[CANCER RESEARCH 42, 956-964, March 1982]0008-5472/82/0042-0000$o2.oo

Isolation of Fluoropyrimidine-resistant Murine Leukemic Cell Lines byOne-StepMutationand Selection1

Mary A. Mulkins and Charles HeideIberger@

University of Southern California, Comprehensive Cancer Center, Los Angeles, California 90033

adenine phosphoribosyltransferase (7, 12, 19), or TK (11, 12)has been extensively studied using clones derived after chemical mutagenesis and one-step selection to analog resistance.

Many of the clinically useful cancer chemotherapeutic agentsare purine or pyrimidine base or nucleoside analogs, and theeffectivenessofthesedrugsisoftenlimitedbythedevelopmentof resistance in the tumor cell population. Classically, themechanisms of resistance to these drugs have been studiedusing cell populations derived in vivo or in vitro by the continuous exposure of the cells to progressively increasing concentrations of a drug (10, 22). Resistant variants obtained by thismultistep selection may have undergone several sequentialchanges that affect the same drug-resistant phenotype (10). Inaddition, many of those cell lines are phenotypically unstableand require the continuous presence of the drug for the maintenance of resistance. This report presents a systematic approach to the study of the biochemical mechanisms of resistance to the fluorinated pyrimidine cancer chemotherapeuticdrugs.

FUra and its nucleoside derivatives, FdUrd and FUrd, weredeveloped by Heidelberger et a!. (18) in 1957. These drugshave been shown to be active against a wide variety of transplantable animal tumors (18) and are used clinically in themanagement of solid tumors of the breast and gastrointestinaltract (17). Like other pyrimidine analogs, FUra, FdUrd, andFUrd must be anabolized to nucleotides by salvage enzymes inorder to exert antineoplastic activity (17). The primary mechanism of fluoropyrimidine cytotoxicity is considered to be theinhibition of DNA synthesis as a consequence of the metaboIism of these drugs to 5-fluoro-2'-deoxyuridylate, which is apowerful inhibitor of thymidylate synthetase. The drugs arealso incorporated, with important biological consequences, intoRNA(i7).

With an early exception (28), most of the previous studies ofthe mechanisms of resistance to FUra have utilized cell linesisolated by a multistep procedure (17). This report describesthe isolation of variant cell lines derived from the murine leukemias, Li 210 and P388, by treatment with chemical mutagens and single-step selection in agarose containing high levelsof FUra, FdUrd, or FUrd. When this research was initiated, thisapproach was novel. During the course of the work, severalrecent and independent examples of mutation and one-stepselection to discern mechanisms of drug resistance have appeared (21 , 23, 31 , 37). The importance of individual biochemical changes to fluoropyrimidine resistance will be described inthe accompanying paper (29). The drug-resistant clones isolated in this study behave as mutants in that they retain theirresistance and characteristic enzyme changes after repeated

dine; EMS, ethylmethane suifonate; IC,@,,concentration of drug that allows a 50%increase in cell number relative to the Duibecco's phosphate-buffered salinecontrols; LD,o, concentration of drug that reduces the cloning efficiency to 50%of that observed with cells plated in untreated dishes.

956 CANCERRESEARCHVOL. 42

ABSTRACT

The effectiveness of the clinically useful fluoropyrimidines inthe treatment of human cancer is often limited by the development of resistance to the drugs by the tumor. In order tosystematically study the mechanisms of resistance to 5-fluorouracil and its nucleoside derivatives, several cell lines resistant to these drugs have been derived from murine leukemiacells by a one-step mutation and selection procedure. Logarithmically growing suspension cultures of Li 210 and P388cells were treated with ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, or ICR-i 91 at concentrations whichresult in 20 to 30% cell survival. After a 10-day expressiontime, mutagenized cells were plated into soft-agarose mediumthat contained 1O@M 5-fluorouracil, 1O@M 5-fluoro-2'-deoxyuridine, or 1O_6M 5-fluorouridine. Twenty stable clones wereisolated and found to be 5- to 28-fold resistant to growthinhibition by 5-fluorouracil, 4,000- to 25,000-fold resistant to5-fluoro-2'-deoxyuridine, or 8- to 220-fold resistant to 5-fluorouridine. The clones retain their drug-resistant phenotypeafter repeated passaging in the absence of selection.

Since the biochemical changes responsible for resistance toone drug can render the cells collaterally sensitive to otherdrugs, the growth-inhibitory effects of antimetabolites that inhibit other steps in pyrimidine metabolism were examined inthe wild-type cells and in the fluoropyrimidine-resistant sublines. Although cross-resistance to 5-azacytidine was found inLi 210 cells selected for resistance to 5-fluorouridine, none ofthe cell lines tested demonstrated collateral sensitivity to methotrexate, 1-fl-D-arabinofuranosylcytosine, 5-azacytidine, or N-(phosphonacetyl)-L-aspartate.

