[CANCER RESEARCH 47, 1297-1301, March 1, 1987]

Atypical Multiple Drug Resistance in a Human Leukemic Cell Line Selected forResistance to Teniposide (VM-26)1

Mary K. Danks, Jack C. Yalowich2, and William T. Beck3

Department of Biochemical and Clinical Pharmacology [M. K. D.. W. T. B.) and Pharmacokinetics and Pharmacodynamics Section, Pharmaceutical Division[J. C. Y], St. Jude Children's Research Hospital. Memphis, Tennessee 38101

ABSTRACT

Resistance to the cytotoxic effects of many natural product drugs afterexposure to a single agent is a common observation. The classes of drugsincluded in the "classic" multiple drug resistance phenotype are Vinca

alkaloids, anthracyclines, epipodophyllotoxins, and antibiotics. We report here the characterization of a human leukemic cell line (CEM/VM-1) with "atypical" multiple drug resistance: despite resistance and cross-resistance to etoposide, anthracyclines, mitoxantrone, and 4'-((9-acridi-

nyl)amino)methanesulphon-in-anisidide (mA.MSA), these cells retainsensitivity to the Vinca alkaloids. Further, even though this cell line is»40-foldcross-resistant to the cytotoxic effect of etoposide (VP-16), itis similar to drug-sensitive CEM cells in the cellular pharmacology of[3H)VP-16 as determined by zero time binding, initial influx rate, steady

state drug concentration, and unidirectional efflux. Our studies suggestthat the resistance of CEM/VM-1 cells to epipodophyllotoxins is due toan altered interaction between drug and its cellular target(s) by a mechanism unrelated to the decreased cellular concentration of drug associatedwith the "classic" multiple drug resistance phenotype.

INTRODUCTION

The simultaneous resistance to several classes of "naturalproduct" drugs in vivo and in vitro after exposure to a chemicallydissimilar agent is termed M DR.4 This is most frequently seen

with cells selected for resistance to alkaloids or to anthracyclines. Compared to drug-sensitive cells, MDR cells usuallyhave a decreased steady state drug level (1-5), decreased retention of drug (1,6, 7), and specific changes in plasma membraneglycoproteins (8-11). The relationship between the pharmacological and biochemical changes is unclear. The mechanism bywhich MDR cells accumulate and/or retain less drug thansensitive cells is also unknown.

Resistance or cross-resistance to the epipodophyllotoxins(semisynthetic derivatives of the "natural product" podophyl-

lotoxin) and its relationship to MDR has been studied in celllines developed in several laboratories (12-20). While most ofthese lines showed decreased drug accumulation and cross-resistance to the Vinca alkaloids as is typical of the MDRphenotype, there are reports of a teniposide-resistant Chinesehamster ovary line with minimal cross-resistance to VLB (17),

Received 8/15/86; revised 11/20/86; accepted 12/4/86.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Research Grant CA30103 (to W. T. B.) and Cancer

Center Support (CORE) Grant CA21765, from the National Cancer Institute,Bethesda, MD; by National Research Service Award CA06795 (to M. K. D.)from the National Cancer Institute; and by American Lebanese Syrian AssociatedCharities.

2Leukemia Society of America, Special Fellow; Recipient of BiomedicaiResearch Support Grant RR05S84-21 from NIH.

3To whom requests for reprints should be addressed, at St. Jude Children's

Research Hospital, Department of Biochemical and Clinical Pharmacology, 332North Lauderdale, P.O. Box 318, Memphis, TN 38101.

4The abbreviations used are: MDR, multiple drug resistance; CEM, parentCCRF-CEM human lymphoblast cell line; CEM/VLB100, CEM subline selectedfor resistance to vinblastine; VLB, vinblastine; CEM/VM-1, cloned CEM sublineselected for resistance to VM-26; VM-26, teniposide [4'-demethylepipodophyl-lotoxin 9-(4,6-O-2-thenylidene-0-r>glucopyranoside)]; VP-16-213, VP-16, [etoposide; 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-/3-r>glucopyrano-side)]; niAMSA. 4'-{(9-acridinyl)amino)methanesuIphon-m-anisidide; DOX,dox-

orubicin; VCR, vincristine; HPLC, high pressure liquid chromatography.

and an mAMSA-resistant P388 line (12) and HL-60 line (20)that are cross-resistant to the epipodophyllotoxins and sensitiveto the Vinca alkaloids.

