(CANCER RESEARCH 48. 6348-6353, November 15. 1988)

Increased mdr Gene Expression and Decreased Drug Accumulation inMultidrug-resistant Human Melanoma Cells1

Jeffrey F. Lemontt,2 Marie Azzaria, and Philippe Gros

Integrated Genetics, inc., Framingham, Massachusetts 01701 [J. F. L.J, and Department of Biochemistry, McGill University, Montreal, Quebec,Canada H3G 1Y6 [M. A., P. GJ

ABSTRACT

Multidrug-resistant clones of a drug-sensitive human malignant melanoma cell line were isolated by single-step selection in culture mediumcontaining either vincristine (4.5 ng/ml or 7.5 ng/ml), vinblastine (3 ng/ml), or colchicine (8 ng/ml). This protocol yielded primary coloniesshowing relatively low (4- to 24-fold) levels of drug resistance. Theseclones exhibit the classical multidrug resistance (MDR) phenotype, beingcross-resistant to Vinca alkaloids, anthracyclines, colchicine, and acti-nomycin D. The appearance of an MDR phenotype in these cells waslinked to a decreased accumulation and increased efflux of the drug|]H|vinblastine when compared to the drug-sensitive melanoma cell line.

This increased drug efflux was dependent on the presence of cellularATP and could be reduced by treatment of the cells with rotenone anddeoxyglucose. A partial human mdr complementary DNA clone was usedto monitor the degree of amplification and the level of transcription ofthis gene in the cloned lines. All 5 MDR sublines expressed increasedlevels of the specific 4.5-kilobase mdr mRNA, but did not show mdrgene amplification. Our results indicate that relatively low levels of drugresistance, similar to those observed clinically and in experimental xeno-grafts, can be achieved by single-step drug selection and result fromincreased expression of at least one member of the mdr gene family.

INTRODUCTION

The appearance of a drug-resistant phenotype in tumor cellsis a major impediment to the effective chemotherapeutic treatment of human cancers (for review, see Ref. 1). MDR3 cells aretypically cross-resistant to a wide range of structurally andfunctionally unrelated cytotoxic agents to which they have notbeen previously exposed (2). Because toxicity for nontumortissues frequently limits therapeutic use of these agents to anarrow dose range, tumors acquiring even low-level drug resistance may become refractory to further clinical treatment. Inorder to design better treatment protocols and drug moleculescapable of circumventing the MDR phenotype, it is importantto develop in vitro MDR cell lines obtained from different tissuetypes and showing relatively low levels of resistance.

Previous in vitro studies of mammalian cell lines have shownthat MDR is most often associated with a decreased intracel-lular drug accumulation, an increased ATP-dependent drugefflux, gene amplification, and overexpression of a group of M,170,000 plasma-membrane glycoproteins designated P-glyco-proteins or gpl70 (2). We and others have shown that theseproteins are encoded by a family of at least 3 closely relatedmdr genes, for which molecular probes have recently beenisolated (3-10). Transfection and overexpression of a singleintact, full-length mdr cDNA clone are sufficient to confer the

Received 4/28/88; revised 8/15/88: accepted 8/18/88.The costs of publication of this article were defrayed in pan by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by National Cancer Institute Contract N43-CM-73717 to Inte

grated Genetics and by grants from the National Cancer Institute and the MedicalResearch Council of Canada to P. G.

1To whom requests for reprints should be addressed, at Integrated Genetics,

Inc., One Mountain Road, Framingham, MA 01701.'The abbreviations used are: MDR. multidrug-resistant or multidrug resist

ance; mdr, MDR-associated gene symbol; cDNA, complementary DNA; PBS,phosphate-buffered saline; RPE, relative plating efficiency; TE. 10 HIMTris-HCl(pH 8.0): 1 mM EDTA; SSC, standard saline citrate; SDS, sodium dodecyl sulfate.

complete MDR phenotype to otherwise drug-sensitive cells (3).Analysis of the predicted amino-acid sequence of the biologically active mouse mdr cDNA indicates that the encodedpolypeptide is most likely a membrane glycoprotein, formed bythe internal duplication of a structural unit that encodes threetrans-membrane loops and one putative ATP binding site. Thispredicted protein is highly homologous to several transportproteins found in the periplasm of Gram-negative bacteria (4).

