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[CANCER RESEARCH 45, 4249-4256, September 1985] Enhancement by Uridine of the Anabolism of 5-Fluorouracil in Mouse T-Lymphoma (S-49) Cells1 William B. Parker2 and Philip Klubes3 Department of Pharmacology, The George Washington University School of Medicine, Washington, DC 20037 with FUra4 are related to the anabolism of the drug to FdUMP ABSTRACT Uridine enhances the growth inhibition due to 5-fluorouracil (FUra) in a cultured mouse T-cell lymphoma (S-49). Using colony formation assays we found that cytotoxicity produced by 24-h continuous exposure to FUra (0.5 to 3.5 /¿M) was increased more than two-fold by simultaneous exposure to 10 /IM undine. Studies were undertaken to explain the mechanism by which undine enhanced the cytotoxicity of FUra in S-49 cells. Uridine (10 UM) increased by about 50% both the anabolism of 1.0 pM [3H]FUra to acid-soluble metabolites and the incorporation of 1.0 ^M [3H]FUra into RNA. However, the incorporation of 1.0 fiM [3H]FUra into these fractions was less than that seen with 2.4 UM [3H]FUra, a dose which was equitoxic to 1.0 tiM [3H]FUra plus 10 ^M and FUTP (1). Recently the incorporation of FUra into DNA (28) as well as the formation of fluorinated analogues of uridine diphosphohexoses (9-13) have also been implicated in the cy totoxicity of FUra. Ullman and Kirsch (14), using a mouse T-cell lymphoma (S-49) cell line in culture, showed that simultaneous administration of uridine with FUra decreases the concentration of FUra required to inhibit cell growth. In S-49 cells FUra is anabolized by OPRTase, and not by either uridine phosphorylase or thymidine phosphorylase (15). Ullman and Kirsch (14) pro posed that the enhancement by uridine of the action of FUra was due to the formation of UTP from exogenous uridine, which acted as a feedback inhibitor of de novo pyrimidine biosynthesis. Presumably, the inhibition of de novo pyrimidine biosynthesis decreases orotic acid levels and thereby increases the availability undine. High-pressure liquid chromatography analysis of the of PRPP and OPRTase for the anabolism of FUra to FUMP. acid-soluble metabolites of FUra did not show any selective Uridine has been shown to be effective as a modulator of the change in specific FUra nucleotides, which could explain the cytotoxicity of FUra. For example, uridine has been used as a increased cytotoxicity associated with 10 ^M uridine. In addition, rescue agent to increase the therapeutic index of FUra against 5-fluoro-2'-deoxyuridine monophosphate levels and the amount the mouse colon tumor 26 (16) and the mouse B16 melanoma of [3H]FUra which was incorporated into the alkali-stable, acid(17). Protection from FUra toxicity afforded by the addition of insoluble fraction were not increased by uridine. Uridine (10 ¿<M) uridine is apparently due to the reduction in the incorporation of inhibited de novo pyrimidine biosynthesis by 70%, while 5- FUra into RNA rather than by reversal of FdUMP inhibition of phosphoribosyl-1-pyrophosphate levels were unchanged. Pre thymidylate synthetase (18). Preliminary clinical trials using uri dine infusion after FUra treatment are in progress (19-21). sumably, the inhibition of de novo pyrimidine biosynthesis de This study explored possible mechanisms by which uridine creased orotic acid levels and allowed more FUra to be anabolmight enhance the action of FUra against S-49 cells in culture. ized to 5-fluorouridine monophosphate via orotate phosphoriWe present evidence which supports the observation of Ullman bosyl transferase. Furthermore, 2.4 /¿M FUra inhibited the incor and Kirsch (14) that uridine enhances the phosphoribosylation of poration of [3H]deoxyguanosine into DNA by 50% after 24 h of FUra. However, this increase in anabolism is restricted to the incubation. In contrast, 1.0 I¿M FUra plus 10 UM uridine did not ribonucleotide pool and can account for, at most, only 50% of inhibit the incorporation of [3H]deoxyguanosine into DNA. The the increased cytotoxicity. A preliminary report of our investiga data suggested that there was a qualitative difference in the tion has already appeared (22). mechanism by which 1.0 UM FUra plus 10 MMuridine killed S-49 cells as compared to 2.4 ¿¿M FUra alone, and that the enhance ment by uridine of the cytotoxicity of FUra was due, in part, to the increased anabolism of FUra to ribonucleotides. MATERIALS AND METHODS Chemicals and Supplies. FUra, MIX. and PALA were obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute. 5Fluoro-2'-deoxyuridine, 5-fluorocytidine, and 5-fluorouridine were ob tained as gifts from Hoffmann-La Roche, Inc. (Nutley, NJ). 5-Fluoro-2'- INTRODUCTION Severe disruptions in DNA and RNA synthesis after treatment 1This investigation was supported by Grant CH-160 from the American Cancer Society, and Training Grant T32-CA09223 from the National Cancer Institute, Department of Health and Human Services. 2 From a dissertation presented to the Department of Pharmacology, The Graduate School of Arts and Sciences, The George Washington University, in partial fulfillment of the requirements for the Ph.D. degree. Present address: Department of Pharmacology, 916 FLOB 231-H, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514. 