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(CANCER RESEARCH54, ‘ts92-4@t98, September 15. 19941 The Inability of the Mouse mdr2 Gene to Confer Multidrug Resistance Is Linked to Reduced Drug Binding to the Protein1 Ellen Buschman and Philippe Gros2 Department of Biochemistry. McGill University. Montreal, Quebec. Canada H3G I Y6 ABSTRACT The mouse mdr gene family is composed of three members designated indri, mdr2, and mdr3. A full-length mdr2 complementary DNA clone has been Introduced in an ampliftable eukaryotic expression vector (pEMC2b1)whichdirectsamplification andoverexpression of a bids tronic mdr2-dihydrofolate selection of transfected reductase mutant mRNA after stepwise methotrexate dihydrofolate ovary DUK cells. Independent reductase cell clones expressing Chinese hamster low to high amounts of mdr2 cellular mRNA and Mdr2 protein in their membrane fraction could be obtained by this selection procedure. Comparison of drug sur vival characteristics of cell clones expressing similar amounts of either Mdrl or Mdr2 proteins revealed that Mdrl but not Mdr2 could confer readily detectable levels of coichicine or vinblastine resistance. Labeling experiments using membrane-enriched fractions and a photoactivatable analogue of ATP showed that the Mdr2 protein was properly inserted in the membrane of transfected cells and could bind this ligand with an apparent affinity similar to that of Mdrl. However, labeling studies with the photoactivatable drug analogue lodoarylazidoprazosin showed consid erably reduced binding of this ligand to Mdr2 as compared to Mdrl. Our findings demonstrate that Mdr2 cannot confer drug resistance and sag gest that this inability is linked to reduced drug binding to the Mdr2 protein. INTRODUCTION MDR3 in cultured cells in vitro and tumor cells in vivo is caused by the overexpression of P-gps (1). P-gps have been shown to bind AlT (2, 3) and drug analogues (4, 5) and to possess ATPase activity (reviewed in Ref. 6), and are believed to function in resistant cells as energy-dependent drug-efflux pumps that prevent intracellular drug accumulation (1). P-gps are encoded by a small family of homologous genes, termed mdr or pgp, which include two members in humans. [MDRJ and MDR2 (also known as MDR3; Refs. 7—9)J,and three members in mice [mdrl, mdr2, and mdr3 (10—13)].Predicted amino acid sequence analyses indicate that P-gps are formed by two se quence homologous halves, each composed of 6 putative TM domains and a NB fold. P-gps show a very high degree of inter- and intraspe cies sequence homology, with 75—85%identity among the 3 mouse proteins. The three mouse mdr genes arose from two successive gene duplication events, the last one creating mdrl and mdr3, which share greater homology to one another than either do to mdr2 (12, 14). Despite this high degree of sequence conservation, striking functional differences have been detected between individual mdr genes in transfection experiments. Mouse mdrl and mdr3 and human MDRJ can directly confer drug resistance to otherwise drug-sensitive cells in transfection experiments (12, 15), while mouse mdr2 (1 1, 16), and human MDR2 apparently cannot (17). Although the normal physiological role of individual human and mouse P-gps remains unclear, their patterns of RNA and protein expression are restricted in an organ- and cell-specific fashion. At the cellular level, P-gps are generally expressed at the apical pole of secretory epithelial cells, suggesting that they may function as trans membrane transporters at these sites (1, 18). Human MDR1 is ex pressed at highest levels in the adrenal gland, epithelia of the kidney, jejunum, colon, and endothelial cells of the blood brain barrier, whereas human MDR2 is expressed almost exclusively in liver (19— 23). In normal mouse tissues, mdrl is most strongly expressed in the pregnant uterus, adrenals, placenta, and kidney, whereas mdr3 is mostly expressed in intestine and lung (24). RNA hybridization cx periments with gene-specific mdr probes have also identified liver as the major site of mdr2 RNA expression (24). Recently, we have used isoform specific anti-P-gp antibodies to show that (a) two P-gp isoforms (Mdr2 and Mdr3) are expressed in liver with Mdr2 being the major species, and (b) P-gp expression is highly polarized in this tissue and restricted to the canalicular side but not the sinusoidal side of the bile canaliculus (25). Furthermore, photolabeling studies with the drug analogue IAAP suggested that Mdr3 but not Mdr2 was capable of binding the drug analogue in canalicular membrane vesi des (25). Although the precise nature of the substrate(s) transported by Mdr2 in CMV remains unknown, the analysis of mutant mice bearing a homozygous null mutation at mdr2 suggests that it may participate in the transport of PC across the canalicular membrane (26). The inability of mouse Mdr2 and its human MDR2 homologue to confer drug resistance and therefore to carry out drug transport has also been