D-Arabinofuranosylcytosine on Pools of Deoxyribonucleoside Triphosphates in L1210 Ascites Cells1

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[CANCER RESEARCH 41. 505-510, February 1981] 0008-5472/81/0041-OOOOS02.00 Effect of Methotrexate and 1-/?-D-Arabinofuranosylcytosine on Pools of Deoxyribonucleoside Triphosphates in L1210 Ascites Cells1

DeWayne Roberts2 and Charles Peck

Department of Biochemical and Clinical Pharmacology. St. Jude Children s Research Hospital. Memphis. Tennessee 38101

ABSTRACT MTX-induced retention of ara-CTP, leading to prolonged ex posure of tumor cells to the nucleotide (27). We now examine, Pools of deoxyribonucleoside triphosphates in L1210 cells in vivo, changes in cellular pools of deoxyribonucleoside tri were assayed for drug-induced changes that might indicate the phosphates during treatment with ara-C and MTX and relate metabolic basis for retention of 1-ÃŸ-o-arabinofuranosylcytosine the findings to our proposed basis for the synergistic activity of triphosphate by these cells after treatment with methotrexate the combination. (MIX) and 1-/?-D-arabinofuranosylcytosine (ara-C). Within 20 To become cytocidal, ara-C must be converted to ara-CTP, min after treatment with MTX, the pool of deoxythymidine which is not only an inhibitor of the synthesis of DMA but also triphosphate (dTTP) had decreased by about 50% and during the precursor for incorporation of ara-CMP into DNA (3, 10, the next 8 hr decreased slowly to 30% of its initial level. When 11, 21, 33). Both the activation and inactivation of ara-C, the MTX-induced decreases in the cellular contents of deoxycyti- latter by conversion to either ara-UMP or 1-/8-D-arabinofura- dine triphosphate (dCTP), deoxyadenosine triphosphate nosyluracil, are catalyzed by enzymes that normally require (dATP), and deoxyguanosine triphosphate (dGTP) were nor either cytidine or deoxycytidine and its analogs as substrates. malized to percentages of the initial levels, they coincided with ara-C and the natural derivatives of cytosine compete for these the second slower phase of decrease in dTTP. During the enzymes, which in some instances are regulated by deoxyri study, levels of both dTTP and dCTP remained constant in cells bonucleoside triphosphates. from mice treated with 0.9% NaCI solution, whereas levels of The cytocidal action of MTX on cultures of human lympho- both dATP and dGTP decreased. MTX caused a significantly blasts has been attributed to a drug-induced deficiency of more rapid decrease in the level of dATP than did 0.9% NaCI dTTP resulting from an inhibition of dihydrofolate reducÃase by solution but not in the level of dGTP. Over a 9-hr period, after the drug (28). Many investigators have reported that MTX injection of ara-C, levels of both dTTP and dCTP doubled while decreases the amount of dTTP in cells (2, 9, 14, 18, 34, 36, levels of both dATP and dGTP remained unchanged. When 37). Changes in the relative amounts of the deoxyribonucleo ara-C and MTX were administered together, levels of dTTP, side triphosphates and, as a result, in the allosteric regulation dATP, and dGTP did not change significantly, and the increase of dCyd-K, dCMP deaminase, and ribonucleoside diphosphate in dCTP was only 25% of the increase after treatment with ara- reducÃase Â¡nL1210 cells would be expected to have both C alone. direct and indirect effecls on the metabolism of ara-C (13, 18, Thus, the most striking change in deoxyribonucleoside tri 24, 25, 35). Measuremenf of Ihe cellular pools of deoxyribo phosphate pools after combined administration of MTX and nucleoside Iriphosphates after the administration of ara-C plus ara-C was an increase in dCTP concentration that reached MTX might therefore indicate the biochemical basis for a MTX- about one-fourth the concentration achieved with ara-C alone. induced delay in the clearance of ara-CTP from tumor cells. We suggest that MTX, by attenuating the ara-C-induced in In the present studies, mice with L1210 leukemia were given crease in dCTP, caused a change in the allosteric regulation of injections i.p. of MTX, ara-C, both drugs, or 0.9% NaCI solution, either deoxycytidine kinase or deoxycytidylate deaminase (or and the amounts of dTTP, dCTP, dATP, and dGTP in the both), thereby potentiating the activity of ara-C. ascites tumor cells were determined at various intervals during the next 9 hr. The most important observation after simultane INTRODUCTION ous administration of both drugs was the absence or attenua tion of changes in the cellular content of these nucleotides The therapeutic synergism of MTX3 and ara-C in combined after treatment with the individual drugs. A preliminary report treatment of L1210 leukemia (1) has been attributed by us to on a portion of these data has been presented (26).

