Role of Uridine Triphosphate in the Phosphorylation of 1-ÃŸ-D- Arabinofuranosylcytosine by Ehrlich Ascites Tumor Cells1

Home , Nucleoside triphosphate, Uridine triphosphate

[CANCER RESEARCH 47, 1820-1824, April 1, 1987] Role of Uridine Triphosphate in the Phosphorylation of 1-ÃŸ-D- Arabinofuranosylcytosine by Ehrlich Ascites Tumor Cells1

J. Courtland White2 and Leigh H. HiÃ±es

Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103

ABSTRACT potent feedback regulation by dCTP (3-9). The level of dCTP in the cell has been shown to be an important determinant of Pyrimidine nucleotide pools were investigated as determinants of the ara-C action in a variety of cell types (10-12). For example, rate of phosphorylation of l-j9-D-arabinofuranosylcytosine (ara-C) by Harris et al. (10) demonstrated that the sensitivity of several Ehrlich ascites cells and cell extracts. Cells were preincubated for 2 h with 10 MMpyrazofurin, 10 imi glucosamine, 50 MM3-deazauridine, or mouse tumor cell lines to ara-C was inversely proportional to 1 HIMuridine in order to alter the concentrations of pyrimidine nucleo- their cellular dCTP level. In addition, these authors observed tides. Samples of the cell suspensions were taken for assay of adenosine that thymidine enhanced ara-C sensitivity in those cell lines S'-triphosphate (ATP), uridine 5'-triphosphate (IIP), cytidine S'-tn- where there was a depression in dCTP levels but not in those phosphate, guanosine S'-triphosphate, deoxycytidine S'-triphosphate cell lines where thymidine did not alter dCTP pools. Cellular (dCTP), and deoxythymidine S'-triphosphate; then l MM[3H|ara-C was pools of dCTP may also be decreased by inhibitors of the de added and its rate of intrazellular uptake was measured for 30 min. 3- novo pathway for pyrimidine synthesis. Grant et al. (13) dem Deazauridine lowered dCTP and stimulated ara-C uptake; however, onstrated both depressed dCTP pools and increased levels of pyrazofurin and glucosamine were potent inhibitors of ara-C uptake ara-CTP in HL-60 cells following exposure to PALA under although they also decreased dCTP levels. Uridine stimulated ara-C some circumstances. Although PALA caused a dose-dependent uptake despite an increase in dCTP. A crude cytoplasmic extract was prepared by a procedure which permitted results of ara-C kinase assays decline in dCTP levels, high concentrations of PALA were less to be expressed as pmol per min per 10* cells as in the cellular uptake effective at enhancing ara-CTP generation than were lower studies. When assayed in the presence of mixtures of ribo- and deoxyri- concentrations. bonucleoside triphosphates at concentrations close to their cellular levels, Although the dCTP level is clearly a key factor in the regu ara-C kinase activity closely approximated the cellular uptake rate for lation of the rate of ara-C phosphorylation, it is not the only the five incubation conditions. Deletion of cytidine 5'-, guanosine 5'-, or determinant. Other pyrimidine nucleotides may play important deoxythymidine S'-triphosphate from the assay mixture had little effect, roles as well. Inhibition of deoxycytidine kinase by dCTP may while deletion of dCTP increased kinase activity 9-fold. Elimination of be at least partially reversed by high concentrations of dTTP ATP also did not alter kinase activity in the presence of the remaining (3,4). Most NTPs are potential phosphate donors (3,4,8), and five ribo- and deoxyribonucleoside triphosphates; however, deletion of at low concentrations of ara-C, the rate of phosphorylation is UTP reduced activity to 22% of the rate with the control mixture. When ara-C kinase was assayed with only 3 HIMATP, dCTP was a very potent more rapid with UTP as the phosphate donor than with ATP inhibitor (50% inhibition concentration = 0.4 ^M). Inhibition was com (5, 6). Addition of uridine to the culture medium enhanced the plete at 10 MMdCTP, a concentration below the intracellular dCTP level sensitivity to ara-C of several B-lymphocyte cell lines which in control cells (25 MM). With 0.9 m\i UTP, enzyme activity was 2-fold were relatively resistant to ara-C (14). Uridine potentiated the greater in the absence of dCTP and the dCTP was 15-fold less potent as therapeutic and toxic effects of ara-C in mice (15) and enhanced an inhibitor (50% inhibition concentration = 6 MM). We conclude that myelosuppression in dogs (16). Thus, inhibitors of pyrimidine the actual phosphate donor for the phosphorylation of 1 MM ara-C in synthesis and uridine have been shown to enhance ara-C phos Ehrlich cells is UTP and not ATP. These observations suggest that phorylation under certain conditions, although these treatments successful combination protocols aimed at stimulating ara-C uptake by would be expected to have opposite effects on cellular nucleotide means of a decrease in dCTP levels must simultaneously preserve or pools. increase UTP pools.

