Biochemical Studies of Resistance to 6_Thioguanine*

D. B. ELLIS AND G. A. LEPAGE

(Life Sciences Research, Stanford Research In.@titute, Menlo Park, California)

SUMMARY

Several 6-thioguanine (TG)-resistant mouse ascites tumors were developed to facilitate the study of possible mechanisms of resistance to this drug. A comparison wasmade of various aspects of purine metabolism in cells of the parent tumor lines and theresistant sublines. It was confirmed that susceptible cells incorporate significantamounts of TG into their nucleic acids, whereas cells of the resistant sublines incorporate only minor amounts of TG under the same conditions. An in vitro assay for synthesis of thioguanylic acid by the nucleotide pyrophosphorylase reaction in soluble enzyme preparations was developed.

Two TG-resistant Ehrlich ascites cell sublines were developed, one of which retainedits capacity for pyrophosphorylase synthesis of thioguanylic acid and one of which hada greatly decreased capacity. The first of these resistant sublines responded to combination therapy of azaserine plus TG. Azaserine pretreatment also greatly increased thequantity of thioguanylic acid formed and the amount of TG found in the nucleic acidfraction of the cells of this subline. The second TG-resistant subline failed to respondto combination therapy ; similarly, azaserine pretreatment had no effect on the metabolism of TG by this line of cells. These findings support the hypothesis that the mechanisms of resistance to a given drug vary and depend upon such features of the experimental approach as the selection pressures used in selecting the resistant populations.

It is well established that many analogs of nucleic acid purines become active by being converted to the nucleotide form (3). The formationof these fraudulent nucleotides can be viewed as alethal synthesis. Earlier investigations of themetabolism and mechanism of action of 6-thioguanine (TG)' demonstrated that drug-sensitiveEhrlich ascites tumor cells convert the analog to6-thioguanine ribonucleotide (TGMP) and thatthese cells also incorporate the analog into theirnucleic acids (12, 13, 16). Drug-resistant cellsformed less TGMP and incorporated much less

* This work was supported in part by Contract No. SA-48-

ph-3068 with the Cancer Chemotherapy National ServiceCenter, National Cancer Institute, National Institutes ofHealth, and in part by grants from the United States PublicHealth Service (CY-4551) and the Gustavus and LouisePfeiffer Research Foundation.

A preliminaryreportof this work has been made (Fed.Proc., 21:164, 1962).

1 Abbreviations are used as follows: TG, 6-thioguanine;

TGMP, 6-thioguanosinemonophosphate;6-MP, 6-mercaptopurine; DNA, deoxyribonucleic acid; PRPP, 5-phosphoribosyl1-pyrophosphate; FGAR, a-N-formyl glycinamide ribotide.

Received for publication September 18, 1962.

TG into their nucleic acids than did drug-sensitivecells (20). These mutant cells, selected by growthin the presence of TG, appear to be defective intheir capacity to accumulate the false nucleotideand in the incorporation of it into their nucleicacids.

The value of purine analogs in cancer chemotherapy is greatly limited by the development ofresistance to these drugs. In general, tumor cellshave been shown to exhibit resistance to purineanalogs in three ways : (a) inability to convert theanalog to the active nucleotide form ; (b) abilityto degrade the drug; and (c) ability to exclude thedrug from the cell. In extensive studies with L1210leukemia cells Brockman et al. (2, 4) have shownthat cells which are resistant to purine analogslack the nucleotide pyrophosphorylases whichcatalyze the lethal synthesis of the fraudulentnucleotides. Sartorelli et a!. (20) have presentedevidence that resistance in an Ehrlich carcinomasubline resulted from an enhanced capacity t@degrade the drug. Recently, Paterson (17, 18) hasindicated that resistance to 6-mercaptopurine (6-MP) may result from an impairment in the trans

436

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ELLIS AND LEPAGE—Resistance to 6-Thioguanine 437