1This investigation was supported by Research Project Grant CA 23337 and MATERIALS AND METHODS Childhood Cancer Center Grant CA 21765 from the National Cancer Institute, NIH, Department of Health, Education and Welfare, and by the American Le Sources of Materials. Female C57BL/6J x DBA/2J F, mice banese Syrian Associated Charities. 2 To whom requests for reprints should be addressed, at the Department of were obtained from The Jackson Laboratory, Bar Harbor, Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Maine. [5-mef/7y/-3H]dTTP, [5-3H]dCTP, and [5-mefr>y/-3H]dThd P. O. Box 318. Memphis, Tenn. 38101. were products of Schwarz/Mann, Orangeburg, N. Y. Nonra- 1 The abbreviations used are: MTX, methotrexate; ara-C, 1-jS-D-arabinofuranosylcytosine; ara-CTP, 1-/J-o-arabinofuranosylcytosine 5'-triphosphate; ara- dioactive deoxyribonucleoside triphosphates were purchased CMP, 1-/?-o-arabinofuranosylcytosine 5'-monophosphate; ara-UMP, 1-/i-D-ara- from Sigma Chemical Co., St. Louis, Mo. Two sources provided binofuranosyluracil 5'-monophosphate; dCyd-K, deoxycytidine kinase; dCMP DNA polymerase. A preparation of enzyme from Micrococcus deaminase. deoxycytidylate deaminase; dThd, thymidine; PBS, 0.9% NaCI so lution adjusted to pH 7.2 with dibasic potassium phosphate. luteus was obtained from Miles Laboratories, Inc., Elkhart, Ind., Received February 20, 1980; accepted October 22, 1980. for the assay of dTTP and dCTP. For the assay of both dATP

