Characterization of the Multidrug Resistance Protein Expressed in Cell Clones Stably Transfected with the Mouse Mdrl Cdna1

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[CANCER RESEARCH 49, 2729-2734, May 15, 1989] Characterization of the Multidrug Resistance Protein Expressed in Cell Clones Stably Transfected with the Mouse mdrl cDNA1

Erwin Schurr,2 Martine Raymond,3 John C. Bell,4 and Philippe Gros5

Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Canada, H3GIY6

ABSTRACT of the nucleotide and predicted amino acid sequences of this clone indicated that the encoded polypeptide was most likely a Structural features of the multidrug resistance protein encoded by the membrane glycoprotein, highly similar or identical to P-glyco mouse mdrl gene were studied in multidrug-resistant cell clones stably protein, which contained 12 putative transmembrane domains transfected with a biologically active cDNA clone. Independently derived and a cluster of /V-linked glycosylation sites near the amino transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, terminus. It is formed by the internal duplication of a structural mitoxantrone, VP-16, and vinblastine but remained sensitive to cis- unit which encodes three transmembrane loops and a putative platinum, 5-fluorouracil, Â¡trabinocytosine, and bleomycin. In drug-resist nucleotide binding fold (7). The duplicated segment is highly ant transfectants the mdrl gene product was greatly overexpressed as a homologous to the energy coupling subunit of bacterial trans polypeptide of apparent molecular weight 160,000-170,000. This protein port proteins particularly HlyB, a protein that participates in was present in membrane enriched fractions and could be metabolically the extracellular transport of haemolysin in Escherichia coli labeled with |3H]glucosamine, confirming that the transfected mdrl gene (14). These predicted features strongly suggest that the mdr encodes a membrane glycoprotein. The protein was found phosphorylated protein(s) participates in energy dependent membrane-associ on serine residues and was shown to be photolabeled by both the calcium ated processes, possibly drug efflux. The cloned mdrl cDNA antagonist azidopine and the ATP analogue 8-azido ATP. Tryptic map was capable of conferring a complete multidrug resistance ping of the ATP-photoaffinity labeled protein indicated that ATP cross- linking was site-specific and limited to two discrete peptide fragments of phenotype when introduced and overexpressed in otherwise drug-sensitive LR73 hamster cells (15). the protein, suggesting that the overexpressed mdr protein is capable of direct and specific ATP binding. The independent contribution of different members of the mdr gene family to the multidrug resistance phenotype remains at present unknown. Cell clones stably transfected with full INTRODUCTION length cDNAs for individual members of the mdr family provide an ideal system to study the structural and functional features The emergence of cell populations resistant to several struc of the corresponding mdr protein on an otherwise drug-sensitive turally and functionally unrelated cytotoxic compounds has cellular background. We have used multidrug-resistant mdrl been termed multidrug resistance. This phenomenon has been transfectants to verify structural and functional features of the traditionally studied in highly multidrug-resistant cell lines overexpressed mdr protein, particularly with respect to the ATP obtained in vitro by stepwise selection with a single cytotoxic binding property, which was originally predicted by computer- drug. In these cells, multidrug resistance is associated with a assisted analysis of the full-length cDNA. Our results indicate decreased intracellular drug accumulation, an increased ATP- that the mdrl gene product is expressed in transfectants as a dependent drug efflux, and concomitant overexpression of a membrane phosphoglycoprotein of molecular weight 160,000- high molecular weight membrane glycoprotein(s), originally 170,000 exclusively phosphorylated on serine residues. We termed P-glycoprotein (reviewed in Refs. 1,2). In drug-resistant show, in ligand binding studies, that the overexpressed protein cells this protein(s) was shown to be capable of combining is capable of binding a calcium channel blocker photoactivatable photoactivatable analogues of Vinca alkaloids (3, 4). This bind analogue, azidopine, and the photoactivatable analogue of ATP, ing was specifically inhibited by certain calcium channel block 8-azido ATP. Our results indicate that binding of 8-azido ATP ers (4) known to partially revert the multidrug resistance phe- to the mdr protein is restricted to two discrete peptide frag notype observed in these cells (5,6). The group of overexpressed ments. proteins appears to be encoded by a small family of at least three related genes, termed mdr or P-glycoprotein genes (7-10), that become amplified during drug selection, in multidrug- MATERIALS AND METHODS resistant cell lines of mouse (11), hamster (12), and human Cell Lines and Tissue Culture Conditions. LR73 Chinese hamster origins (13). cells were transfected with the mammalian expression plasmid We have previously reported the cloning of a full length pDREX4, containing the biologically active cDNA insert of phage A cDNA for a transcriptionally active member of the mouse mdr DR11, as previously described (15). Drug-resistant colonies were ini gene family (7), which will be referred to here as mdrl. Analysis tially selected and subsequently maintained in medium containing Adriamycin at 0.1 fig/ml. Cells were cultured in Â«-minimumessential Received 5/11/88; revised 12/12/88; accepted 2/17/89. medium supplemented with 10% fetal calf serum, penicillin (50 units/ The costs of publication of this article were defrayed in part by the payment ml), and streptomycin (50 ng/ml). For drug-survival experiments, 500 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. cells were plated in 60-mm dishes containing increasing concentrations 1Supported by operating grants from the Medical Research Council (MRC), of individual drugs and further incubated for 7 days at 37Â°Cwithout the National Cancer Institute of Canada, and the Fonds de Recherche en SantÃ© medium change. Colonies were fixed with formaldehyde (4% final), du QuÃ©bec(FRSQ). 2 Recipient of a post-doctoral fellowship from the Deutscher Akademischer stained with mÃ©thylÃ¨neblue,and colonies containing more than 50 Austauschdienst (DAAD), Sonderprogramm "Molekulare Parasitologie." cells were scored. The cellular drug resistance was expressed as the D10 3 Recipient of a studentship from National Science and Engineering Research value which is the drug dose necessary to kill 90% of the cells. Actino Council of Canada. ' Recipient of an MRC scholarship. mycin D was obtained from Merck, Sharp and Dohme; Adriamycin ' To whom requests for reprints should be addressed. Department of Biochem from Adria Laboratories; vinblastine from Sigma; mitoxantrone from istry, McGill University, 3655 Drummond, Room 902, Montreal, Quebec H3G Lederle; m-platinum from Bristol; amsacrine was a gift from Dr. J. 1Y6, Canada. Hancock (Hotel Dieu Hospital, Quebec); and 5-fluorouracil, arabino- 2729