addressed in transfected mammalian cells. In human BRO cells, high level expression of MDR2 failed to confer drug resistance with daunorubicin efflux identical to that of control drug-sensitive cells (17). Likewise, introduction of the mouse mdr2 cDNA into drug-sensitive CHO LR73 cells failed to yield drug-resistant colonies upon direct plating of the transfected cells in medium containing colchicine; DHFR, dihydrofolate reductase; CHO, Chinese hamster ovary cells; DUK, CHO DHFR cell line DUKX-B1; EMC, encephalomyocarditis virus; IAAP, iodoaryla either COL or ADM (1 1, 16). Transfection of CHO cells with chi meric mdrl/mdr2 cDNA molecules created by exchanging homolo gous domains of either protein showed that NB1 and NB2 of mdr2 could functionally complement the drug resistance function of Mdrl, while segments overlapping the predicted TM-associated domains could not (16). Since genetic (27—31)and biochemical analyses (32— 36) have shown that TM domains of P-gp are responsible for drug binding, these results are in agreement with the proposition that Mdr2 transport, by a mechanism similar to Mdrl, different classes of sub strates such as biliary PC, as suggested by Smit et a!. (26). In the present study, we have attempted to understand the molecular basis underlying the functional differences detected between mdrl and mdr2. For this, we have used a novel expression system which zidoprazosin; MEM, minimal essential medium; MTX, methotrexate; NB, nucleotide does Received 5/3 1/94; accepted 7/19/94. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by grants from the Medical Research Council of Canada (P. 0.). P. G. is supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada and is an International Hughes Medical Institute. 2 To whom requests for reprints should be addressed, Research Scholar of the Howard at Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6. 3 The abbreviations used are: MDR, multidrug resistance; ADM, Adriamycin; COL, not rely upon MDR drug selection to achieve high level binding; P-gp, P-glycoprotein; SDS, sodium dodecyl sulfate; TM, transmembrane; VBL, vinblastine; PC, phosphatidylcholine; eDNA, complementary DNA; PBS, phosphate P-gp expression to express high and equivalent amounts of Mdrl and Mdr2 in membrane fractions of transfected cells. The ability of the two Mdr buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 4892 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. DRUGBINDINGTO Mdr2 isoforms to confer drug resistance and to bind drugs as well as Al? photoactivatable analogues was assessed. MATERIALS described previously (3). Purified membrane fractions were further isolated by ultracentrifugation AND METHODS mdr-transfected Cell Clones. 0.1 M NaCI, and 0.01 A full length mdr2 cDNA (nucleotides 200— 4359) (11) was cloned into the blunt-ended EcoRI site of the mammalian expression vector pEMC2b1 (generous gift of R. Kaufman, Genetics Institute, Cambridge, MA) and introduced as a calcium phosphate coprecipitate (37) into drug-sensitive mutant Chinese hamster (150,000 X g for 3 h at 4°C) on a discontinuous density gradient of 60, 45, 35, and 30% sucrose. Membrane vesicles present at the 35 and 45% interfaces were collected, washed, and stored at —80°C in 0.1 MTris, ovary cells (DUK cells) bearing a null concentration (Bio-Rad). M EDTA, pH 7.5 containing Protein Western Blotting. SDS-PAGE was performed according to standard pro tocols (43). Briefly, 12.5—50@g of purified membrane proteins were solubi lized in 2 x Laemmli sample buffer for 5 mm at room temperature before loading onto 7.5% polyacrylamide mutation at the DHFR (dihydrofolate reductase) locus. The pEMC2b1 vector is identical to the pMT2 (38) vector, except for an EMC virus translation 40% glycerol. was determined using an amido black-based commercial assay gels. After electrophoresis, ibrated in transfer buffer (20% methanol-0.76 gels were equil M glycine-2.5 and proteins were transferred to nitrocellulose mM Tris, pH 8) sheets by Western blotting (0.5 reinitiation sequence positioned downstream of the mdr2 cDNA and upstream the MTX resistance gene DHFR. Stable populations of transfectants were A, 100 V, at 4°C)for 1—3 h. The blots were treated to prevent nonspecific isolated binding after selection bonucleotides protocol in a-MEM and containing described culture medium 10% dialyzed previously lacking fetal bovine (39). Mass populations ribo- and deoxyri serum according of cell clones to a surviving this initial selection were harvested as pools 10 days after transfection, ex panded in culture, and subjected to further stepwise selection in the same medium containing 0.02, 0.04, and 0.06 ,.