[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, ¡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 «-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 37°Cwithout the National Cancer Institute of Canada, and the Fonds de Recherche en Santé medium change. Colonies were fixed with formaldehyde (4% final), du Québec(FRSQ). 2 Recipient of a post-doctoral fellowship from the Deutscher Akademischer stained with méthylèneblue,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 ¡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