9 To whom requests for reprints should be addressed, at the Department of Pharmacology, The George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037. Received 11/30/84; revised 5/2/85; accepted 5/13/85. deoxycytidine was obtained from Calbiochem (La Jolla, CA). 5-Fluorocytosine was a gift from Dr. G. Wagner (The George Washington University Medical School, Washington, DC). FUMP and FUTP were obtained from Sierra Bioresearch (Tucson, AR). FdUMP was obtained from Terra Marine Bioresearch (La Jolla, CA). Crude yeast OPRTase and ' The abbreviations used are: FUra, 5-fluorouracil; FdUMP, 5-fluoro-2'-deoxyuridine monophosphate; PRPP, 5-phosphoribosyl-1 -pyrophosphate; FUMP, 5-fluo rouridine monophosphate; FUTP, 5-fluorouridine triphosphate; UTP, uridine triphosphate; PALA, W-<phosphonacetyl)-L-aspartate; MTX, methotrexate; OPRTase, orotate phosphoribosyl transferase; TCA, trichtoroacetic acid; HPLC, high-pressure liquid chromatography; FUrd, fluorouridine; FdUrd, fluorodeoxyuridine; FUDP, 5fluorouridine diphosphate; FCyt, 5-fluorocytosine; FCyd, 5-fluorocytidine; FdCyd, 5-fluorodeoxycytidine. CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4249 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. LIBIDINE ENHANCEMENT orotidine-5'-phosphate decarboxylase, and Crotalus atrox snake venom were obtained from Sigma Chemical Company (St. Louis, MO). [6-3H]FUra (18 Ci/mol) was obtained from Moravek Biochemicals (City of Industry, CA). [5-3H]Orotic acid (20 Ci/mol), [14C]NaHCO3 (46 mCi/mmol), and [2,8-3H]adenosine (35.2 Ci/mmol) were obtained from New England Nuclear (Boston, MA). [8-3H]Deoxyguanosine (7.8 Ci/mmol) and [4,5-3H]- OF FUra ANABOLISM The remaining pellet, which contained the alkali-stable, acid-insoluble fraction, was washed twice with 1.0 ml of ice-cold 5% TCA, and was dissolved in 1.0 ml of 0.3 N NaOH. A 0.5-ml aliquot of each sample was mixed with 10 ml of Liquiscent (National Diagnostics, Somerville, NJ), and the radioactivity was measured in a Beckman LS 6800 liquid scintillation counter. Results were corrected for counting efficiency with an external standard and results were expressed as dpm/107 cells. leucine (58 mCi/mmol), were obtained from ICN Pharmaceuticals (Irvine, CA). All other chemicals were standard analytical grade. Growth Conditions for S-49 Cells. S-49 cells, obtained from Dr. J. Authentic standards of the FUDP-hexoses were not available. In each experiment two radioactive peaks eluted from the column just after FdUMP. The quantity and location of these peaks were similar to those reported for FUDP-hexoses by Pogolotti ef al. (24). Therefore these peaks were assumed to be FUDP-hexoses (presumably FUDP-glucose and FUDP-galactose). In order to identify the [3H]FUra anabolites with retention times greater Maybaum at the University of California School of Medicine, San Fran cisco, were grown as suspension cultures in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 2.2 g NaHCO3/ liter, 2 g glucose/liter, and 10% heat inactivated horse serum (GIBCO), pH 7.4. The cells were kept at 37°Cin a 5% CO2-95% O2 atmosphere. Fresh cells were thawed every 9 months during the course of the study to maintain genetic stability in the culture. The cells were assayed by Biofluids, Inc. (Rockville, MD) and were found to be free from Myco- than 55 min (i.e., the di- and triphosphates), the radioactive peaks were plasma contamination. Effect of Drug Treatment on Cell Growth and Viability. Exponentially growing S-49 cells were added to media containing the desired concen tration of drug(s). Most experiments were begun with 1 x 105 cells/ml. All experiments were carried out in 25-cm2 Falcon culture flasks. One ml atrox snake venom/ml in 0.06 M Tris:0.02 M MgCI2, pH 9. The protein was precipitated by the addition of 50 n\ ice-cold TCA (100 g dissolved collected as they eluted off the column and lyophilized. The samples were then incubated overnight at 37°C with 1.0 ml of 20 mg Crotalus of cell suspension was removed from each flask at the time indicated (usually at 24, 48, and 72 h after drug addition), and cell numbers were counted with a Model Zf Coulter Counter. The data were presented as percentage of control cell growth. To measure viable cells after drug treatment, S-49 cells were cloned in a 0.5% agar solution which contained 20% horse serum and 40% conditioned medium (23). Conditioned medium was obtained from ex ponentially growing S-49 cell suspensions (0.5-1.0 x 106 cells/ml) by centrifugation at 250 x g for 5 min, followed by filtration through a 0.22 urn filter. After 12 to 14 days, colonies (greater than 50 cells) were visible to the naked eye and could be counted. The cloning efficiency of S-49 cells was about 50%. Measurement of the Acid-soluble Metabolites of FUra in S-49 Cells. The method of Pogolotti ef a/. (24) was used to separate the acid-soluble metabolites of FUra. After the addition of drugs, each cell suspension contained approximately 1 x 106 cells/ml in 100 ml of culture medium. The S-49 cells were incubated with 1.0 »M[3H]FUra (1.125 Ci/mmol), 1.0 IM [3H]FUra (1.125 Ci/mmol) plus 10 »Muridine, or 2.4 MM[3H]FUra (1.125 Ci/mmol). After 6 h of incubation, two 45-ml aliquots from each treatment group were centrifugea at 250 x g for 5 min at room temper ature to harvest the cells. The cell pellets were washed once with icecold phosphate-buffered saline, and the acid-soluble metabolites were extracted with 0.25 ml of ice-cold 0.6 M TCA containing 0.10 mg/ml each of FUra, FUrd, FdUrd, FUMP, FdUMP, and FUTP. The samples were then mixed with 0.25 ml of 0.5 M tricaprylyl tertiary amine in trichlorotrifluoroe- in water to 100 ml). TCA was extracted by mixing with 1.0 ml of 0.5 M tricaprylyl tertiary amine in trichlorotrifluoroethane and centrifuging at 1000 x g for 5 min. The aqueous layer was collected, evaporated under vacuum at 43°C, and resuspended in 250 n\ of water plus 50 ¡Aof solution containing FUra, FCyt, FUrd, FCyd, FdUrd, and FdCyd (0.16 mg/ml each). Twenty ^l of the sample were counted for radioactivity and 200 n\ were analyzed for fluoropyrimidine bases and nucleosides using a Waters C-18, radial compression lO-^m HPLC column. The mobile phase was 10 ITIMsodium acetate, pH 4.5, with a flow rate of 2.0 ml/ min. Measurement of Intracellular PRPP Levels. The method of May and Krooth (26) was used, with some modification, to measure intracellular PRPP. Approximately 2 x 10s exponentially growing S-49 cells were treated with 10 MMuridine, 300 /¿M PALA, 0.04 IM MTX, or 0.4 MMMTX for 6 h. The cell pellet, which was collected by centrifugation at 250 x g for 5 min at 4°C,was mixed in 1.0 ml of water and disrupted by sonication at 4°C.The supernatant fluid (0.9 ml) was collected after centrifugation (18,000 x g for 5 min at 5°C)and was assayed for PRPP. The assay utilized OPRTase and orotidine-5'-phosphate decarboxylase to convert [3H]orotic acid to [3H]uridylate. PRPP is made rate limiting in this reaction, and the amount of [3H]uridylate that is formed is equal to the amount of PRPP that is present. [3H]Uridylate was separated from [3H]orotic acid by paper chromatography (26). The [3H]uridylate spot was cut from the paper and placed in a scintillation vial with 10 ml of Liquiscent, and the radioactivity was determined by extrapolation from a standard curve (nmol PRPP added versus [3H]UMP formed) which was prepared for thane to extract the TCA (25). Two hundred n\ of this solution were injected onto a Waters C-18 radial compression 10-MM HPLC column each experiment. Measurement of the Rate of de Novo Pyrimidine Biosynthesis. The method of Karle ef al. (27) was used to determine the effect of drug treatment on de novo pyrimidine biosynthesis. S-49 cells (5x106) were (Milford, MA). During each analysis two solvents were mixed by a Waters Associates Model 660 solvent programmer according to a concave upward program (Curve 8). Solvent A contained 5 mw tetrabutylammon- incubated with 10 UM uridine, 1.0 /¿MFUra, 1.0 JIM FUra plus 10 UM uridine, or 2.4 ^M FUra for 6 h in 5 ml of Dulbecco's modified Eagle's medium which contained 6.5 mM NaHCO3. Twenty-five ^Ci of [14C]- ium hydrogen sulfate and 5 ITIM potassium phosphate, pH 7. Solvent B is a mixture of Solvent A plus methanol (60/40, v/v). Solvent A was replaced entirely by Solvent B over a 60-min period by the solvent programmer, followed by a 10-min run with Solvent B. The column was NaHCOs were added to each incubation during the last hour. At the end of the incubation period, the cells were collected by centrifugation at 150 x g for 5 min, and the macromolecules were precipitated with ice-cold reequilibrated with Solvent A for 10 min before another injection. The flow rate was 2 ml/min. FUra and its metabolites were detected by their absorbance at 254 nm. To measure the incorporation of [3H]FUra into RNA and the alkali- 5% TCA. The TCA was extracted by mixing with 0.5 M tricaprylyl tertiary amine in trichlorotrifluoroethane, and the uridine nucleotides were de graded to uridine by Crotalus atrox snake venom. Pyrimidine nucleosides were added to each sample for detection purposes, and they were separated using a Waters C-18 radial compression 10 ^m HPLC column stable, acid-insoluble fraction, the TCA precipitate of the cell extracts was washed twice with 1.0 ml of ice-cold 5% TCA, and resuspended in 3 ml of 0.3 N NaOH. After incubation in a 37°Cshaking water bath for 3 (Milford, MA). The mobile phase was 10 mM sodium acetate, pH 4.5, flow rate 2.0 ml/min. The nucleosides were detected by their absorbance at 254 nm. Fractions containing each pyrimidine (uridine, cytidine, de- h, 300 ¿ilof ice-cold 5.2 N HCIO«were added, and the samples were centrifugea at 18,000 x g for 5 min at 5°C.The supernatant fluid from oxycytidine, or deoxyuridine) were collected from the column, and the radioactivity was determined as described above. In control cells the incorporation of [14C]NaHCO3 into uridine nucleotides was linear with each sample was removed and counted for radioactivity (RNA fraction). CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4250 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. URIDINE ENHANCEMENT OF FUra ANABOLISM respect