@M MTX. Individual colonies growing in 0.06 p.M MTX were harvested, expanded in culture, and frozen in 90% fetal calf serum and 10% dimethyl sulfoxide. Drug-sensitive DUK or LR73 (40) cells, used as negative controls in all experiments, by incubation overnight at 4°C in 10 mM Tns, which recognizes all P-gp isoforms with goat anti-mouse antibody M NaCl (44), for 1 h at room temperature, conjugated to alkaline and then phosphatase. Specific antigen-antibody complexes were revealed by incubation with 5-bromo-4chloro-3-indoyl phosphate Immunoprecipitation. p-toluidine and nitro blue tetrazolium (Bio-Can). Control and mdr-transfected cells (1.5 X 106) growing in 60-mm dishes were metabolically and all other cell clones pH 8-0.15 0.02% Tween 20-1% bovine serum albumin,followed by incubationwith a 1:300 dilution of mouse anti-P-gp monoclonal antibody C219 (Centocor), labeled for 12 h at 37°C in were grown in a-MEM supplemented with 10% fetal calf serum, glutamine (2 a-MEM lacking methionine (Flow Laboratories), supplemented with 10% mM), penicillin (50 units/ml) and streptomycin dialyzed fetal calf serum, L-glutamine (2 mM), and L-[35S]methionine at 35 @Ci/ml(specific activity, 1000 Ci/mmol; Amersham). Labeled cells were indicated. mdrl Multidrug-resistant (clone control 1-1) or mdr3 (clone (50 p@g/ml),unless otherwise cell lines expressing 3-5) were maintained either wild type in medium washed containing twice with PBS, harvested with a Pasteur pipet, solubilized bation levels of a chimeric SDS, and sonicated (3 X for 20 s) on ice. Next, 0.8 ml of a solution containing Mdr2 introduced Mdrl/Mdr2 (clone 1-2) consisting of the linker domain of in Mdrl, was grown in medium containing ng/ml (16). Finally, the mdrl-transfected clone EX4N-7, which for 5 mm at 0°C in 0.2 ml of buffer containing by incu VBL at 50 ng/ml (28). A multidrug-resistant cell clone stably expressing high 50 mM Tris pH 7.5-1% ADM at 100 1.25% Triton X-100, 200 mM NaCI, and 50 mM Tris (pH 7.5) was added and expresses the mixture was flushed low amounts of Mdrl protein, was maintained in medium lacking MDR drugs but containing Genetycin (0.5 mg/ml) (41). gently tamed protease inhibitors (2 pepstatin; Sigma). through a 26-gauge needle. All buffers con @g'mltrasylol, 5 @tWmlleupeptin, 0.04 @.tg/ml The solution was clarified by centrifugation at 15,000 X g Northern Blotting Experiments. Total cellular RNA was prepared by for 15 mm at 4°Cand immunoprecipitation with the anti-P-gp monoclonal homogenizing transfected and control cells in a solution containing guani dinium hydrochloride (6 M), followed by serial ethanol precipitations and complexes were purified using protein A-Sepharose (Pharmacia); washed phenol/chloroform extensively analysis, extractions, as described previously 10 @.tgof each RNA sample [denatured (14). For Northern antibody C219 with 150 mr@i NaCl, in 7 X standard saline-citrate (1:100 dilution) a solution 50 mM Tris was carried containing (Boerhinger-Mannhein); and resolved amide gels, as described above. transferred onto Gene Screen Plus (DuPont New England Nuclear) by capillary blotting. Membranes were baked for 2 h at 70°C,prehybridized, and hybrid ized sequentially in a solution containing 1 M NaCI, 1% SDS, and 10% dextran Kodak XAR film. sulfate at 65°C for 16 h each. citrate)-7.5% The mdr2-specific formaldehyde hybridization for 15 2276 (14). The hybridization 108 was labeled with [a-32P]dATP X-100, bovine by SDS-PAGE The gels were fixed, 0.03% serum SDS, albumin on 7.5% polyacryl dried, and exposed to PhotoafTinity Labeling. Purified membrane fractions from control and mdr-transfected cells were incubated with IAAP (specific activity, 2000 Cu was a mmol; DuPont New England Nuclear) and crosslinked described previously (35). For AlT-labeling, 100 with UV light, as @.tgof purified membrane extracts were incubated with [a-32Pj8-azidoadenosine-5'-triphosphate (Dupont, of Ic activity, @pm/p@gDNA. The blot was washed to a final stringency of Labeled P-gps were recovered by immunoprecipitation with C219 and ana Nuclear) 0.5 x standard saline by random primer extension citrate, 1% SDS at 65°C and exposed film for 24 h at —80°Cwith an intensifying Drug Survival to a specific to Kodak 2—10 Ci/mmol; ICN Biomedicals), as described (specif activity New England 5 x probe Triton 1869— probe 410-base pair Hinfi fragment of clone A-DR29 including nucleotides 0.1% (pH 8), and 5 mg/ml mm at 65°C]were fractionated in formaldehyde-containing agarose gels and (1 X SSC = 0.15 M NaCl, 0.015 M sodium out for 16 h at 4°C. Immune previously (3). lyzed by SDS-PAGE exactly as described above. Gels were fixed, dried, and exposed to Kodak XAR films with an intensifying screen (Kronex, Dupont de XAR screen. Assay. A modification of a cell survival assay (42) based Numours) at —70°C for 1—2 weeks. on sulforhodamine B staining of total cell protein was used. Briefly, 5 X i03 drug-sensitive DUK or LR73 control cells or mdr-transfected cells were plated in 96-well titer plates in complete medium containing increasing concentra tions of VBL or COL and incubated for 72 h at 37°C.Cells were then washed RESULTS once in ice-cold PBS, fixed in 17% trichloroacetic acid in PBS for 1 h at 4°C, and then washed extensively in tap water. Cellular proteins were stained with As opposed to its mdrl and