In vitro studies of the mechanism of multidrug resistance inmammalian cell lines have relied mostly upon the use of rodentcell lines selected for high-levels of drug resistance (2). Fewstudies have attempted to identify putative cellular mechanismsresponsible for low, clinically relevant levels of multidrug resistance. Although elevated mdr gene expression has been observed in some human tumors and normal tissues (11, 12),other mechanisms unrelated to increased P-glycoproteinexpression may also contribute to the occurrence of MDR inhuman tumor cell lines (13, 14). In addition, the degree ofinitial responsiveness and the likelihood of appearance of MDRcells might differ greatly with tumor cells from different anatomical sites.

To tackle this problem, we have decided to create and characterize a series of multidrug-resistant cell lines starting withdrug-sensitive parental lines of different tissue types, usingselection in media containing low concentrations of anthracyclines. Vinca alkaloids, or other drugs. In this paper we reportthe isolation and characterization of 5 multidrug-resistantclones, each independently derived from a human malignantmelanoma cell line (Bowes melanoma).

MATERIALS AND METHODS

Cell Culture. We have used the established human malignant melanoma cell line called Bowes melanoma, originally isolated as cell lineRPMI 7932 by Dr. George E. Moore (Denver General Hospital) in1972 from pleural fluid of a patient (female, age 26) with disseminatedmalignant melanoma (IS). Cells were grown in ..minimal essentialmedium containing 10% fetal calf serum (Gibco), 5 mM L-glutamine(Giòco),penicillin (50 units/ml), streptomycin (50 ^g/ml), as well asribonucleotide and deoxyribonucleotide precursors. Cells were fed every72 h and were grown in a standard tissue culture incubator at 37°Cin

the presence of 5% CO2. Cells were passaged every 6 days at a densityof 5 x 10* cells/ml in 75-cm2 flasks. Confluent cell cultures werewashed once with prewarmed PBS-citrate and then treated for 2 minat 37"C with PBS-citrate containing trypsin (0.5 mg/ml) and EDTA

(pH 8.0) (0.2 mg/ml). Cells were washed twice with complete mediumbefore seeding to new flasks. For frozen stocks, cells were prepared asabove and then resuspended at a concentration of 2 x IO'1cells/ml in

ice-cold complete medium containing 10% dimethyl sulfoxide. Cellstocks were kept frozen at —¿�90°C.Mouse leukemic LI210 cells and

their daunorubicin-resistant L1210 DN derivative were obtained fromDr. J. Croop (M. I. T., Cambridge, MA).

Cytotoxic Drugs. Stock solutions of the following drugs were prepared in sterile distilled water, aliquoted, and kept frozen at -20°Cin

the dark: 2 mg/ml of daunorubicin (Rhône-Poulenc); 1 mg/ml ofdoxorubicin hydrochloride (Adria Laboratories); 1 mg/ml of vinblastinesulfate (Eli Lilly); 1 mg/ml of colchicine (Sigma); 0.1 mg/ml of vincristine sulfate (Eli Lilly); and 0.05 mg/ml of actinomycin D (Merck,Sharp, and Dohme). Stocks were quickly thawed, used immediately,

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MULTIDRUG-RESISTANT HUMAN MELANOMA CELLS

and refrozen not more than 10 times, after which the tube was discarded.Determination of Drug Resistance Levels. Exponentially growing cell

cultures (5 day) were harvested by trypsinization, washed, counted in ahemocytometer, and resuspended at 5 x IO3 cells/ml. Five hundred

cells were seeded in 6-well titer plates containing 3 ml of mediumsupplemented with increasing concentrations of cytotoxic drugs. Allplatings of cells were in triplicate. The drug-containing medium wasreplaced every 4 days, the cells being allowed to grow for a total of 14days. Colonies were fixed for 4 h at 20"C in a solution containing 3.7%

formaldehyde and stained for 5 min with 0.1% méthylèneblue. Plateswere washed with tap water and allowed to dry. Colonies containing50 cells or more were scored as survivors.

The RPE of each cell line was calculated by dividing the meannumber of colonies observed at a given drug concentration by the meannumber of colonies formed by the same cell line in control mediumlacking the drug. This ratio was expressed as a percentage and plottedversus the drug concentration. The DIOvalues correspond to the drugconcentrations needed to reduce the RPE to 10%. The relative resistance of a subline at 10% RPE was calculated by dividing the Du, valueby that of the parental Bowes melanoma line.