to time over a 90-min period. Measurement of Intracellular DTP Levels. Thirty-five ml of S-49 cells (7.14 x 105 cells/ml) were incubated with 1.0 //M FUra, 2.4 ¿IM FUra, 10 MMundine, 1.0 UM FUra plus 10 ^M uridine, or no drug. After 6 h the cells from each treatment group were collected by centrifugaron and washed with 1.0 ml of ice-cold phosphate-buffered saline, pH 7.4. Cells were Õ 80- harvested by centrifugation, and the cell pellets were then mixed with 0.5 ml of ice-cold 10% TCA. The acid-insoluble material was removed by centrifugation and the acid-soluble extract was mixed with 0.5 ml of 0.5 M tricaprylamine in trichlorotrifluoroethane to remove TCA. The suspensions were centrifuged at 1000 x g for 5 min at 4°Cand 0.4 ml Fln pl.s 10«*Uni of the aqueous layer was removed and filtered through a 0.45-^m Millipore filter. Samples were stored at -70°C until they could be analyzed by HPLC. The nucleoside triphosphates were separated using a 10-Ã-Ã-Ã-Ti SAX column in a radial compression HPLC system (Waters Associates, Milford, MA). The mobile phase was 0.4 M Na2HPO4, pH 3.6, at a flow rate of 2.0 ml/min. The amount of UTP in each sample was determined by extrapolation from a standard curve relating UTP peak height to the concentration of UTP. Measurement of Nucleic Acid Synthesis. Cells were incubated with no drug, FUra (1.0 or 2.4 ^M), 1.0 ¿JM FUra plus 10 MMuridine, or 10 MM uridine alone for 2, 6, or 24 h. One h before the end of each incubation, 1 fiCi/ml of either [3H]adenosine or [3H]deoxyguanosine was added to each cell suspension. Thirty min before the end of each incubation two 0.25-ml aliquots were removed and the cell numbers were determined using a Model Zf Coulter Counter. At the end of each incubation two 2- 00 25 05 10 20 FUra (,») Chart 1. Enhancement by uridine (Urd) of the growth inhibition due to FUra. S49 cells (1 x 10* cells/ml) were incubated with FUra (0.125-2.0 ¡iu)either atone or with 10 fiu undine. After 72 h of incubation, two 10-m l aliquots were obtained from each treatment and cell numbers were determined using a Model Zf Coulter Counter. The original cell number was subtracted from the 72-h cell count of each treatment and the results were presented as a percentage of control growth. There were approximately 1.3 x 10°cells/ml in control flasks after the 72-h incubation. Points, mean of three experiments; oars, SE. ml aliquots were transferred from each cell suspension to microfuge tubes containing 2.0 ml of ice-cold 10% TCA. These samples were frozen until further analysis. Each sample was thawed and centrifuged at 1000 x g for 5 min at room temperature. The supernatant fluid from each sample was dis carded and the pellets were washed twice with ice-cold 0.2 N HCIO4. The pellets were incubated for 2 h in 0.3 N NaOH at 37°C, and then 0.3 ml of 5.2 N HCIO4 was added. The suspensions were centrifuged, and the pellets were washed twice with 1.0 ml of 0.2 N HCIO4. The super natant fluid and two washes were combined and an aliquot was mixed with 10 ml of Liquiscent. The radioactivity was then determined as described above. This fraction represented the incorporation of [3H]adenosine or [3H]deoxyguanosine into RNA. The pellet was dissolved in 1.0 ml of 0.3 N NaOH and transferred to a scintillation vial. The solutions were acidified with 0.1 ml of 100% TCA and counted for radioactivity in 10 ml of Liquiscent. This fraction represented the incorporation of [3H]adenosine or [3H]deoxyguanosine into DNA. In control cells the incorporation of either [3H]deoxyguanosine or [3H] adenosine into either RNA or DNA was linear with respect to time over a 90-min period. RESULTS 100-1 FUra o 110- 2 o FUra pi» lOpM Urd z o o 2 HI O C 01- Effect of Undine on the Cytotoxicity of FUra. Ullman and Kirsch (14) reported that the concentration of FUra required to inhibit the growth of S-49 cells by 50% was decreased from 0.8 to 0.5 UM in the presence of 50 ^M uridine. We examined the effect of various concentrations of uridine (2.5 to 100 ^M) to increase the action of FUra against S-49 cells. Uridine (10 ^M) produced the maximum enhancement of cell growth inhibition due to FUra (data not shown). In addition, 10 /¿M uridine alone did not affect cell growth. Therefore, in all further experiments, 10 ¿¿M uridine was used with FUra. The effect of 10 pu uridine on the inhibition of cell growth by various concentrations of FUra was examined. As can be seen (Chart 1), 10 MM uridine decreased the concentration of FUra required to inhibit the growth of S-49 cells by 50% from 0.73 to 0.4 MM(P < 0.01, Student's t test). CANCER RESEARCH 0.5 1.0 1.5 2.0 2.5 I 30 FUraOiM) Chart 2. Effect of uridine (Urd) on the cloning efficiency of cells treated with FUra. S-49 cells (1 x 105 cells/ml) were incubated with FUra (0.5-3.0 t¡u)either alone or with 10 /¿M uridine. After 24 h, cells from each treatment were collected and cloned. Twelve to 14 days later the colonies were counted. The cloning efficiency from each treatment was determined and the results are presented as percentage of control cloning efficiency The cloning efficiency of control cultures was about 50%. Points, mean