40 C. for 5 minutes. The swollen cells were then

disrupted by being homogenized in a PotterElvehjem homogenizer with a Teflon pestle. Thesupernatant obtained after centrifugation at 105,-000 X g for 60 minutes at 4°C. was used as thesoluble enzyme preparation. Similar cell-free extracts were made from TG-sensitive and -resistanttumor cells. The protein content of these preparations was estimated according to the method ofLowry et a!. (14). Enzyme solutions (0.1 ml.) prepared in this manner were incubated at 37°C. for60 minutes with 0.6 @molesof TG-8-C'4 (3 X I 0@counts/min/@imole), 1.5 moles of PRPP presentas the dimagnesium salt, and 50 @molesof Trisbuffer (pH 7.6) in a final volume of 1 ml. Enzymeaction was terminated by placing the tubes in aboiling water bath for 3 minutes. After centrifugation, aliquots of the reaction were analyzed bydescending paper chromatography, with 5 per centNa2HPO4 as the developing solvent. Radioactivecompounds were located with the aid of a Vanguard Instruments Company chromatogram scanncr. Quantitative results were obtained by elutingthe radioactive spots with 2 N NH4OH for counting. The above cell-free extracts were also testedfor purine deaminase activity toward guanine andTG according to the method of Kalckar (10). Thismethod involves measurement of xanthine produced in the reaction by oxidation with xanthineoxidase. Oxidation was followed spectrophotometrically by measuring the increase in opticaldensity at 290 mj@corresponding to the formationof uric acid. When TG was used as substrate,change in optical density at 355 mj@ was used toindicate formation of thiouric acid (16).

The in rivo tracer experiments with TG-8-C'4and adenine-8-C'4 were carried out in the mannerdescribed in previous reports (12).

RESULTS

Metabolism of thioguanine-8-C'4 in vivo.—Inprevious reports (12,20) it was demonstrated thatEhrlich ascites cells in the presence of TG accumulated thioguanylic acid and incorporated TGinto their nucleic acids and that the latter processis responsible for the tumor-inhibitory propertiesof TG (13), whereas cells of the TG-resistantsubline were much less efficient in these reactions.Consequently, the conversion of TG to its ribonucleotide and its incorporation into nucleic acidswere compared in the four sensitive and resistanttumor lines for differences which might be relatedto TG resistance.

The amounts of thioguanylic acid found in theacid-soluble fractions and incorporated into thenucleic acids following a single intraperitoneal

port of 6-1'wIP across the cell membrane. Thesediffering explanations for development of resistance to purine analogs of closely related structures were obtained from different tumor lines.The fact that only TG, of the purine analogs in usetoday, is incorporated into the DNA of susceptiblecells may be the basis for the dev@1opment offurther mechanisms of resistance to this drug. Toascertain whether more than one mechanism couldbe responsible for resistance to TG, several mousetumors resistant to TG have been developed. Thepresent report is part of a study of TG resistancein such model systems. Experiments are describedin which various biochemical characteristics of theTG-resistant and -susceptible tumor cells werecompared. The properties studied include themetabolism of TG in sensitive and resistant ascitescells in vivo and a comparison of the capacity of cellfree preparations from sensitive and resistant cellsto catalyze the reaction of TG with 5-phosphoribosyl-1-pyrophosphate (PRPP). Measurementswere also made of the deaminase activity of theseenzyme preparations.

MATERIALS AND METHODSThe tumors used (Ehrlich, Sarcoma 180, Adeno

carcinoma 75.5, and L1210 leukemia ascites cells)and the mouse lines in which they were carriedhave been described previously (12). The resistanttumor lines used in this study were originallyachieved by consecutive passages of ascites cellsin mice given intraperitoneal injections of 2 mg/kgof TG2 once daily on 4 successive days after tumortransplantation. After 7 days, tumor cells fromone such mouse of each line were used for transplantation. This procedure was continued for tentransfers. From this time on the resistant tumorswere treated once with 2 mg/kg of TG 1 day aftertumor transplantation. Resistance to TG was assessed at frequent intervals throughout the transplantation history of these resistant lines. Thetumor cells were used for biochemical studies 5—6days after inoculation.

Cell-free extracts used to catalyze the reactionof TG with PRPP were prepared as follows : After5—6days of growth, ascitic fluids were removedfrom the mice with the aid of Pasteur pipettes,pooled in ice-cold centrifuge tubes, and spun at1400 X g for 2 minutes to collect the tumor cells.The cells were then dispersed in 4 volumes of icecold glass-distilled water and allowed to stand at

2 Thioguanine was supplied by Dr. R. K. Robins. Thiogua

nine-8-C'4 was synthesized in this laboratory (12). Guanine-8-C14@ in the preparation of radioactive TG was purchasedfrom Isotope Specialties Company. The dimagnesiumsalt ofPRPP wasobtainedfromSigmaChemicalCompanyandxanthine oxidase from Nutritional BiochemicalsCorporation.