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. MULTIDRUG RESISTANCE PROTEIN EXPRESSION cytosine, and bleomycin was obtained from Dr. C. Shustik (Royal presence of buffers adjusted to pH 1.9 (88% formic acid, acetic acid, Victoria Hospital, Montreal). water, at a 50:156:1794 ratio) and pH 3.5 (as above), respectively. Membrane Preparation. Cells grown to confluency in roller flasks were harvested by gentle treatment with Dulbecco's PBS6 containing sodium citrate (20 HIM) and lacking magnesium and calcium. Cells RESULTS were washed twice, lysed in hypotonie medium (10 mM TRIS, pH 8.0; 1 mM MgClj) using a Dounce type of homogenizer and nuclei were Drug-sensitive LR73 cells were transfected with the biolog eliminated by centrifugation through a cushion of 45% sucrose. In some ically active cDNA clone of X phage DR11, constructed in the experiments membranes were further purified by ultracentrifugation of expression vector pDREX4 (15), and drug-resistant colonies the crude membrane extract on a step gradient containing sucrose at were selected in Adriamycin. Three independent colonies concentrations of 60, 45, 35, and 30%. Membranes floating on the 45 (clones 1A, 5, 8) were expanded in culture and their degree of and 35% interfaces were collected and stored frozen at -80"C in PBS cross-resistance to several drugs was quantitated. Results of containing 10% glycerol. Membrane-enriched fractions prepared by this analysis are shown in Table 1 for clone 1A. Similar results this procedure showed no detectable lactate dehydrogenase activity, a cytoplasm Â¡cenzyme,and a 25-fold increase of 5'-nucleotidase activity, were obtained for the two other independent transfectants (data not shown). Adriamycin-resistant cell lines stably transfected a marker enzyme of plasma membranes (as measured by assay systems purchased from Sigma diagnostics). with the mouse mdrl cDNA expressed the classical pleiotropic drug-resistance phenotype: they were resistant to anthracy- Metabolic and Photoaffinity Labeling. For metabolic labeling with [35S]methionine, cell cultures at 50 to 60% confluency were incubated clines, Vinca alkaloids, actinomycin D, VP-16, amsacrine, and in methionine free a-MEM medium containing 10% dialyzed fetal calf mitoxantrone but remained susceptible to drugs that are not serum, 5 mM L-glutamine and [35S]methionine at 100 /jCi/ml for a traditionally part of the multidrug resistance spectrum such as minimum of 4 h. For glucosamine labeling, the cells were incubated for bleomycin, civ-platinum, arabinocytosine, and 5-fluorouracil 12 h in low glucose (2 mM) RPMI medium, containing 10% dialyzed (Table 1). Although small clone-to-clone variations were ob fetal calf serum and [3H]glucosamine at 25 nC\/m\. For phosphorylation served, the overall level of drug resistance of the transfectants experiments, cells were labeled in phosphate-free medium containing varied between 7- (amsacrine) and 30-fold (mitoxantrone). 600 ÃÃCi/mlof[32P]orthophosphate for 12 h. In all experiments labeled Extensive cDNA sequence homology indicates that the cells were washed twice with PBS before immunoprecipitation. For cloned mouse mdrl cDNA and hamster pgp cDNAs (P-glyco- azidopine photoaffinity labeling, membranes (2 mg/ml) were incubated in the dark at 20' ( for 1 h in a buffer containing 0.04 M potassium protein genes) are members of the same gene family (8). There phosphate (pH 7.4), calcium chloride (1 x 10"' M), dimethyl sulfoxide fore, we used the anti-hamster P-glycoprotein monoclonal an (0.1%) and [3H]azidopine (0.7 x IO"6 M; specific activity, 3 Ci/mmol). tibody C219 (18) in an attempt to identify in transfectants the For competition experiments, 0.7 ^M ['HJazidopine was incubated for protein encoded by the mdrl cDNA. LR73 hamster control 1 h at 20Â°Cwith membrane fractions (2 mg/ml) in the presence of 50 cells and drug-resistant transfectants 1A, 5, and 8 were labeled with [35S]methionine and total cellular proteins were immuno MMnifedipine, 50 Â¿iMverapamil, or 50 ^M vinblastine. The mixtures were then irradiated with UV light 20 min at 0 or 20Â°C,according to precipitated with the C219 antibody (Fig. \A). A specific