[CANCER RESEARCH 34, 1883-1888, August 1974]

Effect of Methotrexate on Deoxyribonucleotide Pools and DNA Synthesis in Human Lymphocytic Cells1

Arnold Fridland Laboratories of Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

SUMMARY INTRODUCTION

When added to cultures of human lymphocytic cells MTX,2 a synthetic antimetabolite of folie acid, is used (subline CCRF-CEM), methotrexate produced a rapid routinely in the treatment of various human cancers (5). It is decrease in intracellular pools of deoxythymidine Iriphos- thought to inhibit cell growth by inactivating dihydrofolate phate and deoxycytidine triphosphate, an initial increase in reducíase,the enzyme that converts folie acid lo dihy- the deoxyadenosine triphosphate pool, and no change in the drofolic and telrahydrofolic acids (15, 30). This disruption deoxyguanosine triphosphate pool. After 3 hr of incubation, of folie acid melabolism leads lo a reduclion in cellular all four deoxyribonucleotide pools were greatly reduced. conlenl of folale coenzymes (23) and lo an inhibilion of ihe Addition of adenosine and methotrexate together resulted in biosynlhesis of dTMP and of purine ribonucleolides (3, 29). a rapid three-fold increase in the size of the deoxyadenosine MTX can also inlerfere wilh amino acid melabolism in cells triphosphate pool, which lasted through 9 hr of incubation. (13). Finally, receñísludieshave shown lhal MTX may The pools of deoxyguanosine triphosphate, deoxycytidine inhibil enzymes olher than dihydrofolate reducíase,suggest triphosphate, and deoxythymidine triphosphate in these ing lhal ihe inhibilion of cell growlh by ihe drug in cullure cells were decreased, as in cells incubated with methotrexate could involve inhibilion al separale enzymalic siles (7, 25). alone. Methotrexate inhibited RNA synthesis, but this Aboul 4 years ago, Soller and Handschumacher (28) inhibition was completely prevented by the addition of described an enzymalic procedure for delermining concen adenosine. The loss of deoxyadenosine triphosphate corre trations of dNTP's. Since MTX may exert its antitumor sponded with the inhibition of RNA synthesis by methotrex activily by inhibiling dTTP synlhesis, Ihis procedure pro ate, indicating a reduction of purine ribonucleotide concen vides a means of defining in grealer delail ihe mode of MTX trations, which serve as precursors for deoxyribonucleolides aclion in cells. Adams et al. (1) found lhal ihe addilion of and RNA. These results show that methotrexate produces a MTX lo L929 cells resulled in a decrease in size of the purineless and thymineless state in CCRF-CEM cells, in dTTP pool. However, Beaumonk and Friedman (4) re support of the concept that an action of this antimetabolite ported lhal Ihe drug did not affecl dTTP pools in HeLa involves depletion of tetrahydrofolates by inhibition of cells. They poslulaled, instead, the exislence of a large, dihydrofolate reducíase. slowly lurning over dTTP pool lhal remained unaffecled by The kinetics of incorporation of tritiated deoxycytidine MTX in HeLa cells. into acid-soluble and -insoluble fractions of methotrexate- In this sludy, I delermined ihe effecl of MTX on treated and untreated cells was also studied. In untreated concenlralions of DNA precursors in human lymphocytic cells, about 97% of the intracellular soluble radioactivity cells. These experiments show lhal ihe drug produces large was associated with deoxycytidine nucleotides, mainly de changes in ihe pool sizes of all 4 NTP's. In addilion, oxycytidine triphosphate and deoxycytidine monophos- radioaclive precursors were used to study MTX action on phate. In methotrexate-treated cells, however, 83 to 97% of the 4 dNTP pools, and kinetic studies with tritiated this radioactivity was associated with deoxyuridine mono- deoxycytidine