[CANCER RESEARCH 45.978-982, March 1985]

Selective Toxicity of a New Lipophilic Antifolate, BW301U, for Methotrexate-

resistant Cells with Reduced Drug Uptake

Ian W. Taylor,1 Peter Slowiaczek, Michael L. Friedlander, and Martin H. N. Tattersall

Ludwig Institute tor Cancer Research (Sydney Branch), Blackburn Building, University of Sydney, Sydney, New South Wales 2006, Australia

ABSTRACT

Three methotrexate (MTX)-resistant Å“il lines and their MTX-sensitive counterparts have been used to examine 2,4-diami-no - 6 - (2,5 - dimethoxybenzyl)- 5 - methyl - pyrido[2,3 - d]pyrimidine(BW301U), a novel lipophilic antifolate, and compare its cytotox-

icity with MTX and metoprine. Collateral sensitivity for bothBW301U and metoprine was observed in CCRF-CEM/MTX R-

cells, where MTX resistance appeared to be primarily due to adeficiency in drug uptake. This was particularly pronounced withBW301U which proved to be as effective in killing CCRF-CEM/MTX R as was MTX with the parental CCRF-CEM cell line. This

effect was not seen in other cell lines, L5178Y/MTX or L1210/MTX R, where resistance to MTX was correlated with either anoverproduction of 5,6,7,8-tetrahydrofolate:nicotinamide adenine

dinucleotide phosphate oxidoreductase EC 1.5.1.3 (DHFR) orwith combined uptake defect and increased DHFR levels, respectively. In each case, however, BW301U and metoprine,especially at high concentrations, were more effective than MTXin treating MTX-resistant cells.

INTRODUCTION

MTX2 has been successfully used in the treatment of a number

of commonly occurring tumors in humans. However, the development of resistance to further treatment with MTX in tumorswhich have initially responded to the drug remains a major clinicalproblem. The evolution of this acquired resistance to MTX hasbeen shown, primarily in experimental systems, to occur by avariety of mechanisms which include: (a) decreased net uptakeof MTX into cells (11, 12, 15, 30, 37); (o) increased cellularproduction of the target molecule DHFR (1, 2, 10, 18, 25, 30,37); and (c) reduced affinity for MTX of an altered form of DHFR(1,13,23,30).

Of these mechanisms of resistance to MTX, those involvingdrug transport and overproduction of DHFR have been examinedin most detail. At low concentrations of MTX, the drug is thoughtto be transported into the cell by the same active transportsystem used for the uptake of naturally occurring reduced folates(28, 32, 35, 36). In transport-resistant cells, both the Vmaxand

Kn, of this transport system for MTX can be altered in such away as to reduce the net accumulation of intracellular MTX (36).Attempts to overcome this type of resistance have involved theuse of high-dose MTX or lipophilic MTX analogues such asDDMP where the primary route of cellular uptake is by passivediffusion (6, 7, 9, 16, 17, 21, 29, 32, 33, 36, 41). In addition, it

1To whom requests for reprints should be addressed, at Queensland Institute

of Medical Research, Bramston Terrace, Herston, Brisbane, OLD, 4006, Australia.2The abbreviations used are: MTX, methotrexate; DHFR, 5,6,7,8,-tetrahydro-

folate:NADP* oxidoreductase EC 1.5.1.3; BW301U, 2,4-diamino-6-{2,5-dimethox-ybenzyl}-5-methyl-pyrido[2,3-d]pyrimidine; DDMP, metoprine.

Received October 14, 1983; accepted November 9,1984.

has been suggested that such resistant cells also exhibit changesin transport kinetics of naturally occurring reduced folates. Thesechanges may potentiate the effects of antifolates capable ofpenetrating such folate-compromised cells (32, 36).

Overproduction of DHFR, on the other hand, has been shownto occur by increased enzyme synthesis due to selective ampli-

cation of DHFR genes (for reveiw, see Refs. 2 and 20). This typeof MTX resistance can also be partially overcome by the use oflipophilic antifolates such as DDMP where membrane diffusionoccurs so rapidly that even high levels of DHFR can be saturated(19). More recently, a novel technique involving the use of tritiateddeoxyuridine has also been reported as a means of overcomingresistance due to increased levels of DHFR, but this has yet tobe clinically evaluated (40).