Drug Transport Determinations. Cells were harvested with trypsinand allowed to stand 2 h at room temperature in complete mediumbefore starting the assays. Depletion of ATP was performed by incubating the cells 15 min at 37°Cin PBS with 20 ng/ml of rotenone,

followed by addition of 2-deoxyglucose to 2 mM for another 10 min at37°C.The extent of ATP depletion was determined by measuring levels

of intracellular ATP with a luciferase-based assay (16). Samples thatwere not ATP depleted were incubated at 37°Cin PBS supplemented

with 1 mM glucose and 1 mM glutamine. The ATP depletion treatmentsand control treatments were performed at 37°Cto optimize metabolic

ATP depletion of cells, while minimizing exposure of the cells torotenone and 2-deoxyglucose. By contrast, all uptake and efflux measurements were performed on cells incubated at room temperatureinstead of 37°C,so that the resulting slower transport rates could be

measured more accurately. In all cases, control and drug-resistant celllines were treated identically.

Uptake. At specific intervals after mixing the cells in PBS with[3H]vinblastine (specific activity, 11.5 Ci/mmol; Amersham) to a finalconcentration of 75 nM, 0.5 ml of the mixture (IO6 cells) were layeredonto 0.2 ml of a silicone oil:mineral oil mixture (4:1, v/v) and centri-fuged 10 s at 12,000 x g. The aqueous phase and oil were removed,and the cell pellet was digested overnight at room temperature in NNaOH. Samples were removed the next day for analysis of tritiumactivity and protein concentration. Uptake data were expressed as pmolof vinblastine per mg of protein in the cell pellet.

Efflux. All cells were depleted of ATP before loading them with 20nM [3H]vinblastine for l h at room temperature. The cells were centri-

fuged, and efflux was initiated by resuspending the cell pellet in PBS(either containing or lacking 6 mM glucose and glutamine) and incubating at room temperature. At specific times 0.35 ml of the cellsuspension (1.5 x IO6cells) were layered onto the silicone/mineral oil

mix and centrifuged for 10s at 12,000 x g. The aqueous medium (0.2ml) remaining over the oil was removed for tritium counting. Effluxdata were calculated as the concentration of vinblastine appearing inthe supernatant per julof intracellular water (water-space estimate) andexpressed as the percentage of maximum drug that could be expelled.The initial I min time point was subtracted from all time course pointsto eliminate the counts due to the rapid nonspecific binding of the drugto cell membranes.

For water-space estimates, an aliquot of each cell suspension wasincubated with either [14C]carboxymethyldextran or [3H]water for 5 min

at room temperature. Cells were centrifuged through silicone/mineraloil, and aliquots of the supernatant were analyzed for tritium activity.Cell pellets were processed as described previously. The intracellularwater space was calculated by subtracting the counts associated withthe [l4C]carboxymethyldextran (extracellular space) from the counts

arising from the tritiated water (intracellular and extracellular spaces).Human MDR Hybridization Probe. The probe used in Northern and

Southern analyses was an approximate 600-bp human mdr cDNAclone, isolated from a human fetal liver cDNA library constructed inphage Xgtl 1. The library was screened with the 4.3-kilobase full-length

mouse mdr cDNA insert of phage ADR11 (3). Several hybridizingclones were identified. The £coRIinsert of a positive human phageclone corresponding to approximately 600 bp of structural sequence atthe 5' end of the mRNA was subcloned into plasmid pUC18. Forlabeling, the cDNA insert was purified by electrophoresis on a low-melting agarose gel and radiolabeled by nick translation with [a-32P]-

dATP (approximately 3000 Ci/mmol; New England Nuclear), according to standard procedures (17). The estimated specific activity of thisprobe was greater than 10" cpm per ¿ig-

Nucleic Acids Isolation. Approximately 5x10'' cells were lysed in

100 mM Tris-HCl (pH 8.0):50 mM NaCl:0.1% SDS:2 mM EDTA:100Mg/ml of proteinase K. Lysates were incubated overnight at 37°C,

followed by 2 phenol extractions and 2 chloroform extractions. DNAwas precipitated with 2 volumes of isopropyl alcohol and dissolved in1.0 ml of TE. Following digestion with both EcoRl and //mdlll(New England Biolabs), DNA samples were extracted withphenol:chloroform (1:1), precipitated, washed in ethanol, lyophilized,and finally dissolved in TE. DNA concentration was estimated byoptical absorbance at 260 nm. Total RNA was extracted from exponentially growing cells as previously described (18). MDR sublines weregrown in the respective drug-containing culture medium used for colonyisolation. Total RNA from multidrug-resistant mouse LI210 DN cellswas used to provide a size marker for 4.5-kilobase mrfr-specific mRNA.