INTRODUCTION

Since it was demonstrated in 1968 by Kao and Puck (20)and by Chu and Mailing (9) that the frequency of phenotypicvariants in cultured Chinese hamster cells could be increasedby treating the cells with alkylating agents, chemical mutagenesis has been used to study many genetic loci in somatic cells.Resistance to purine or pyrimidine analogs has often beenchosen as a genetic marker, since the salvage enzymes required for the anabolism of these drugs to their cytotoxicnucleotide forms are not essential for cells possessing a functional de novo pathway for nucleotide biosynthesis (5, 12). Theloss of such salvage enzymes as HGPRT3(5, 8—10, 12, 38),

Received July 10, 1981 ; accepted December 8, 1981.,SupportedinpartbyagrantfromtheHoffmann-LaRocheFoundation.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: HGPRT, hypoxanthine-guanine phosphoribo

syitransferase; TK, thymidine kinase; FUra, 5-fluorouracil; FdUrd, 5-fluoro-2'-deoxyuridine; FUrd, 5-fluorouridine; MTX, methotrexate; are-C, 1-@-o-arabinofuranosylcytosine; 5-aza-CR, 5-azacytidine; PALA, N-(phosphonacetyl)-t.-aspartate; DFCS, dialyzed fetal calf serum; MNNG, N-methyl-N'-nitro-N-nitrosoguani

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Mutation to F!uoropyrimidine Resistance

passaging in the absence of selective agents.As a tumor cell develops resistance to a cytotoxic drug, the

associated biochemical changes may make it collaterally sensitive to a second drug, and selective cytotoxicity is likely toresult if a drug-resistant tumor is treated with a compound towhich it is collaterally sensitive (30). Twelve representativeclones resistant to FUra, FdUrd, and FUrd were compared tothe wild-type cell lines with respect to their relative sensitivityor resistance to other chemotherapeutic drugs. Because collateral sensitivity is most likely to be demonstrated amonginhibitors of related steps in a single metabolic sequence (30),drugs which inhibit various steps in pyrimidine biosynthesiswere tested, including MTX, ara-C, 5-aza-CR, and PALA. Thesestudies were undertaken to determine the effect of a partial ortotal deletion of an enzyme involved in pyrimidine metabolismon the cytotoxicity of chemotherapeutic drugs which affect thismetabolic pathway.

MATERIALS AND METHODS

Materials. FUra, FdUrd, and FUrd were generously provided byHoffman-LaRocheInc., Nutley, N. J. ICR-i91 was purchasedfromPolysciences, Inc., Warrington, Pa. PALA (NSC 2241 31 ) was obtainedthroughthe NationalCancerInstitute,Bethesda,Md. All mediaandsera were obtainedfrom Grand Island Biological,Co. Santa Clara,Calif.All other chemicalswere purchasedfrom SigmaChemicalCo.,St. Louis,Mo., andplastictissueculturewarewasfromCorningGlassWorks,Corning,N. V.

Maintenance of Cell Lines. The Li 21 0 murine leukemia cell linewasobtainedfrom Dr. RichardMoran,and the P388 murinelymphocytic leukemia line was provided by the Arthur D. Little Corp., Cambridge,Mase.Freshcellswerethawedfroma Mycop!asma-freestockevery 2 monthsduring the course of this study to maintaingeneticstabilityin the culture.Stocksof resistantcloneswerefrozenin liquidnitrogenimmediatelyafter isolation,andtestsfor Mycop!asmausingamodIficationof the cultureprocedureof Hayflick(16) werenegative.All cell lines were maintainedin suspensionculture in RoswellParkMemorialInstituteMedium1640withoutantibiotics,supplementedwith10% DFCS. Modal chromosome numbers were determined for thewild-type cells and for some resistant clones according to a modification of the procedureof Bloch-ShtacherandSachs(4).

Dstsrmlnetlon of Cloning Efficiencies in Soft Agarose. As a measure of cytotoxicity, the colony-forming ability of individual cells in thepresence of selective agents was assayed in soft agarose, using amodificationof theprocedureof Macpherson(25).Cells(1O@to 1O@)in2 ml of mediumwith 10% DFCS,10% dialyzedhorseserum,2 mp@ipyruvate, and 0.24% agarose (Sigma type II) were layered over 5 ml of0.4%agarosein thesamemedium,whichhadbeenpouredpreviouslyinto a 60-mm plastic Petri dish. Various concentrations of FUra , FdUrd,or FUrdwereaddedto someof thedishes,whichwerethenincubatedfor 2 to 3 weeks in a humidified37°incubatorunder 5% CO2.Thenumber of colonies in dishes containing drug was compared to that inuntreated dishes, plated at the same cell density where possible. Onlycolonies which contained at least 50 cells were scored. At higher celldensities, several 4-sq mm areas were counted using a stereomicroscope with a grid, and the average colony number was determined forthe entireplate.

The agarosecloningefficiencyassaywas also usedto determinethecytotoxicityof thechemicalmutagensusedin thisstudy.Mutagenized cells were plated into nonselective agarose medium 24 hr aftertreatment, and the cloning efficiencies were compared with those ofcellstreatedwith0.5%acetoneonly.

Mutagsnsls and SelectIon Procedure. Mutagen treatment wasbased on the methodof Thompsonand Baker (38). Exponentiallygrowingcultures(4to5 x 1O@cells/mI)weretreatedincomplete

medium buffered with 16 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 8 mM morpholinopropanesulfonic acid. Cells weretreatedwith: 2.7 to 6.8 ,@MMNNGdissolvedin acetone(0.1% finalconcentration) for 24 hr, 1.1 to 2.2 zM ICR-191 dissolved in dimethylsulfoxide (0.5% final concentration) for 24 hr, or 10 mM EMS for 2 hr.Theseconcentrationsof mutagensresultedin 20 to 30%cell survival,relative to solvent-treated controls. The cultures were incubated in arotating 37°incubator after the addition of the mutagen. After 24 hr,aliquotsof thecellsuspensionwereplatedinsoftagarosetodeterminethe cytotoxicity of the mutagen. Cultures were fed every 2 days withfresh nonselective medium for 8 to 10 days. After this time, 4 dishes of1@ cells/dish were plated in nonselective agarose for cloning efticiency determinations, and 5 to 20 dishes containing 1o@cells/dishwereplatedin eachof the selectivedrugs,FUra,FdUrd,or FUrd.Theconcentrationof eachdrug usedfor selectionwaschosenfromcytotoxicity determinations as that concentration which completely prevented colony formation of 1O@sensitive cells/dish. Cells treated withacetoneor dimethylsulfoxidewere run in parallelas controls in allexperiments.