INTRODUCTION MATERIALS AND METHODS The cytotoxicity of ara-C3 has been correlated with the cel Chemicals. [uC]Sucrose, [3H]ara-C, and 3H2O were obtained from lular accumulation of ara-CTP (1,2). The steady-state intracel Amersham Corp. PALA was obtained from Dr. Narayanan, Develop lular ara-CTP concentration is determined by the relative rates mental Therapeutics Program, National Cancer Institute. Pyrazofurin was obtained from Calbiochem-Behring. 3-Deazauridine, 6-azauridine, of synthesis and breakdown. These are both multistep processes and all other biochemicals were obtained from Sigma Chemical. Dow the rates of which are subject to regulatory controls. The rate-limiting step for the phosphorylation of ara-C in Corning silicon oils Nos. 550 and 200 were obtained from William F. Nye, Inc., Bedford, MA, and blended 84:16 to a density of 1.3 g/ml. murine tumor cells is the initial phosphorylation of ara-C to ara-C Accumulation Velocity. Ehrlich ascites tumor cells were main ara-CMP by deoxycytidine kinase. This enzyme is subject to tained in CF-1 mice and prepared for experimentation as previously described (17). Washed cells were suspended at 1 x IO7 cells/ml in Received 10/15/86; revised 1/5/87; accepted 1/7/87. Eagle's minimal essential medium (Flow Laboratories) supplemented The costs of publication of this article were defrayed in part by the payment with 2 HIM glutamine and buffered with 20 HIM 3-(A'-morpho- of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lino)propane sulfonic acid at pH 7.4. The cell suspensions were prein 1Supported by Grants CH-289 and CH-289A from the American Cancer cubated in stoppered flasks in a shaking water bath at 37Â°C.The time Society. 2Leukemia Society of America Scholar. To whom requests for reprints should course of uptake was initiated by the addition of 0.3 /tCi/ml of [3H]ara- be addressed. C plus unlabeled ara-C to a total ara-C concentration of 1 MM.At 3The abbreviations used are: ara-C, 1 .arabimit'uranosylcy tosine: ara-CMP, intervals, 150-^1 samples of the cell suspension were removed and 1 .f-i>-arabinufiminosylcy tosine monophosphate; ara-CTP, 1-/J-D-arabinofurano- layered over 100 /nl of silicon oil in a 40(1//I tube, and the cells were sylcytosine triphosphate; NTP, unspecified nucleoside triphosphate; PALA, N- (phosphonacetyl)-L-aspartate; U'V1, concentration for 50% inhibition; HPLC, separated from the medium by microcentrifugation (8000 x g for 30 high performance liquid chromatography; HEPES, 4-(2-hydroxycthyl)-l -pipera- s). After the media and oil were aspirated, the tip of the polyethylene zineethanesulfonic acid. tube containing the cell pellet was cut off and placed in a 7-ml scintil- 1820