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438 Cancer Research Vol. 23, March 1963

dose of 10 mg/kg of TG are shown in Chart 1. Onehour after the injection of TG-8-C'4, the cells wereharvested, and the acid-soluble fraction was extracted with cold 2 per cent perchioric acid. Theacid-insoluble residue was extracted with hot 10per cent trichloroacetic seid to give the pu.rine

NUCLEIC ACIDS

@ SENSITIVE0 RESISTANT

x ii,C.) It):5 r@

Caurr 1.—Conversionof thioguanine-8-C'4 to its ribonucleotide and incorporation into the nucleic acids of TGsensitive and -resistant ascites tumor cells. Tumor-bearing mice

were each given a single intraperiteneal dose of 10 mg/kgof thioguanine-8-C―(specificactivity approximately 2 X 10counts/mm/mg), and 1 hour wasallowedfor metabolicutilization. TG was isolated from the tumor cells as described previously (12). Each bar represents the average of several experiments in each of whichanalysesweremade on pooledcellsfromthree mice.

components of the nucleic acids. TGMP-C'4 andTG-C@@were isolated by ion-exchange chromatography and measured with a Nuclear-Chicago gasflow counter (12). These results show that resistant cells of the Ehrlich, 5-180, and Ca-755tumor lines do form nucleotide and incorporateTG into their nucleic acids but only of the order of

30 per cent of that found in the correspondingsusceptible lines. In contrast, the L1210 TG-resistant line failed to form significant amounts ofnucleotide, and only negligible amounts of TGwere found in the nucleic acids of these cells.

Thioguanylic pijrophosphorylase activity of cellfreeextractsof8en.ntiveandresistantascitestumorce&—The observations by Brockman (2, 4), thatL1210 tumor cells resistant to 6-mercaptopurine,8-azaguanine, and 6-thioguanine are deficient inthe pyrophosphorylases that catalyze the reactionsof these drugs with PRPP to yield the corresponding nucleotides, suggested that a comparison of thethioguanylic pyrophosphorylase activity for thefour sensitive and resistant ascites tumor cellsshould be undertaken. The data summarized inChart 2 present a comparison of the capacities of

I It)C) IC)

:i F;.@ 0@ C.)

CHART 2.—Enzymatic synthesis of thioguanylic acid by

cell-free preparations from TG-sensitive and -resistant ascitestumor cells. 0.1 ml. of enzyme extracts (approximately 300 pg.protein) were incubated for 1 hour at 37°C. in a medium contaming so pmoles Tris buffer (pH 7.6), 0.6 pmoles TG-8-C'4, and1.5 pmoles PRPP in a final volume of 1 ml. Reaction mixturesafter termination of the incubation were analyzed as describedin the text.

cell-free extracts prepared from the TG-sensitiveand -resistant tumor lines to catalyze the reactionof TG-8-C'4 with PRPP. The assay system employed in these experiments is described under“Materials and Methods.― Incubations were terminated by heat denaturation. However, preliminary experiments indicated that taking the incubation mixture from 37°C. to 100°C. resulted insome conversion of TG to ribonucleotide. Consequently, zero time incubations were also denatured by boiling for S minutes, and the amountof nucleotide formed in these controls was subtracted from that formed in the test samples. It is

(1)

wC-)

-j@ 4.

C')

C)wC-)

zz

I-

25Cl)-I-IwC)

-I!.15Q.

U)

(@5I—

D0.@.0.8zUiI-0

0

@0.4a.

CDI-.

Cl)Ui..10

ACID-SOLUBLES

0 0

U) @I

0 0

U)

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TumorAcid-solublefraction5NA-adenine

(counts!min/@

moleXiO-'Sensitive

Resistant83,000 86,0002.4 2.7

ELLIS AND Li@fAGE—Resistance to 6-Thioguanine 439

apparent from Chart 2 that loss of thioguanylicpyrophosphorylase activity in the L1210 resistantcells is responsible for failure of these cells to formthioguanylic acid in vivo and that this is the basisof resistance in these cells, as was the case in thelines observed by Brockman (2, 4). However, insharp contrast to the results obtained with L1210extracts it was found that, with the Ehrlich, S-180,and Ca-755 tumor lines, extracts prepared fromsensitive and resistant cells had similar thioguanylic pyrophosphorylase activities. Paterson (17, 18)in his study of resistance to 6-NP in Ehrlich ascites cells also found that extracts of resistant andsensitive cells had similar capacities for PRPPdependent synthesis of thioinosinate. From thesedata it is evident that resistance to TG in Ehrlich,S-180, and Ca-755 occurs by a different mechanismfrom that in the L1210 resistant cells, where lossof activity of the enzyme catalyzing the formationof thioguanylic acid is responsible for the development of resistance.