were conducted lo determine the effecls of phosphate. These experiments indicate that the presence of MTX on DNA replicalion. methotrexate in CCRF-CEM cells leads to a marked increase in deamination of deoxycytidine nucleotides, which probably accounts for the depletion of deoxycytidine tri MATERIALS AND METHODS phosphate in these cells. Incorporation of tritiated deoxycy Cells. Human lymphoblasl cells (subline CCRF-CEM) tidine into DNA was rapidly decreased by methotrexate but were grown al 37°as suspension cullures (12). Periodic continued at a low rate for at least 9 hr after addition of the drug. assays of ihe cullures by Dr. Leonard Hayflick have shown lham lo be free of Mycoplasma conlaminalion. 1This work was supported in part by Grant DRG-1172 from the Damon Chemicals. MTX was provided by the drug developmenl Runyon Memorial Fund, Grant NP-106 from the American Cancer Society, and Cancer Center Grant CA-08480 from the National Cancer ''The abbreviations used are: MTX, methotrexate; dNTP, deoxyribonu- Institute, NIH, and by ALSAC. cleoside 5'-lriphosphate; TCA. trichloroacetic acid: TdR, deoxythymidine: Received November 5, 1973; accepted April II, 1974. CdR, deoxycytidine: UdR, deoxyuridine.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1974 American Association for Cancer Research. A. Fridland branch. Drug Research and Development, Chemotherapy, once with 95% ethanol and ether, and then was dried in air. National Cancer Institute, Bethesda, Md. The drug was The precipitate was dissolved in 0.2 ml of 0.5 N KOH, and dissolved in sterile water and stored at -20° until used. the RNA was hydrolyzed to acid-soluble fragments by Tritiated nucleosides and nucleotides were obtained from incubation at 37°for 60 min. Then, 0.1 ml of 6 N HC1 was Schwarz/Mann, St. Louis, Mo. Unlabeled nucleosides and added to precipitate DNA and protein, and the sample was nucleotides were products of Sigma Chemical Co., St. spotted on a Whatman GF/A glass-fiber disc. The filtrate Louis, Mo. Polyethyleneimine-impregnated cellulose sheets was collected in liquid scintillation-counting vials and its (Brinkmann Instruments, Inc., Des Plaines, 111.) were radioactivity was determined by counting in PCS (Amer- washed with water:methanol (3:1, v/v), dried, and stored at sham/Searle Corp., Arlington Heights, 111.).Incorporation 4°until used. of label was measured as the difference between a sample Extraction of Acid-soluble Nucleotides. About 2 x 10' taken at time zero and a sample taken after a given time of CCRF-CEM cells were cooled to 0°and centrifuged for 5 incubation. Preliminary experiments with TdR-3H showed min at 800 x g. The cells were washed with ice-cold that DNA and RNA were well separated by this procedure. phosphate-buffered saline and recentrifuged at 800 x g. After cell suspensions were incubated with CdR-5-3H (0.5 Ice-cold 0.5 M HC1O4, 0.2 ml, was added to the cell pellet. Ci/mmole), as indicated in the appropriate experiments, The mixture was suspended with a glass rod, kept on ice for 5-ml replicates of each suspension were mixed with ice-cold 10 min, and centrifuged at 800 x g for 10 min. To the phosphate-buffered saline containing 5 mM KCN, 5 mM supernatant were added 20 jul 4 M KOH, 15 to 20 /¿ll M glucose, and 0.01 mM unlabeled CdR. KCN was for the KOH, and 10 jil l M potassium phosphate buffer, pH 7.4. purpose of preventing further conversion of radioactivity This procedure provided an extract at pH of about 7.0. The between dCyd nucleotides after stopping the reaction. The KC1O4 precipitate was spun down, and the supernatant was cells were centrifuged and the cell pellet was suspended in measured and stored at -20°prior to assay. The method of 0.2 ml of ice-cold 5% TCA. This mixture was kept on ice for extraction of these nucleotides