of three experiments; oars, SE. The effect of drug treatment on colony formation is a more sensitive indicator of cell kill than is the inhibition of cell growth. Therefore, S-49 cells were treated with various concentrations of FUra (0.5 to 3.0 MM),with or without 10 MM uridine for 24 h and cloned in nutrient agar (Chart 2). Uridine alone had no effect on cell viability. However, uridine decreased the concentration of FUra required to kill 50% of the cells from 1.85 MMto 0.7 MM. VOL. 45 SEPTEMBER 1985 4251 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. URIDINE ENHANCEMENT OF FUra ANABOLISM MM [3H]FUra plus 10 MM uridine, or 2.4 MM [3H]FUra, and the intracellular levels of FUra, its anabolites, and its incorporation into RNA were determined (Chart 3). There was no difference in the amount of FUra in cells treated with either 1.0 MM [3H]FUra or 1.0 MM [3H]FUra plus 10 MM uridine. In contrast, 2.4 MM [3H]- OfjM »H-FUra Qi ,¡v»HFUra 30-20-10-r1TREATMENTDi URO• plus lOuM 'H-FUra|M .:,.'-' FUra increased the level of intracellular FUra approximately 150% as compared to 1.0 MM[3H]FUra. Similarly, 2.4 MMFUra increased the levels of FUrd, FUMP, FdUMP, FUDP-hexoses, and FUTP as compared to 1.0 MM [3H]FUra. Treatment with 1.0 MM [3H]FUra plus 10 MMuridine increased the levels of the FUDP-hexoses and FUra FUrd À JÕ FUMP FdUMP FUTP as compared to 1.0 MM FUra alone. There were trends, which were not statistically significant, toward increases in FUrd, FUMP, FdUMP, and FUDP after treatment with 1.0 MM[3H]FUra plus 10 MM uridine as compared to 1.0 MM [3H]FUra alone. \\300-200-100-+rffÃ-fI* FUDP FUDP MEXOSES FUTP RNA Chart3. Effect of undine (URD) on the anabolism of FUra to acid-soluble metabolites. S-49 cells (1 x 10e cells/ml) were incubated with 1.0 /IM [3H]FUra, 1.0 /IM [3H]FUra plus 10 AIMuridine, or 2.4 /IM (3H]FUra. After 6 h, two 45-ml aliquots were collected from each treatment and the acid-soluble metabolites of FUra as well as its incorporation into RNA was determined. Columns, mean of three experiments; oars, SE. *, significantly different from either 1.0 /JM |3H]FUra alone, or 1.0 itM [3H]FUra plus 10 »IM uridine, P < 0.05; Newman Keuls' multiple range test. +, significantly different from 1.0 /IM [3H]FUra, P < 0.05; Newman Keuls' Furthermore, 10 MM uridine did not increase the anabolism of any acid-soluble metabolite of 1.0 MM[3H]FUra to the level seen with 2.4 MM [3H]FUra. After 24 h of incubation, the effect of 10 MMuridine on the anabolism of 1.0 MM [3H]FUra to its individual metabolites was qualitatively similar to that seen in the 6-h incubation (data not shown). Uridine (10 MM) also increased the incorporation of 1.0 MM [3H]FUra into RNA by 40% as compared to 1.0 MM [3H]FUra multiple range test. alone; however, this incorporation was less than that seen with 2.4 MM [3H]FUra alone (Chart 3). Uridine (10 MM)caused similar increases in the incorporation of 1.0 MM[3H]FUra into RNA after 2, 4, or 24 h of incubation. In addition, at these time points the amount of [3H]FUra in RNA of cells treated with 1.0 MM[3H]FUra plus 10 MMuridine was also less than that seen with 2.4 MM[3H]FUra (data not shown). The FUDP and FUTP peaks were collected to confirm the identity of the radioactivity associated with these peaks. The nucleotides were degraded to nucleosides by Crotalus atrox snake venom, and rechromatographed in a HPLC system used to separate fluoropyrimidine nucleosides. More than 85% of the radioactivity coeluted with FUrd, indicating that the radioactivity in the FUDP and FUTP peaks was due to [3H]FUDP and [3H]Chart 4. Effect of uridine (Urd) on the incorporation of [3H]FUra into the alkalistable, acid-insoluble fraction. S-49 cells (1 x 10e cells/ml) were incubated wth 1.0 ,,M [3H]FUra, 1.0 nu [3H]FUra plus 10 JIM uridine, or 1.7 nM [3H]FUra for 24 h. At 0, 2, 4, 6, and 24 h two 45-ml aliquots from each treatment were collected and the radioactivity in the alkali-stable, acid-insoluble fraction was determined. Points, mean of three experiments; bars, SE. Absence of SE bars indicates that the SE was less than the size of the symbol. *, significantly different from cells treated with either 1.0 *iM [3H]FUra plus 10 ^M uridine or 1.0 »u[3H]FUra alone, P < 0.05; Newman Keuls1 multiple range test. These results, which were consistent with the growth inhibition experiments (Chart 1), indicated that 10 UM uridine enhanced the cell-killing action of FUra by about 2.5-fold. FUra (1.0 MM)alone did not decrease cell viability, whereas either 1.0 MM Fura plus 10 MM uridine or 2.4 MM FUra alone were equitoxic in that they both decreased cell viability approximately 85%. Effect of Undine on the Anabolism of FUra. One mechanism by which uridine could enhance the cytotoxicity of FUra would be to increase the anabolism of FUra to active nucleotides (14). To test this hypothesis we determined the effect of uridine on the anabolism of FUra to acid-soluble metabolites, as well as its incorporation into RNA and into an alkali-stable, acid-insoluble fraction. The alkali-stable, acid-insoluble fraction should consist of [3H]FdUMP bound to thymidylate synthetase and [3H]FUra