Cellsplatedintotheagaroseselectivemediumwereincubatedfor 3to 4 weeks,with 1 mlof freshmedium(containingtheselectiveagent)added at the end of the second week to replace nutrients and moisturelost to evaporation. Clones appearing in the selective dishes wereisolatedby cutting a smallsquarearoundeach clone in the agaroseand incubatingit in a small test tube containingfresh nonselectivemedium.Cellsthatgrewoutof theagaroseweregrownin nonselectivemediumfor 1 month and were then tested in the growth inhibition assaydescribedbelow,sothatonlythosecloneswithastable,drug-resistantphenotype would be further studied.

Determinationof Growth Inhibition. As a rapid and convenientdeterminationof the growth-inhibitoryeffectsof variouschemotherapeutic drugs on the resistant clones isolated in this study, the techniqueof Grindey and Nichol (14) was used. Drugs were dissolved in Dulbecco's phosphate-buffered saline, and 200 @dof each drug concentrationto betestedwereaddedin duplicateto sterile12- x 75-mmtesttubes. Control tubes contained 200 @lof Dulbecco's phosphatebufferedsalineonly.A cellsuspensionof 2 x 1O@cells/mI in completemediumbufferedwith 16 m@N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid and 8 mMmorpholinopropanesulfonicacid wasprepared, and 1.8 ml were pipetted into each tube using a Cornwallautomaticpipet.Thetubeswerestopperedwith No.00 latexstoppersandincubatedat 37°.Whenthecontrolcellshaddoubled4 to 5 times(48 to 60 hr), the tubeswerevortexed,and the cell numberin eachwasdeterminedusinga ModelB CoulterCounter.

RESULTS

Expression Time. After mutagen treatment, Sometime mustelapse before selection to permit expression of new phenotypes and to allow the cells to resume their normal doublingtime and cloning efficiency. The optimal expression time inLi 2i 0 and P388 cells after treatment with MNNG (i .tg/ml)was determined for each of the 3 selective agents, FUra,FdUrd, and FUrd. Every day for i 2 days, i 06 cells/dish wereplated into each selective medium, and the number of coloniesobserved was corrected for cell survival. The results are presented in Chart i . For both Li 2i 0 and P388 cells, with all ofthe selective agents, the maximum plateau region of theexpression time curves included Day 10. Since it was convenient to plate mutagenized cells into all 3 selective media on thesame day, i 0 days was chosen as the standard expression

timefortheselectionoffluoropyrimidine-resistantmutants.Frequencies of Fluoropyrimidine-resistant Colonies. Using

the conditions described in â€M̃aterials and Methods,' â€w̃hichwere experimentally determined to be optimal for mutagen

MARCH 1982 957

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Mutation frequencies of IIuoropyrimidineresistanceCell

lineTreatmentaSurviving fraction@'Resistant

clones! 1o@survivorsCi

o@ PAFUra1 o@ M FdUrd1 0_UMFUrdL1210Acetone

MNNG(3.4@,,i)MNNG(6.8@M)EMS(lOmM)ICR-191 (2.2MM)1

.00.30.20.30.30.004

(8)d0.09 (11)1.15 (19)0.11 (5)0.084 (5)0.14

(10)1.5 (11)7.8 (20)1.7 (6)0.48 (6)0.02

(10)0.20 (ii)1.16 (19)0.29 (4)0.17(4)P388Acetone

MNNG (3.4 @LM)MNNG(6.8@iM)EMS(lOmM)ICR-191 (2.2@uM)1

.00.30.20.30.30.002

(9)0.048 (11)0.22 (14)0.083 (9)0.018 (6)0.006

(8)0.1 15 (10)0.185 (11)0.182(16)0.15 (5)0.002

(8)0.052 (10)0.12 (13)

<0.006 (8)0.005 (6)

M. A. Mu!kins and C. Heide!berger

treatment and selection, clones resistant to FUra, FdUrd, andFUrd were isolated. The observed mutation frequencies tofluoropyrimidine resistance from 4 to 20 independent experi

U)

aU

C

U)

U)0

aU)aC00(2

Chart1. Phenotypicexpressiontimeof fluoropyrimidineresistancein Li 2i 0(•)andP388(0) cells.CulturesweretreatedonDay0 wIth6.8 @ø.iMNNGinsuspension medium, as described in the text, and were plated in soft agarose onsuccessive days with 10@ N FUra (A), 10@ PAFdUrd (B). or 10@ PAFUrd (C).

ments are summarized in Table 1. In Li 210 cells, the spontaneous frequency of FUra-resistant colonies was about 4/i O@cells. When FdUrd was used as the selective agent, this figurewas much higher, about 1/1 0@cells; while for FUrd resistance,the spontaneous frequency was 2/i O@cells. The treatment ofLi 210 cells with 3.4 ,sMMNNG induced a 20-fold increase incolonies resistant to FUra and a i 0-fold increase in clonesresistant to FdUrd and FUrd. When the concentration of MNNGwas raised to 6.8 LM,the induction of FUra-resistant colonieswas 300 times the spontaneous rate, while clones selectedwith FdUrd or FUrd were 60 times more frequent than in theacetone-treated controls. Treatment of Li 210 cells with 10 mp@iEMS or 2.2 JLMICR-i 91 gave the same cell survival (about30%) as did treatment with 3.4 @MMNNG. Treatment with the3 mutagens at equitoxic levels also induced approximately thesame frequency of resistant colonies in the 3 selective agents

(i/1O6 cells with FUra, i/i05 cells with FdUrd, and 2/10@cells with FUrd).