mdr3 homologues, introduction of the mouse mdr2 cDNA in LR73 CHO cells fails to yield drug-resistant colonies on plating the transfected cells in cultured medium contain ing COL or ADM (11, 16). Although these experiments suggest that mdr2 cannot confer MDR, appropriate expression of the full-length protein in the membrane fraction of these cells needs to be demon strated to sustain this conclusion. Therefore, the aim of the present study was to express the Mdr2 protein in stably transfected cells, initiate a biochemical analysis of the protein, and try to identify the basis for its apparent inability to confer drug resistance. Initial exper iments using mdr2 cDNA inserted in the eukaryotic expression vector pMT2 and cotransfected with indicator plasmid pSV2neo produced stable G41& colonies which expressed either no or only very low a solution of 0.4% sulforhodamine B in 1% acetic acid for 15 mm at room temperature, followed by 4 washes with 1% acetic acid to remove excess stain. After the plates were dried, the stain was dissolved in 10 mM Tris (pH 9.0) and quantification was carried out using an automated enzyme-linked immunosor bent assay plate reader (Bio-Rad Model 450) set at 490 nm. The relative plating efficiency of each clone was calculated by dividing the absorbance observed at a given drug concentration by the absorbance detected in the same clone in medium devoid of drug and is expressed as a percentage. The drug dose required to reduce plating efficiency of each clone by 50%, was also calculated. Isolation of Membrane tured cell clones Fractions. Crude were prepared by homogenizing membrane extracts cells in hypotonic from cul buffer as 4893 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. DRUG BINDING TO Mdr2 amounts of the Mdr2 protein, insufficient for biochemical analysis (data not shown). To increase the level of protein expression in transfected cells, we turned to the eukaryotic expression vector pEMC2b1. The expression cassette of pEMC2b1 is similar to that of pMT2 (38), except that the mdr2 cDNA is fused to DHFR cDNA with an EMC virus translation reinitiation sequence separating the two cDNAs. Transfection of such constructs into DHFR mutant CHO (DUK) cells, followed by selection in increasing concentrations of MTX, yields cell clones producing very high levels of bicistronic recombinant DHFR mRNAs allowing high level expression of the foreign protein encoded by the introduced cDNA. The pEMC2b1-mdr2 construct was introduced into DUK cells, and transfected cell clones were selected first in a-MEM lacking ribo- and deoxyribonucleotides, followed by stepwise selection in increasing concentrations of MTX up to 0.06 ,[email protected] independent clones were isolated, expanded in culture, and examined for the presence of mdr2 mRNA and protein expression. Northern blotting analysis with an mdr2-specific hybridization probe of a representative cell clone fairly abundant (Fig. 1B; Lanes 2 and 6). Nevertheless, the amount of Mdr2 protein present in the highest P-gp-expressing clone was still considerably lower (10—20%)than the amount of P-gp expressed in multidrug-resistant cell clones transfected and overexpressing either wild type Mdrl or Mdr3 proteins (Fig. 1C; Lanes 2 and 6, respec tively) or the Mdrl-Mdr2 chimera (Fig. 1B; Lane 11). In agreement with our previous analysis of Mdr2 protein expression in normal bile canalicular membrane vesicles (25), the apparent electrophoretic mo bility of Mdr2 was distinct from that of Mdrl and Mdr3 expressed in the same cells (Fig. 1C). We noted a marked difference between the level of mdr2 mRNA expressed in our cell clones (Fig. IA) and the final amount of Mdr2 polypeptide produced in these cells (Fig. 1C). Although the reason of this discrepancy remains unclear, it could result from (a) a poor translatability in these cells of the mdr2-DHFR hybrid mRNA produced from intact or rearranged copies of the transfected plasmid, (b) impaired processing or posttranslational mod ification of the Mdr2 protein leading to degradation by cellular proteases, or (c) cellular toxicity of the Mdr2 protein, resulting in (2—6) obtainedbythisselectionidentifiedveryabundant mdr2-DHFR negative selection against high levels of expression. chimeric transcripts of heterogeneous size (Fig. IA, Lanes 2 and 3). We next analyzed the functional consequences of Mdr2 protein These heterogeneous mRNAs probably reflect the use of several expression on cellular resistance to the MDR-type drugs VBL and cryptic polyadenylation signals within the 3' ends of the mdr2 and COL. For this, the drug dose necessary to reduce the plating efficiency DHFR cDNAs, as well as the SV4O polyadenylation signal engineered of mdr2 transfected clones by 50% was established in a cell survival downstream the expression cassette. The hybridization signal was assay and was compared to that of control drug-sensitive nontrans specific, as shown by (a) the lack of hybridization of the mdr2 probe fected DUK