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

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Role of Uridine Triphosphate in the Phosphorylation of 1-ß-D- Arabinofuranosylcytosine by Ehrlich Ascites Tumor Cells1 [CANCER RESEARCH 47, 1820-1824, April 1, 1987] Role of Uridine Triphosphate in the Phosphorylation of 1-ß-D- Arabinofuranosylcytosine by Ehrlich Ascites Tumor Cells1 J. Courtland White2 and Leigh H. Hiñes 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 37°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 .<i>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 thèradiolabel was solubilized in 500 n\ of l N NaOH RESULTS plus 0.6% sodium lauryl sulfate for 1-2 h at 60°C.The digest was neutralized with 500 ¿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 ¿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.
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