High-dose MTX has been used to overcome acquired MTX

resistance; however, in head and neck cancer, at least thistreatment can result in severe normal tissue complications andmarginal clinical benefit (43). Of the lipid-soluble antifolates used

in clinical practice, DDMP has a limited efficacy at tolerateddoses and is associated with undesirable side effects which arethought to be related to its effects on histamine metabolism andrelatively long half-life in vivo (8, 29).

A new lipid-soluble inhibitor of DHFR, BW301U (7, 9,17, 33),

has recently been reported to be effective in overcoming MTXresistance due to overproduction of DHFR (33). In addition,BW301U has a relatively short half life in vivo and only minimalinfluence on histamine synthesis, suggesting a possible clinicalrole as an alternative to DDMP (7, 8).

In this study, we have examined the cytotoxicity of BW301Uon a number of MTX-resistant cell lines and their MTX-sensitive

counterparts and have compared these results with the cellularresponse to MTX and DDMP treatment.

MATERIALS AND METHODS

Cell Lines

All cells were maintained as static suspension cultures at 37°in RPMI

Medium 1640 supplemented with 4-(2-hydroxyethyl)-1 -piperazineethane-sulfonic acid buffer (2 x 10~2 M), L-glutamine (2 x 10~3 M), penicillin/

streptomycin (100 units/ml), and 10% fetal calf serum. Nondialyzedserum was used throughout this study. All cell counts and determinationsof cell viability were made by phase-contrast microscopy. MTX-resistant

cell lines were cultured in the absence of methotrexate for several monthsprior to study.

CCRF-CEM. This is a human leukemic T-cell line (14) which has beenmaintained in our laboratory for several years. Its MTX-resistant sublineCCRF-CEM/MTX R was derived by continuous exposure to 2 x 10~8 M

MTX for a period of 4 weeks. Our previous studies (39) have shown thatat this concentration of MTX, cell death and cell division are balanced,resulting in no net increase in cell numbers. After 1 month, cells in 2 x10~" M MTX were growing at the same rate as non-drug-treated controls.The MTX concentration was then increased from 2 x 10~8 M to 4 x 10~7

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BW301U AND MIX RESISTANCE

M over a further period of 6 weeks. At this time, the cells could maintaincontrol growth in 4 x 10~7 M MIX, and samples of them were stored at

-70°. Subsequent subculturing of CCRF-CEM/MTX R was made in

MTX-free medium.

The cell population doubling time for both cell lines was about 22 hr.In our hands, the CCRF-CEM cell line does not clone reliably in soft agar,

and so determinations of cell viability after drug treatment were made byphase-contrast microscopy.

L1210 and L5178Y. These are mouse leukemic cell lines which havebeen maintained in our laboratory for a number of years. Their MTX-

resistant counterparts, L1210/MTX R and L5178Y/MTX R, were obtained several years ago from Dr. J. Berlino, Yale University, New Haven,CT. The cell population-doubling times for all 4 cell lines were 12 to 14

hr. All 4 cell lines were cloned in soft agar, although both the L1210 celllines had a very much higher plating efficiency (90 to 100%) than theL5178Y cells (15 to 20%). Conditions used for soft-agar cloning have

been fully described elsewhere (38).

Drug Treatment

MTX was supplied by Dr. A. Hellestrand, Cyanamid (Australia) Pty,DDMP, and BW301U were supplied by Dr. C. A. Nichol, BurroughsWellcome Co., Research Triangle Park, NC.

All drugs were made up as concentrates at 30-fold of the final required

concentration in distilled water and sterilized by Millipore filtration. Solutions of BW301U were solubilized by the addition of NaOH (1 M). Thesolubility of DDMP was increased by the addition of 1 to 2 molarequivalents of lactic acid.

For all experiments, cells were set up at 10s cells/ml (CCRF-CEM) or5 x 104 cells/ml (L1210 and L5178Y) 24 hr before drug addition. For all

cytotoxicity data shown, the drug exposure time approximates 2 cellpopulation-doubling times, i.e., 48 hr for CCRF-CEM and 24 hr for L1210

and L5178Y, in order to facilitate comparison between cell lines.

MTX Uptake Studies

Prior to uptake experiments, [3',5'7-3H]MTX (Amersham Australia,

Pty. Ltd.) was diluted to a specfic activity of 1200 Ci/mol and purified bygel permeation chromatography on Fractogel TSK 40 (F) (BDH Australia)in a manner similar to that of Whitehead et al. (42).