cells; multidrug-resistant mdrl transfectant (EX4N-7) to control RNAs from multidrug-resistant transfectants expressing expressing low levels of the Mdrl protein (41), which are comparable high levels of mdrl or mdr3 (Fig. IA, Lanes 5 and 6), and (b) positive to those detected in our highest expressing mdr2 transfectants (see Fig. 3A), and clone 1-2, which expresses high levels of the Mdrl/ hybridization to RNA from a multidrug-resistant transfectant (clone 1-2) expressing a chimeric mdrl/mdr2 gene (16) containing an mdr2 Mdr2 chimeric protein. The results of a typical drug survival assay are segment overlapping the hybridization probe (Fig. IA, Lane 1). Re shown for one mdr2 transfectant (clone 2-6), control DUK cells, EX4N-7 cells, and highly drug-resistant 1-2 cells in Fig. 2; the hybridization of the Northern blot with mdrl and mdr3 gene-specific probes confirmed that the mdr mRNA expressed in clone 2-6 was combined analysis of 6 individual mdr2 transfectant clones is tabu indeed mdr2 (data not shown). The level of Mdr2 protein expression lated in Table 1. High level Mdrl-Mdr2 chimeric protein expression in clone 1-2 resulted in 52- and 38-fold resistance to VBL and COL, in 10 independent cell clones obtained by this selection protocol was respectively, while low level Mdrl protein expression in EX4N-7 determined by immunoprecipitation using the monoclonal anti-P-gp antibody C219 (Fig. 1B). This antibody is directed against the peptide produced moderate but readily detectable levels of resistance to the epitope VQE/AALD (45), which is precisely conserved in all three same drugs (VBL, 6-fold; COL, 4-fold). By contrast, none ofthe mdr2 transfectants showed any detectable degree of VBL or COL resistance mouse Mdr proteins. This analysis showed variable levels of Mdr2 over the background levels measured in the control drug-sensitive protein expression in these clones ranging from barely detectable (Fig. DUK cells. Similar results were also obtained with MDR drugs 1B; Lanes 1, 3, and 4) to intermediate (Fig. 1B; Lanes 5 and 7—10)to 1 2 3 4 5 6 ‘i@ I 2 3 4 5 6 7 8 9 10 11 12 I 2 3 456 ( 200 : 200 C 100 100 4.5. A B C Fig 1. Expression of mdr mRNAs and proteins in control and mdr-transfected Chinese hamster ovary cells. (A) Northern analysis of total cellular RNA of tndr-transfccted cell clones. Ten @sgof total cellular RNA from a cell clone transfected and overexpressing an mdrl/mdr2 chimeric eDNA containing the linker domain of mdr2 inserted into mdrl (clone 1-2, Lane I); transfected cell clones expressing either mdr2 (clone 2-6; Lanes 2 and 3), mdrl (clone 1-1; Lane 4), or mdr3 (clone 3-5; Lane 5); or control untransfected LR73 cells (Lane 6) wereanalyzedby Northernblottingwitha [32P]-labeled 410-basepairmdr2gene-specific probeoverlapping thelinkerdomain(nucleotides 1869—2276). Ordinate,estimated size of hybridizing mRNAs in kilobases (Kb). (B) P-glycoprotein iinmunoprecipitation in independent mdr2-transfected cell clones. [35S]Methionine metabolically labeled total cellular proteins from 10 independent mdr2-expressing clones (Lanes 1—10), the drug-resistant clone 1-2 expressing an MdrlfMdr2 chimeric protein (Lane I I), and drug-sensitive DUK cells (Lane 12) were immunoprecipitated with the anti-P-glycoprotein monoclonal antibody C219 (dilution 1:100) and separated by SDS-PAGE on a 7.5% gel. Molecular mass standards in kilodaltons were myosin (200 kilodaltons) and phosphorylase B (97 kilodaltons). The gel was fixed, dried, and exposed to X-ray film for 2 weeks (C). Immunoprecipitation of P-glycoproteins encoded by mdri, mdr2, and mdr3. Immunoprecipitation methods and cell clones were as described in A. Lane 1, LR73 control cells, Lane 2, clone 1-1 (nsdri), Lanes 3 and 5, DUK cells, Lane 4, clone 2-6 (mdr2) and Lane 6, clone 3-5 (mdr3). The gel was fixed, dried, and exposed for 3 days. 4894 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. DRUG BINDING TO Mdr2 The M,., 90,000—100,000 C219-reactive peptide present in these mem brane fractions represents a degradation product of the intact protein that has been described previously (34). Photolabeling studies of Mdrl and Mdr2 with [32P]8-azido-ATP showed that both proteins could be labeled to comparable levels by this photoaffinity probe, suggesting that they can bind Al? with a similar affinity (Fig. 3C; mdrl clone EX4N-7, Lane 1; mdr2 clone 2-6, Lane 2). In this experiment, a membrane-enriched fraction from an independent mdr2-transfected cell clone (clone 2-G7; Fig. 3C, Lane 3) also bound Al? at an equivalent level to Mdrl expressed in EX4N-7 (Fig. 3C, Lane 1). These results are in agreement with our previous observa tions that both nucleotide-binding folds of Mdr2 are functional and can complement the biological activity of Mdrl in chimeric molecules constructed between the two proteins (16). These results also indicate that the Mdr2 protein is properly targeted to the membrane fraction of transfected cells and that it is folded in an appropriate configuration