Northern and Southern Blot Hybridizations. All solutions and procedures involving electrophoresis, blot transfers, and hybridizationswere as described (17) except as noted. For Northern blots, approximately 5 /¿gof total RNA from cell lines were fractionated by electrophoresis on 1.2% agarose containing 2.2 M formaldehyde in 20 mMmorpholinopropanesulfonic acid (pH 7.0): 5 mM sodium acetate: 0.1mM EDTA. RNA was transferred to a nitrocellulose membrane withoutfurther treatment. The filter was baked (2 h at 80°C)and prehybridizedat 42°Cfor 30 min in 5x SSC, 50% deionized formamide, 25 mM

sodium phosphate buffer (pH 6.5), 0.02% bovine serum albumin, 0.02%Ficoll, 0.02% polyvinylpyrollidone, 100 Mg/ml of tRNA (Escherichiacoli), and 0.1% SDS. Hybridization was at 42°Cfor 16 h in the samesolution containing a heat-denatured probe (IO6 cpm/ml). The filterwas then washed at 65°Cin 2x SSC and 0.1% SDS, followed byexposure for 14 days at -80°Con X-ray film with intensifying screens.

The X-ray film was scanned in a Model 620 Video Densitometer (Bio-Rad Laboratories) for total density in the 4.5-kilobase bands.

For Southern blots, the DNAs were digested with restriction enzymesunder conditions specified by the supplier and loaded in duplicate wells(approximately 5 Mgeach) of a 1% agarose gel containing 89 mM Tris-borate:89 mM boric acid:2 mM EDTA (17). Following electrophoresis,the gel was stained with ethidium bromide, denatured, neutralized, andthen blotted to a nitrocellulose membrane according to standard conditions (17). The filter was baked at 80°Cunder vacuum for 2 h.Hybridization with the human mdr probe was for 16 h at 65°Cin 5x

SSPE (0.15 M NaCl: 1 mM EDTA: 10 mM NaH2PO4) (pH 7.4), 0.1%SDS, 0.1% Ficoll, 0.1% polyvinylpyrollidone, 0.1% bovine serum albumin, and 0.01% torula yeast RNA. The filter was then washed at65°Cin 2x SSC and 0.1% SDS, followed by exposure for 5 days at—¿�80°Con X-ray film with intensifying screens.

RESULTS

Selection of Drug-resistant Melanoma Sublines. To selectdrug-resistant colonies in a single step, Bowes melanoma cellswere first exposed to several concentrations of each of 6 different drugs (vincristine, vinblastine, colchicine, doxorubicin, dau-norubicin, or actinomycin D) to determine the DIO for eachdrug. Thus, drug concentrations that would give rise to resistantcolonies were established empirically. The selection for drug-resistant colonies was initiated in medium containing a singlecytotoxic drug at a concentration between 1 and 5 times greaterthan the D,0 value observed for the drug-sensitive Bowes parental cells. Under those conditions viable drug-resistant coloniesappeared at a frequency of approximately 1 in IO7 cells. Approximately 3 x IO6 cells were plated in 150-mm dishes containing 30 ml of drug-supplemented medium. Since all platings

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Fig. 1. The effects of 6 different cytotoxicdrugs (A to F, as shown) on 6 different melanoma cell lines. RPE is plotted versus drugconcentration for Bowes melanoma (O), VCR4.5/4 P), VCR 7.5/4 (•),VBL 3 (A), VBL 4(A), and COL I (•).

100

80

£ 60LU

è4020

100

80

£ 60

KI 40

20

\02 05 I 23 5 7 IO 1520 30 50

[Vincristine] (ng/ml)

05 I 23 5 7 10 1520 30 50[Colchicine] (ng/ml)

02 05 I 23 5 7 10 1520 30 50[Daunorubicin] (ng/ml)

100

80

Q.CC40

20

02 05 I 2 3 5 7 10 15[Vinblastine] (ng/ml)

D

02 05 I 2 ÃŽ 5 7 IO 15[Doxorubicin] (ng/ml)

02 05 I 2 3 5 7 10 15[Actinomycin D] (ng/ml)

were in triplicate, approximately IO7 cells were screened atonce. Cells were fed every 4 days with fresh drug-containingmedium, and the cultures were incubated and monitored forthe presence of colonies for up to 6 wk.