We report here the development and characterization of ateniposide-resistant human leukemic lymphoblastic cell linethat expresses "atypical" MDR. This line (CEM/VM-1) has apattern of cross-resistance similar to cells having the "classic"

MDR phenotype with the exception that it is completely sensitive to the Vinca alkaloids. Also, in contrast to the "classic"

MDR phenotype, the CEM/VM-1 cells accumulate nearly asmuch drug as do drug-sensitive CEM cells, suggesting a mechanism of resistance other than decreased drug retention. Comparison of the CEM/VM-1 line and our well-characterized"classic" MDR cell line CEM/VLBIOO is also reported. A pre

liminary account of this work has been presented (21).

MATERIALS AND METHODS

Chemicals and Supplies. Eagle's minimal essential medium, withEarle's spinner salts, was purchased from Grand Island Biological Co.

(Grand Island, NY) and fetal bovine serum was from Hyclone Laboratories (Sterile-Systems, Inc., Logan, UT). VCR and VLB were generousgifts of Eli Lilly and Co. (Indianapolis, IN). DOX was from AdriaLaboratories (Wilmington, DE), and bleomycin, VM-26, and VP-16were provided by Bristol-Myers Laboratories (Syracuse, NY); dimethylsulfoxide was from Fisher Scientific Co. (Fair Lawn, NJ). The drugsolutions were prepared as reported previously (22); mAMSA, obtainedfrom the Drug Synthesis and Chemistry Branch, DTP, DCT, NationalCancer Institute, was dissolved in 100% dimethyl sulfoxide and thendiluted with 0.9% sodium chloride to a concentration of 1 HIMbeforeits dilution to final concentration with 0.9% sodium chloride. Methanoland acetonitrile were purchased from Burdick and Jackson Laboratories(Muskegon, MI). | 'II|VI'-I6 was purchased from Moravek Biochemi-cals. Brea, CA; and [car¿ary/-uC]inulin was from New England NuclearProducts, Boston, MA. Propidium iodide was obtained from Calbi-ochem (San Diego, CA). AHother chemicals and supplies were obtainedfrom commercial sources.

Cells and Culture Conditions. Teniposide-resistant cells were selectedfrom the original parental CCRF-CEM cell line by growth in theintermittent presence of sublethal concentrations of drug as describedfor our VCR-resistant cells (22). The CEM/VM-1 line was cloned bylimiting dilution (23) and retained its resistance to VM-26 for at least2 months in the absence of drug. All cells used for experiments weregrown in drug-free media for at least 1 week. We have described theselection and maintenance conditions for the (TAI/VI,HH>), cell lineelsewhere (11).

Growth Inhibition Assay. The concentration of drug required toinhibit growth of cells by 50% (ICjo) in 48 h was determined for eachcell line, as described previously (22). Fold resistance is calculated bydividing the K '<„of the resistant line by the K \(>of that drug in the

CEM parent line.DNA Content. CEM, CEM/VLB100 and CEM/VM-1 cells were

analyzed by now cytometry for DNA content and cell cycle distributionas described previously (24).