which allows Al? binding. However, these experiments do not cx dude the possibility that the Al?ase activity of Mdr2 may be quan titatively or qualitatively different from that of Mdrl. On the other hand, photolabeling studies on serial dilutions of membrane fractions from Mdrl and Mdr2-expressing cells with the drug analogue IAAP produced strikingly different results. IAAP labeling of Mdr2 protein was much less pronounced than labeling of Mdrl for an equal amount of protein loaded (Fig. 3B). These differences in IAAP labeling of Mdrl and Mdr2 proteins were consistently detected in independent experiments. Taken together, these results show that although Mdrl and Mdr2 can combine an Al? analogue with similar efficiency, binding of a drug analogue to Mdr2 is greatly reduced, suggesting that reduced drug binding may underlie the incapacity of Mdr2 to transport these types of substrates. VINBLASTINE ---0--j DUK 112 2.6 EX4N7 U a U E a a 4@ a 10 100 1000 Vinblastine (ng/ml) COLCHICINE ---0--S DUK 112 —P1-— 2-6 —.-—0-- EX4N7 U 5 U E as a a DISCUSSION a 10 100 1000 10000 Colchicine (ng/ml) Fig 2. Drug survival characteristics of cell clones expressing Mdrl or Mdr2 proteins. A total of 5000 cells from mdr2-transfected clone 2-6, the mdrl-transfected clone EX4N-7 (both expressing equivalent amounts of protein), the highly multidrug-resistant clone 1-2 (112) expressing the chimeric Mdrl-Mdr2 protein, and control DUK cells (DUK) were plated in medium containing increasing concentrations of VBL and COL and grown for 3 days. Plating efficiency represents the percentage of cells surviving at a given drug concentration compared with the same cells grown in medium without drug. actinomycin @ D, ADM, and gramicidin D (data not shown). Several studies have established that as opposed to the mouse mdrl and mdr3 and human MDRJ genes, transfection of full-length cDNAs for either mouse mdr2 or its human counterpart MDR2 into otherwise drug-sensitive cells fails to yield multidrug-resistant clones on plating the transfected cells into drug-containing medium (1 1, 16, 17). In the case of human MDR2, stable expression of the protein in transfected BRO cells fails to increase the resistance of these cells to MDR-type drugs (17). The goals of the present study were (a) to determine the phenotypic consequences on drug resistance of stable mouse Mdr2 protein expression in transfected cells, and (b) to investigate the biochemical basis for the apparent lack of biological activity of Mdr2 These Table 1 Drug survival characteristics of control and independent mdr transfected results indicate that as opposed to low level expression of Mdrl (41) (EX4N-7, Fig. 2) or Mdr3 (28), low level Mdr2 protein expression fails to confer multidrug resistance in transfected CHO cells. These observations are in agreement with our previous inability to di rectly select multidrug-resistant mdr2 transfectants in drug containing medium. We next wished to investigate the biochemical basis for the func tional differences detected between Mdrl and Mdr2 proteins in trans fected cell clones. In particular, we compared the capacity of Mdrl and Mdr2 to bind the photoactivatable drug analogue IAAP and the photoactivatable Al? analogue 8-azido-ATP. For this, we used mem brane-enriched fractions prepared from one of the Mdr2-expressing clones (2—6)and from the Mdrl-expressing clone EX4N-7. Western blotting analysis of serial dilutions of these membrane fractions with the monoclonal anti-P-gp antibody C219 confirmed that both cell clones produced similar amounts of the respective proteins (Fig. 3A). Chinese hamster ovary cell clones, expressing either Mdrl or Mdr2 proteins The various cell clones analyzed are described in the text. VBL (ng/ml) COL (ng/ml) DUK EX4N-7 1/2 Cell clone 3.9° 25 (6x)― 210(53X) 70 (4X) 2700 (38X) 2-3 2-6 2-8 3.1 (0.8X) 5.3(1.3X) 2.9 (0.7X) 2-64 3.1 (0.8X) 25 (0.4X) 2-68 2-G7 2.9 (0.7x) 6.0 (1.5X) 30 (0.4x) 50 (0.7X) 35 (0.5X) 70(1X) 35 (0.5 X) a Drug concentrationrequired to reduce plating efficiency of control and mdr-trans fected cell clones by 50%, as determined in a 3-day cytotoxicity assay. Measurements are the means of two experiments carried out in duplicate. b The degree of drug resistance of individual mdr-transfected cell clones is expressed as a fold resistance (in parentheses) and was obtained by dividing the drug concentration required to reduce plating efficiency by 50% value measured for each clone by the drug concentration required to reduce plating efficiency by 50% of the control drug-sensitive parental cells. 4895 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. DRUG BINDING TO Mdr2 Mdr.Z 12.5 I 25 MdrJ. 50 12.5 25 I C 50 2 3 4 5 6 50 II (200 200 j'g iii @2O0 I 100 60 100 ..