Resistant colonies were scraped and aspirated off the plasticsurface using a sterile plugged Pasteur pipet under microscopicobservation. Colonies were tranferred in 24-well titer platescontaining 1.5 ml of drug-free medium for 48 h, at which timethe same drug concentration as used in selection was addedback to the medium. Confluent cultures were serially transferredto 25-cm2 and 75-cm2 flasks and grown further. Aliquots were

preserved as frozen stocks.Of approximately IO7cells screened, 12 vincristine-resistant

colonies, 5 vinblastine-resistant colonies, and 4 colchicine-re-sistant colonies were isolated. Some of these clones were characterized further. VCR 4.5/4 was selected in 4.5 ng/ml ofvincristine (approximately 2.4 x DIO).VCR 7.5/4 was selectedin 7.5 ng/ml of vincristine (approximately 4 x D10).VBL 3 andVBL 4 were each selected independently in 3 ng/ml of vinblas-tine (approximately 3.3 x D)0). COL 1 was selected in 8 ng/mlof colchicine (approximately 3.5 x DIO). Under experimentalconditions corresponding to between 3 and 10 times the D,0,we were unable to isolate colonies in medium containing dox-orubicin, daunorubicin, or actinomycin D, even after the screening of approximately 30 million cells.

Relative Drug Resistance of Melanoma Sublines. Each of thefive clonal isolates described above was tested for relative plating efficiency in the presence of each of the six drugs, with theexception that VBL 3, VBL 4, and COL 1 were not tested indoxorubicin. The results of a single experiment are shown in

Table 1 Relative drug resistance levels of cloned melanoma sublines at 10% RPE

DrugVincristine

VinblastineColchicineDoxorubicinDaunorubicinActinomycin DBowes

melanoma1.0

1.01.01.01.01.0VCR4.5/49.7

4.72.23.11.52.3Cell

lineVCR7.5/423.7

9.23.92.31.58.0VBL317.4

7.14.7a3.3

9.6VBL415.8

6.03.23.010.0COL113.74.33.92.05.0

" —¿�,not tested.

Fig. 1, in which each point represents the mean of 3 platingswith drug divided by the mean of 3 platings without drug.Similar results were obtained in other independently performedexperiments (data not shown). The data of Fig. 1 are summarized in Table 1, which displays calculated dose ratios at 10%survival.

All 5 isolates exhibit the classical multidrug resistance phe-notype. All the sublines exhibit various degrees of cross-resistance to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D (Fig. 1). All sublines exhibit the greatest level ofresistance to vincristine (Table 1), irrespective of the drug usedfor selection. The greater resistance of VCR 7.5/4 to vincristine,vinblastine, colchicine, and actinomycin D, compared withVCR 4.5/4, seems to correlate with the higher vincristineselection pressure used in isolation.

Kinetics of Drug Transport in Melanoma Sublines. The Bowesmelanoma, VCR 7.5/4, and VBL 4 cell lines were examinedfor their ability to accumulate and retain tritium-labeled vinblastine. Both MDR sublines tested exhibit reduced drug ac-

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cumulation, compared to the Bowes melanoma line, over a 90-min period (Fig. 2A). At 90 min drug-sensitive Bowes cellsaccumulated (in medium containing glucose) approximately 5times more drug than both multidrug-resistant derivativestested. Reduction of cellular ATP levels by treatment withrotenone and 2-deoxyglucose increased the accumulation ofdrug in the Bowes parental line and in the drug-resistant VBL4 isolate.

Direct drug-efflux measurements (Fig. 2B) in initiated vin-blastine-labeled cells indicate a much greater efflux activity(efflux of 60% of preloaded drug at 60 min) in the drug-resistantderivatives tested (VBL 4 and VCR 7.5/4) than in the drug-sensitive parental line (10% released at 60 min). This enhancedefflux was reduced if the efflux occurred under conditions ofreduced intracellular ATP levels. It is interesting to note thatthe ATP dependence of the drug-efflux mechanism was mostevident in the parental and in the VBL 4 cell lines, while lessevident in VCR 7.5/4 (which is more resistant to the two Vincacompounds). This observation correlates well with the fact thatonly partial ATP depletion (67% and 76% in 2 experiments)could be achieved in VCR 7.5/4 by our experimental procedure,while significantly greater levels of ATP depletion occurred inthe parental (86%, 94%) and VBL 4 (71%, 87%) lines. It is