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. ROLE OF UTP IN ara-C PHOSPHORYLATION lation vial, and thÃ¨radiolabel was solubilized in 500 n\ of l N NaOH RESULTS plus 0.6% sodium lauryl sulfate for 1-2 h at 60Â°C.The digest was neutralized with 500 Â¿ilofl N HC1 prior to counting in 4 ml of Safety- Radiolabeled nucleosides are rapidly transported by murine Solve (Research Products International) on a Beckman 1801 liquid tumor cells, and intracellular nucleoside rapidly reaches a scintillation counter with automatic quench correction. Comparable steady-state concentration. Net uptake of nucleosides beyond samples at the same cell density were mixed with 3H2O or [l4C]sucrose the extracellular concentration is due to their intracellular and processed as above to determine the volume of total and extracel metabolism to forms which cannot penetrate the cell mem lular water associated with the pellet. The volume of intracellular water brane. The initial step in the trapping of [3H]ara-C within the was calculated from the difference between total and extracellular water. cell is phosphorylation to ara-CMP by deoxycytidine kinase. The radioactivity associated with the cells that were incubated with When Ehrlich cells are incubated with 1 Â¿tM[3H]ara-C,uptake [3H]ara-C was corrected for [3H]ara-C in the extracellular space. The of radiolabel is linear for the period from 5-30 min. Analysis data from 5-30 min were fit by linear regression to determine the rate of intracellular radiolabel by HPLC after a 25-min incubation of accumulation. with [3H]ara-C revealed predominately ara-CTP (64%) and Analysis of Intracellular Radiolabel. After 25 min incubation with 1 unchanged ara-C (25%). The intracellular concentration of ara- UM[3H]ara-C, 150 pi of the cell suspension were layered over 100 pi of C (0.85-1 pM) was close to the extracellular concentration; this silicon oil which were in turn layered over 100 pi of 10% trichloroacetic acid in a 400-pl microcentrifuge tube. After centrifugation, the trichlo indicated that membrane transport capacity did not limit the uptake rate. ara-C kinase was apparently the rate-determining roacetic acid layer was extracted with 150 n\ of 0.5 M trioctylamine in Freon by a modification of the method of Khym (18). Samples incu step for the synthesis of ara-CTP from intracellular ara-C since bated with 'HjO or [l4C]sucrose were processed similarly to permit the immediate product of ara-C kinase, ara-CMP, did not calculation of the volume of intracellular water in the cell extract. [3H]- accumulate. ara-C metabolites were separated by anion-exchange HPLC (19). The The activity of ara-C kinase is known to be sensitive to the elution positions of radiolabel were compared with standard solutions concentrations of nucleotides, particularly dCTP. To examine of ara-C, ara-CMP, UMP, CDP, CDP-choline, and ara-CTP, and the the role of pyrimidine nucleotides in the regulation of ara-C counts under each peak were summed. kinase in situ, Ehrlich cells were preincubated for 2 h with Intracellular Nucleotide Concentrations. Duplicate 1-ml samples of the cell suspensions prepared for [3H]ara-C accumulation were layered agents likely to alter pyrimidine nucleotide pools prior to the measurement of the rate of ara-C uptake. A representative over 300 ÃŸ\ofsilicon oil which were in turn layered over 100 pi of 10% trichloroacetic acid in a 1.5-ml microcentrifuge tube. After microcen- experiment is shown in Fig. 1. Both pyrazofurin, an inhibitor trifugation for 30 s, the media and oil layers were aspirated, and the of orotidylate decarboxylase (24), and glucosamine, which se questers uridine nucleotides in the form of UPD-hexosamines trichloroacetic acid layer was extracted as above. Cell suspensions incubated with 3H2O or [l4C]sucrose were processed similarly to deter (25), inhibited the net uptake of 1 pM [3H]ara-C. Other inhibi mine the volume of intracellular water in the cell extract. Ribonucleo- tors of de novo pyrimidine synthesis, PALA and 6-azauridine, side triphosphates were separated by anion-exchange HPLC as de also inhibited ara-C uptake in Ehrlich cells (data not shown). scribed by Moyer and Handschumacher (20) and quantitated by peak 3-Deazauridine, an inhibitor of CTP synthetase (26), and uri heights relative to standards. DeoxyCTP and dTTP were assayed dine both stimulated the uptake of ara-C. In a separate experi enzymatically as described by Kinahan et al. (21). ment with similar conditions, radioactivity accumulated by the Preparation of Cell Homogenate. A crude cell homogenate was pre pretreated cells was analyzed by HPLC. The level of [3H]ara- pared for assay of ara-C kinase activity. Washed cells were suspended CTP at 25 min correlated closely with the rate of uptake; at 10* cells/ml in 50 HIMHEPES, pH 7.4, and lysed by three cycles of however, the concentration of unchanged intracellular [3H]ara- freezing and thawing. After clarifica! Â¡onbycentrifugation at 35,000 x C was similar for all treatment groups (0.84-0.98 JÂ«M).These K for 60 min, small molecules such as nucleotides were removed by column centrifugation (22, 23). Columns of Sephadex G-50 were pre data indicate that membrane transport capacity was sufficient pared in the barrels of 5-ml disposable syringes equilibrated with 50 to permit rapid equilibration of ara-C across the cell membrane HIM HEPES, pH 7.4, and centrifuged at 1000 x g for 5 min. After under all treatment conditions. application of 1 ml of homogenate, the columns were placed in fresh In the experiment shown in Fig. 1, samples of the cell suspensions were removed just prior to the addition of [3H]ara- tubes and centrifuged at 50 x g for 10 min, and then at 1000 x g for 5 min. Enzyme activity was completely recovered in the original volume. C and analyzed for ribonucleotides, dCTP, and dTTP as de Since there was no dilution, this procedure allowed enzyme activity per scribed in "Materials and Methods" (Table 1). A crude cell pi of homogenate to be converted to activity per IO6cells. homogenate was prepared from Ehrlich cells and small mole- ara-C Kinase Assay. The procedure for assay of [-'Hjara-C was 12r adapted from that of Cheng et al. (8). Homogenate was added last to + 3-Deazauridine an assay mix containing nucleoside triphosphates with equimolar MgCl2, 0.05 pCi [3H]ara-C, 1 pM ara-C, 50 mM HEPES (pH 7.4), 0.5