In P388 cells, the spontaneous frequency of resistant colanies in the 3 selective agents was <6/ i O@cells (Table i).Treatment with 3.4 @MMNNG, which reduced cell survival to30%, produced a 20- to 25-fold increase in the number ofcolonies observed in all 3 selective agents. When this concentration of MNNG was doubled (6.8 sM),the frequency of FdUrdor FUrd-resistant variants was also doubled, while the numberof colonies selected with FUra was increased 4-fold. WhenP388 cells were treated with 10 mp,iEMS, the frequency ofcolonies capable of growth in FUra or FdUrd was 30 to 40times the spontaneous rate, but no colonies were observedamong cells selected with FUrd. In some experiments, 2.2 @sMICR-i 9i was used to induce mutations. The induction of FUraresistant clones by this mutagen was 9 times the spontaneousrate, while FdUrd-resistant colonies appeared at 25 times thefrequency of the acetone-treated controls. Clones of P388cells surviving FUrd selection after treatment with 2.2 @ip,iICR191 appeared at only 3 timesthe spontaneousfrequency.

Thus, under these selective conditions, the spontaneousfrequency of variants resistant to FUra, FdUrd, or FUrd wasless than 1O@ (except for LI 210 cells selected with FdUrd,which had a frequency of 1O_6),and an increased frequencyof resistant colonies was observed after treatment with theknown chemical mutagens, MNNG, EMS, or ICR-i 91.

Isolation of FluoropyrlmidIne-reelstant Colonies. Coloniesof Li 210 and P388 cells that survived in the 3 selective media

Table 1

78

Days after treatment

aCellsweretreatedin suspensionasdescribedin@ MaterialsaridMethods―at 2 hr for EMSand24 hr for all othertreatments.b Fraction of cells which formed colonies in agarose 24 hr after mutagen treatment, relative to solvent-treated controls.C Observed total number of colonies per total number of surviving cells.

d Numbers in parentheses, number of independent experiments used to derive mutation frequencies.

CANCERRESEARCHVOL. 42958

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Fluoropyrimidine-resistantclones isolated from L 1210cellsMutagen

treatment°Selective agentbDoublingtime

(hr)Li2lOwlld-typell.9±O.7cL12i0

FUralL1210 FUra2L121OFUra3L121OFUra4MNNG

(3.4 @zM)MNNG (3.4 @M)MNNG(1.4p@@i)EMS(i0mM)FUra

(10@ N)FUra (lOs M)FUra(105M)FUra(105 PA)13.5

±2.8

13.1 ±2.511.5 ±0.5L1210

FdUrdlMNNG (3.4 @tM)FdUrd (10@ M)13.0 ±2.4LI21OFUrd1

L1210 FUrd2L121OFUrd3L1210 FUrd4L121OFUrd5MNNG(3.4@i)

MNNG (3.4 @M)MNNG(3.4@ø.i)MNNG (3.4 @M)IcR-191 (1.1 ,@M)FUrd(106M)

FUrd(10.8M)FUrd(10°M)FUrd(106 M)FUrd(10M)12.3

±0.9

13.4 ±2.2

13.5 ±2.7

Fluoropyrimidine-resistant dones isolated from P388cellsMutagen

treatmentaSelective agent@'Doublingtime

(hr)P388

wild-type1 1.3±P388

FUralMNNG (2.7 @ø,l)FUra(105 M)14.3 ±1.0P388FUra2MNNG (5.4 @zM)FUra(105 M)15.1 ±0.7P388FUra3MNNG (6.8 @*M)FUra (10@M)P388FUra4EMS (7.5 mM)FUra (i05M)P388FUra5EMS(lOmM)FUra(105M)13.8

±0.6P388

FdUrdlMNNG (2.7 uM)FdUrd (10@M)P388FdUrd2MNNG (4.1 @M)FdUrd (10@ M)14.0 ±0.5P388FdUrd3EMS (10 mM)FdUrd (10@M)P388FdUrd4

P388 FdUrd5ICR-191(1.1tiM)

ICR-i 91 (2.2 tiM)FdUrd(1O@M)

FdUrd (1o@ M)15.2±1.8

Growth inhibition studies of L 12 10cloneslC@

[1o@ x concentration(M))―FUraFdUrdFUrdLl2lOwild-type360±

700.53± 011b4.1±1.2Li

210 FUrai4,600 ± 300 (13)C0.98 ± 0.17 (0)65 ±14(16)Ll2iOFUra27,200±100(20)0.80± 0.15(0)2.2±0.3(0)Ll2iOFIJra310,000±1,000(28)0.83±

0.04 (0)15±1(4)L121OFUra45,100±400(14)0.99± 0.33 (0)10±2(2)L1210

FdUrdi410 ± 10 (0)1800 ±500 (3600)9.2±0.1(2)Li210

FUrdi490 ± 230 (0)0.59 ± 0.12 (0)860±130(220)L1210FUrd2440 ± 160 (0)0.36 ± 0.03 (0)370±100(90)L1210FUrd3890 ± 360 (2)0.86 ± 0.21 (0)770±350(190)Li210FUrd4370 ± 210 (0)0.40 ± 0.05 (0)31±18(8)Li210FUrd5440 ± 230 (0)0.51 ± 0.10 (0)370 ±220(90)

Mutation to F!uoropyrimidine Resistance

were then Isolated as described in â€M̃aterials and Methods.―Both of these cell lines are very density dependent for cellgrowth and do not survive in liquid medium at concentrationsof less than I 04 cells/mI. Consequently, only about 5% of theobserved colonies (usually the largest) survived the procedureof removal from the soft agarose cloning medium and growthin suspension culture. No clones were isolated from experiments in which spontaneously arising colonies were seen inthe solvent-treated controls.