pro the procedure described by Safa et al. (16). For 8-azido ATP photo- tein species of apparent molecular weight of 160,000-170,000 affinity labeling, membranes (1 mg/ml in PBS containing [32P]8-azido was detected in the three transfectant cell clones but was un ATP at 100 nCi/ml; specific activity, 8 Ci/mmol) were irradiated for 5 min at 0Â°Cwithan ultraviolet source at 254 nm (General Electric) at a detected in the control, drug sensitive, LR73 cells. To determine whether mdrl was expressed as a membrane protein in trans distance of 4 cm. fectants, metabolic labeling with [35S]methionine was carried Immunoprecipitation, SDS-PAGE, and Immunoblotting. All inumi out on cells from clone 1A and membrane enriched fractions noprecipitations were carried out essentially according to the protocol described by Greenberger et al. (17) with the anti-P-glycoprotein mono were prepared by ultracentrifugation on discontinuous sucrose clonal antibody C219 (18) which was generously provided by Dr. V. gradients. The labeled membrane fractions were immunopre Ling (Ontario Cancer Institute, Toronto). After incubation of radiola- cipitated and analyzed for the presence of the mdr protein by beled protein mixtures with the C219 antibody, immune complexes SDS-PAGE. Results presented in Fig. IB (Lanes 1 and 2) were isolated with protein A-sepharose beads. SDS-PAGE was carried indicate that the specific protein was present in membrane- out according to Laemmli (19), except that the samples were not heated enriched fractions sedimenting on the 35 and 45% sucrose prior to loading on the gel. Immunoblotting was carried out with the gradient fractions. Moreover, when equal amounts of proteins C219 monoclonal antibody according to Towbin et al. (20). Immune obtained from whole cell lysates and membrane enriched frac complexes were visualized by incubating the blot with I25l-protein A tions were analyzed by immunoblotting, an approximately 20- followed by autoradiography. fold enrichment for the mdrl gene product was observed in the Tryptic Mapping and Phosphoamino Acid Determination. For tryptic membrane-enriched fraction (Fig. 1C). Finally, the radiolabeled mapping, the ATP cross-linked immunoprecipitated protein was elec- mdr protein could also be immunoprecipitated from membrane- troeluted from the gel, trichloroacetic acid-precipitated, and incubated enriched fractions prepared from cells grown in medium con with performic acid for 1 h on ice. After removal of residual performic taining [3H]glucosamine (Fig. IB, Lanes 3 and 4). Taken to acid, the oxidized protein was digested with trypsin (5 mg/ml) at 37Â°C for 16 h. Digestion products were spotted onto cellulose thin-layer gether these results confirm that in the mdrl transfectants, the plates (Polygram eel 400; Machery-Nagel) and electrophoresed for 30 encoded protein is expressed as a membrane glycoprotein. min at 1000 V, in buffer containing pyrid inc. acetic acid, and water at Phosphorylation has been suggested as a posttranslational a ratio of 10:100:1890 (pH 3.5). The second-dimensional thin-layer modification implicated in the regulation of P-glycoproteins chromatography was carried out in the presence of n-butanol, pyridine, (21, 22). Metabolic labeling of LR73 and drug-resistant clone water, and acetic acid in a 4:3:3:1 ratio. For phosphoamino acid 1A cell cultures with [32P]orthophosphate followed by immu determination, the immunoprecipitated 32P-labeled protein was re noprecipitation of whole cell lysates with the C219 antibody covered from the gel and hydrolyzed at 100Â°Cfor 2 h with double- indicated that the overexpressed mdr protein encoded by the distilled 6 N HC1. Phosphoamino acids were separated by two-dimen transfected gene was phosphorylated in this clone (Fig. 2A). sional electrophoresis on cellulose thin-layer plates (eel 300) in the Subsequent phosphoamino acid analysis shown in Fig. 2/f 'The abbreviations used are: PBS: phosphate buffered saline; SDS-PAGE, indicated that the protein was phosphorylated exclusively on sodium dodecyl sulfate polyacrylamide gel electrophoresis. serine residues whereas no radioactivity was found associated 2730