HIMtetrahydrouridine, 10 mM sodium fluoride, and 10 mM levantisele + Uridine in a final volume of 50 pi in 13- x 75-mm test tubes. After incubation for 30 or 45 min at 37Â°C,the tubes were transferred to a 0Â°Cbath, and 8 6 30 n\ were immediately spotted onto numbered 2- x 2-cm squares of 'o Whatman DE81 filler paper. The squares were washed three times with Control I 1 mM ammonium formate and twice with 95% ethanol. The squares 3 were dried at 60Â°Candtransferred to 7-ml scintillation vials. The filters + Pyrazofurin + Glucosamine were extracted for at least 20 min with 1.5 M NaCl/0.5 M HC1 before addition of 4 ml Safety-Solve scintillation fluid. Samples were counted 10 20 30 after a delay of at least 6 h in order to maximize counting efficiency. To test extraction and counting efficiency, samples of [3H]dCTP were Minutes spotted on DE-81 squares and processed as above, or aliquots were Fig. 1. Effect of modulators of pyrimidine nucleotide pools on the rate of uptake of 1 *tM[3H]ara-C by Ehrlich ascites cells. Cells were incubated for 2 h in added directly to a vial and counted. There was no difference in the control media (â€¢),orwith 10 *iMpyrazofurin (O), 10 pM glucosamine (D), 50 pM dpm values after correction of the quench by an external standard 3-deazauridine (â€¢),or 1 mM uridine (A) prior to the addition of 1 Â«Â¿M[3H]ara-C. method. Total cellular radiolabel was determined as described in "Materials and Methods." 1821 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. ROLE OF UTP IN ara-C PHOSPHORYLATION

Table I Effects of modulators on intracellular nucleotide concentrations senting control cells (Table 3). Elimination of dTTP, CTP, or Cells were preincubated with modulators for 2 h prior to assay. GTP from the mixture had little effect on the activity of ara-C % of concentration kinase. As anticipated, enzyme activity increased 9-fold when dCTP dTTP UTP CTP ATP GTP dCTP was eliminated. Deletion of ATP, which had the highest (25 MM) (25 UM) (0.35 HIM) (0.2 IHM) (2.0 IHM) (0.5 IHM) intracellular concentration of any potential phosphate donor, ControlPyrazofurin(10 had no effect on the activity of the enzyme in the presence of the five remaining NTPs. Deletion of UTP, however, reduced MM)Glucosamine(1(1 enzyme activity to 22% of the control. m\i)l IvÃ©sandDurham (3) have shown that at low concentrations M)3-Deazauridine(50'ridilli' (1 m of deoxycytidine, high concentrations of dTTP (2 HIM) can