The fluoropyrimidine-resistant clones of Li 210 cells arelisted In Table 2, with the mutagen treatment used to inducethem and the agent used to select them. Also shown are thepopulation-doubling times determined for each cell line grownin suspension culture with Roswell Park Memorial InstituteMedium 1640 plus 5% DFCS. None of the resistant clones hada doubling time that was more than i 3% (1.6 hr) longer thanthat of the wild-type Li 2i 0 cells (11.9 hr). Chromosome numbers were determined from 30 to 45 metaphase cells fromeach line. Li 210 cells are pseudodiploid, with a modal chromosome number of 40, while in the resistant clones the modalchromosome numbers vary from 39 to 40.

Table 2

The fluoropyrimidine-resistant clones of P388 cells are listedin Table 3. In this cell line, none of the colonies that wereselected in 1O@ M FUrd could be isolated. These coloniesappeared at a lower frequency, and those that were observedin soft agarose could not be transferred to growth in liquidmedium. The population-doubling time for P388 cells was i 1.3hr in medium supplemented with 5% DFCS, and the doublingtimes determined for the FUra- and FdUrd-resistant cloneswere 20 to 35% longer than for the wild-type cells. Analysis of30 to 45 metaphase cells from each line determined that P388cells have a modal chromosome number of 40, while theresistant clones vary in modal chromosome number between36 and 40.

Growth Inhibition by the Fluoropyrimidines In IsolatedClones. After isolation, the clones were grown in nonselectivesuspension medium for i month. The growth inhibition assaydescribed in @‘Materialsand Methods' ‘was then used to determine the relative sensitivity of the resistant clones to the fluoropyrimidines. Since it was of interest to determine whetherany of the clones was resistant to more than one of the drugs,the lCso5for FUrs, FdUrd, and FUrd were determined for eachof the isolated resistant clones. Table 4 presents a summary of

Table3

a Mutagen treatment as described in Materials and Methods.@

b@ after mutagen treatment in soft agarose cloning medium in thepresence of the indicated drug.

C Mean ± S.E. of 2 to 4 determinations of population-doubling time, measured

in suspension culture in Roswell Park Memorial Institute Medium 1640, supplemented with 5% DFCS.

a Mutagen treatment as described in@ Materials and Methods.@b Selected after mutagen treatment in soft agarose cloning medium in the

presence of the Indicated drug.C Mean ± SE. of 2 to 4 determinations of population-doubling time, measured

in suspension culture in Rosweli Park Memorial Institute Medium 1640, supplemented with 5% DFCS.

Table 4

a@ determinedasdescribedin‘MaterialsandMethods.'@b Mean ±S.D. of 3 to 5 independent experiments.C Numbers in parentheses, fold increase in concentration of drug required to inhibit cell growth by 50%, relative to

wild-type cells.

MARCH 1982 959

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Growth inhibition studies of P388clonesIC50

[10' xconcentration(M)rFUraFdUrdFUrdP388

wild-type500 ± 1700.56 ± O.l9@'3.6 ±0.8P388

FUralP388 FUra2P388 FUra3P388FUra4P388 FUra51

2.000 ±2,000 (24)'2,500 ± 400 (5)3,400 ± 300 (6)5,600 ± 500 (11)4,200 ± 800 (8)1

.0 ± 0.10.38 ± 0.19.5 ± 0.31.1 ± 0.10.97 ± 0.18(0)

(0)(17)

(2)(0)1

3 ±1 (3)4.8 ±0.3 (0)

21 ±1 (5)12 ±3 (3)12 ±1(3)P388

FdUrdlP388FdUrd2P388 FdUrd3P388 FdUrd4P388 FdUrd52,600

± 200 (5)440 ± 120 (0)

1.000 ± 300 (0)860 ± 120 (0)670 ± 110 (0)1

1 000 ±3,00011 000 ±2,00014,000 ±9,00011.000 ±4,00011.000 ± 1 000(20,000)

(20,000)(25,000)(20,000)(20,000)9.2

±1.1 (2)3.2 ±1.2 (0)9.6 ±0.6 (2)8.4 ±2.7 (2)7.2 ±0.5 (0)

M. A. Mu!kins and C. Heide!berger

IC50values for wild-type Li 2i 0 cells and resistant clones.Clones selected with FUra required i 3 to 28 times more FUrafor growth inhibition than did wild-type Li2iO cells and werenot cross-resistant to FdUrd. However, one clone, Li 210FUrai , was equally resistant to FUra and FUrd. Li 2i 0 FdUrdiwas as sensitive to FUra and FUrd as were the wild-type cellsbut was 3600-fold resistant to FdUrd, its selective agent. Theclones selected in FUrd had lCsosfor FUrd that were 8 to 220times higher than that observed for the wild-type cells, but nochanges were seen in the IC50sfor FUra or FdUrd.

