[CANCER RESEARCH 50. 1779-1785. March 15, 1990] Multidrug Resistance Phenotype of Human BRO Melanoma Cells Transfected with a Wild-Type Human mdrl Complementary DNA1 Carsten R. Lincke, Alexander M. van der Blick, Gerrit J. Schuurhuis, Trijntje van der Velde-Koerts, Jaap J. M. Smit, and Piet Borst1 Division of Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Aether/ana's [C. R. L., A. M. v. d. B., T. \: d. V-K., J. J. M. S., P. B.]; Department of Biochemistry; L'niversity of Amsterdam, 1105 AZ Amsterdam, The Netherlands f( '. K. I..J; and Department of Oncology, Free L'nirersily of Amsterdam, 1007 MB Amsterdam, The Netherlands [G. J. S.¡ ABSTRACT We have transfected a eukaryotic expression vector containing a mdr\ complementary DNA isolated from normal human liver into human BRO melanoma cells to study the drug-resistant phenotype produced by the exclusive overexpression of normal human mdr\ P-glycoprotein. The drug resistance pattern of mdr\ -transfected clones includes relatively high resistance to gramicidin D (about 300-fold), vincristine (about 100- fold), and actinomycin D (about 100-fold) and a lower degree of resistance to doxorubicin (about 10-fold), VP16-213 (about 10-fold), and colchicine (about 6-fold). The transfectants did not exhibit resistance to trimetrex- ate, CIS-platinum, mitomycin C, l-/3-i)-arabinofuranosylcytosine, bleo- mycin, G418, or magainin-2-amide; they were slightly more sensitive to verapamil (2-fold) but not to Triton \-100. As in other multidrug- resistant cell lines, resistance to vincristine could be reversed by verapa mil and, more effectively, by cyclosporin A. Chloroquine only marginally increased drug sensitivity in mdr\ -transfected cells. Gramicidin D resist ance was also reversed by verapamil, suggesting that the mechanism of resistance to this polypeptide antibiotic is similar to that of other drugs transported by P-glycoprotein. Thus, expression of the wild-type mdrl complementary DNA induces a drug-resistant phenotype similar to that induced by mdrl complementary DNAs isolated from drug-resistant cell lines with relatively low colchicine resistance. As other cell lines may display a different pattern of drug resistance, it is clear that other resistance mechanisms or cell type-specific factors may modulate the resistance. mdr\ -transfected cell lines provide a convenient tool for the identification of P-glycoprotein-mediated phenomena. INTRODUCTION The MDR4 phenotype of mammalian cell lines selected for drug resistance in vitro by exposure to a single chemotherapeu- tic agent can be brought about by overproduction of a P- glycoprotein (1-4). Direct experimental evidence for this has been obtained by DNA- and chromosome-mediated transfer of the MDR phenotype from drug-resistant to drug-sensitive re cipient cells (5-9) and by transfection of cDNAs of a mouse mdr gene (the homologue of hamster pgp2) and the human mdr\ gene derived from drug-resistant cells (9-14). P-Glyco- proteins appear to act as membrane-bound ATP-consuming drug-efflux pumps, capable of transporting a wide variety of structurally and functionally unrelated substances across the plasma membrane (10, 15-17). Received 8/2/89; revised 12/1/89. 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. 1This work was supported in part by Grant NKI 88-6 of The Netherlands Cancer Foundation to P. B. : Present address: Division of Biology. 156-29, California Institute of Tech nology, Pasadena. CA 91125. 3To whom requests for reprints should be addressed, at the Division of Molecular Biology, The Netherlands Cancer Institute. Plcsmanlaan 121. 1066 CX Amsterdam. The Netherlands. 4The abbreviations used are: MDR. multidrug resistance; SDS, sodium do- decyl sulfate; SSC, standard saline citrate (150 mM NaCI:15 mM sodium citrate. pH 7); PBS. phosphate-buffered saline (137 mM NaCI:2.68 mM KCI:8.06 mM NajHPO.:1.47 mM KH^O«); cDNA. complementary DNA; 1C,», concentration of drug at 50% growth inhibition compared with cells grown in the absence of drugs as determined in a 5-day growth inhibition assay in the continuous presence of drugs. The cross-resistance patterns and the relative resistance of MDR cell lines that overexpress P-glycoprotein may vary con siderably, but include resistance to colchicine, anthracyclines, Vinca alkaloids, and actinomycin D in all cases examined so far (1). In a few cases cross-resistance to various other drugs, like melphalan (18, 19), methotrexate (20). and trimetrexate (21) has been found in MDR cell lines selected for drug resist ance in vitro. Furthermore, reversal of MDR by calcium channel blockers like verapamil, most likely due to competition for binding to P-glycoprotein, is consistently observed in MDR cells (reviewed in Refs. 3 and 4). Thus far, it is not clear what factors contribute to or deter mine the variability of the MDR phenotype. A point mutation in the human mdrl gene has been shown to selectively increase the degree of resistance for the drug used for selection (22). Other mechanisms expected to modulate the cross-resistance pattern of MDR cells are the differential (over-)expression of multiple or differentially spliced P-glycoprotein genes (23, 24) that might differ in substrate specificity. "Atypical" MDR cell lines have also been described. These do not overexpress P- glycoprotein, and their cross-resistance patterns only partially match the P-glycoprotein-mediated pattern (25-28). Although this type of drug resistance can be due to altered topoisomerase II activity (29, 30), this does not appear to explain the atypical MDR phenotype of all cell lines described (27, 28). The cross- resistance pattern of a MDR cell might also be a composite of several of these mechanisms acting in concert. To distinguish between effects mediated by the wild-type mdr\ gene and those of other alterations we transfected a full- length human mdrl cDNA from normal human liver into BRO melanoma cells. Here we describe the MDR phenotype of these transfectants. MATERIALS AND METHODS Cell Culture. The transfection of human BRO melanoma cells (31) with a full-length mdrl cDNA was previously reported (32). This cDNA was inserted into a eukaryotic expression vector providing the imme diate early gene promoter-enhancer of human cytomegalovirus up stream of the cDNA and, downstream, the hepatitis B virus polyade- nylation signal, kindly provided by Dr. F. Meyer (Zurich, Switzerland). BRO cells were grown in Dulbecco's modified Eagle's medium supple mented with 4 mM l.-glutamine, penicillin (50 units/ml), streptomycin (50 Mg/ml), and 10% fetal calf serum in the presence of 5% CO2. Subculture was every 3 to 4 days. Transfected clones were selected and expanded in the presence of 10 n\t vincristine. The non-small cell lung carcinoma cell lines SW-1573, originally isolated by Dr. A. Leibovitz (Temple, TX), and SW-1573-500 (33) were cultured as described (34). The human ovarian carcinoma cell lines A2780 and 2780AD (35) were obtained from Dr. R. F. Ozols (Bethesda. MD), and were grown in RPMI 1640 with 10% fetal calf serum, the latter in the presence of 2 JIMdoxorubicin. Chemicals. Doxorubicin (doxorubicin hydrochloride), chloroquinc. colchicine, cytarabine (1-ß-D-arabinofuranosylcytosine), gramicidin D, lidocaine. melphalan, and vincristine sulfate were purchased from 1779 Research. on December 3, 2021. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from