Metaboli#m of ad,enine-8-C'4.----The observationthat extracts from several TG-sensitive and -resistant tumor cells in the presence of PRPP possesssimilar pyrophosphorylase activities in vitro maynot be a true indication of their activity in vivo,where the concentration of PRPP in the resistantcells may become limiting. To determine whetherthe resistant cells lack adequate resources ofPRPP for the synthesis of thioguanylic acid iniyit@o,the rate of incorporation of adenine-8-C'4 intothe acid-soluble fraction and into the nucleic acidsof TG-sensitive and -resistant 5-180 ascites cellswas followed (Table 1) . The “salvage―pathway(11) by which adenine is converted to ribonucleotide requires the participation of PRPP, and itwas reasoned that a measure of the availablePRPP in the tumor cells would be provided byfollowing the rate of incorporation of adenine-8-C―into the nucleic acids. No difference in either theamount of radioactivity found in the acid-solublefraction or in the specific activity of nucleic acidadenine was observed between the two tumorlines, as shown in Table 1. These results indicatethat, relative to the parent susceptible line, theTG-resistant S-180 cells were not deficient in theircapacity to synthesize adenylic acid, confirmingthat the resistant cells contain an adequate supplyof PRPP for the ribonucleotide syntheses whichutilized preformed purines.

Deaminase activity of cell-free extracta.—Theohservations of Sartorelli et at. (19, 20) indicated thatincreased degradation of the drug can be responsible for the development of resistance to TG.When large doses of TG were given to mice bearingsensitive or resistant Ehrlich ascites cells, larger

amounts of the metabolites thioxanthine and thiouric acid were found in the resistant tumor cells(@0). Hirschberg et at. (9) have shown that severaltumors resistant to 8-azaguanine contained anenzyme system capable of deaminating the drugto 8-azaxanthine, a compound having no carcinostatic action, whereas three of four tumors susceptible to 8-azaguanine were low in this enzyme.Shacter and Law (21), in contrast, found no distinct differences in enzymatic activity betweenleukemic tumors susceptible to the action of 8-azaguanine as compared with sublines of the neoplasms showing resistance to 8-azaguanine. Whenextracts prepared from both TG-sensitive and -resistant cells were tested for nucleotide pyrophos

TABLE 1

UTILIZATION OF ADENINE-8-C'4 BYTG-sENsrrivE AND-RESISTANT

S.180 ASCITES CELls

a Total counts in acid-soluble fractionper gram wet weight of cells.

Tumor-bearingmicewere givenan intraperitoneal injection of 55 pg. adenine8@C14(0.5 pe.; 5.8 X 10@ counts/min/mg)and 1 hour was provided for metabolicutilization. Each figure represents the average of results obtained from the separateanalyses of two to three mice.

phorylase activity (Chart 2), no evidence of catabolic metabolites of TG was detected even whenPRPP was omitted from the medium. Despite thisapparent failure of the extracts to deaminate TG,enzyme preparations from sensitive and resistantEhrlich ascites cells were tested for guanase andTG deaminase activity according to the method ofKalckar (10). Both enzyme preparations deaminated guanine yet had no effect on TG as measured by change in optical density at 355 m@ corresponding to the formation of thiouric acid. (Xanthine oxidase is capable of converting thioxanthineto thiouric acid [16].) Guanine-deaminase activityof TG-resistant Ehrlich ascites cells is presented inTable 2. On addition of the enzyme extract therewas a marked increase in optical density at 290m@, corresponding to the formation of uric acid,which remained unchanged in the presence ofadded TG. This result indicates that TG does notcompete with guanine for the enzyme which deaminates guanine. It would appear, therefore,

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ADDITIONBGUANASE

ACTIVITYXanthine

ozidaseCell-free extractTG(pg/flask)+

+++—

+++—

-

751500.09

0.670.740.65

TIME AFTERASASKIUNENUCLEOTIDE*N@

CLEICACIDS*SensitiveResistantSensitiveResist@tntControl

10mm.30mm.2hours8hours19.1 24.06.1

7.88.9

10.819.24.0 6.11.3

1.62.63.05.5

440 Cancer Research Vol.23,March 1963

that factors other than TG deaminase activitymust now be involved in the difference of responseof these TG-sensitive and -resistant ascites tumorcells to the drug.