Cells from exponentially growing cultures were adjusted to approximately 5 x 106 cells/ml in fresh medium, and MTX uptake into these

cells was monitored over a 4-hr period. Mean incorporation was determined for duplicate 1-ml cultures to which MTX (600 Ci/mmol) was

added in 10 ¿il(final MTX concentration, 2.5 //M). Cultures were maintained at 37°in a shaking water bath and at the end of specified time

intervals, terminating cultures were placed on ice and quickly diluted with9 ml of ice-cold Dulbecco's phosphate-buffered saline. Cultures were

immediately centrifuged at 1700 x g for 1 min at 3°and after decanting

the supernatant, the cell pellet was subjected to 2 further washes withice-cold Dulbecco's phosphate-buffered saline in quick succession (1

min at 1700 x g). Control cultures were placed on ice, and 10 M!of [3H]MTX were added and then allowed to stand at 4°for up to 1 hr prior to

washing as described above.The cell pellets were hydrolyzed at 70°for 2 hr after the addition of

100 A<Iof 3 M NaOH. Subsequently, 200 ¡Aof 2 M HCI were added, andthe total sample was counted on an LKB 1217/Rackbeta liquid scintillation counter. Results were expressed as pmol of [3H]MTX per 106 cells.Uptake of MTX in control cultures at 4°was minimal even after 60 minand represented predominantly a temperature-independent "instantaneous" uptake (34).

DHFR Assay

Crude extracts from cell cultures were assayed for DHFR activityusing a modification of the spectrophotometric technique described byMathews et a/. (27).

Approximately 5 x 10e cells were harvested and washed twice with

10-ml volumes of ice-cold phosphate-buffered saline. Cell pellets were

resuspended with 5 ml of 0.1 M potassium phosphate buffer at pH 7.0and homogenized by sonication. Homogenates were stored at -70°until assayed. After thawing, the homogenate was centrifuged at 4°for

1 hr (48,000 x g), and the clear supernatant was used in the assay. Thispreparation was stable with respect to DHFR activity for three dayswhen stored at 4°.

Assays were carried out at 37°in disposable plastic cuvets, and the

decreasing absorbance at 340 nm was measured in a Cary 219 spectra-

photometer. The final assay volume was 1.2 ml containing 60 ^mol ofpotassium phosphate buffer (final pH, 7.0), 1.2 mg of bovine serumalbumin, 12 //mol of 2-mercaptoethanol, 720 Mmol of KCI, 20 M'of enzyme

preparation, 48 nmol of NADPH, and 12 nmol of dihydrofolate. The assaywas initiated by the addition of dihydrofolate, and the resulting ratecorrected for a small background reaction where dihydrofolate wasomitted. Inhibition of DHFR was assayed by the addition of MTX (finalconcentration, 10~10 - 5 x 10~9 M) to the assay mixture. Protein

concentrations in the cell extract were estimated by the method of Lowryer al. (26).

RESULTS

Net uptake of MTX and levels of DHFR activity were examinedin all 3 MTX-resistant cell lines and compared with their MTX-

sensitive counterparts in order to determine the primary modeof MTX resistance prevailing in each case (Chart 1; Table 1). The

4- 3

l 2"o 1

Ea.

CCRF-CEM

r L 1210

Q5 1

Time (hr)Chart 1. Uptake of [3H|MTX by MTX-resistant cells (•)and their Mix-sensitive

counterparts (O).

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BW301U AND MIX RESISTANCE

Table 1DHFR activities in MTX-resistant and -sensitive cell lines

% of MTX inhibition"DHFR

CelllinesCCRF-CEMCCRF-CEM/MTX

RL1210L1210/MTXRL5178YL5178Y/MTXRactivity86101126653220IO"10

M868610-« M2337634436635x1 0~BM5695

"Activity expressed in nmol/mg/min at 37°using 10 ¡IMdihydrofoíateand 40

,.MNADPH.6 Expressedas percentageof remainingDHFRactivity in the presenceof MTX.