FEBRUARY 1981 505

and dGTP, purified enzyme from Escherichia coli was obtained Turnover of Intracellular Pools of Thymidylate and Its from Boehringer Mannheim Corp., Indianapolis, Ind. ACS, a Analogs. On Day 3 after the i.p. injection of 1 million L1210 liquid scintillant for aqueous solutions, was a product of the cells and 5 min after the i.p. administration of equivalent Amersham/Searle Corp., Arlington Heights, III. Sodium chlo volumes of 0.9% NaCI solution, MTX, ara-C, or MTX plus ara- ride for injection (U.S.P. 0.9% solution) was obtained from C, mice were given injections i.p. of 40 juCi of tritiated dThd (6 Invenex, Chagrin Falls, Ohio, and was opened on the day of Ci/mmol) in 0.2 ml of 0.9% NaCI solution. At various intervals, study. Both MIX and ara-C were provided by the Division of the mice were killed, and the ascites tumor cells were removed. Cancer Treatment of the National Cancer Institute, and both The tumor cells were collected by centrifugation and subse were dissolved in 0.9% NaCI solution. quently washed 4 times by resuspension in ice-cold PBS, after Preparation of L1210 Ascites Cells. Unless indicated oth which the cells were resuspended again in PBS and divided erwise, mice were given injections i.p. of 2 million L1210 into fractions for cell counts and for determination of the ascites cells and, 3 days later, were treated with 12 mg of residual amount of radioactivity in the acid-soluble fraction MTX, 1200 mg of ara-C, both drugs, or an equivalent volume from the cells. In other similar studies at 20 and 60 min after of 0.9% NaCI solution per kg of body weight; all mice received the administration of tritiated dThd, the acid-soluble pools of a total volume of 0.02 ml/g of body weight. At various intervals residual radioactivity in L1210 cells were collected and frac thereafter, the mice were killed by dislocation of the cervical tionated on 4-cm anion-exchange columns from Bio-Rad Lab spine and given injections i.p. of 3 ml of a solution that con oratories, Richmond, Calif., by the previously described pro tained 5 g of glucose, 0.9 g of NaCI, and 100 mg of disodium cedure of Chou ef al., cited in Roberts ef al. (27). EDTA per 100 ml at 4Â° and pH 7. The ascites cells were Statistical Methods. Regression equations were calculated removed rapidly, diluted to a total volume of 10 ml with PBS, with the assistance of Model SR-51-II electronic calculator and collected by centrifugation for 5 min at 200 x g and 4Â°. from Texas Instruments Inc., Houston, Texas. For comparison The pellets of cells were resuspended in PBS, and aliquots of changes in the levels of various nucleoside triphosphates, were removed for determination of the number of cells in the the values in Charts 1 to 4 were normalized to percentages suspension and of the amount of deoxyribonucleoside triphos- from which regression equations were derived. The variation phates in the cells. between slope constants from these equations was tested for For analyses of the deoxyribonucleoside triphosphates, the significance by the method of Dixon and Massey (5). The ascites cells were collected again by centrifugation and resus equations for the clearance of dTTP from L1210 cells in Chart pended in 1 ml of 60% methanol solution. After storage at 5 were derived by the method of Efroymson-Tukey (7). -20Â° until the next day, the methanol-soluble fraction was removed, diluted with 1.5 ml of H2O to raise the freezing point, and lyophilized. The residue from the extraction of approxi RESULTS mately 50 million L1210 cells was resuspended in 0.5 ml of H2O immediately before the deoxyribonucleoside triphos dTTP Content of L1210 Cells. Within 20 min after i.p. phates were assayed. injection of MTX, 12 mg/kg, the cellular content of dTTP DMA Polymerase Assay of Deoxyribonucleoside Triphos decreased to about 50% of the mean level Â±S.D. (17.2 Â±5.0 phates. For the assay of dTTP, dCTP, and dGTP, 8 /il of the pmol/million cells) in mice treated with 0.9% NaCI solution extract from L1210 cells were added to 65 /il of reagent, and (Chart 1A). The content of dTTP continued to decrease, but 4 /il of extract were used in the assay of dATP. The reagent much more slowly, throughout the remaining observation pe contained 90 mw Tris-HCI at pH 7.4, 4 HIM MgCli, 1.3 HIM 2- riod and by 9 hr was about 30% of the level in cells from 0.9% mercaptoethanol, 73 /ig of DNA, 3.5 units of DNA polymerase, NaCI solution-treated mice. By contrast, treatment with ara-C, 145 ng of dTTP (0.3 /iCi), and 3 jug of 2 other deoxyribonucle 1200 mg/kg, increased the amount of dTTP in L1210 cells oside triphosphates (9). The omitted deoxyribonucleoside tri- (Chart 1B). The increase was linear with time for 9 hr, at which phosphate was provided by the cell extract, and, when dTTP point the content of dTTP had approximately doubled. Neither was assayed, tritiated dCTP was substituted for dTTP. Before the i.p. injection of both drugs nor treatment with 0.9% NaCI these studies were begun, cellular ara-CTP was expected to solution caused appreciable changes in the content of dTTP interfere with the assay; thus, the DNA polymerase reaction (Chart 1, C and D). was allowed to proceed at 37Â°for 1 hr rather than for 20 min, We attribute the rapid initial decrease in levels of dTTP after which from preliminary studies was judged to be adequate. treatment with MTX to a block of the synthesis de novo of The tritiated DNA from the polymerase reaction was precipi dTMP by MTX and to the subsequent incorporation of preex tated by the addition of 9 /il of a solution containing 33% isting dTTP into DNA (28). Inhibition of the synthesis of DNA trichloroacetic acid and 0.1 M Na^O?. Immediately after re- by ara-C was followed by a gradual increase in the amount of suspension of the sediment, an aliquot was placed on a 2.4- dTTP contained by the population of tumor cells. With admin cm Whatman GF/A filter disc that was prewashed with 5% istration of both drugs, the drain on the preexisting pools of trichloroacetic acid plus 0.01 M Na4P2O7; subsequently, the dTTP, after the administration of MTX, was apparently blocked filters and their contents were washed with the same solution. by ara-C with the inhibition of the synthesis of DNA. The The filters were dried at 55Â° before they were placed in maintenance of a constant amount of dTTP throughout the aqueous-counting scintillant. A standard curve, constructed period of observation after the administration of both drugs with known amounts of the same nucleoside triphosphate pro could result from either the absence of a turnover by these vided in the cell extract and omitted from the reagent, was pools in L1210 cells or a scavenging of dThd or reduced used in each assay. folates by these cells in the absence of the synthesis of DNA.