Cellular Pharmacology of VP-16. Influx, efflux, accumulation, andretention of ['HJVP-16 by the cell lines were determined by the cold

buffer wash method, as previously described (25). Intracellular watervolume was determined from the difference between the wet and dryweights of cell pellets, taking into account the extracellular volume, asdetermined with [14C]inulin. Intracellular water (ml/g dry weight) was

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DRUG RESISTANCE TO TENIPOSIDE IN A HUMAN LEUKEMIC CELL LINE

determined to be 4.3, 4.5, and 3.9 for CEM, CEM/VM-1, and CEM/VLBioo cells, respectively. Data are expressed either as nmol VP-16/gdry weight or /imol VP-16/liter cell water as previously reported (25).Some drug accumulation studies were also done using a standardsilicone oil technique (26) for separating the cells from the extracellularmedium. At indicated times, aliquots of cell suspension were layeredover 0.5 ml Nyosil 50 oil (William F. Nye, Inc., New Bedford, MA) ina microcentrifuge tube and centrifuged for 5 min at room temperature.The [3H]VP-16 used in the experiments was 93-97% pure by HPLC

(27).HPLC Analysis for Possible Cellular Metabolism of |3H)VP-16. Cell

pellets were prepared for drug metabolism studies after a 60-minincubation at 37*C using an initial extracellular VP-16 concentrationof 170 pM. After incubation with drug, cell pellets containing [3H]VP-

16 were treated twice with 100 M' of 100% methanol; this treatmentextracted 100% of the radiolabel. Analysis for potential metabolites of[3H]VP-16 was by the HPLC method of Sinkule and Evans (27).

Statistics. Comparison of rates of influx of VP-16 in the three celllines was done with a Wald \- analysis (28). /' values for simple

comparisons of drug accumulation, retention, and r were obtained byStudent's r test (28).

RESULTS

Drug Cytotoxicity. Forty-eight-h IC50 values of several drugsfor the parent drug-sensitive (CEM), VLB-resistant (CEM/VLB,oo), and VM-resistant (CEM/VM-1) cell lines are shownin Table 1. Each of the resistant lines is equally resistant orcross-resistant to VM-26 and VP-16, permitting direct comparison of degrees of cross-resistance and cellular pharmacology data. They are also each =20-fold cross-resistant to mitox-antrone and 80- to 150-fold cross-resistant to DOX. The CEM/VM-1 line is resistant to mAMSA, but sensitive to the Fincaalkaloids VLB and VCR. By contrast, the CEM/VLBIOO cellsare resistant to Vinca alkaloids. Both resistant lines are sensitiveto bleomycin.

Flow Cytometric Findings. Since similarities or differences indegrees of resistance to the drugs in Table 1 could be due todifferences in growth rates or cell cycle distribution, we determined doubling times for each line and analyzed, with cells inlog phase growth, the DNA content of each line. Results ofthese experiments are shown in Table 2. The similarity of boththese measurements in all three lines suggests that growthkinetics do not account for contrasting cross-resistance patternsin these lines, and lends further support to the comparability ofthe other data presented in this paper.

Influx, Efflux, Steady State Accumulation, and Retention of|3H|VP-16. The uptake of [3H]VP-16 was measured from 0.5 to

Table 1 Cross-resistance ofCCRF-CEMsublines selected for resistance toVM-26 or VLB

Table 2 Doubling times and cell cycle distribution of drug-sensitive and -resistantcell lines

CCRF-CEMDegree of resistance*

DrugsVM-26VP-16VLBVCRDOXMitoxantronemAMSABleomycinICwdiM)«48.0

±39.0416±2002.76±2.502.40

±2.1029.0±24.012.2±14.756

±71.26 ±0.84°CEM/VM-I47413'0.3"841630*3.2GEM/VLB,«324418620231522161.6

" 50% inhibitory concentration in a 48-h growth inhibition assay ±SD. Values

shown for CEM cells are means of 14 to 18 separate experiments. For CEM/VM-1 and CEM/VLB,oo cells, values are means of 3 lo 12 separate experiments.

* 1C,,,of the resistant cell line divided by that of the parent CCRF-CEM line.' Units/liter.d Degree of resistance significantly different (P < 0.001) from CEM/VLB,«,

cells.' Degree of resistance significantly different (/> < 0.02) from CEM/VLB,,»

cells.

CelllineCCRF-CEMCEM/VM-1

CEM/VLB.ooDoubling

time(h)"31

±533±6

32 ±7Distribution

of cellsin phases of cycle

(% ±SDfG,44±4

53 ±850 ±7S45

±538 ±837 ±8Gj

+M11±3'

10±314 ±4

" Determined graphically and defined as the number of hours required for cells

in log phase growth to double in number. Values are the mean ±SD of four tofive experiments.