incorporation into DNA (28, 29). S-49 cells were incubated for 6 h with 1.0 MM [3H]FUra, 1.0 CANCER RESEARCH FUTP, respectively. No radioactivity was detected in the fractions corresponding to FCyd, FdCyd, or FdUrd, which indicated that their di- or trinucleotides were not formed. This method was sensitive enough to detect 0.13 pmol of a fluorinated nucleotide (data not shown). Uridine (10 MM) did not increase the incorporation of 1.0 MM [3H]FUra into the alkali-stable, acid-insoluble fraction of cells (Chart 4). The alkali-stable, acid-insoluble fraction should consist of the thymidylate synthetase: FdUMP:/v5,/V10-methylenetetrahydrofolate complex plus the incorporation of [3H]FUra into DNA (28, 29). Since the incorporation of [3H]FUra into DNA accounts for only a small percentage (less than 3%) of the amount of radioactivity found in this fraction,5 this fraction primarily repre sents FdUMP which is bound to thymidylate synthetase. Fur thermore, the results seen in Chart 4 are consistent with the observation (Chart 3) that the formation of [3H]FdUMP was similar in cells treated with either 1.0 MM [3H]FUra plus 10 MM uridine or 1.0 MM[3H]FUra alone. Effect of Uridine on the Level of PRPP. It has been hypoth esized that uridine enhances FUra action due to its conversion to UTP and a subsequent feedback inhibition by UTP of de novo pyrimidine biosynthesis (14). Decreases in the intracellular levels 5W. B. Parker, unpublished observation. VOL. 45 SEPTEMBER 1985 4252 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. URIDINE ENHANCEMENT OF FUra ANABOLISM of orotic acid should increase PRPP which is necessary for the 120conversion of FUra to FUMP by OPRTase (30). The increased anabolism of FUra could then contribute to, or account for, the increased cell kill seen with this combination. 100-O To test this hypothesis, the effect of uridine on intracellular PRPP levels was measured. S-49 cells were incubated for 6 h 1C£ with either 10 MM uridine, 300 MM PALA, 0.04 MM MTX, 0.4 MM 80-O MTX, or no drug, and intracellular PRPP levels were measured. üLU° In five experiments which were carried out, 10 MMuridine did not 60-Z increase intracellular PRPP levels (data not shown). Since uridine did not affect PRPP levels, the effect of either PALA or MTX on LUO PRPP levels was examined to confirm that increases in PRPP or UJ 40-1 could be detected. PALA (300 MM)or 0.40 UM MTX both caused o.20a 90% decrease in growth of S-49 cells over a 72-h period (data not shown), which was similar to that of 1.0 MMFUra plus 10 MM uridine (Chart 1). However, neither PALA (300 MM)nor MTX (0.04 MM)increased intracellular PRPP levels in S-49 cells. However, 0.4 MMMTX did increase intracellular PRPP levels. 0T*URDTT*FUra FUra(10,iM) FUra +URD One experiment was carried out to determine the effect of (1.0MM) (1.0„M;10»iM) (24/jM] either 10 MMuridine, 1.0 MMFUra, 1.0 MMFUra plus 10 MMuridine, Charts. Effect of uridine (URD) on the incorporation of ["C]NaHC03 into or 100 MM uridine on intracellular PRPP levels. Only 100 MM individual pyrimidine nucleotides. S-49 cells (1 x 10* cells/ml in 5 ml) were incubated in the presence of FUra (1.0 or 2.4 ^M). 10 ^M uridine, or 1.0 ^M FUra plus 10 ¡it* uridine appeared to increase intracellular PRPP levels (148% of uridine. After 5 h of incubation, 25 ß\of ['4C)NaHC03 were added to each treatment control), whereas 10 MM uridine, 1.0 MM FUra, or 1.0 MM FUra group. After an additional 1 h of incubation the incorporation of ["CINaHCOs into acid-soluble pyrimidine nucleotides was determined. The data are presented as plus 10 MM uridine had little, if any, effect on intracellular PRPP percentage of control pyrimidine synthesis. Columns, mean from three experiments, levels (111, 113, and 122% of control, respectively; data not except for 2.4 ,.M FUra. which is the result from only one experiment; oars, SE. *, significantly different from control, P < 0 05; Newman Keuls' multiple range test. shown). Effect of Undine on de Novo Pyrimidine Biosynthesis. The observation that 10 MM uridine failed to increase intracellular Table 1 Comparison of FUTP and UTP levels, and the RNAnmol/107 incorporation of FUra into PRPP levels did not support the concept that uridine enhanced cellsTreatment1 the cytotoxicity of FUra by increasing PRPP levels and thereby increasing the anabolism of FUra. Therefore, the effect of uridine, x FUra, or FUra plus uridine on de novo pyrimidine biosynthesis 1000.94(1.0)1.14(1.2)2.25 was examined. As can be seen (Chart 5) treatment of cells with .0FUra10 /IM (1 .Of .0)0.045(1.8)0.061 (1 either 10 MMuridine or 1.0 MMFUra plus 10 MMuridine decreased 3.91 (1.5)2.71 0.085(1.4)0.138(2.3)FUTP:UTPratio the incorporation of [14C]NaHC03 into total pyrimidine nucleolM uridine 2.4 MMFUraUTP"2.67 (1.0)FUTP00.025(2.4)FUra-RNA00.060(1.0) (2.4) " Values were obtained from Chart 7. 