Effect of aza8erine on the metaboli,sm of thioguanine-8-C14 by TG-sensitive and -re.ii@tant ascite8cells in vivo.—The TG-resistant line of cells developed from the Ehrlich ascites tumor by Sartorch et at. could be made susceptible when azaserine was combined with TG (20). Combinationtreatment with azaserine3 and TG, besides pro

TABLE 2

GUANASE AcTIvITY OF TG-RESISTANT

EIIRLICH ASCITES CELL EXThACTS

was markedly increased in the resistant cells to alevel comparable to that found in the TG-sensitivecells. A similar result was observed with the incorporation of TG into the nucleic acids of thesecells. This increased accumulation of TGMP afterpretreatment with azaserine confirms the in vitrostudies with cell-free extracts indicating that theappropriate pyrophosphorylase for TG is still present in TG-resistant S-180 ascites cells. Similar results to those shown in Table 3 were obtained withthe TG-resistant Ehrlich and Ca-755 ascitestumor lines. The 8-hour delay between azaserineand TG doses produced a marked effect, in contrast to the results of Sartorelli et cii. (20) where animmediate effect on TG metabolism was observed.No explanation of this effect has been found. Sincethe results of Sartorelli et at. (20) were obtained,the TG-resistant Ehrlich ascites line has been carned in approximately 175 generations of TG

TABLE 8

EFFECT OF AZASERINE PRETREATMENT ON NUCLEOTIDE

FORMATION AND INCORPORATION OF THIOGUANINEINTO THE NUCLEIC AcIDs IN TG-SENSITIVE AND -RESISTANTS-ISO ASCITESCELLS

a Increase in O.D. at 290 mp/hour.Ascites cells disrupted by homogenization in water were

centrifuged for 60 minutes at 105,000 X g at 4°C., and thesupernatant fraction was used as the enzyme. Enzymic activityof freshly prepared extracts was determined as follows: Flasks,containing 7.0 ml. of 0.067 M phosphate buffer (pH 7.6), 0.5ml. guanine solution containing 150 pg/ml guanine and waterto 10.5 ml., were incubated for 60 minutes at 37°C.; 1.0 ml.of diluted xanthine oxidase,1.0 ml. of enzyme extract, and 0.5ml. or 1.0 ml. of a TG solution (150 pg/mI) were included inthe reaction medium as indicated; 8-ml. samples were taken at0, 20, and 60 minutes and inactivated with 0.2 ml. of 26 percent perchloric acid. The samples were centrifuged, and theirabsorbancies at 445 mp and 290 mp were measured.

ducing a synergistic response on the survival timeof mice bearing TG-resistant cells, resulted in amarked increase in the accumulation of TGMPand its incorporation into the nucleic acids of theresistant cells. Further work in this direction hasnow shown that, with the present resistant lines,azaserine has to be given 8 hours prior to TG toproduce these effects. Table 3 shows the effect ofpretreatment with azaserine at different time intervals on the formation of ribonucleotide fromTG and its incorporation into the nucleic acids ofTG-resistant 5-180 ascites cells. When azaserinewas given at a short time prior to TG no significant increase in nucleotide formation or increasedincorporation into the nucleic acids was observed.However, when 0.2 mg/kg azaserine was given 8hours prior to TG, the concentration of TGMP

3 The azaserine used in these experiments was obtained from

the Cancer Chemotherapy National Service Center, NationalCancer Institute.

a Expressed as pg. TG-8-C― found in this form per gramwet weight of cells.

Ascites tumor-bearing mice were given injections of 0.2mg/kg of azaserine. At intervals groups of mice received 2.50pg. (10 mg/kg) of TG-8-C― (4 X 10@counts/mm/mg) permouse, and 1 hour was allowed for metabolic utilization. Eachfigure represents the mean of several analyses of pooled cellsobtained from two mice.

treated mice. From these results and the deaminase studies it is now evident that TG-resistantcells different from the resistant variants in theoriginal sensitive population have appeared duringthis time and that this subline, which reacts todelayed treatment with azaserine, has becomedominant. In addition to its effect on purine poolsizes, azaserine may cause the marked increase inTG incorporation in the resistant cells by the indirect action of a-N-formyl glycinamide ribotide(FGAR), which accumulated after treatment withazaserine (15). Such an effect would be greatestwhen the maximum amount of FGAR had accumulated.