* Determined by DNA content, as indicated in "Materials and Methods."' Percentages do not equal 100 when added, since these are mean values of

three separate experiments.

Fig. 1. Initial uptake of I3H]VP-16 by CEM, CEM/VM-1, and CEM/VLB,«,cells. External concentration of [3H]VP-16 was 5 »IM.Each point is the mean oftriplicate determinations from three experiments run on separate days. liur\.mean •SE. See -Materials and Methods" for experimental details.

10 s to determine initial influx rates in the three cell lines (Fig.1). Using the first three time points (0.5-2.0 s), comparison ofthe rates of drug influx (slopes) showed only a 2-fold differencebetween CEM and CEM/VM-1 cells, but a greater than 10-fold difference between the CEM and CEM/VLB100 cells. Theseinitial influx rates are given in Table 3. There is no significantdifference (P = 0.91) in zero-time binding (Y intercept value)among the cell lines (Fig. 1).

Despite the fact that the two drug-resistant lines are equallycross-resistant to VP-16, they accumulated different amountsof drug at steady state, as shown in Fig. 2. CEM/VM-1 cellsaccumulated 70% as much [3H]VP-16 as the CEM cells, but

the CEM/VLBioo cells accumulated less than 10% as much[3H]VP-16 as the drug-sensitive controls. These steady state

values are listed in Table 3.Removal of [3H]VP-16 from the extracellular compartment

results in the rapid loss of drug from all cell lines to a plateau(Fig. 2). Statistically, these levels of nonexchangeable drug(Table 3) were significantly different only when comparing thetwo resistant cell lines.

Unidirectional efflux of [3H]VP-16 was determined in thesecell lines after "loading" for 30 min with 5 /IM [3H]VP-16 to

achieve steady state drug levels (Fig. 2). As observed for influx,there was a similar rate of efflux of VP-16 from the parentalCEM and CEM/VM-1 cells (Table 3). (Because of the smallstandard error, these rates are statistically different; but thedifference is of doubtful biological significance.) However, ac-

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DRUG RESISTANCE TO TENIPOSIDE IN A HUMAN LEUKEMIC CELL LINE

Table 3 Analysis of VP-16 influx, efflux, accumulation, and retention inCEM, CEMjVM-l, and CEM/VLBIM cells

Influxrate(nmol/g

Cell line dry weight/s)°Efflux(i»,s)'VP-16

concentration(nmol/g dry weight)Nonex-

Total changeable

CEM 0.230 ±0.084 102 ±1CEM/VM-1 0.123 ±0.026 94 ±IeCEM/VLB.oo 0.021 ±0.046'' 256±33C''

21.39 + 0.80 0.73 + 0.2114.63 ±0.94 0.41 ±0.04

1.79 ±0.1S*' 0.20 ±0.03'

" Includes first three points from 0.5-2.0 s. Data are presented as mean ±SE

from three separate experiments run on separate days. Three to four measurements at each of five different times were taken as previously described (25).

* Cells were incubated with 5 /IM [3H]VP-16 for 30 min, after which they werewashed two times in ice cold buffer. Total VP-16 was measured in triplicate andthe cells were resuspended in two to three times the original volume of VP-16•free buffer at 37'C. Over the next 300 s, at least 12 measurements were made ofcellular VP-16 concentration. The log of the fraction of exchangeable VP-16(determined from the difference between total and nonexchangeable VP-16)remaining in the cell as a function of time after resuspension was plotted (notshown) and the resulting linear efflux rate was used to calculate the i, valuesshown. Nonexchangeable VP-16 was determined from measurement of VP-16concentration taken 30-45 min after resuspension of cells in VP-16-free buffer.Data are presented as the mean ±SE from three experiments run on differentdays.