80 100 120 140 160

Time (min.)Fig. 2. A, uptake of tritium-labeled vinblastine (f'HjyBL) by drug-sensitive

Bowes human melanoma cells (O, •¿�)and the multidrug-resistant derivatives VBL4 (A, A) and VCR 7.5/4 (D, •¿�).Cells in suspension were exposed to f'HjyBL at

a concentration of 75 nM. At different times, cells were separated from thenonincorporated drug by centrifugation through a mineral oil cushion. The cell-associated radioactivity in the cell pellet was determined and is expressed as thenumber of pmol of['H]VBL per mg of cellular protein. Drug uptake was measured

under conditions of normal (closed symbols) or reduced (open symbols) levels ofintracellular ATP, as described in "Materials and Methods." B, for drug efflux,

cells were at first treated to reduce levels of intracellular ATP, as described in"Materials and Methods," and then loaded with [^HJVBL. Efflux was allowed to

take place under conditions of normal (closed symbols) or reduced (open symbols)levels of intracellular ATP. At predetermined intervals, the radioactivity releasedfrom the cells into the extracellular medium was measured and is expressed asthe percentage of the maximum cell-associated radioactivity obtained immediatelyafter loading the cells (see "Materials and Methods"). Symbols are the same as

in Fig. 2A.

possible that rotenone, the metabolic inhibitor used in our ATPdepletion protocol, may be affected by the drug-efflux pump,leading to less inhibition in drug-resistant cell lines havinggreater pump activity.

Analysis of nulr DNA Sequences in Melanoma Sublines. Theobservation that multidrug-resistant derivatives of human

Bowes melanoma cells, obtained in this study, show an increased ATP-dependent drug efflux is reminiscent of othermultidrug-resistant cell lines in which mdr genes are amplifiedand overexpressed. To determine whether possible amplification of human mdr genes could account for the drug-resistancephenotype of the Bowes multidrug-resistant derivatives, wecarried out Southern blotting experiments with a cloned humanmdr cDNA probe. The probe used in this analysis (Fig. 3)detected a unique fcoRI-7/indIII fragment of approximately 5kilobases in all the cell lines. The degree and pattern of hybridization in MDR sublines are not significantly different fromthose in the Bowes melanoma cell line. Ethidium-bromidestaining of the gel prior to blotting confirmed that all lanescontained comparable amounts of DNA (data not shown).These results indicate that, in the MDR sublines, mdr genesequences are neither amplified nor rearranged.

Expression of mdr mRNA in Melanoma Sublines. Northernanalysis indicates (Fig. 4) that MDR sublines express a muchgreater amount of mRNA hybridizing with the human probethan does the parental Bowes melanoma cell line. Ethidium-bromide staining of 18S and 28S ribosomal RNA subunits (datanot shown) ascertains that Lanes 2 to 7 contained equalamounts of total RNA. The light bands appearing in Lanes 1and 8, which contain RNA from a multidrug-resistant mousecell line LI210 DN, serve as a size marker for the 4.5-kilobasemouse mdrl mRNAs known to be overexpressed in multidrug-resistant mouse cell lines. Although this mouse RNA is considerably enriched for this 4.5-kilobase message, compared to thedrug-sensitive parental mouse line L1210,4 hybridization is very

weak on this blot, suggesting considerable sequence divergencebetween the human probe and the analogous mouse sequencein the 5' structural region. Densitometer scanning of the 4.5-

kilobase bands corresponding to the human mdr gene mRNAtranscript present in Lanes 2 to 7 indicates that VCR 4.5/4,VCR 7.5/4, VBL 3, VBL 4, and COL 1 expressed approximately 6-, 10-, 10-, 14-, and 6-fold more mdr RNA, respectively,than the drug-sensitive Bowes melanoma parental cell line.

DISCUSSION

In view of the need for a better understanding of the mechanisms underlying drug resistance observed in different types ofhuman solid tumors, we have examined the mechanistic basisof multidrug resistance in independently derived cell clones ofa human malignant melanoma, following single-step selectionfor low levels of resistance to 3 cytotoxic drugs. We have focusedon the molecular events associated with the initial acquisitionof relatively low levels of drug resistance in these cells. Low-level drug resistance is a significant problem during in vivochemotherapeutic treatment of human tumors.