Table 1 Drug sensitivity (DIO) of cell line LR73 and a drug resistant cell clone transfected with mdrl cDNA Drug-sensitive LR73 Chinese hamster cells and the mdrl transfectant clone 1A were examined for their level of susceptibility to various drugs as described in "Materials and Methods." The DIDwas defined as the drug dose necessary to kill 90% of the cells plated in Adriamycin (ADM), vinblastine (VBL), etoposide (VP- 16), actinomycin D (ACTD), amsacrine (AMSA), mitoxantrone (MIT), cu-platinum (C1SP), 5-fluorouracil (5FU), arabinocytosine (ARAC), and bleomycin (BLEO). Drug doses for ADM, VBL, VP-16, ACTD, AMSA, and MIT are expressed in nanograms per milliliter and in micrograms per millilitri for CISP, 5FU, ARAC, and BLEO. Drugs tested Cell type ADM VBL VP16 ACTD AMSA MIT CISP 5FU ARAC BLEO LR73 LR73/1A28 25022 3604.5 2376 6403.5 210.64 210.95 1.030.9 2.13.4 4.51.10 1.05 with phosphothreonine or phosphotyrosine (Fig. 2B). tides could be identified after digestion with trypsin, suggesting Multidrug resistance can be partially overcome by exposing that coupling of the ATP analogue to the protein was site- drug-resistant cells to a group of calcium channel blockers, specific and restricted to two binding sites in the molecule. The including verapamil (5, 6). Since these compounds can specifi difference in intensity of the two labeled tryptic peptides sug cally compete with the binding of photoactivatable drug ana gests that the probe binds to the two sites with different affinity. logues to P-glycoprotein (4, 27), it is speculated that they share The participation of sequences overlapping one or both of the a common or overlapping binding site on this protein. To predicted nucleotide binding folds in the binding of 8-azido determine whether the M, 170,000 polypeptide encoded by the ATP is currently investigated by site directed mutagenesis ex transfected mdrl gene is capable of binding calcium channel periments. blockers, photoactivatable [3H]azidopine (16) was cross-linked by UV irradiation to membrane-enriched fractions from both drug-resistant 1A mdrl transfected cells and drug-sensitive DISCUSSION LR73 control cells. Separation of the total labeled protein A large body of biochemical data on multidrug resistance has fraction by SDS-PAGE shows one predominant band at = M, been obtained from the study of highly drug-resistant cell lines 170,000 specifically in the transfected cells (Fig. 3A, Lane 2). produced by stepwise selection procedures (1,2). These studies Immunoprecipitation of these labeled membrane extracts with have consistently linked the emergence of drug resistance to the the C219 antibody confirmed that the protein predominantly amplification and overexpression of one or more members of labeled in 1A cells was expressed by the transfected mdr gene the mdr/P-glycoprotein gene family. We have shown that a full- (Fig. 3A, Lane 4). These results suggested a high specificity of length cDNA clone for one of these genes, mdrl, can efficiently [3H]azidopine binding to the mdrl gene product. The specificity confer drug resistance to otherwise drug-sensitive cells in trans- of azidopine binding was further investigated in a series of fection experiments (15). We feel that the study of multidrug competition experiments employing nifedipine and verapamil, resistant cell clones obtained by direct transfection of a biolog and the chemotherapeutic drug vinblastine as specific compet ically active cDNA presents distinct advantages: (a) drug-re itors (Fig. 3Ã„).As is shown in Fig. 3Ã„all