â€žM)10067731872710075651437110042195201431007771380851001071079911210013294170128 partially overcome the inhibition of deoxycytidine kinase by dCTP. Addition of dTTP at concentrations up to 150 MM,a "â€¢1 level that is 6-fold higher than the dTTP level in control cells, to an ara-C kinase assay mixture containing 10 MMdCTP, 0.5 + 3-Deazuridine HIMUTP, and 2.5 HIMATP did not increase the rate of ara-C phosphorylation by extracts from Ehrlich cells. 0) 0 ATP, UTP, and most other NTPs have been shown to be active as phosphate donors for ara-C kinase (8). The lack of an 02 + Uridine effect on ara-C kinase activity when ATP, CTP, GTP, or dTTP were individually deleted from the assay mixture in Table 3 Control suggested that dCTP might be more potent against intracellular - Pyrazofurin concentrations of these NTPs than against UTP. Fig. 3 shows + Glucosamine the effect of a range of deoxycytidine or dCTP concentrations 0.2 0.4 on the phosphorylation of 1 MM[3H]ara-C when assayed with 3 HIM ATP or 0.9 HIM UTP. The rate of phosphorylation of 1 pmol / min / 10* cells MM[3H]ara-C was higher with cellular concentrations of UTP Cell-Free Phosphorylation Rate Fig. 2. Correlation of the rate of uptake of 1 MM[3H]ara-C by cells treated than with ATP. The ICSo of deoxycytidine was the same with with modulators of pyrimidinc nucleotide pools with the rate of ara-C phospho- ATP or UTP (0.25 MM). However, dCTP was 15-fold more rylation by a cell-free extract assayed in the presence of mixtures of nucleoside potent as an inhibitor of ara-C kinase with ATP than with triphosphates representative of modulator-treated cells. Cellular uptake rate was determined from the slopes of uptake data in Fig. 1. UTP. The IC50 for dCTP versus 3 HIMATP was 0.4 MM.Thus, phosphorylation by ATP would be completely blocked by 10- cules were separated from the protein fraction as described in 20 MMdCTP, i.e., at concentrations typical of Ehrlich cells. By "Materials and Methods." The procedure for preparation of contrast, the 1CÂ«,fordCTP when assayed with UTP (6 MM)is the extract was designed to allow enzyme activity data to be in the range of cellular dCTP concentrations. Phosphorylation expressed as pmol per min per IO6 cells. ara-C kinase activity of ara-C by UTP would therefore be only partially inhibited by in this extract was measured in the presence of mixtures of the cellular levels of dCTP. six NTPs (Table 1). Pyra/ofurin and glucosamine depleted all pyrimidinc nucleotide pools. Although dCTP levels were sig nificantly reduced, the activity of ara-C kinase when assayed DISCUSSION with the mixture of NTPs from pyrazofurin- or glucosamine- ara-CTP is the cytotoxic metabolite of ara-C. The intracel treated cells was only about 50% of the activity in the presence lular synthesis of ara-CTP requires transport of ara-C across of the NTP levels found in control cells. Activity with the the cell membrane by a facilitated transport system followed by nucleotide mixture resulting from 3-deazauridine or uridine three sequential phosphorylation steps. Plagemann et al. (27) treatment was greater than in the control NTP mixture. In have shown that in Novikoff rat hepatoma cells, the rate of ara- creased ara-C kinase activity was observed in the mixture rep resenting uridine-treated cells despite an increase in the dCTP Table 2 ara-C kinase activity when assayed with nucleotide concentrations pool. representative of intact cells When assayed with NTP mixtures that result from ant Â¡me Nucleotide mix ara-C kinase activity (%) tabolite treatment, the activity of ara-C kinase in cell extracts Control cells 100 correlated closely with the rate of ara-C uptake by intact cells Pyrazofurin (10 MM) 86 Glucosamine (10 HIM) 63 (Table 2, Fig. 2). This complete experiment consisting of in Uridine (1 HIM) 185 cubation of cells with an tÂ¡metabolites,measurement of cellular 3-Deazauridine (50 MM) 331 phosphorylation rates, measurement of NTP levels, and assay of a cell-free enzyme extract with these NTP mixtures has been Table 3 Rule of individuai nucleotides in the control of ara-C kinase activity repeated four times. The NTP levels and phosphorylation rates varied significantly between experiments; however, there was Complete mixture" 100* excellent correlation between cellular and cell-free phosphoryl ation within each experiment and all slopes were near unity. NodCTP 934 Thus, the measurements of cell-free ara-C kinase activity in the NodTTP 132 No UTP 22 presence of cellular concentrations of ribo- and deoxyribonu- No CTP 108 cleotides closely approximated ara-C kinase activity in situ. No ATP 107 No GTP 111 In order to determine which NTPs were important for the " 25 MMdCTP, 25 MMdTTP, 0.35 mM UTP, 0.2 HIMCTP, 2.0 mM ATP, and regulation of ara-C kinase activity, individual NTPs were de 0.5 HIMGTP. leted, one at a time, from the mixture of six nucleotides repre * 100% = 0.105 pmoI/min/106 cells. 1822