was found to be very 10 min and centrifuged at 1000 x g for IOmin. TCA in the important because the substitution of 5% TCA for HC1O4 supernatant was extracted with ether, and the acid-soluble resulted in significant breakdown of the purine deox- radioactivity in a portion of the aqueous phase was counted. yribonucleotides. The acid-insoluble material was washed 3 times with 5% Assay of Deoxyribonucleoside Triphosphates. Micrococ- ice-cold TCA and dissolved in 0.5 ml NCS (Amersham/ cus lysodieikticus DNA polymerase at a specific activity of Searle Co.) by incubating at 37°for 60 min. A sample (0.1 420 units/mg and a concentration of 450 units/ml was to 0.3 ml) was taken and placed in PCS for radioactivity supplied by Miles Laboratories, Inc., Kankakee, 111.The determination. reaction mixture contained, in a total volume of 0.3 ml: 1 Isolation of Deoxyribonucleotides. Ten or 20 /¿Iofacid- nmole of each of 2 dNTP's, including dTTP-3H or dATP- soluble fraction and 5 /il of a marker solution containing 3H (65 nCi, each); 2 units of DNA polymerase; 15 /¿gof dTTP, dCTP, dCDP, dTMP, dCMP, CdR, and TdR were denatured DNA (14); 20 Amólesofglycine buffer, pH 9.0; 2 applied to a prewashed cellulose sheet impregnated with /¿molesof MgCl2; 0.3 //mole of mercaptoethanol: and 20 /il polyethyleneimine. The sheet was developed first in acetic of the neutralized acid-soluble extract. The mixture was acid at room temperature, with the solvent front ascending 2 incubated for 45 min at 37°andthe reaction was terminated cm above the origin. Then the sheet was transferred, without by the addition of 15/tl of ice-cold 33% TCA containing 0.1 drying, to a chromatography tank containing l N acetic acid M sodium pyrophosphate. The reaction tubes were kept in and 3 M LiCl (2:1, v/v). The solvent front was allowed to an ice-bath for 10 to 15 min to allow precipitation to occur. ascend approximately 15cm above the origin. The sheet was The acid-insoluble material was collected on a Whatman then dried and the spots absorbing UV were marked, GF/A glass fiber disc, washed with 5% TCA:0.01 Msodium scraped off, and extracted with 1.5 N NaCl. pyrophosphate, and dried. The disc was placed in toluene- Radioactivity Determinations. Two scintillation fluids based scintillation fluid for radioactivity determination. were used. For nonaqueous material, a solution containing 3 Preliminary experiments were conducted to obtain stan g of PPO and 0.1 g of POPOP (Packard Instrument Co., dard curves for each of the 4 dNTP's. These studies showed Downers Grove, 111.)per liter of toluene was counted in a that the assay used in this work was sufficiently sensitive to Packard Tri-Carb liquid scintillation spectrometer. The detect about 10 pmoles of each of the 4 dNTP's. Incorpora radioactivity in aqueous material was counted in PCS. tion was linear for at least up to 100 pmoles of dNTP. Values were corrected for background. Incorporation of dATP-3H into acid-insoluble material was found to be linear for at least 40 /¿Iof cell extract and additive with a dNTP standard. RESULTS Incorporation of Radioactivity into RNA and DNA. Cell suspensions were incubated with 6 /tCi/ml of uridine-5-3H Effect of MTX on Thymidylate Synthesis and dNTP (28 Ci/mmole) for 20 min at 37°.Thesuspensions were then Pools. First experiments were carried out to determine the centrifuged and the cells were resuspended in 0.2 ml of extent of inhibition of thymidylate synthesis in CCRF-CEM growth medium to which 2 ml of ice-cold 5% TCA had been cells incubated with 5 x 10~7MMTX. Several investigators added. The protein in the medium served as carrier. The (7, 20, 24) have shown that the incorporation of UdR-3H precipitate was washed twice with ice-cold 5% TCA and into acid-insoluble fraction of cells may be used as a