three drugs success sistant transfectants are obtained by single-step drug selection, fully competed with azidopine for binding to the mdrl protein therefore minimizing the risk that additional genetic altera suggesting that these compounds bind to identical or overlap tions, resulting from long periods of exposure to high drug ping sites on the mdrl protein. concentrations, may alter the phenotypic expression of mdr Nucleotide sequence analysis of the mouse mdrl cDNA (7) proteins; (Â¿>)multiplemembers of the mdr family are amplified indicated the presence of two consensus sequences for nucleo- in drug resistant cell lines (11-13) and it is necessary to intro tide binding folds (23), which have also been reported in a duce and express individual mdr cDNA clones in drug sensitive number of bacterial and mammalian proteins associated with cells to clarify the independent contribution of each of these energy dependent functions (23, 24). To determine whether the genes to the final resistance phenotype; (c) functional analysis mature mdr protein is indeed capable of combining ATP, we of the individual mdr protein(s) can be greatly enhanced by the have used the photoactivatable ATP analogue 8-azido ATP to use of site-directed mutagenesis of the corresponding cDNA photoaffinity label the protein. Membrane-enriched fractions and subsequent testing of mutant clones in transfection exper of clone 1A drug-resistant cells were prepared and the photo- iments. affinity probe ["P]8-azido ATP was cross-linked to membrane In this paper we report on the characterization of the mouse proteins by irradiation with UV light. Immunoprecipitation of mdrl gene product in cell clones stably transfected with the the reaction mixture indicated the presence of a strongly labeled corresponding cDNA. In these cells the protein is overexpressed polypeptide in the membrane fraction of 1A cells (Fig. IB, Lane as a membrane glycoprotein of approximately Mr 170,000, in 7) which was not detected in LR73 control cells (data not agreement with the predicted sequence of the protein deduced shown). In this experiment, [35S]methionine and [3H]glucosa- from the cDNA (7). The protein encoded by the transfected mine labeled mdr proteins were used as positive controls in mdrl gene is recognized by the anti-hamster P-glycoprotein Lanes 5 and 6, respectively. The presence of 1 mM ATP inhib monoclonal antibody C219 (18), confirming that the two pro ited cross-linking of the photoaffinity probe, whereas 10 mM teins are encoded by closely related members of the same gene ADP had no effect on 8-azido-ATP coupling (data not shown). family, as previously suggested by the strong nucleotide se To ascertain that coupling of the ATP analogue was specific quence homology of the respective cloned cDNA (8). P-glyco- and restricted to a discrete portion(s) of the protein, the pho- proteins(s) have been previously reported to be phosphorylated tolabeled protein was eluted from the gel, submitted to digestion in highly multidrug-resistant cell lines: in multidrug-resistant with trypsin, and the digestion products were separated by derivatives of the mouse macrophage line J774 (25), phospho- sequential electrophoresis and chromatography on cellulose amino acid analysis reveals that the protein is phosphorylated plates. Results presented in Fig. 4 were reproduced in three on both serine and threonine residues, while in human multi- independent experiments. Only two discrete radiolabeled pep- drug-resistant K562/ADM leukemic cells, phosphorylation is 2731