6 Values were obtained from Chart 3. c Numbers in parentheses, ratio of drug treatment to treatment with 1.0 »uFUra tides to 30% of control. In contrast, FUra (either 1.0 or 2.4 MM) alone had no effect on the incorporation of [14C]NaHCO3 into pyrimidine nucleotides. Effect of Undine on UTP Levels. Presumably the inhibitionof de novo pyrimidine biosynthesis by uridine (Chart 5) was due to increase in intracellular UTP, which acts as a feedback inhibitor of carbamoyl phosphate synthetase II (30). In order to determine if intracellular UTP levels were increased, UTP was measured after incubation of S-49 cells for 6 h with 10 MMuridine, 1.0 MM FUra, 2.4 MM FUra, or 1.0 MM FUra plus 10 MM uridine. None of these treatments changed intracellular UTP levels (data not shown). However, while controls had 2.54 nmol of UTP/107 cells, treatment with either 1.0 MM FUra plus 10 MM uridine or 10 MM uridine alone resulted in 4.00 nmol of UTP/107 cells. Although the increases in UTP due to either of these treatments were not shown to be statistically significant, the data suggested a trend toward increased UTP levels which might account for the ob served inhibition in de novo pyrimidine biosynthesis (Chart 5). Comparison of FUTP, UTP, and Incorporation of FUra into RNA. A comparison of the effect of uridine on UTP levels, FUTP levels, and the incorporation of FUra into RNA can be seen in Tabble 1. FUra (2.4 MM)increased both the levels of FUTP and its incorporation into RNA about 2.4 times as compared to cells treated with 1.0 MM FUra (Chart 3; Table 11 This indicated that when cells were treated with FUra alone the fer .-nation of FUra- CANCER alone, which is taken as 1.0. RNA correlated with the levels of FUTP. FUra (2.4 MM) had no effect on intracellular UTP pools (Table 1). In cells treated with 1.0 MMFUra plus 10 MMuridine, the FUTP levels were 1.8 times that seen in cells treated with 1.0 MMFUra alone (Chart 3; Table 1). However, the incorporation of FUra into RNA in cells treated with the combination was only 1.4 times that of cells treated with 1.0 MM FUra alone. Treatment with 1.0 MM FUra plus 10 MM uridine resulted in less incorporation of FUra into RNA than would be expected based on FUTP levels. It is possible that the trend toward increased UTP after treatment with 1.0 MM FUra plus 10 MMuridine accounted for the decreased incorporation of FUTP into RNA. Thus, the formation of FUra-RNA was more closely related to the FUTP: UTP ratio than it was with FUTP levels. Effect of Drug Treatment on Macromolecular Synthesis. The results shown above (Chart 3) indicated that uridine in creased the anabolism of 1.0 MM FUra to levels which were approximately halfway between those seen with 1.0. MM FUra and 2.4 MM FUra (the latter of which was equitoxic to 1.0 MM FUra plus 10 MMuridine; see Chart 2). Therefore, the increased RESEARCH VOL. 45 SEPTEMBER 1985 4253 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. URIDINE ENHANCEMENT anabolism of FUra may not account for all of the enhanced cytotoxicity of the combination of 1.0 MMFUra plus 10 MMuridine. In a further attempt to localize the site at which uridine enhances the cytotoxicity of FUra, the effect of either FUra or FUra plus uridine on protein, RNA, and DMA synthesis was determined. Incubation of S-49 cells for 2, 6, or 24 h with 1.0 MMFUra, 2.4 MM FUra, or 1.0 MM FUra plus 10 MM uridine did not affect the incorporation of either [3H]leucine into total protein or [3H]adenosine into total RNA (data not shown). However, FUra (2.4 MM) inhibited the incorporation of [3H]deoxyguanosine into DNA by 50% after 24 h of incubation, respectively (Chart 6). In contrast, during 24 h of incubation, neither 1.0 MM FUra nor 1.0 MM FUra plus 10 MM uridine inhibited DNA synthesis. It was of interest that at 24 h this result was qualitatively different from that obtained with 2.4 MMFUra. This suggested that the mechanism of cell kill for 1.0 MM FUra plus 10 MM uridine might be different from that of the equitoxic dose of FUra (2.4 MM)alone. DISCUSSION Enhancement by Undine of the Anabolism of FUra. Ullman and Kirsch (14) reported that uridine enhances both the growth inhibitory effects of FUra against S-49 cells, as well as the phosphoribosylation of FUra. They suggested, but did not at tempt to prove that uridine increases the action of FUra by increasing its anabolism. Our data (Chart 1) confirmed their observation, and in addition, we showed that uridine enhanced the cell killing action of FUra by about 2.5-fold, as determined by colony formation assay (Chart 2). It is of interest that the con centration of uridine in the serum of mice is approximately 10 MM (31), and the concentration of uridine in human serum ranges from 2 to 9 MM (32). The results reported herein indicate that physiological levels of uridine can affect the anabolism and action of FUra. Furthermore, these results suggest that in in vitro studies it may be more meaningful to study the mechanism of action of the antimetabolites in the presence of relevant physio logical metabolites. In order to test the hypothesis that uridine increases the OF FUra ANABOLISM cytotoxicity of FUra by increasing the anabolism of FUra to FUMP, the effect of uridine on the anabolism of FUra to active nucleotides was examined. Uridine (10 MM) increased both the anabolism of 1.0 MM FUra to acid-soluble metabolites and the incorporation of FUra into RNA (Chart 3). If the increase in cytotoxicity