18X4 CANCER RESEARCH VOL. 34

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1974 American Association for Cancer Research. Effect of M TX on Deoxynucleotides and DNA 20 serum. Thus, it may be assumed that in these studies purines and pyrimidines are not present in the growth medium to interfere with the MTX block. The addition of 10 * M adenosine together with MTX increased the dATP pool size about 3-fold during the 1st hr of incubation; this elevated level was maintained for at least 8 hr (Chart 1). The dCTP, dTTP, and dGTP pools decreased as in cells incubated with MTX alone (Chart 1). The concentrations of dNTP's varied about 50% in 4 different experiments, but the effect of MTX and of MTX and adenosine on the dNTP's was always the same. The fact that the addition of adenosine together with MTX increased dATP pool size suggested that the decrease of dATP and dGTP pool size in the presence of MTX alone was due to a decrease in the intracellular concentration of purine ribonucleotides. Table 1 shows that, 3 hr after incubation with MTX, uridine-3H incorporation into RNA was completely inhibited. In cultures incubated with MTX and adenosine, the incorporation of uridine-3H into RNA was similar to that of cells incubated with adenosine alone. These results are in agreement with those of Hryniuk ( 14) in L5178Y cells in suggesting that the intracellular concentra tion of purine ribonucleotides, which serve as precursors of their respective deoxyribonucleotides, is reduced within 3 hr after the addition of MTX. Effect of MTX on CdR-3H Incorporation into Acid- soluble and -insoluble Cell Fractions. In order to compare the effect of MTX on dNTP pools and DNA synthesis, tritiated CdR was added at 10~5M, together with MTX, to

o exponentially growing cells. The finding that MTX inhibits 8 IO u 2 8 10 the synthesis of dCTP (Chart 1) made it important to HOURS OF INCUBATION determine whether MTX affected the intracellular conver Chart 1. Effectof MTX on dNTP pools in CCRF-CEM cells. Cultures sion of CdR-3H into acid-soluble nucleotides. The kinetics (5 x IO5cells/ml) were incubated in the absence (O) or presence (•)of5 x of labeling of the acid-soluble and -insoluble cell fractions is 10 ' MMTX or in the presence (A) of MTX with 10 4Madenosine at 37°. shown in Chart 2, A and B, respectively. The rate of At the indicated times, samples of cells were taken, and the concentration of each of the 4 dNTP's was determined as described in "Materials and CdR-3H incorporation into the acid-soluble fraction of Methods." Points, averages of duplicate samples of 2 separate experi MTX-treated cells was about twice that of the control and ments. increased for at least 8 hr after the addition of MTX (Chart 2A). measure of thymidylate synthesis. Thirty min after expo The incorporation of CdR-3H into the acid-insoluble nentially growing CCRF-CEM cells were incubated with 5 fraction was decreased by about 50% within 30 min after the X 10~7 M MTX, UdR-3H incorporation into the acid- addition of MTX (Chart 2B). However, the incorporation of insoluble fraction of cells was inhibited by 97% (data not shown). Trypan blue exclusion tests showed cell viability to Table I be about 96% during 10 hr of incubation with this same Effect of MTX on uridine-*H incorporation into RNA in presence and concentration of MTX. absence of adenosine Studies were then conducted to determine the effect of 5 CCRF-CEM (5 x 10scells/ml) were incubated with and without MTX X 10~7 M MTX on the 4 dNTP pools of exponentially (5 x 10~7 M) and/or adenosine (10~* M). At the indicated times, 3-ml samples of cells were incubated with uridine-3H (o^Ci/ml). After 20 min. growing CCRF-CEM cells. During the 1st 2 hr of incuba the cell suspension was cooled to 0°.The RNA fraction was isolated and tion, the pools of dTTP and dCTP decreased about 70 and counted as described in "Materials and Methods." 80%, respectively (Chart 1). Further incubation with MTX Radioactivity in RNA fraction led to further reduction of the dTTP pool to about 5% of its (cpm/10" cells) initial size by 7 hr after addition of the drug. Conversely, the intracellular concentration of dATP increased about 45% Additions Ohr 3hr 6hr during the 1st 2 hr of incubation, while that of dGTP remained constant (Chart 1). Thereafter, the dATP pool NoneMTXAdenosineMTX diminished rapidly while the dGTP pool decreased more gradually. The kinetic profile of MTX action on dNTP pool and adenosine145511631251108410120262828841634024252717 sizes was unchanged in cultures of cells containing dialyzed