' Significantly different from CEM (P < 0.01).' Significantly different from CEM (P < 0.05).' Significantly different from CEM/VM-1 (p < o.OI).f Significantly different from CEM/VM-1 (P < 0.001).

16 r

Minutes

Fig. 2. Accumulation and retention of VP-16 in CEM, CEM/VM-1, andCEM/VLB,oo cells. Cells were incubated with 3.6 MM| '111V]' 16 and intracellularVP-16 was determined at the times indicated. After 25 min, cells were harvested,washed twice in ice-cold 0.85% sodium chloride, and resuspended at 37°Cin twoto three times the original volume of drug-free incubation buffer. Over the next20 min the cell-associated epipodophyllotoxin concentration was measured.

cumulated [3H]VP-16 leaves the CEM/VLB100 cells moreslowly, the fy, being 2.5-fold greater than that observed for theother two lines (Table 3).

The accumulation of [3H]VP-16 by the three cell lines was

also studied using the silicon oil method (Fig. 3). By thistechnique, both CEM and CEM/VM-1 lines accumulated VP-16 to a similar steady state concentration. This contrasts withthe results using the cold buffer wash method where VP-16concentrations were 30% lower in CEM/VM-1 cells than inCEM (Fig. 2). The steady state concentration of VP-16 in theCEM/VLBioo cells was about 25% that of the parent line [versus10% by the buffer wash method (Fig. 2)].

At 5 MMextracellular [3H]VP-16, CEM cells accumulated

similar concentrations of epipodophyllotoxin regardless of thetechnique used to quantitate drug uptake (5.0 MMversus 5.9 MMfor wash method and silicon oil method, respectively). Accumulation experiments were also done by each method with anextracellular concentration of [3H]VP-16 3.6 MM.Under these

conditions intracellular accumulation was approximately 3.6MMin each cell line.

VP-16 Metabolism in CEM/VM-1 Cells. Since the CEM/VLBioo cells and the CEM/VM-1 cells were equally resistantto VP-16, but the cellular drug concentration in the CEM/VM-1 cells was »8-foldhigher than in the CEM/VLB100 cells, thequestion of drug metabolism was addressed. If the parent drugwere metabolized to an inactive or less active form in the CEM/VM-1 cells, the effective drug concentration in the two linesmight be similar. We therefore examined our CEM/VM-1 cellsfor metabolites of VP-16. No metabolites were found by HPLCanalysis (data not shown), suggesting that the mechanism ofresistance in the CEM/VM-1 cells is not due to increasedmetabolism of the parent compound.

DISCUSSION

The two CEM sublines in this report are resistant to at leastfive of the eight drugs studied; therefore, each line could be saidto be "multiply drug resistant." All the drugs are natural prod

ucts or their semisynthetic derivatives, and all except bleomycinhave been reported to be involved in MDR (2, 8, 12, 14). Theproposed mechanisms of cytotoxicity of the drugs in Table 1include DNA damage (29-32) (DOX, mitoxantrone, bleomycin, and mAMSA), tubulin binding (33) (VLB and VCR), andalteration of topoisomerase activity (34-37) (mAMSA, DOX,VM-26, and VP-16). However, the CEM/VLB100 line is resistant to the Vinca alkaloids, while the reverse is true of the CEM/VM-1 line. These contrasting cross-resistance data suggest thateven though the lines are equally resistant to the epipodophyl-lotoxins, the mechanisms of resistance differ. In support of thisconclusion, the resistance of the CEM/VLBioo line to VP-16was likely due to the characteristic decrease in steady state drugconcentration associated with the "classic" MDR phenotype.

This was probably not the case for the CEM/VM-1 line. Therefore, despite resistance to several classes of natural productdrugs, the MDR of the CEM/VM-1 cells is "atypical" because

the cellular pharmacology of VP-16 in these cells is similar tothat in the CEM cells and also because the CEM/VM-1 cellsare sensitive to the Vinca alkaloids.