1234 1 2 A B 200- IIÂ» 200-

97-

69- P-S- 97- P-T-

68-

P-Y-

43-

30-

Fig. 2. Phosphorylation of the mdrl protein. .-I. drug-sensitive LR73 and multidrug-resistant Â»i

restricted to serine residues (26). In multidrug-resistant mdrl M i-i transfectants the overexpressed mdr protein appears to be phos phorylated solely on serine residues. A possible explanation for differences in the phosphoamino acid content is that threonine phosphorylation of the protein is specific to the macrophage line used in those studies. Alternatively, threonine phosphoryl ation may be limited to an antigenically related protein species encoded by another member of the mÃ¼rgene family. Indeed, drug-resistant J774 cells selected with different drugs are known to express antigenically cross-reactive but distinct mdr proteins (17). â€¢¿- Multidrug resistance can be partially overcome if cells are grown in the presence of calcium channel blockers (S, 6). Such

sensitive LR73 cells. B, cells from the m

Fig. 3. Cross-linking of azidopine to the mdrl protein. A, membrane-enriched fractions were prepared from both drug-resistant 1A and B drug-sensitive LR73 cells and UV-irradiated in the presence of [3H]azidopine. Lanes 1 and 1234 12345 2, profiles of labeled membrane proteins from LR73 and 1A cells, respectively. Proteins from |'H]azidopine cross-linked membrane frac tions were immunoprecipitated with the anti body C219: Lane 3, LR73 cells; Lane 4, drug- resistant 1A cells. Arrow, location of a trans fectant-specific [3H]azidopine-labeled protein with apparent molecular weight of 170,000. B, competition of azidopine binding to membrane fractions from multidrug resistant 1A cells. Lanes 4 and 5, protein profiles of membranes from 1A and LR73 cells, respectively, which were cross-linked to azidopine by UV irradia tion at 20Â°C.The binding of azidopine to a M, 170,000 protein in IA membranes (arrow) could be competed out by verapamil (Lane 1), vinblastine (Lane 2), and nifedipine (Lane 3).

yet to determine if these two peptides correspond to fragments overlapping the two predicted nucleotide binding folds in the mdr protein or correspond to photoaffinity labeling of two TLC peptides surrounding a single functional nucleotide binding fold. It is interesting to note that the tryptic map of the photoaffinity labeled mdr protein reported here is similar (showing a weakly and a strongly labeled peptide) to that of 8- azido ATP photolabeled E. coli RecB and RecD proteins (30), two proteins which each contains the consensus sequence for a nucleotide binding fold present in mdrl (31, 32). Studies presented in this paper describe the model system we Fig. 4. Tryptic map of 8-azido-ATP coupled mdrl protein. ATP-photoaffinity wish to utilize to characterize the functional role and structural labeled mdr protein was subjected to complete digestion with trypsin and digestion features of individual members of the mdr gene family: we have products were separated by electrophoresis (first dimension) and chromatography (second dimension). For electrophoresis, the origin (O) and positions of the anode recently obtained the complete nucleotide sequence of a second and cathode are indicated . Arrow, direction of thin layer chromatography (TLC); transcriptionally active member and have identified a third arrow heads, two photoaffinoty labeled tryptic peptides. transcriptionally active mdr gene.1 The systematic study of these genes in transfection experiments using full length cDNA treatment results in an increased intracellular drug accumula clones should clarify their role in the establishment of multidrug tion in these cells, presumably because calcium channel blockers resistance and may help clarify their role in normal physiolog directly compete with drug molecules for the drug efflux pump. ical processes. The suggestion that calcium channel blockers and chemotherapeutic drugs share a common or overlapping binding site on ACKNOWLEDGMENTS the efflux pump is supported by the observation that either group of compounds can compete with the binding of photoac- We thank Dr. Victor Ling for the gift of antibody C219, Drs. Cliff tivatable analogues of the other to P-glycoprotein (4, 27). In Stanners, Gordon Shore, and Rose Johnstone for critical review of this these studies, [3H]azidopine bound specifically to at least two manuscript and Margaret Licorish for skillful secretarial assistance. (16) and possibly three (27) antigenically related overexpressed protein species. Moreover, binding of [3H]azidopine was highly REFERENCES specific and limited to one tryptic fragment of the protein (27). It is tempting to speculate that the proteins identified in those 1. Gerlach, J. H., Kartner, N., Bell, D. R., and Ling, V. Multidrug resistance. Cancer Surv., 5: 25-46, 1986. studies are encoded by closely related but distinct members of 2. Pastan, I., and Gottesman, M. M. Multiple-drug resistance in human cancer. the mdr gene family. Results from our study clearly indicate N. Engl. J. Med., 316: 1388-1393, 1987. that the protein encoded by the transfected mdrl cDNA is 3. Safa, A. R., Glover, C. J., Meyers, M. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. Characterization of the Multidrug Resistance Protein Expressed in Cell Clones Stably Transfected with the Mouse mdr1 cDNA

Erwin Schurr, Martine Raymond, John C. Bell, et al.

Cancer Res 1989;49:2729-2734.

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