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1974 American Association for Cancer Research. A. Fridland during the conversion of dUMP-5-3H to dTMP via thymi- dylate synthetase (27). The presence of MTX greatly affected the distribution of radioactivity among the nucleotides (Table 3). Thirty min after the addition of CdR-3H and MTX, 83% of the label was associated with dUMP. Only 6% of the radioactivity was associated with CdR and UdR in cell extracts or in the medium in which cells were incubated with CdR-3H showed that 97% of the label in the nucleosides was associated" with CdR in the presence or absence of MTX (data not shown). Thus, deamination of CdR to UdR was slight and, further, was not increased by the addition of MTX to the cells. These data indicate that MTX in CCRF-CEM cells reduces the concentration of deoxycytidine nucleotides by increasing the deamination of dCMP to dUMP.

DISCUSSION

After Solter and Handschumacher (28) described their enzymatic assay for dNTP, others were able to study the effect of antimetabolites on pool sizes of DNA precursors (1, 4, 26). Skoog and Nordenskjold (26) showed that hydroxyurea, an inhibitor of ribonucleoside diphosphate reducíase,causes a loss of dGTP and dATP pools and an HOURS OF INCUBATION increase in dCTP and dTTP pools in mouse embryo cells. Chart 2. Effect of MTX on the incorporation of CdR-3H into acid- Removal of the drug from these cells resulted in a rapid soluble and -insoluble fractions of CCRF-CEM cells. Cultures (5 x 10s increase of dGTP and dATP pools to control levels. These cells/ml) were incubated with CdR-3H in the presence (O) or absence (•) effects on the pools were attributed to the allosteric of 5 x 10 ' M MTX at 37°.The specific activity and concentration of regulation, by dNTP's, of ribonucleotide diphosphate re- labeled precursor were 0.5 Ci/mmole and 10 ^M, respectively. At the ductase, which reduces all 4 ribonucleoside diphosphates indicated times, samples of cells were taken, and the radioactivity in the acid-soluble (A) and acid-insoluble (B) fractions was determined as (26). described in "Materials and Methods." Points, averages of 2 separate MTX inhibits isolated dihydrofolate reducíase from experiments. mammalian and bacterial cells (10). It is thought that Ihis inhibition reduces the tetrahydrofolale pools and leads lo CdR-3H into the acid-insoluble fraction of MTX-treated Ihe inhibilion of ihymidylale synlhesis (6). Adams et al. (1) cells continued for at least 7 hr after the addition of MTX to found lhat 1-hr incubation with MTX reduced the dTTP cultures. Between 1 and 7 hr, the radioactivity incorporated pool in L929 cells by 50% and slighlly increased Ihe other into the acid-insoluble fraction was 9 cpm/min/10~6 cells in dNTP pools. In Ihe present kinetic studies, soon afler MTX the presence of MTX and 40 cpm/min/10 6 cells in its was added lo cell cullures, both dTTP and dCTP pools absence. Thus, despite the effective inhibition of dNTP decreased rapidly, Ihe dATP pool increased inilially, and synthesis (Chart 1), cells can continue to synthesize DNA Ihe dGTP pool remained unchanged. Afler 2 hr of incuba several hr after incubation with MTX. Similar observations tion, bolh purine dNTP pools slarled lo decrease. The have been reported by Wise and Prescott (31) and Ockey addilion of adenosine and MTX logelher resulled in a 3-fold (24). expansion of Ihe dATP pool and reversed Ihe inhibition of Chromatography of Acid-soluble Nucleotides from CdR- uridine-3H incorporation inlo RNA by MTX alone. There 3H-labeled Cells. Chromatographie analysis of the acid- fore, ihese dala imply lhal Ihe addilion of MTX to soluble fraction from CdR-3H-labeled cells (Chart 2A) was CCRF-CEM cells resulls in a rapid reduclion of the cellular carried out to determine the distribution of radioactivity conteníof lelrahydrofolales, so thai Ihe synlhesis of bolh associated with the various nucleotides. Between 0.5 and 8 purine ribonucleolides and thymidylale synlhesis, which are hr after labeling with CdR-3H, the acid-soluble nucleotides dependenl on lelrahydrofolales, becomes impaired. in the control cultures contained 35 to 56% of the radioactiv The 45% increase in dATP pool size during Ihe Isl 2 hr of ity associated with dCTP, 16 to 20% ofthat associated with incubalion was probably noi Ihe resull of dATP accumula- dCDP, 48 to 26% ofthat associated with dCMP, and 3.7 of lion due lo synchronizalion of cells in early S phase, since 0.8% of that associated with dUMP and dUTP, (Table 2). dATP pool size does noi change greally during G¡andearly Although thymidine phosphates and deoxyuridine phos S phases of Ihe cell cycle (8). A more plausible explanalion phates are not separated by the solvent used in these is offered by Ihe following hypolhetical scheme. The pool of experiments, only the latter nucleotides contained radioac dTTP, being only about 1% as large as purine ribonucleo tivity, because the label at 5-position of CdR would be lost tide pools in asynchronous cells (11), may be expected lo