1.27: we have focused on the rate of ara-CTP formation rather than on the steady-state ara-CTP level. Exposure of Ehrlich cells to relatively high concentrations of 8 Â°-9 pyrazofurin, glucosamine, uridine, or 3-deazauridine for 2 h Ã´ caused major alterations in pyrimidine nucleotide pools and in the rate of net accumulation of 1 ^M [3H]ara-C to [3H]ara-CTP. Ã«0.6? When ara-C kinase activity in a crude cell extract was assayed with a mixture of six nucleotides at concentrations which closely 0.3 approximated their intracellular levels, there was remarkable qualitative and quantitative agreement between the cellular and cell-free phosphorylation data. This result suggests that the 10' 10"' 10"' 10-s 10"' 10" average intracellular nucleotide concentrations must be close to [Deoxycytidine] the local nucleotide concentrations at the enzyme site. It also indicates that other nucleotides and potential modulators which were not included in the assay mix probably have only a minor 1.2 role in the regulation of ara-C kinase in situ. The regulation of deoxycytidine kinase in the test tube may be more complex than it is in the intact cell. When assayed 2 0.9 with ATP as the phosphate donor, high concentrations of dTTP (2-5 mM) have been shown to inhibit phosphate transfer, but these dTTP levels may also reduce the inhibition by dCTP 0.6 0.9 mM UTP = 6 under certain circumstances (3, 4). The activity of ara-C kinase from Ehrlich cells, when assayed at cellular concentrations of 0.3 dCTP, ATP, and UTP, was not discernibly altered by concen trations of dTTP up to 150 /Â¿M,that is, at levels up to 6-fold greater than those in control cells. Most nucleoside triphos 10" 10" 10" IO" phates are able to function directly as phosphate donors; how [dCTP] ever, selective deletion of individual nucleotides from the mix Fig. 3. Inhibition of the phosphorylation of 1 fiM [3H]ara-C by deoxycytidine ture indicated that only dCTP and UTP were key determinants (A) or dCTP (/(). Enzyme activity in a crude homogenate was assayed in the of the rate of ara-C phosphorylation. presence of 0.9 mm UTP (O) or 3 mM ATP (D)- Although ATP is present at the highest concentration of any intracellular nucleotide, it does not appear to play a major role C transport is in great excess as compared to the rate of in the phosphorylation of l ^M ara-C in Ehrlich cells because phosphorylation. Our observation that in the Ehrlich cell the cellular concentrations of dCTP completely block phosphoryl intracellular concentration of unchanged ara-C is close to the ation of ara-C by ATP. Phosphorylation by UTP is only par extracellular concentration indicates that transport is suffi tially inhibited. Kowal and Grindey (9) have described the ciently rapid to allow equilibration of ara-C across the cell difference in the sensitivity of ATP- versus UTP-mediated ara- membrane. Thus, in these cells, transport is not a determinant C phosphorylation to dCTP in terms of "metabolic deinhibi- of the rate of ara-CTP accumulaton. Wiley et al. (28-30) have tion." According to their model, dCTP binds more weakly to shown, however, that transport rates for ara-C in human leu the enzyme-UTP complex than to the enzyme-ATP complex. kemia cells taken directly from patients are much lower than They reported much higher K, values for dCTP with UTP as in experimental cell lines. Analysis of the relative capacities of the phosphate donor rather than ATP. This result supports transport and intracellular metabolism have indicated that their model for the enzyme involving separate sites for dCTP membrane transport is the rate-determining step for net uptake and the phosphate donor. Momparler and Fischer (7) and in patient leukemia cells at concentrations of ara-C below 1 JÃ•M Cheng et al. (8) have reported competitive inhibition by dCTP (31-32). with respect to ATP. This suggests that the enzyme has a single The steady-state level of ara-CTP is determined by the rela nucleoside triphosphate binding site. Cheng et al. (8) also observed that the Km for UTP (0.8 ^M) was 20-fold lower than tive rates of anabolism and catabolism. The rate-determining step for the synthesis of ara-CTP from intracellular ara-C the Km for ATP (16 HÂ¿M).Thus,one might anticipate that at cellular concentrations of ATP and UTP, dCTP would be less appears to be the initial phosphorylation step catalyzed by effective against UTP than ATP. Additional kinetic and struc deoxycytidine kinase. This enzyme is known to be subject to tural studies with punned enzyme may be necessary to deter complex controls by nucleotides (3-9). Exposure of tumor cells mine whether dCTP binds at the phosphate donor site or at an to a variety of antimetabolites, normal metabolites, and other allosteric site. agents has been shown to alter steady-state ara-CTP pools (33- In summary, these data indicate that both UTP and dCTP 36). These alterations in ara-CTP levels are often attributed to levels are critical determinants of the rate of ara-C phosphoryl alterations in the activity of ara-C kinase; however, other steps ation in intact Ehrlich ascites cells. Therefore, attempts at in the metabolism of ara-C should also be considered. Abe et biochemical modulation of ara-C phosphorylation designed to al. (37) have shown that the rate of dephosphorylation of ara- deplete dCTP should simultaneously preserve UTP pools. The CTP is decreased by hydroxyurea. The deamination of ara- use of inhibitors of de novo pyrimidine biosynthesis, or the CMP by dCMP deaminase, an enzyme known to be sensitive opposite approach, supplementation of pyrimidine nucleotide to nucleoside triphosphates, especially dCTP and dTTP (38), pools with uridine, are likely to have unpredictable cell-specific may also be a determinant of the steady-state level of ara-CTP. and dose- and time-dependent effects since one of the above Thus, in order to study the regulation of ara-C kinase in situ, goals must be sacrificed for the other. 1823