MTX

0 10

Drug concentration ( M )

Chart 2. Responseof CCRF-CEM(O)and CCRF-CEM/MTXR (•)cells to MTX,ODMP,and BW301U. CCRF-CEM/MTX R cells are MTX-resistant primarilydue toa drug uptake defect. Cell counts are of viable cells estimated by phase-contrastmicroscopy. Points, means of 2 to 3 individual experiments. The range betweenexperiments was not greater than 20%.

CCRF-CEM/MTX R-cells were found to have a much reduced

uptake of MTX and no major change in DHFR levels. In contrast,while little variation in initial MTX uptake was seen between theL5178Y cell lines, L5178Y/MTX R-cells had a 600-fold increasein DHFR levels compared to MTX-sensitive controls. This fact

would probably account for the higher ¡ntracellularlevels of MTXobserved in the resistant subline after approximately 30 min ofdrug uptake (Chart 1). These cell lines were, therefore, judgedto be resistant primarily through a transport defect and through

0.1

0.01

qoon\DDMP

E 0.1

0.1

0.01

BW301u

10° 10' 10° 10"

Drug concentration (M

Chart 3. Responseof L5178Y (O)and L5178Y/MTX R (•)cells to MTX, DDMP,and BW301U. L5178Y/MTX R cells are MTX-resistant due to the overproductionof DHFR. Surviving fraction was estimated from a colony-forming assay in softagar. Points, meansof 2 individualexperiments.

DHFR overproduction, respectively. The L1210/MTX R-cell line,

on the other hand, had both a reduced MTX uptake and anincreased level (24-fold) of DHFR activity and is therefore prob

ably resistant to MTX by virtue of both mechanisms. We foundno evidence to suggest that there was any substantial differencebetween sensitive or resistant cells in the degree to which DHFRwas inhibited by MTX (Table 1).

Chart 2 shows that CCRF-CEM/MTX R-cells are approximately 100-fold resistant to MTX compared with controls, and

that this resistance can be overcome by high concentrations ofMTX. When, however, these MTX-transport defective cells are

treated with either DDMP or BW301U (Chart 2), they appear tobe more sensitive to these agents than normal CCRF-CEM cells.

This contrasts with the response of the DHFR overproducingL5178Y/MTX R-cell line which is extremely resistant to MTX(~ 10,000-fold) and also shows comparable resistance to both

DDMP and BW301U although at high concentrations, both ofthese agents are superior to MTX (Chart 3).

The doubly resistant L1210/MTX R-cells (Chart 4) show re

sponses to all 3 agents intermediate to those described abovefor CCRF-CEM/MTX R and L5178Y/MTX R. This cell line demonstrates a 5000-fold increase in resistance to MTX and a

marked sensitivity to both DDMP and BW301U compared to

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0.1

."0.1

0.1

DDMP

BW 301U

IO'8 10'7 10'6 105

Drug concentration ( M

10'

Chart 4. Response of 11210 (O) and L1210/MTX R (•)cells to MIX. DDMP,and BW301U. L1210/MTX R cells are MTX-resistant due to a combined reduced

drug uptake and overproduction of DHFR. Surviving fraction was estimated froma colony-forming assay in soft agar. Points, means of 2 individual experiments.

Table 2

Comparative toxicity of MTX, DDMP, and BW301U

CelllinesCCRF-CEM

CCRF-CEM/MTXRL1210

L1210/MTXRL5178Y

L5178/MTX RMTXio-7

io-«5x1Cr7io-3*5x10-«»io-4D,o8DDMP10-»10-«10-«-io-45x10-«10-BW301U10-«io-75

x 10-75X1Q-«3X10-410-«

" D,o is the molar drug concentration required to decrease cell viability to 10%

of control values.0 Estimated by extrapolation.

MTX, but not to the extent of being more sensitivethan theparentallineas is thecasewith CCRF-CEM/MTXR.

Theeffectsof all 3 agentson eachcell linecanbeseenmostreadilyby comparisonof the valuesof the molarconcentrationrequiredto decreasecellviabilityto 10%of controlvalues(Table2). This shows that while both DDMPand BW301Uare moreeffectivethan MTX on MTX-resistantcell lines,particularlyathigh drug concentrations,collateralsensitivityto these compoundsis only found when MTX resistanceis due primarilytodefectiveaccumulationof thedrug.