The occurrence of toxicity for nontumor tissues often precludes the use of 2-fold increases in chemotherapeutic drugdoses, such that tumors rarely reach the high levels of drugresistance classically observed in vitro in highly resistant celllines. High-level MDR, obtained previously in human androdent cells by multistep selection protocols, has been frequently associated with amplification of one or more mdr genes,

4 P. Gros, unpublished data.

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Fig. 3. Autoradiograph of Southern blot(1% agarose gel) containing X/£coRI///mdIIImarkers (Lanes I and 14) and DNA (5 fig perlane) digested with both EcoRl and HináUlfrom each of the following melanoma celllines: Bowes melanoma (Lanes 2 and 3); VCR4.5/4 (Lanes 4 and 5); VCR 7.5/4 (Lanes 6and 7); VBL 3 (Lanes 8 and 9); VBL 4 (Lanes10 and //); and COL 1 (Lanes 12 and 13). Anick-translated human mdr cDNA containingapproximately 600 bp of 5' structural sequence

was used as probe.

23456 789 IO 12 13 14 M-21.7

-5.1-4.3

-2.0

2345678

-28S

-I8S

Fig. 4. Autoradiograph of Northern blot (1.2% agarose/formaldehyde gel)containing total RNA from a daunorubicin-resistant mouse L1210 cell line (LanesI and A'.4 ng per lane) and from each of the following melanoma cell lines (5 ¿ig

per lane): Bowes melanoma (Lane 2); VCR 4.5/4 (Lane 3); VCR 7.5/4 (Lane 4);VBL 3 (Lane 5); VBL 4 (Lane <S);and COL 1 (Lane 7). The probe was the sameas that used in Fig. 3. The position of 18S and 28S ribosomal RNA subunits,visualized by ethidium bromide staining, is shown.

leading to overexpression of the P-glycoprotein drug-effluxpump (2). We have recently shown that increased transcriptionof a single full-length mouse mdr cDNA clone transfected intodrug-sensitive cells is sufficient to confer the complete MDRphenotype (3, 19). However, the roles oímdr gene overexpression and/or amplification in low-level drug resistance need tobe clarified. It would be important to establish whether lowlevels of drug resistance arise in drug-resistant human tumorsby transcriptional activation of specific mdr genes or by otherunrelated mechanisms.

As part of our ongoing studies of low-level drug resistance inhuman tumor cell lines derived from different tissue types, wehave isolated and analyzed drug-resistant variants of humanmelanoma cells. Single-step selection for resistance to a singledrug yielded several independent cloned isolates, 5 of whichwere studied in detail. All 5 expressed relatively low levels ofcross-resistance to drugs not used in the selection (Table 1) andshowed increased levels of wi/r-specific mRNA (Fig. 4) withoutany detectable mdr gene amplification (Fig. 3).

With respect to drug concentrations used in isolation exper

iments, it is interesting to note that, in the two vincristine-selected cell clones, increased mdr mRNA expression and drug-resistance levels (towards several but not all drugs tested) bothseem to correlate with the selection pressure (vincristine concentration) used. Specifically, VCR 4.5/4 and VCR 7.5/4,isolated in 4.5 ng/ml and 7.5 ng/ml of vincristine, respectively,express approximately 6-fold and 10-fold increases in mdrmRNA, respectively, when compared to the parental melanomacell line mRNA level. Further, VCR 7.5/4 exhibits greaterlevels of resistance to vincristine, vinblastine, colchicine, andactinomycin D than is exhibited by VCR 4.5/4 (Table 1), whilecross-resistance to doxorubicin and daunorubicin does not showthe same correlation. VBL 3 and VBL 4 were both isolatedwith the same selection pressure (3 ng/ml of vinblastine), andboth exhibit comparable levels of cross-resistance to the otherdrugs tested (Table 1).

All 5 MDR sublines were found to be most resistant tovincristine, irrespective of the nature of the different drugs usedfor isolation. Although dose-effect data (Fig. 1) and deriveddrug resistance levels (Table 1) are from a single experiment(platings in triplicate), other independent determinations ofdose effect were performed with the same results. The levels ofsignificance of a small difference between two low levels of drugresistance, however, are more difficult to quantitate, requiringa larger number of independent experiments. In conclusion, itappears that, within the limits of our ability to measure resistance of individual clones to single drugs, the level of cross-resistance of the clones parallels the degree of selection pressureused in the initial selection procedure and the level of specificmdr mRNA expressed by the cells.