19. Rustum, Y. M., and Preisler, H. D. Correlation between leukemic cell REFERENCES retention of 1-0-D-arabinofuranosylcytosine 5'-triphosphate and response to 1. Kufe, D. W., Major, P. P., Egan, E. M., and Beardsley G. P. Correlation of therapy. Cancer Res., 39:42-49, 1979. cytotoxicity with incorporation of ara-C into DNA. J. Biol. Chem., 255: 20. Moyer, J. D., and Handschumacher, R. E. Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by A'-(phosphonacetyl)-L-aspar- 8997-9000, 1980. 2. Kufe, D., Springs. D., Egan, E. M., and Munroe, D. 1984. Relationships tate. Cancer Res., 39:3089-3094, 1979. among ara-CTP pools, formation of (ara-C)DNA, and cytotoxicity of human 21. Kinahan, J. J., Often, M., and Grindey, G. B. Evaluation of ribonucleoside leukemia cells. Blood, 64: 54-58, 1984. and deoxyribonucleoside triphosphate pools in cultured leukemia cells during 3. IvÃ©s,D. H., and Durham, J. P. Deoxycytidine kinase: III. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. Role of Uridine Triphosphate in the Phosphorylation of 1-β -d-Arabinofuranosylcytosine by Ehrlich Ascites Tumor Cells

J. Courtland White and Leigh H. Hines

Cancer Res 1987;47:1820-1824.

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