‘100 A B C Fig 3. Ligand-binding characteristics of Mdrl and Mdr2 proteins expressed in membrane fractions from transfected cell clones. (A) Western blot analysis of membrane fractions from cell clones expressing equivalent amounts of Mdrl and Mdr2 proteins. Purified membrane fractions (12.5, 25, and 50 @g)isolated from mdr2-transfected clone 2-6 (Mdr2), mdrl-transfected clone EX4N-7 (Mdrl), and untransfected LR73 control cells (C) were analyzed by Western blotting. The immunoblot was incubated with the anti-P-gp antibody C219 (1:300 dilution), and specific antigen-antibody complexes were visualized using a goat anti-mouse IgG antisera coupled to alkaline phosphatase. Ordinate, positions of molecular mass markers in kilodaltons. (B) Photoaffinity labeling of Mdrl and Mdr2 isoforms with IAAP. Purified membrane fractions from the mdrl-transfected clone EX4N-7 (Lanes 1—3) and the mdr2-transfected clone 2-6 (Lanes 4—6)were incubated with IAAP (30 nti, final concentration) and cross-linked with UV. The amounts of purified photolabeled membrane proteins loaded onto the gel were 40 (Lanes 1 and 4), 80 (Lanes 2 and 5) and 160 (Lanes 3 and 6) @.tg. Mdr proteins were immunoprecipitated using the mouse monoclonal anti-P-gp antibody C219. Reaction products were separated by SDS-PAGE and the gels were dried and exposed to X-ray film for 1 week. No specific IAAP-labeled product was detected by C219 in 160 @.tg of purified membranes from control LR73 cells (not shown). Right ordinate, positions of molecular mass standards in kilodaltons. (C) Photoaffmity labeling of Mdrl and Mdr2 isoforms with [a-32P]8-azidoadenosine-5'-triphosphate (Al?). Purified membrane fractions (100 @.tg)from the mdrl-transfected cell clone EX4N-7 (Lane I), mdr2-transfected clones 2-G7 (Lane 2) and 2-6 (Lane 3) and control untransfected LR73 cells (Lane 4) were incubated with [a-32P18-azido-ATP and cross-linked with UV. Mdr proteins were immunoprecipitated with the mouse anti-P-gp monoclonal antibody C219 and reaction products were separated by SDS-PAGE as described in B. The gel was exposed to X-ray film for 3 weeks. Left arrows, different migration of the two AlT-labeled Mdr isoforms (upper arrow, Mdrl ; lower arrow, Mdr2). The length of the resolving gel in A (5 cm) was different from that used in gels shown in B and C (15 cm). toward MDR drugs, in particular to compare the ATP and drug binding characteristics of Mdr2 to those of the biologically active Mdrl protein. The isolation of transfectants expressing readily detect able amounts of Mdr2 protein in cell membrane fractions proved difficult by standard transfection procedures using a cotransfected dominant selectable marker such as pSV2neo (data not shown). To circumvent this difficulty, it was necessary to introduce the full-length mdr2 cDNA in an expression vector which directs the synthesis of high levels of a bicistronic mdr2-DHFR mRNA with a viral EMC translation reinitiation sequence positioned immediately upstream the DHFR portion of the mRNA. Transfection of this construct into DHFR mutant CHO cells, followed by stepwise selection of trans fected cells in MTX-containing cultured medium, allowed the isola tion of several cell clones stably expressing fairly abundant amounts of the Mdr2 protein. Drug cytotoxicity assays showed that Mdr2 expression in independent cell clones failed to increase their levels of resistance to MDR drugs such as VBL and COL, while similar levels of Mdrl protein expression in the same cells readily conferred mul tidrug resistance as measured in the same assay. Biochemical analysis of the Mdr2 and Mdrl proteins in cross-linking experiments using membrane-enriched fractions from transfected cells, photoactivatable analogues of Al?, and the known P-gp ligand LAAP showed that although both proteins bound the ATP analogue to the same extent, binding of I.AAP to Mdr2 was significantly reduced when compared to Mdrl. These data indicate that as opposed to Mdrl, Mdr2 does not modulate drug efflux in these transfectants, and that this inability to confer drug resistance is linked to decreased drug binding to Mdr2. It is also interesting to note that abolition of ADM and COL resistance in an mdrl mutant bearing a unique substitution at position 941 (TM1 1) results in approximately 75% loss of IAAP binding to the protein (46), a reduction similar to the differences detected here between wild type Mdrl and Mdr2 proteins (Fig. 3B). Several lines of evidence suggest that our findings on Mdr2 in transfected CHO cells are an accurate reflection of functional differ ences between Mdr2 and Mdrl/Mdr3 proteins, rather than a particu targeting, posttranslational protein modification, or folding of an otherwise biologically active protein. Transfection of mdr2 in several additional cell types from distinct anatomical origins, including hepa tocytes derived cell lines (the site of normal mdr2 expression) fails to yield drug-resistant cell clones. Expression of equivalent amounts of Mdrl and Mdr2 proteins in the same cell type confer increased drug resistance only in the Mdrl-expressing cells (Fig. 2, Table 1). Western blotting analysis of highly enriched membrane fractions from mdr2 transfectants shows abundant Mdr2 protein expression in