The lC@sfor wild-type P388 cells and resistant clones aresummarized in Table 5. Clones selected with FUra required 5to 24 times more FUra for growth inhibition than did the wildtype cells. Four of the clones were 3- to 5-fold resistant toFUrd, and one clone, P388 FUra3, was also cross-resistant toFdUrd. P388 clones selected with FdUrd were 20,000- to25,000-fold resistant to that drug. One clone, P388 FdUrdi,was also 5-fold resistant to FUra, but none of the other cloneswas more than 2-fold resistant to FUra or FUrd.

Stability of the Fluoropyrimidine-resistant Clones. The stability of the drug-resistant phenotypes was measured afterpassaging the resistant clones in nonselective medium for 90days. The agarose cloning assay was then used to determinethe cytotoxicity of the agent with which each clone had beenselected. For each resistant line, i 0@to i 06 cells/dish wereplated at increasing concentratiOns of the selective agent, andthe cloning efficiencies were compared to those seen in theabsence of drug. The cytotoxicity curves for several resistantclones are compared to those for the wild-type cell lines inCharts 2 and 3.

The relative cell survival with increasing concentration ofFUra, FdUrd, or FUrd is shown for wild-type Li 2i 0 cells andfor several resistant cell lines in Chart 2. The LDso for FUra,which was 3 x i O-@M in wild-type Li 2i 0 cells, was shifted to4 x i06MfortheLi2i0FUrai celllineand i.5 x iO5MforLi 2i 0 FUra3 cells (Chart 2, top). When FdUrd was used asthe selective agent (Chart 2, midd!e), the LDso for wild-typeLi 210 cells (3 x iO1° M)was shifted to 6x i07 Mfor Li 2i0FdUrdi cells. Chart 2 (bottom) presents cell survival as afunction of increasing FUrd concentration. The LDsofor wildtype Li 21 0 cells (5 x i O@ M) was shifted more than 200-fold

(to 8 x 10-@ M) in the 2 FUrd-resistant clones tested.

In a similar study, Chart 3 (top) demonstrates that both P388FUra2 and P388 FUra5 had LD@sfor FUra of 4 x i O_6 M,compared to 7 x i 0@ M for wild-type P388 cells. Likewise,the LD50was i 0,000-fold higher for 2 FdUrd-resistant clones,P388 FdUrd2 and P388 FdUrd4, than for the wild-type P388cells (Chart 3, bottom).

For all 3 drugs, the LD50values determined by the agarosecloning assay agree closely with the ICsovalues (Tables 4 and5) measured by the growth inhibition assay.

Collateral Sensitivity Studies. The relative sensitivity orresistance of the fluoropyrimidine-resistant clones to growthinhibition by chemotherapeutic agents that affect different partsof the pyrimidine-biosynthetic pathway was studied, and theresults are shown in Table 6. Li 210 and P388 clones selectedfor resistance to FUra, FdUrd, and FUrd were not significantlyaltered in their sensitivity to MTX or PALA. Two clones, Li 2i 0FUrai and Li 2i 0 FUrdi , were slightly (4- and 3-fold, respectively) cross-resistant to ara-C, but inasmuch as the other FUraor FUrd-resistant clones were equally as sensitive as the wildtype line, no general conclusions on the mechanism of crossresistance can be made without further studies. Li 2i 0 clonesselected for resistance to FUrd (Li 210 FUrdi , FUrd3, andFUrd5) were cross-resistant to 5-aza-CR, as was a clone selected for resistance to FUra (Li 2i 0 FUrai ), which demonstrated cross-resistance to FUrd (Table 4). In the cell linesselected for resistance to FUrd, the kinase that phosphorylatesFUrd has significantly lower activity (29), and this enzyme isapparently also responsible for the activation of 5-aza-CR.

DISCUSSION

The Li 210 and P388 murine leukemia cell lines were selected to study the mechanisms of resistance to the fluoropyrimidines for several reasons. Grown as transplantable ascitestumors in DBA/2 mice, these lines are thoroughly validatedmodel systems in which to quantitatively study the activity ofchemotherapeutic drugs (35). !n vitro, these lines have a shortdoubling time and grow to high cell densities in suspensionculture, which enable large batches of cells to be grown forbiochemical analysis. The cells are clonable in soft agarose,so that quantitative cytotoxicity studies can be done. BothLi 2i 0 and P388 cells have a relatively stable, pseudodiploid

Table 5

a @@50determined as described in@ Materials and Methods.@b Mean ±S.D. of 3 to 5 independent experiments.C Numbers in parentheses, fold increase in concentration of drug required to inhibit

cell growth by 50% relative to wild-type cells.

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Mutation to F!uoropyrimidine Resistance

Chart 3. Top, relative cloning efficiencies of wild-type P388, P388 FUra2, orP388 FUra5 cells as a function of increasing concentrations of FUra in the softagarose medium during a 2-week exposure. Bottom, relative cloning efficienciesof wild-type P388, P388 FdUrd2, or P388 FdUrd4 cells as a function of increasingconcentrations of FdUrd in the soft agarose medium during a 2-week exposure.

karyotype, which should allow a recessive phenotype to bemore easily expressed than in a polyploid cell line (6, 33).