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Table 2 Chromatographie analysis of acid-soluble fraction from CdR-3H-labeled cells The procedure of incubation of CCRF-CEM cells with CdR-3H is described in the legend to Chart 2. At the indicated times, aliquots of each acid-soluble fraction were applied to a sheet of cellulose impregnated with polyethyleneimine, and the radioactivity associated with each nucleotide fraction was determined as described in "Materials and Methods." Each value represents the average of 2 separate experiments. cells)period Radioactivity in nucleotide fractions (cpm/ IO6

%"0.51.02.04.06.08.080000808000000.60.8dCTP22428449251611921144%34.635.537.634.548.456.0dUDP000000%000000dCDP108148228300440332%16.718.517.420.017.916.2dUMP0048404816%003.72.71.90.8dCMP316368540640768536%48.846.041.342.831.226.2(hr) dUTP

Percentage of total radioactivity in nucleotides.

Table 3 Chromatographie analysis of acid-soluble fraction from MTX-treated cells labeled with CdR-3H The procedure of incubation of CCRF-CEM cells with CdR-3H and the method for analyzing the amount of radioactivity associated with each nucleotide fraction are described in Table 2. Each value represents the average of 2 separate experiments.

Incu bation cells)dUTP000000%-000000dCTP14410824822012492%5.84.24.53.01.61.1dUDP000000%000000dCDP6464100724424%2.62.51.81.00.60.3dUMP206021804860665676767876%83.485.287.392.196.097.7dCMP20420435028015672%8.28.06.43.92.00.9in nucleotide fractions (cpm/ IO6 period(hr)0.51.02.04.06.08.0Radioactivity