In an attempt to demonstrate multiple media

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TUMORSURVIVAL

TIMESPRPPASE

ACTIVITY OF

CELI.-FREE

EXTRACTStNUCLEOTIDENuci.zic

Acms@Control.

AzasermepretreatedControl.

AzasermepretreatedDose

.(mg/kg Xdaily dose)5Average

.survival

(days)TGAzaserineEhrlich

ETGR I

ETGR II0

0.6X20

0.6X2

00.6X2

00.6X2

00.6X2

00.6X20

00.2X20.2X2

00

0.2X20.2X2

00

0.2X20.2X212.6±2.6@

19.6±6.720.4±2.142.6±8.8

13.4±8.111.2±3.520.5±2.341.8±8.4

14.0±2.313.5±3.325.8±8.426.6±7.70.55±0.04@

0.52±0.05

0.0524.8

6.7

1.937.1

32.1

1.58.5

0.9

0.15.4

6.5

0.1

ELLIS AND LEPAGE—Resistance to 6-Thioguanine 441

nisms of resistance with one tumor, a further TGresistant subline of the Ehrlich ascites carcinomawas developed. The selection of this resistantpopulation was achieved by consecutive passages,with inocula of 10@ascites cells per mouse, in animals treated with large doses (10 mg/kg) of TGonce daily for 4 days after tumor transplantation.After 7 days, tumor cells from one such mousewere used for transplantation. This procedure wascontinued for fifteen generations. From this timeon the resistant tumor was maintained on a dose

activity of ETGR II extracts was less than 9 percent of that of the other two lines. From theseobservations it was apparent that a TG-resistantEhrlich ascites tumor had been selected which wasresistant owing to a decreased capacity of theenzyme catalyzing the formation of TGMP. Atthis time further biochemical studies and survivalexperiments with TG and azaserine were carriedout. A comparison of such studies in Ehrlich,ETGR I, and ETGR II tumor lines is presented inTable4.

TABLE 4

CoMPARIsoN OFSuRvxv@LDATA AND BIOCHEMICALSTuDiEs WITH TG ANDAZASERINE IN TG-SENSITIVE AND -RESISTANT Emiucn ASCITES C@Lis

a Tr@-'@@@for 6 days beginning 24 hours after tumor transplantation.

t pmolesTGMPformedpermg.proteinperhour.Experimentalconditionsasdescribedin Chart2.@ Expressed as pg. TG-8-C― found in this form/gram wet weight of cells. Tumor-bearing mice were each given a single intra

peritoneal dose of 10 mg/kg of TG-8-C'4 (2 X 10' counts/mm/mg), and 1 hour was allowed for metabolic utilization. Azaserinepretreatment was 0.2 mg/kg 8 hours prior to the dose of TG.

§Averagedeviationfrommean.

of 10 mg/kg of TG 1 day after tumor transpiantation. This subline is now designated ETGR II,and the subline selected under much less severeselection pressure (2 mg/kg of TG), which hasbeen used in all previous experiments, as ETGR I.The first suggestion that ETGR II was differentbiochemically from ETGR I was observed duringa routine investigation of nucleotide pyrophosphorylase activities of cell-free extracts, when theactivity of the ETGR II subline was found to beless than 0.14 moles TGMP formed/mg protein!hour as compared with values of 0.60 and 0.55for the parent susceptible line and ETGR I, respectively. After further treatment with 10 mg/kgof TG once weekly for seven more generations, the

The most striking difference between ETGR Iand ETGR LI is their response to combinationtreatment with azaserine and TG and the effect ofazaserine pretreatment on the formation of TGMPand incorporation of TG into the nucleic acids ofthese cells. ETGR II fails to respond to combination treatment; a biochemical explanation of thisis afforded with the observation that azaserine pretreatment failed to cause an increase in the amountof TG incorporated in the nucleic acids following asingle intraperitoneal injection of TG-8-C―. Aninteresting facet of Table 4 is the example of collateral sensitivity shown by the ETGR II sublinewhere an apparent increase in sensitivity to azaserine was observed. The results of this study also

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442 Cancer Research Vol. 23, March 1963

illustrate that, although it is possible to circumvent resistance to a given agent through the use ofcombinations of drugs as in the case of the ETGRI tumor, another sublime resistant to the sameagent does not necessarily respond to this attemptat circumvention (ETGR II).