The detailed molecular basis for different patterns of cross-resistance to drugs is not known but could involve mutationalalteration of P-glycoprotein structure (20) and differential over-expression of other mdr genes. Indeed, we have recently obtained evidence that, in multidrug-resistant derivatives of amouse macrophage cell line, the 3 mouse mdr genes are differentially amplified and overexpressed (data not shown). SI nu-clease protection or Northern blotting types of experiments,using gene-specific probes able to distinguish between transcripts from individual members of the human mdr gene family,should help clarify the independent contribution of each ofthese genes to the final phenotype observed in the differentMDR melanoma sublines. Moreover, the ability to correlatecross-resistance spectra with differential mdr gene expressionpatterns in clinical samples of human tumors, or in drug-resistant cell lines obtained from other tissue types by similarsingle-step selection protocols, would help elucidate the roles

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MULTIDRUG-RESISTANT HUMAN MELANOMA CELLS

played by other members of the mdr gene family in the multi-drug-resistance phenotype.

As suspected from the observations of elevated mdr mRNAlevels, VCR 7.5/4 and VBL 4 both exhibited altered energy-dependent drug transport (Fig. 2): decreased intracellular accumulation and increased efflux. We expect that the other threeMDR sublines would express a similar drug-transport phenotype. These results suggest that decreased drug accumulation,resulting from increased drug efflux mediated by P-glycopro-tein, is the major cause of multidrug resistance in these drug-resistant melanoma lines.

Taken together, the data indicate that these melanoma cellsacquire low-level drug resistance by a "classic" MDR mecha

nism involving overexpression of one or more related M,170,000 transmembrane drug-efflux pump mdr genes (P-gly-coproteins). We have shown that transcriptional activation ofone or more mdr genes is likely to be responsible for the earlyevents leading to low-level MDR in this human tumor cellsystem.

Our human mdr probe, isolated from a human fetal livercDNA library, exhibits strong sequence identity with the 5' end

(putative transmembrane region) of the human mdrl cDNA(8). The cDNA sequence in the corresponding region at the 5'

end of the human mdr3 cDNA (10) differs significantly fromthat oímdrl (21). Thus, it seems likely that, in our melanomasublines, unamplified mdrl sequences are being detected on theSouthern blot (Fig. 3). Similarly, the increased mdr mRNA inmelanoma sublines likely represents mdrl transcripts, ratherthan a mixture of mRNAs from different mdr genes.

Recent evidence (Ref. 22; Footnote 5) suggests that the P-glycoprotein gene family contains 3 members in rodent cellsbut only 2 members in human cells. Moreover, while expressionof a transfected complete mdrl cDNA coding sequence (derivedfrom human liver RNA) confers drug resistance on drug-sensitive human melanoma cells, transfection with a similarlyderived complete mdr3 cDNA sequence did not lead to drugresistance (21). The possible functional role of mdr3 in multi-drug resistance is not currently known.

Further studies of the molecular alterations in our melanomasublines may prove useful in helping to elucidate mechanismsby which human tumor cells acquire clinically relevant low-level multidrug resistance. All five independently isolated sub-lines exhibit an increase in mdr-specific mRNA, suggestingthat, at least in these cells and using our selection procedure,mechanisms unrelated to /ní/i'7-encodedP-glycoprotein expres

sion may not play a major role in this type of drug resistance.Although we do not know whether all five sublines carry thesame genetic change, we may speculate on several potential e/sor frans-acting alterations affecting transcription of mdr genes.These include mutations in untranslated regions leading togreater mRNA stability (half-life) or rate of synthesis, as wellas additional mutations in protein-encoding regions. Mutationin the 5' regulatory sequences of a human mdr gene was recently

suggested as a cause for the appearance of a new transcriptioninitiation site in a vinblastine-resistant derivative of the humanKB carcinoma cell line (9). Future characterization of mdr genesisolated from our melanoma sublines, along with RNA metabolism and DNA-protein binding studies, will make it possible

*V. Ling, personal communication.

to understand the nature and function of positive- and negative-control sequence elements of mdr genes and the protein products that regulate their transcription.

ACKNOWLEDGMENTS

We thank Dr. David Housman and Dr. Gordon Moore for criticalreview of this manuscript and William Dackowski for assistance withcDNA cloning.

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1988;48:6348-6353. Cancer Res   Jeffrey F. Lemontt, Marie Azzaria and Philippe Gros  Accumulation in Multidrug-resistant Human Melanoma Cells

Gene Expression and Decreased DrugmdrIncreased

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