" Percentage of total radioactivity in nucleotides. decrease faster. This decrease in intracellular concentration ily was associaled wilh CdR nucleolides. Since Ihe deami- of dTTP may change the substrate specificity of the nation of CdR to UdR in the presence of MTX is slighl, Ihe ribonucleotide reducíase,causing an initial increase of the observed increase in concentration of dUMP is probably dATP pool. Then, as the purine ribonucleotide concentra caused by an increase in dCMP deaminase activily. Such an tion decreases, dATP and dGTP synthesis is inhibited by a increase could resull from Ihe removal of feedback inhibi- lack of substrates; this in turn leads to a depletion of dATP lion of enzyme aclivily by a decreasing dTTP pool. Sludies and dGTP. wilh purified dCMP deaminase have shown thai dTTP is a The addition of adenosine and MTX to cells resulted in a potent inhibitor of Ihis enzyme, while dCTP acls as an striking expansion of dATP but not dGTP pools. Similarly, aclivalor (18, 19). This aclivalion-inhibilion conlrol of Neuhard and Munch-Peterson (22) showed that the thymine dCMP deaminase may help lo regúlalethe relalive sizes of auxotroph of Escherichia coli, when deprived of intracellu CdR and TdR nucleolides in vivo. Thus, an external lar thymine deoxyribonucleotides by thymine starvation, slimulus lo reduce Ihe dTTP pool may decrease Ihe accumulates dATP but not dGTP. dATP is thought to act inhibilion of dCMP deaminase aclivily, so lhal Ihe rale of as an inhibitor of ribonucleotide diphosphate reducíase(9). synthesis of TdR nucleolides via Ihis palhway is increased. The rapidly increasing dATP pool, after addition of adeno In Ihe presence of MTX, however, Ihe reaction is stopped at sine and MTX, may therefore prevent dGTP synlhesis. Ihe level of ihymidylale synthetase, resulting in the accumu- The presenl dala show that MTX depresses dCTP pools lalion of dUMP. Allernalively, the mechanism for Ine as readily as the dTTP pool in CCRF-CEM cullures. When elevalion of enzyme aclivity could be more a direcl effecl of cells were incubaled simullaneously wilh MTX and CdR- MTX action in cells. The receñíobservalionthai MTX 3H, incorporalion of radioaclivily inlo Ihe acid-soluble leads to an increase in cytidine deaminase aclivily in HeLa nucleolide fraclion was increased 2- lo 3-fold. Direct cells seems compalible with this suggestion (21). measuremenl of Ihe radioaclivily associaled wilh CdR and These studies also show thai, despite 97% inhibition of UdR nucleotides of Ihe acid-soluble fraclion showed lhal, in thymidylale synlhesis within 30 min after addilion of MTX, Ihe presence of MTX, 83 lo 97% of Ihe label was associaled DNA synlhesis continues for several hr after addition of the wilh dUMP. In Ihe conlrol cullures, 97% of the radioacliv drug. Allempls lo eslimale more accurately the rale of Ihis

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residual DNA synthesis by determining the specific activity 14. Hryniuk, W. M. Purineless Dealh as a Link belween Growth Rate and of the dCTP-3H pool were unsuccessful. The major diffi Cylotoxicily by Metholrexale. Cancer Res., 32: 1506-1511, 1972. culty was that MTX treatment reduced the dCTP pool size 15. Karnofsky, D. A., and Clarkson, B. D. Cellular Effects of Anlicancer to concentrations that were too low for accurate determina Drugs. Ann. Rev. Pharmacol., 3: 357 428, 1963. tion with the enzyme assay. 16. Larsson, A., and Richard, P. Enzymatic Reduction of Ribonucleo- tides. Progr. Nucleic Acid Res., 7: 303 347, 1967. Previous studies in Chinese hamster ovary cells have 17. Magnusson, G. Hydroxyurea-lnduced Accumulation of Short Frag- shown also that MTX fails to hold cells at the GrS border menls during DNA Replication. I. Characlerization of Fragments. J. of the cell cycle (24, 31). Recent studies (17) in polyoma- Virol., 12: 600 608, 1973. infected 3T6 cells showed that inhibition of DNA synthesis 18. Maley, F., and Maley, G. F. On the Mechanism of Feedback by hydroxyurea resulted in the accumulation of DNA Inhibition of Deoxycytidylate Deaminase by Deoxythymidine Tri- fragments that may be precursors of growing DNA chains. phosphale. J. Biol. Chem., 240: 3226-3227, 1965. It was suggested that a late step during the discontinuous 19. Maley. G. F.. and Maley, F. Nucleolide Interconversions. IX. The Regulatory Influence of Deoxycytidine 5'-Triphosphate and Deoxy synthesis of polyoma DNA may be selectively inhibited by thymidine 5:-Triphosphales on Deoxycytidylate Deaminase. J. Biol. hydroxyurea (17). I am presently attempting to determine the rate of DNA synthesis by use of different precursors and Chem., 237: 3311 3312, 1962. 20. Margolis, S., Phillips, F. S., and Sternberg, S. S. 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1888 CANCER RESEARCH VOL. 34

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Arnold Fridland

Cancer Res 1974;34:1883-1888.

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