Mutations at loci that confer resistance to other purine andpyrimidine analogs have usually been recessive for the drugresistant phenotype (i 2, 36). In diploid cells, the genes codingfor salvage enzymes should be present in 2 copies (i 2, 36),and both alleles must be made nonfunctional before enzymeproduction ceases. Therefore, the most commonly studiedanalog resistance locus is that coding for HGPRT, since this

Chart 2. Top, relative cloning efficiencies of wild-type Li 210, Li 210 FUrai,or Li 210 FUra3 cells as a function of increasing concentrations of FUra in thesoft agarose mediumduring a 2-week exposure. Middle, relative cloning efficiencies of wild-type Li 2i 0 or Li 210 FdUrdi cells as a function of increasingconcentrations of FdUrd in the soft agarose medium during a 2-week exposure.Bottom, relative cloning efficiencies of wild-type Li 210, Li 210 FUrdi , or Li 210FUrd3 cells as a function of increasing concentrations of FUrd in the soft agarosemedium during a 2-week exposure.

z0I-.

U.(9z5'>

C/),

io_4

[FUrdl

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IC@[i 0)( concentration(M)rMTXara-C5-aza-CRPALALi

210 wild-type1 4 ±2b71 0 ± 3302i .000 ± i000Ll2iOFUrai

Li2iOFUra3Li2iOFUra49.2

±2.716 ±616 ±5226

±79(4)C87±4360±238,700

± 5,100 (i2)720± 530370± i6039,000

±27,00039,000± 4,00037,000±2,000Li2i0FdUrdi15

±466± 17670± 27022,000±8,000Li

210 FUrdiL12i0 FUrd3Li 210 FUrd58.4

±0.223 ±517 ±4i

87 ±55 (3)49 ±2796 ±3436,000

±15,000 (5i )18,000 ± 8,000 (25)20,000 ±i i .000 (28)29,000

±24,00026,000 ± 8,0002i 000 ±1,000P388

wild-type5.0 ±2.049 ±24680 ± 2i 01 5,000 ±6,000P388

FUraiP388 FUra2P388 FUra54.3

±0.54.2 ±2.13.6 ±0.93i

± 745 ± 235 ±14780

± 480580 ± 390630 ± i8027,000

± 7,000i 7,000 ± i 00027,000 ±5,000P388

FdUrd2P388 FdUrd44.7

±0.85.7 ±3.339

± 744 ±15330

± 80470 ± 2024,000

± 7,00015,000 ± 3,000

M. A. Mu!kins and C. Heide!berger

Table 6Growth inhibition by other chemotherapeutic agents in wild-type and fluoropyrimidine-resistant clones

a IC@ determined as described in@ Materials and Methods.b Mean ±S.D. of 3 to 4 independent experiments.C Numbers in parentheses, fold increase in concentration of drug required to inhibit cell growth by 50%, relative to

wild-type cells.

gene is located on the X-chromosome in mouse, Chinesehamster, and human cells and consequently is functionallyhemizygous (24). Nevertheless, recessive mutations have beenmeasured in salvage enzyme genes that are thought to bepresent as 2 functional alleles in the pseudodiploid cell linesstudied. Clive et a!. (1 i ) have shown that the loss of TK inL5i 78Y mouse cells occurs in 2 discrete steps. The first stepproduces cells with reduced activity of this salvage enzyme,accompanied by partial resistance to the selective analog,while the second step yields highly resistant cells with nodetectable TK activity. The spontaneous frequency observedfor the second step was approximately 2 x i O@, a value inthe range of those obtained for spontaneous mutation frequencies at the X-linked HGPRT locus. Treatment with chemicalmutagens can increase the spontaneous frequencies observedat the TK locus i 00- to 1000-fold (i i).

As shown in Table 1, the observed one-step mutation frequencies to fluoropyrimidine resistance were higher in Li 210and P388 cells than would have been predicted for recessiveloci in pseudodiploid cells. In Li 2i 0 cells, the spontaneousmutation frequencies to resistance were approximately 4 xi 0-8 for FUra, 2 x i O@ for FUrd, and slightly greater thani 0_s for FdUrd. The observed spontaneous frequencies ofresistance to each of the 3 drugs ranged between i08 andi O-@ in the P388 cells. Chemical mutagens were used toinduce a 3- to 300-fold increase in fluoropyrimidine-resistantvariants (Table i ). The spontaneous and chemically inducedmutation frequencies at loci involved in fluoropyrimidine resistance are therefore in the same range as those thought to resultfrom a change at a single allele (e.g., loss of HGPRT or loss ofone allele for TK).

An unexpectedly high frequency of phenotypically recessivemutations has been observed in other pseudodiploid cell lines(15, 27, 36), and it might be argued that such phenotypicchanges are not genetic in nature (i 5, 27). Although epigeneticmechanisms have not been ruled out, several mechanisms canbe proposed for the mutational origin of fluoropyrimidine-re

sistance in the clones isolated in this study. One or more of theloci involving resistance to these drugs could be located on theX-chromosome or could have existed as heterozygous allelesintheanimalsfromwhichtheoriginalcelllineswerederived(8). Furthermore, Siminovitch (36) has proposed that, duringthe establishment of permanent cell lines from primary cultures,rearrangements of genetic material may result in regions offunctional hemizygosity. This may also be true for the highlymalignant Li 2i 0 and P388 cells. A single mutation could alsobe dominant if it affected a regulatory gene, which couldinactivate more than one structural gene (i 0).

