or hybridoma supematants of MAbs QCRL-1, QCRL-2, or QCRL-3 (diluted 1:6). Immune complexes were precipitatedwith protein A-Sepharose (left panel), or protein0-Sepharose (right panel). lmmunoprecipitated proteins were resolved by SDS-PAGEfollowed by fluorography. The autoradiographs shown are 20-h (left panel) and 16-h(right panel) exposures. B, proteins from membrane-enriched fractions were separated bySDS-PAGEthrough5 or 7%polyacrylamidegels andelectroblottedontomembranes.Leftpanel, the gel was loaded with proteins from MRP-transfected T5 and vector-transfectedCl cells (10 @g,1ane),multidrug-resistant H69AR cells, parental H69 and revertantH69PRcells (5 g@g/1ane),and from P-gp overexpressing8226/Dox4Omyelomacells (25@tg/lane).The blot was probed with MAb QCRL-l hybridoma supernatant (diluted1:5000). When corrected for protein loading of gels, the H69AR cells were estimated toexpress 5-fold higher levels of MRP than T5 cells by densitometry. Middle panel, the gelwas loaded with protein from 15 cells (15 pg/lane) and H69PR cells (75 p@g/lane),and theblot was probed with MAb QCRL-1 ascites (diluted 1:5000). Right panel. the gel wasloaded as in the left panel, and the blot was probed with anti-P-gp MAb C219 (1 pg/mI).Antibody binding was visualized by an enhanced chemiluminescence detection system.The immunoblotsshown are from gels run underreducingconditions. Identicalresultswere obtained with nonreducing conditions.

MAbs reacted strongly with MRP-rich membrane fractions from bothdrug-selected H69AR cells and MRP-transfected T5 cells and weaklyor not at all with parental H69, revertant H69PR, or control Cl cellmembranes (Fig. 1). These data, particularly the specificity for theMRP-transfected T5 cells, strongly suggested that the MAbs werereacting with MRP. None of the MAbs cross-react with P-gp, sincethey showed no reactivity with membrane fractions from 8226fDox40cells, which are known to overexpress this Mr 170,000 protein (25)and which reacted with the P-gp-specific MAb C219.

To confirm the MRP specificity of these MAbs, immunoprecipitation and immunoblot analyses were carried out. MAb QCRL-3 im

munoprecipitated a single Mr 190,000 protein from [35S]methioninelabeled H69AR cells when protein A was used to bind immunecomplexes (Fig. 14, left panel). MAb QCRL-2 also precipitated thisMr 190,000 protein but only very poorly (data not shown). This

precipitated protein had the same electrophoretic mobility as theprotein precipitated by the polyclonal antiserum MRP-2, which wasraised against an MRP-derived peptide.4 To immunoprecipitate MRP

with MAb QCRL-1, protein G was required to bring down immunecomplexes (Fig. 2A, right panel). Protein 0 was also effective inprecipitating MRP with MAbs QCRL-2 and QCRL-3. A Mr 190'000protein was precipitated with all three MAbs from the MRP-trans

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MRP-SPECIFIC MONOCLONAL ANTIBODIES

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Fcytometric analyses of fixed cellsIA. Cellswerefixedwith0.5%

@permeabilizedwith 0.1% Triton@ with MAb QCRL-3 (ascites,.

@byincubation with fluoL goat anti-mouseIgG. Shown

Irelative fluorescence intensity@ H69PRcells(A)andMRP

vector-transfected Cl HeLa

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@ to produce and isolate hybridomas secreting MAbsIconformation-dependent epitopes, we deliberately omitted-__—__-fromthemembranesusedforimmunizationand:-@ :@— blottingscreeningprocedure.Thisstrategyhas

@ successful since it appears likely that only one of the@ obtained, MAb QCRL-1, recognizes a linear epitope, as

demonstrated by its reactivity with denatured protein in immunoblots.In contrast, the other four MAbs only detect MRP in nondenaturingimmunodot blots or under relatively nondenaturing conditions inimmunoprecipitations and in fixed cells. These observations stronglysuggest that these latter MAbs recognize conformation-dependentepitopes. Because of its unique ability to detect MRP in immunoblots,it may be inferred that MAb QCRL-1 reacts with an MRP epitopedistinct from those recognized by the other four MAbs. The ability of

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1@@-@-. .-- .. labelingofcellswithMRP-specificMAbQCRL-3.Cellsfixedin0.5%paraformaldehydewerelabeledusingMAbQCRL-3ascites(dilutedgoat anti-mouseIgO. Cells were centrifugedonto microscopeslides andexaminedusing a Leitz Aristoplanmicroscope.A, parentalH69 cells; B,

@@@JARC, vector-transfectedcontrolCl cells; D, MRP-transfectedmultidrug-resistantT5 cells.

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MRP-SPECIFIC MONOCLONALANTIBODIES

MAbs QCRL-2 and QCRL-3 to immunoprecipitate MRP, whileMAbs QCRL-4 and QCRL-6 are unable to do so under the sameconditions, suggests that these two pairs of MAbs also recognize atleast two different epitopes. Mapping studies to identify the epitopesequences of these MAbs are in progress.

Most studies to date on MRP detection in normal and malignantcells have relied solely on the measurement of MRP mRNA (26—30).Interpretation of results from such studies may be limited becauselevels of expression of MRP mRNA may not always correlatewithlevels of Mr 190,000 protein (7). Therefore, the MAbs described inthis report will be valuable reagents for clinical and basic researchinvestigations requiring the specific detection of the protein encodedby the MRP gene. The high degree of sensitivity of MAb QCRL-1 inimmunoblot analysis and the ability of MAbs QCRL-1, QCRL-2, andQCRL-3 to recognize MRP in cells treated with several commonfixatives enables the detection and quantitation of low levels of MRP.These MAbs should, therefore, be useful for detecting the presence ofMRP in tumor samples and normal tissues. The availability of MAbsagainst different epitopes on the MRP molecule should also facilitatestudies of the secondary structure and membrane topology of thisnovel drug resistance protein.

Acknowledgments

We thankK. C. Almquist,G. Valdimarsson,andS. E. L MirSkifor helpfuladvice and assistance. The technical assistance of K. Sparks, K. Fraser, J.Johnson,and M. Arnoldis also gratefullyacknowledged.

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1994;54:5788-5792. Cancer Res   David R. Hipfner, Stephan D. Gauldie, Roger G. Deeley, et al.   with Monoclonal Antibodies

190,000 Multidrug Resistance Protein, MRP,rMDetection of the

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