506 CANCER RESEARCH VOL. 41

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â€¢ara-C(l,200 mg/kg)Y 20E| =21.3112.09Xr =0.464df=32p

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Chart 1. Variations in the levels of dTTP in L1210 cells. On Day 3 after the i.p. injection of 2 million L1210 ascites cells, the mice were treated i.p. with 12 Chart 2. Variations in the levels of dCTP in L1210 cells after treatment with mg of MTX. 1200 mg of ara-C, both drugs, or an equivalent volume of 0.9% NaCI 12 mg of MTX, 1200 mg of ara-C, both drugs, or equivalent volumes of 0.9% solution per kg body weight. At various intervals thereafter, the ascites tumor NaCI solution per kg body weight. The data were obtained with the same cell cells were collected, and the cellular content of dTTP was determined. Data were fractions used to measure dCTP levels in Chart 1 pooled from either 4 or 6 studies with one animal per each point in time from each study. In addition, 2 fractions of cells were collected from each animal, and each fraction was assayed in triplicate. The curves were derived from the regression equation presented in each section.

I2S 125 dCTP Content of L1210 Cells. Administration of MTX pro duced a linear decrease with time in the amount of dCTP in the L1210 cells (Chart 2). After 7 hr, the intracellular content of IOO dCTP was about 35% of the level in 0.9% NaCI solution-treated cells (/.e., 12.5 Â± 2.9 pmol/million cells). Administration of 75 75 ara-C produced a linear increase with time in the content of 50 50 dCTP, and, by 9 hr after treatment, the content of dCTP was MTX (I2 mq/kg) ora C(1,200 mg/kq) about twice the initial level in L1210 cells. The slight increase d Y = I08-6.3X Y = 100 + I.6X LÃœ r --0.346 with time in the level of dCTP after administration of both drugs O 25 r = H 0.809 25 z df = 22 df = 21 o is compatible with a summation of responses to the individual p < 0.0l P 0.05 drugs, and, as will be shown, dCTP was the only deoxyribo- IO OI OI nucleoside triphosphate to change in concentration as a result C. D. of treatment with the combination. Treatment with 0.9% NaCI IOO IOO solution did not significantly change the amount of dCTP in L1210 cells. 75 The observed decrease in dCTP after the administration of MTX is compatible with a relaxation of the inhibition of dCMP 50 deaminase by dTTP and a diversion of the precursors of dCTP MTX + O'oC 50 NoCI(0.9%) Y =92.4 + 0.74X Y = 92-2.5 X to the formation of dUMP (9); however, the temporal pattern is r =0.158 r = (-10.445 similar to the slower secondary decrease in dTTP. Again, as 25 df = 20 25 df = 2l with dTTP, inhibition of the synthesis of DNA by ara-C appar p 0.05 p 0.05 ently altered the normal balance between anabolism, catabo- 01 357901 3579 lism, and the incorporation of dCTP into DNA, resulting in a HOURS slow increase in the amount of dCTP. dATP Content of L1210 Cells. As indicated in Chart 3, the Chart 3. Variations in levels of dATP in L1210 cells after treatment with 12 mg of MTX, 1200 mg of ara-C, both drugs, or equivalent volumes of 0.9% NaCI pool of dATP in L1210 cells was larger than the individual solution per kg body weight. These data represent 3 or 4 animals per point in pools of the 3 other deoxynucleoside triphosphates (see Chart time and were obtained from 4 studies as indicated in Chart 1.