DISCUSSION

The present study has confirmed the previouslyreported observation that resistance to TG can becorrelated with the diminished capacity of theresistant sublines to convert TG to its ribonucleotide form. The data given here and that publishedby others illustrate the great diversity in the possible mechanisms of resistance to a single agent.Brockman and his associates (2, 4) have explained resistance to 6-MP in L1210 tumor cells byloss of the nucleotide pyrophosphorylase whichcatalyzes the synthesis of thioinosinate. In contrast, Paterson (17, 18) has reported that cells of a6-MP-resistant sublime of the Ehrlich ascites carcinoma possess thioinosinate pyrophosphorylasein apparently undiminished concentration. In thiscase, resistance appears to have been acquiredthrough loss of the transport mechanism or permease by which 6-MP enters the cell (17). In invitro studies with L1210 ascites tumor cells Davidson (7), however, found that sensitive and resistant cells were equally permeable to 6-MP. Afurther possibility for a resistant mechanism isafforded by the results of Balls et al. (1), who consider that a greater capacity for de novo purinesynthesis may be responsible for resistance to 6-MP in a Streptococ@rtiafaecalt@ mutant. A similardiversity in the mechanisms of resistance to TGhas now been shown to exist in ascites tumor cells.

In agreement with Brockman's observations,resistance to TG in L1210 leukemia cells is a consequence of loss of activity of the nucleotide pyrophosphorylase responsible for activating the drugto the nucleotide level. This deletion of the enzymecatalyzing the synthesis of fraudulent nucleotidesis the simplest form of resistance to purine analogsand has been well documented in drug-resistantleukemia cells (2, 4), bacterial mutants (5), and ahuman epidermoid carcinoma growing in tissueculture (6). Emphasis on this mode of resistance topurine analogs has overshadowed studies of otherpossible mechanisms, although Davis and Maas(8) have stated the possibility of mutation-selection providing a basis for changes in the functionof the cell that could result in multiple resistantmechanisms.

The results obtained with TG-resistant sublinesofEhrlich, S-180, and Ca-755 ascites tumors, where

no loss in nucleotide pyrophosphorylase activitywas found, are consistent with Paterson's observations with 6-MP-resistant Ehrlich ascites cells (17,18) . These three TG-resistant tumor lines are susceptible to combination treatment with azaserineand TG, and show a marked increase in theamount of TGMP accumulating after treatmentwith azaserine (Table 8), indicating that the nucleotide pyrophosphorylase remains intact. Fromthese data it would appear that loss of pyrophosphorylase could not account for the developmentof resistance to TG in these three tumors and thatpermeability to TG could not be involved. Although thioguanylic pyrophosphorylase is stillpresent in these resistant cells, it is possible that,in vivo, the enzyme is partially blocked and theresultant low level of intracellular metabolism ofTG to nucleotide does not prove inhibitory to thegrowth of the cell. Restoration of enzyme activityin these resistant cells in vivo would be possible ifloss of activity were due to the presence of an inhibitor which could be removed. The addition ofazaserine may result in a removal of this inhibitionof enzyme action, causing an increase in TGMPformation and consequently in the quantity of TGfound in the nucleic acid fraction. This, togetherwith the effect of azaserine on de novo purine synthesis, presumably is responsible for the synergistic response of the TG-resistant cells to combination therapy with azaserine and TG. Such aneffect did not occur with the TG-resistant L1210tumor cells presumably because of complete lossof the appropriate nucleotide pyrophosphorylase,an effect which could not be remedied by the use ofazaserine.

The data presented above further emphasize thedifficulties facing successful cancer chemotherapyand also help to reconcile the widely different explanations which have been suggested for resistance to a given drug (1, 2, 7, 17, 18). Differingresistant mechanisms have been demonstrated fora single drug (TG) in lines of tumor cells developedfrom the same susceptible population on differentoccasions. Use of a high selection pressure (highdose) might be expected to favor the type of resistance involving loss of an activating enzyme, as inthe above example. The three resistant lines developed earlier, using low selection pressures, cannot be ascribed to any of the three modes of resistance to purine analogs previously described (2,4, 17, 18, 20). Although no definite biochemicalexplanation is available for these examples of resistance to TG, it is important to note that resistance to TG was overcome by means of combination therapy involving azaserine and TG.

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ELLIS AND L@PAGE—Resistance to 6-Thioguanine 443

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