Our observations with the CEM/VLBioo line (Table 1) areconsistent with those in the literature in that in each MDR lineshowing decreased accumulation of natural drug products, thereis cross-resistance to both the Vinca alkaloids and anthracy-clines (4-6, 38-40). Comparable studies with lines similar tothe CEM/VM-1 line are limited: of the three lines reported

CEM

= 50 -

200 400

Seconds600

Fig. 3. Determination of |3H)VP-16 accumulation by CEM, CEM/VM-1,CEM/VLBiao cells using the silicone oil rapid separation method. Cells wereincubated in buffer containing 5 MM[3H]VP-16. At the indicated times, portionsof the cell suspension («Sx to' cells) were centrifuged through silicon oil asdescribed in "Materials and Methods." The cell pellet was analyzed for associated

radioactivity after correction for small amounts of contaminating buffer carriedwith cells through the silicon oil (25).

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DRUG RESISTANCE TO TEN1POSIDE IN A HUMAN LEUKEMIC CELL LINE

with an "atypical M DR" phenotype (12, 17, 20), single point

drug accumulation studies have been done on one line (12,41).Consistent with our results, Kessel étal.(41) showed no changein accumulation of mAMSA or daunorubicin in the P388/AMSA line.

Comparison of results shown in Figs. 2 and 3 suggest thatthere is a fraction of accumulated VP-16 in both resistant lines(but not in the parent line) that is lost from the cell by washingat 0°C;this fraction may be intracellular free drug (i.e., osmot-

ically active) or drug loosely bound in or on the cell. Theseresults are consistent with another study reporting loss of VM-26 during cold buffer washing of LI210 cells resistant to VM-26 (40).

Our findings are unique in that the data suggest a mechanism^) of epipodophyllotoxin resistance in CEM/VM-1 cellsdifferent from that in the CEM/VLBIOO cells. Resistance to VP-16 in the CEM/VLB100 cells probably involves a "classic" MDR

mechanism with decreased drug accumulation and retentionassociated with characteristic membrane changes (1). TheCEM/VM-1 cells, in contrast, show only a slight decrease fromcontrol in cell-associated drug and no detectable metabolism ofparent compound that might result in a lower effective drugconcentration. Other data5 indicate that these cells do notoverexpress the MDR "marker" glycoprotein (gplSO), or its

mRNA.Kinetic data (Table 3) in this paper present the first evidence

that, in contrast to cells with the "classic" MDR phenotype

(CEM/VLBioo), alterations in transport and accumulation ofVP-16 most likely do not account for epipodophyllotoxin resistance in our human leukemic cell line (CEM/VM-1). Preliminary experiments with VM-26 (data not shown) and VCR (21)produced similar results. Further, we showed that resistance toVP-16 in the CEM/VM-1 cell line is likely not due to drugmetabolism. We suggest, therefore, that the mechanism ofepipodophyllotoxin resistance in the CEM/VM-1 line is alteration of a cellular drug target(s). The compounds to which thisline is cross-resistant (epipodophyllotoxins, anthracyclines,mAMSA) implicates changes in topoisomerase II (34-37) or inother factors involved in topoisomerase II activity (42, 43); butthis remains to be determined.

We conclude that the CEM/VM-1 line is resistant to thecytotoxic action of multiple natural drug products, but becauseof its sensitivity to the Vinca alkaloids and minimal alterationin drug accumulation, this line expresses an "atypical" MDR

phenotype. Studies are currently in progress in our laboratoriesto define the mechanism of this "atypical" MDR.

ACKNOWLEDGMENTS

The authors gratefully acknowledge and thank Margaret Cirtain,Susan Benton, and Ed Wingfield for technical assistance; Dr. RichardAshmun for analysis of cytofluorograms; Dr. Diane Fairclough forstatistical analysis; Dr. Valentino Conter for help in developing theCEM/VM-1 cell line; and Vicki Gray for manuscript preparation.

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1987;47:1297-1301. Cancer Res   Mary K. Danks, Jack C. Yalowich and William T. Beck  Line Selected for Resistance to Teniposide (VM-26)Atypical Multiple Drug Resistance in a Human Leukemic Cell

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