DISCUSSION

ThisstudyshowsthatbothDDMPandBW301Uareselectivelycytotoxicto cellsthat are primarilyresistantto MTXby virtueofreducednet drug uptake.Similarfindingshavebeen reportedpreviouslyfor DDMP and other lipophilicantifolatesand arethought to be a consequenceof the alteredtransportkineticsfor naturallyoccurringreducedfolates in these cells (32, 36).This collateralsensitivityassumespotentialimportancein thelightof otherstudieswhereit was foundthat transportdefectsformedthemajorcategoryof acquiredresistanceto MTXinbothhumanand murinecell lines(30,37). In this respect,BW301Uappearsto be more effectivethan DDMPwith a molar drugconcentrationrequiredto decreasecellviabilityto 10%of controlvaluesfor CCRF-CEM/MTXR-cellsidenticalto that of MTXforthe MTX-sensitiveparent line. Approximately10-fold moreDDMPwas requiredundercomparablecircumstances.This increasein molarefficiencyof BW301Ucomparedto DDMPwasalsoobservedto a variabledegreeinallothercelllinesexaminedin this study.

It must be noted, however,that the collateralsensitivityofbothDDMPandBW301Uto transportdefectphenotypesis lostwhenthe modeof resistanceis compoundedby overproductionof DHFR.Underthesecircumstances,althoughdoublyresistantcells are more sensitiveto DDMPand BW301Uthan to MTX,these cells are relativelyresistant to both compoundswhencomparedwith the MTX-sensitiveparent line. In the studiesmentionedpreviously(30,37),theamplificationof DHFRactivityin the transportdefectphenotypewas foundto occurat a highfrequency,particularlyin humanlymphoblastoidcells.

In circumstanceswhere over productionof DHFRappearedto bethereasonfor MTXresistance,DDMPandBW301Uprovedto be more cytotoxic than MTX only at high dose levels,andsuchcellswererelativelyresistantto bothdrugswhencomparedto their MTX-sensitivecounterpartsas reportedpreviously(19,33).

Ourexperimentaldatado not ruleout thepossibilitythat MTXresistancehas developed,in part, throughthe mechanismsofalteredDHFRaffinityof the drug (13)or decreasedpolygluta-mylation(31).However,anypresumedchangesin DHFRinhibition kineticswould probablybe of minorsignificancegiventhedata presentedin Table1 and the extent of the alteredDHFRactivityand/orMTXuptakethatareapparentinresistantvariantsthat we describe.Furthermore,assumingthat MTX polygluta-mates may enhanceMTX cytotoxicity by virtue of retained¡ntracellulardrug after removalof the externaldrug supply(31)an alterationin polyglutamylationabilitywould be of little or noselectiveadvantageto any of the thesecell lineswhereresistancehasbeenestablishedin a systemwhereextracellulardrugis constantlyavailable.Therefore,it wouldappearmostunlikelythat the polyglutamylationstatusof theseMTX-Rlineshasanybearingon our basic observationswhich can be reasonablyaccountedfor withoutinvokingmechanismsotherthandefectivedruguptakeand/orincreasedDHFRactivity.

Todate,therehavebeenfewstudiescharacterizingthemechanismsinvolvedin the developmentof clinicallyrelevantresistance to MTX in humans,although it appearsthat transportdefects, amplifiedDHFRactivity and altered DHFRinhibitionkinetics,have been identified(3-5, 22, 24). The relevanceofmodelsof resistancesuchas we describehere,basedas they

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BW301U AND MTX RESISTANCE

are on immense selective pressures (e.g., constant drug exposure) and resulting in predictably dramatic changes in phenotype,can be questioned. Currently, we are investigating resistancemechanisms as they arise in intermittent drug treatment protocols that are more closely related to the clinical schedules usedin cancer treatment.

The data presented in this study suggest that lipophilic anti-

folates such as DDMP and BW301U have a potential role in thetreatment of MTX-resistant tumors. These drugs may be particularly effective in MTX-resistant tumors exhibiting defective MTX

uptake. The combined administration of MTX and a lipophilicantifolate such as BW301U may be advantageous as initialtreatment, particularly in those tumor types where MTX transportdefects are prone to occur.

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CANCER RESEARCH VOL. 45 MARCH 1985

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1985;45:978-982. Cancer Res   Ian W. Taylor, Peter Slowiaczek, Michael L. Friedlander, et al.   Methotrexate-resistant Cells with Reduced Drug UptakeSelective Toxicity of a New Lipophilic Antifolate, BW301U, for

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