FEBRUARY 1981 507

4 for dGTP). This observation of a relatively large pool of dATP

MEAN Ã®SE was unexpected because, in general, the levels of dATP are â€¢MTX* ARA-C Log VI-100I043X +4706 lower than those of dTTP; however, the ratios of nucleotide I rt ' 28 78 min r â€¢I-I0.929 pools do vary for different types of cells. Levels of dATP in L1210 cells decreased during the subse quent 9 hr after administration of 0.9% NaCI solution (p < 0.05). The administration of MTX was followed by a more rapid decrease in the level of dATP in L1210 cells than occurred in cells from 0.9% NaCI solution-treated mice, and the variation in responses to MTX and to 0.9% NaCI solution was significant (p < 0.02). Although MTX presumably inhibited the synthesis o Aro-C Log, -I-IO.CH43X4830 de novo of purine nucleotides and of dTMP simultaneously IV,' 2105 mÂ«! r-l-)0959 df'22 (28), an immediate decrease in the content of dATP, similar to e- Â¡lili the decrease in dTTP, was not observed. In contrast to the 2- fold increases in both dTTP and dCTP produced by ara-C, this 60 120 180 240 TIME FROM THYMIDINE ADMINISTRATION (mm) drug did not change the amount of dATP in the cells. Again, as with dTTP, the administration of both drugs did not change the Chart 5. Clearance of dThd and its nucleotides from the acid-soluble pool in L1210 cells. At 5 min after pretreatment with 12 mg of MTX, 1200 mg of ara-C. amount of dATP in L1 210 cells. both drugs, or equivalent volumes of 0.9% NaCI solution per kg body weight, dGTP Content of L1210 Cells. The amount of dGTP in mice with L1210 leukemia were given injections i.p. of 2 fiCi of [5-mefhy/-3H]- L1210 cells decreased after treatment with MTX (Chart 4). A dThd (specific activity, 6 Ci/mmol) per g body weight. At various intervals after the administration of dThd, the ascites tumor cells were collected, and the similar significant decrease in the amount of dGTP was noted residual radioactivity in the acid-soluble pools was determined. The equations also after treatment with 0.9% NaCI solution (p < 0.01); com for the clearance of radioactivity were derived by the Efroymson-Tukey procedure parison of the slope constants for the decrease in dGTP after of stepwise regression (7). the administration of either MTX or 0.9% NaCI solution indi cated that a common factor might have caused the changes in both instances (p > 0.1). Again, as with dATP, treatment with drugs or 0.9% NaCI solution in order to prelabel the pool of ara-C, either alone or in combination with MTX, did not signif dTTP; results from both studies were very similar. After mice were treated with MTX, ara-C, both drugs, or icantly change the amount of dGTP in cells. Turnover of dTTP. Two types of studies were used to 0.9% NaCI solution, the clearance of tritiated dThd from the demonstrate that the pool of dTTP does indeed turn over after acid-soluble pool of L1210 cells followed one of 2 patterns treatment with MTX plus ara-C. For the study presented in (Chart 5). The clearance of radioactivity after the administration Chart 5, the 2 drugs were administered in rapid succession 5 of either MTX or 0.9% NaCI solution was described by quad min before i.p. injection of 2 /tCi of [5-me?fty/-3H]dThd per g ratic equations and did not differ significantly for these 2 treatments. After treatment with ara-C, either alone or with body weight. In the other study, for which data are not included, the tritiated dThd was administered 20 min before either the MTX, the clearance of radioactivity from L1210 cells was proportional with time and did not differ significantly for these 2 treatments. At 20 min after the administration of tritiated 40 A. MTX (I2mg/kg) aro C(1,200 mg/kg) dThd, 70% of the radioactivity in the acid-soluble pool chro- Y = 16.8- I.I X Y = 19.6+ 0.28X matographed as dTTP, as did 60% of the residual radioactivity r = (-) 0.624 r = 0.236 3020co0 df = 2l df = 2l after 60 min. 0.01--*-l^^i p '0.05-, p The clearance of radioactivity from L1210 cells after treat â€¢i' -i ' Ã® ment with ara-C, either alone or in combination, indicates a turnover of cellular pools of dTTP. From this study, we con ioÃˆ cluded that the pool of dTTP in L1210 cells remained in a dynamic equilibrium after treatment with MTX plus ara-C and