One of these mechanisms may account for the high frequency of FdUrd-resistant variants observed in this study.These clones were highly resistant to FdUrd (4,000- to 25,000fold increase in lCsoas shown in Tables 4 and 5) and, as willbe seen in the accompanying paper (29), they have no detectable TK activity. If Li 2i 0 and P388 cells were hemizygous forTK, a single mutation at the remaining functional allele wouldgive a totally TK-deficient line, as is seen in the FdUrd-resistantclones isolated here. This system would be analogous to thesecond step of TK loss reported by Clive (1 i ) in heterozygousL5178Y cells and occurs within the same range of mutationfrequencies. A similar study of 2-step mutation to FdUrd resistance has been reported, involving the loss of TK (37).

The high frequency of resistance to FUra or FUrd may beexplained on a different basis. The IC50for FUra is elevatedonly 5- to 28-fold in clones selected with that drug, while thelCsofor FUrd is 8- to 220-fold higher in FUrd-resistant clonesthan in the wild-type cells (Tables 4 and 5). As will be shown inthe accompanying paper (29), resistance to FUra or FUrd isassociated with partial, but not total, loss of the activity ofpyrimidine phosphoribosyltransferase or uridine kinase, respectively. The quantitatively lower degree of resistance observed in clones selected with FUra or FUrd may be accountedfor by the substantial amount of residual salvage enzymeactivity. The high frequency of variants resistant to FUra orFUrd may result from a single allele mutation in one of the

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structural genes for the salvage ,enzymes or by a regulatorymutant that could control the total amount of active enzymesynthesized.

A recent study by Levinson et a!. (23) describes the isolationof a variant, after mutagenesis and selection in FUra, whichhas lost 50% of the pyrimidine phosphoribosyltransferase activity found in the wild-type cell line. That resistant line hasproperties similar to those isolated in this study. The totaldeletion of this enzyme might be lethal, since it is involved inboth salvage and de novo pathways of pyrimidine biosynthesis.Patterson (31) and Krooth et a!. (2i ) have reported the isolationof variants totally deficient in phosphoribosyltransferase activity, but the survival of these lines depends on the addition ofexogenous uridine to the medium to bypass this metabolicblock. Since the selective medium used in the present studydid not include a pyrimidine source, similar variants would nothave survived.

The fluoropyrimidine-resistant clones isolated in this studydisplay many characteristics expected in phenotypic variantsof a genetic origin (10, 38). These variants arise at spontaneousfrequencies of i O@ to i O_6, which can be increased up toseveral hundred-fold by treatment of the cultures with knownbacterial mutagens. The clones are stably resistant after 90days of growth in the absence of selective pressure and, aswill be shown in the accompanying paper (29), have stablealterations in the activities of certain enzymes involved inpynmidine metabolism.

The decreased activity of these pyrimidine salvage enzymesdid not Increase the sensitivity of the variant cell lines to MTX,ara-C, 5-aza-CR, or PALA. It is important to note, however, thatthe biochemical alterations also did not result in cross-resistance to any of these drugs (except for 5-aza-CR in FUrdresistantLi 210 cells). Therefore, the concurrent administrationof these drugs with the fluoropyrimidines would be expected tobe effective (30), since the simultaneous development of resistance to both inhibitors in the treatment protocol was notseen in these 2 cell lines. In addition, very few of the clonesselected for resistance to FUra, FdUrd, or FUrd in this studyare cross-resistant to the other 2 selective agents, making itlikely that different genetic loci are involved in resistance to thethree drugs.

The spontaneous frequencies measured in this study for theemergence of resistance to the fluoropyrimidines are highenough to indicate that some fluoropyrimidine-resistant cellsmight be present in a clinically apparent tumor of i O@cells. Inaddition, the fluoropyrimidines and other antimetabolites areoften used In combination chemotherapy protocols with othercancer chemotherapeutic drugs, such as cyclophosphamide,melphalan, and Adriamycin, which have been shown to bemutagenic (3). Treatment with these mutagenic drugs couldconceivably induce mutations in the tumor that could conferresistancetosomeoftheotherdrugsusedinthecombination.The system of mutation and one-step selection used to isolatefluoropyrimidine-resistant variants in this study is, therefore, amodel for a phenomenon which may be clinically important.This system can be used to isolate variants that are resistant toother chemotherapeutic drugs; similar studies have been recently published on resistance to MTX (13) and to ara-C (26,34).

Although all of the resistant cell lines isolated in this studywere deficient in one of the pyrimidine salvage enzymes, other

Mutation to F!uoropyrimidine Resistance

mechanisms of drug resistance have been reported. In a MTXresistant Chinese hamster ovary line (i ), a 200-fold increase indihydrofolate reductase activity has been correlated with a200-fold amplification of the gene coding for this enzyme. Thatcell line was selected by exposure to increasing concentrationsof MTX, and such a gradual selection procedure may benecessary to isolate cells with increased enzyme activity ifgene amplification is an obligatory step. FdUrd-resistant celllines, isolated using a similar multiple-step selection, have beendescribed (2, 32) which have increased thymidylate synthetaseactivity. It may be unlikely that such variants would have beenisolated by the one-step mutation and selection procedureused in the study reported here. Each of the 2 methods forisolating drug-resistant cell lines is therefore valuable in elucidating mechanisms of resistance to chemotherapeutic drugs.

ACKNOWLEDGMENTS

The authors would like to thank J. S. Johnston for dedicated technicalassistance and J. Hubert, I. Romo, and R. Millstein for the careful preparation ofthis manuscript.

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1982;42:956-964. Cancer Res   Mary A. Mulkins and Charles Heidelberger  Lines by One-Step Mutation and SelectionIsolation of Fluoropyrimidine-resistant Murine Leukemic Cell

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