this fraction, suggesting appropriate membrane targeting of the protein (Fig. 3A). The photoactivatable Al? analogue 8-azido-Al? labels to the same extent Mdr2 and Mdrl both expressed at the same level in these membrane fractions (Fig. 3C), suggesting appropriate Mdr2 protein folding in the membrane of these transfected cells. The reduced binding ofthe drug analogue IAAP to Mdr2 detected in the membrane fractions of transfected cells (Fig. 3B) is in agreement with the absence of IAAP binding to Mdr2 expressed in normal liver canalic ular membrane vesicles reported previously by our group (25). Our previous studies with chimeric Mdrl/Mdr2 molecules con structed by exchanging homologous protein domains have indicated that both NB sites of Mdr2 are functional and can complement the biological activity of Mdrl in transfection experiments (16). Although these findings do not establish that the Al?ase activity of Mdr2 is similar to that of Mdrl, they do indicate that both NB sites are interchangeable in these proteins. Likewise, both the highly divergent @2termi@ segment and the linker region of Mdr2 can be substi tuted in Mdrl without loss of Mdrl biological activity (16).@ How ever, the substitution of small or large protein segments overlapping the membrane-associated regions of Mdrl by the homologous do mains of Mdr2 completely abolished the ability of the resulting chimeras to confer MDR, suggesting that these regions are function ally distinct in the two proteins. Similar results have also been obtained with a chimeric human MDR1 protein bearing a small TM2-TM3 segment derived from human MDR2 (47). These data, larity of the in vitro cell expression system used here where the specific cellular background could lead to inappropriate membrane 4896 4 P. Gros and E. Buschman, unpublished observations. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. DRUG BINDING TO Mdr2 together with those reported here, suggest that reduced IAAP binding to Mdr2 compared to Mdrl may be linked to functional differences in the membrane-associated segments of Mdr2. Indeed, the genetic anal ysis of mutant P-gps showing altered substrate specificity has shown that mutations in or near predicted TM domains in both halves of the proteins affect drug transport by modulating the drug-binding char acteristics of the protein, including those of IAAP (27—31,46, 48). Recently, the binding site of IAAP in Mdrl (Mdrlb) has been dc gantly localized by epitope mapping of proteolytic radiolabeled frag ments to two small peptides near predicted TM6 (minor site) and TM12 (major site) of the protein (32, 35, 36). A comparison of the predicted amino acid sequences of Mdrl and Mdr2 in these regions reveals very few nonconservative substitutions (jositions 968—969 and 1002—1006). It is tempting to speculate that these amino acid residues may play a key role in forming a binding pocket for IAAP, which may be disrupted in Mdr2. The functional role of these amino acid residues in drug binding can now be addressed by site-directed mutagenesis and by using the expression system described in this study. Finally, our finding that (a) the ability of Mdr2 to bind 8-azido-Al? is equivalent to that of Mdrl, and that (b) both predicted NB sites of Mdr2 can functionally complement the homologous NB sites of Mdrl (16) are in agreement with the proposal that Mdr2 may act on non-drug substrates. In normal mouse liver, the very abundant and polarized expression of Mdr2 in the canalicular membrane of epithe hal cells of the biliary ductules (25), and the specific phenotype of mouse mutants lacking mdr2, indeed suggest that Mdr2 may transport PC by a lipid flippase mechanism (26). ACKNOWLEDGMENTS The authors thank France Talbot for technical assistance and Dr. R. Kaufman (Genetics Institute, Cambridge, MA) for the generous gift of plas mids pEMC2b1 14. Raymond, M., Rose, E., Housman, D. E., and Gros, P. Physical mapping, amplifi cation, and over-expression of the mouse mdr gene family in multidrug resistance cells. Mol. Cell. Biol., 10: 1642—1651, 1989. 15. Ueda, K., Cardarelli, C., Gottesman, M. 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K@,fhoi, K@,Agresti, M., Tamai, L, Metes, N. D., and bacteriophage T4. Nature (Land.), 227: 680—685,197. 44. Kartner, N., Evernden-Porcelle, D., Bradley, 0., and Ling, V. Detection of P. Roninson, I. B. Molecular basis of preferential resistance to colchicine in multidrug resistant human cells conferred by Gly185 to Val185 substitution in P-glycopmtein. Proc. NaIl. Aced. Sd. USA, 87: 7225-7229, 1990. 4898 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research. The Inability of the Mouse mdr2 Gene to Confer Multidrug Resistance Is Linked to Reduced Drug Binding to the Protein Ellen Buschman and Philippe Gros Cancer Res 1994;54:4892-4898. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/18/4892 Sign up to receive free email-alerts related to this article or journal. 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