i) i i i i) i i i that the pool of dTTP in these cells must be replenished from Â°(i 13579" 13579- some source in the mouse. 40O p MTX+ oro C D NoCI(0.9 %) â€¢o0 Y = 17.5+ 0.08X Y = 2l. 1- 0.67 X r --0.058 r=HO538p DISCUSSION 500.20IOi" df= 22 0.05- p 0.01i < At the outset of these studies, we postulated that metabolic .â€¢ responses related to the therapeutic interaction between MTX â€¢.i and ara-C caused the retention of ara-CTP by L1210 cells (27). Since MTX has been reported to decrease the amount of both dTTP and dCTP in cells (2, 9), and since these 2 nucleotides i) i i i iD i i i allosterically regulate enzymes that are related directly to the 1 35 7940302010403020100~B.9HOURS 1 3 5 7 metabolic disposition of ara-C, the present studies of the deoxyribonucleoside triphosphates were begun. Four distinct pat Chart 4. Variations in levels of dGTP in L1210 cells after treatment with 12 terns of change in deoxyribonucleoside triphosphate pools mg of MTX, 1200 mg of ara-C, both drugs, or equivalent volumes of 0.9% NaCI solution per kg body weight. Cell fractions used in these studies were the same were noted. as those used in Chart 3. (a) Levels of dATP and dGTP decreased in cells from 0.9%

508 CANCER RESEARCH VOL. 41

NaCI solution-treated mice, whereas amounts of dTTP and cells during the S phase of the cell cycle. dCTP remained constant. When levels of the purine nucleotides Hence, the changes in levels of dCTP observed in this study were normalized to percentages of their original amounts, i.e., could have affected the allosteric regulation of dCyd-K and their respective intercepts, the regression constants for the dCMP deaminase and thereby the metabolism of ara-C in decreases were similar (p > 0.5). It could be argued from these L1210 cells. The cellular level of dCTP, moreover, is a direct results that 0.9% NaCI solution affected nucleotide pool sizes determinant of the sensitivity of cells to ara-C; Harris ef al. (13) in a nonphysiological manner, making comparisons with results observed an inverse relationship between the levels of dCTP in in drug-treated cells invalid. However, since both MIX and ara- cell lines in culture and the dosage of ara-C required to inhibit C were administered in a 0.9% NaCI solution, any such effect cell division by these lines. Two other groups of workers have by 0.9% NaCI solution would not probably interfere with the also reported an association between resistance to ara-C and interpretation of findings. increases in the cellular levels of dCTP (22, 34). (to) MIX produced a biphasic change in the level of dTTP in Although the cellular concentration of dTTP contributes to L1210 cells. The early decrease in dTTP indicates a rapid the allosteric regulation of both dCyd-K and dCMP deaminase, onset of the inhibition by MTX of the synthesis de novo of dTTP it is uncertain from the present observations whether the MTX- and corresponds with the inability of the cells to synthesize induced retention of ara-CTP is a direct consequence of dTMP. In earlier studies, the administration of 1.5 mg of MTX changes in the levels of dTTP. This uncertainty stems from the per kg inhibited completely the activity of dihydrofolate reduc- following considerations, (a) dTTP competes poorly with dCTP tase within less than 5 min and, at the earliest time point tested for both dCyd-K and dCMP deaminase, and, since the change (i.e., 1 hr), 85% of the capacity of L1210 cells to incorporate in the levels of dTTP is of the same magnitude as the change deoxyuridine into DNA in vivo (29). The slower secondary in dCTP, these increases in dTTP may contribute little to phase of decrease in the content of dTTP corresponded with counteracting the variation in the levels of dCTP. (to) The decreases in the levels of the 3 other nucleotides (p > 0.1 after increase in level of dTTP after treatment with ara-C alone normalization). should facilitate the retention of the drug by L1210 cells, and (c) ara-C increased significantly and coordinately the content the maintenance of normal levels of dTTP after treatment with of both dCTP and dTTP in L1210 cells but did not increase the both drugs should facilitate clearance of the drug; these pre content of either dATP or dGTP; however, the drug did prevent dicted responses to dTTP are the reverse of those observed, the decrease that occurred in the purine nucleotides after (c) The kinetics of the decrease in levels of dCTP after treat treatment with 0.9% NaCI solution. Since the responses of the ment with MTX followed the same general pattern as that of 4 nucleotides to ara-C are qualitatively similar, i.e., increased the purine nucleotides and of the secondary decrease in the levels of the nucleotides relative to the controls, some general level of dTTP rather than the initial immediate decrease in effect by the drug appears responsible. An inhibition of the dTTP. The latter observation contrasts with the temporal cor synthesis of DNA by ara-C might be expected to lead to an relation between the decrease in dCTP and dTTP observed accumulation of these nucleotides in cells in S phase, of cells with a cell line of human lymphoblasts (9). with these nucleotides at the Gi-S interphase, or of both Our observations also pose questions concerning the effect responses. of ara-C on the oncolytic action of MTX. In the survival studies, (d) The simultaneous administration of both drugs blocked the dosage of MTX contributed independently and through an the decreases produced by MTX alone, and only the cellular interaction with ara-C to increase the life span of mice with content of dCTP increased significantly during the period of L1210 leukemia (1). The observation of normal or near normal observation (0.05 > p > 0.01). The apparent reversal of the levels of all 4 nucleotides in L1210 cells for the first 9 hr after action of MTX by ara-C is contrary both to the therapeutic the administration of both drugs is puzzling, since the oncolytic synergism observed between the drugs and to the present action of MTX is considered generally to result from a defi understanding of the pharmacodynamics of MTX and is better ciency in one or more of these nucleotides. It must be consid understood as a consequence of both an inhibition by ara-C of ered, however, that these drugs, alone as well as in combina the synthesis of DNA and a scavenging of nucleosides and/or tion, are oncolytic and that the therapeutic effectiveness of the reduced folates by L1210 cells. After treatment with MTX combination may result from both independent and synergistic alone, the early decrease in the levels of dTTP would be actions of the drugs on various subpopulations of leukemia expected to slow the rate of synthesis of DNA, and apparently, cells. the scavenging of metabolites was almost sufficient to maintain normal levels of the 3 other deoxyribonucleoside triphosphates ACKNOWLEDGMENTS in cells with the slower rate of synthesis of DNA. Two enzymes of potential importance in regulating the me The authors gratefully acknowledge the editorial suggestions of John Gilbert tabolism of ara-C are dCyd-K, which converts ara-C to ara- and the assistance of Delores Anderson and Fabrienne Holloway in preparation of the manuscript. CMP (6, 17, 23), and dCMP deaminase, which converts ara- CMP to ara-UMP (30). The activity of dCyd-K is inhibited by dCTP, and this inhibition can be prevented or partially reversed REFERENCES by dTTP (4, 15, 16, 17, 19, 23, 32). On the other hand, dCMP 1. Avery, T. L, and Roberts, D. Dose-related synergism of cytosine arabinoside deaminase is activated by dCTP and inhibited by dTTP (8, 20, and methotrexate against murine leukemia L1210. Eur. J. Cancer, Â¡0 425- 31). Normally, these 2 enzymes plus ribonucleoside diphos- 429, 1974. 2. Cadman. E., and Eiferman, F. Mechanism of synergistic cell killing when phate reductase, also allosterically regulated by deoxyribonu methotrexate precedes cytosine arabinoside. J. Clin. Invest., 64: 788-797, cleoside triphosphates (38), control the amount of dCTP in 1979.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. Effect of Methotrexate and 1-β-d-Arabinofuranosylcytosine on Pools of Deoxyribonucleoside Triphosphates in L1210 Ascites Cells

DeWayne Roberts and Charles Peck

Cancer Res 1981;41:505-510.

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