due to uridine was solely due to increased anabolism of FUra, then the levels of the acid-soluble metabolites of FUra as well as the amount of FUra incorporated into RNA in cells treated with 1.0 MM FUra plus 10 MM uridine should be equal to that of cells treated with 2.4 MMFUra. However, these increases in FUra anabolism due to 10 MM uridine were not equal to the levels seen with 2.4 MM FUra alone (Chart 3). Therefore, the increase in FUra anabolism due to uridine, which was observed, did not appear to account for all of the increase in the cytotoxicity of 1.0 MMFUra plus 10 MMuridine. The effect of uridine on the formation of indvidual anabolites of FUra was studied to determine if uridine selectively increased any particular FUra nucleotide. After treatment with 1.0 MMFUra plus 10 MM uridine, both FUTP and the FUDP-hexoses were increased above the levels in cells treated with 1.0 MM FUra alone, while the other acid-soluble metabolites showed a trend (which was not statistically significant) toward increases (Chart 3). Furthermore, in cells treated with 1.0 MM FUra plus 10 MM uridine the levels of each of the acid-soluble metabolites of FUra (including FUTP, FdUMP, and FUDP-hexoses) were significantly less than from cells treated with 2.4 MM FUra alone. Therefore, uridine did not selectively increase the formation of any particular acid-soluble metabolite of FUra to a level which could account for the increase in cytotoxicity of this combination. Uridine did not affect the incorporation of FUra into the alkalistable, acid-insoluble fraction of S-49 cells (Chart 4). Most (ap proximately 97%) of the radioactivity in this fraction consists of the thymidylate synthetase:FdUMP:A/5,W10-methylenetetrahydrofolate complex.5 Similarly acid-soluble FdUMP was not increased by uridine (Chart 3). This suggests that the enhancement by uridine of FUra action is directed toward the fluororibonucleotide pool and not the fluorodeoxyribonucleotide pool. Effect of Undine on the Levels of PRPP. The increase in the anabolism of 1.0 MM FUra by 10 MMuridine was not associated with elevated intracellular PRPP levels. Other investigators have also shown that uridine does not necessarily increase PRPP levels. For example, treatment of S-49 cells for 4 h with 5 MM uridine plus 5 MMerythro-9-(2-hydroxy-3-nonyl)adenine, an adenosine deaminase inhibitor, does not affect PRPP levels (33). Treatment of rat liver and spleen slices for 3 h with 10 mw uridine has no effect on PRPP levels (34). Furthermore, neither 0.04 MM MTX nor 300 MM PALA affected PRPP levels after 6 h of incubation. Both 0.04 MM MTX and 300 MM PALA inhibited the growth of S-49 cells by about 90% after 72 h of incubation (data TIME (hours! Chart 6. Effect of either FUra or FUra plus uridine (URD) on the incorporation of |3H]deoxyguanosine into DNA. S-49 cells (3 x 105 cells/ml in 5 ml) were incubated with FUra (1.0 or 2.4 MM), 1.0 i¡uFUra plus 10 (¿M uridine, or no drug for 2, 6, and 24 h. pHJDeoxyguanosine (1.0 /jCi/ml) was added to two flasks of each treatment 1 h prior to the end of the incubation. At 2, 6, and 24 h two 2.0-ml aliquots from each treatment were collected, and the incorporation of [3H]deoxyguanosine into DNA was determined. The radioactivity (dpm) in DNA from each treatment was divided by the total number of cells in that treatment, and the data were presented as percentage of control DNA synthesis. Points, mean from three experiments; bars, SE. *. significantly different from 1.0 vu FUra. P < 0.05; Newman Keuls' multiple range test. CANCER RESEARCH not shown), which indicated that MTX and PALA, at concentra tions which inhibited cell growth did not increase PRPP levels. It is of interest that Ullman and Kirsch (14) observed that 250 MM inosine decreases PRPP levels 50% in S-49 cells, which coin cides with a decrease in the anabolism of FUra and protection from growth inhibition by FUra. The results of Ullman and Kirsch suggest a positive correlation between PRPP levels and FUra anabolism. However, the data reported herein indicated that 10 MMuridine increased both the anabolism of 1.0 MM FUra and its cytotoxicity, although PRPP levels were not simultaneously in creased. VOL. 45 SEPTEMBER 1985 4254 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. URIDINE ENHANCEMENT Effect of Undine on de Novo Pyrimidine Biosynthesis. Un dine (10 MM) inhibited de novo pyrimidine biosynthesis in S-49 cells by 70% (Chart 5). This inhibiton probably accounted for the increase in FUra anabolism by undine. Uridine also inhibits de novo pyrimidine biosynthesis in other systems. For example, incubation of rat liver and spleen slices with 10 mw uridine for 3 h inhibits de novo pyrimidine biosynthesis by 75%, while there were no increases in PRPP levels (34). Furthermore, de novo pyrimidine biosynthesis in rat spleen slices was inhibited only 20% with 1.0 HIM uridine. The apparently high concentration of uridine (i.e., 10 mw) needed to inhibit de novo pyrimidine biosyn thesis in this system may be due to the rapid degradation of uridine by liver or spleen. Tsuboi ef al. (35) found that treatment of HT-29 cells with 10 UM uridine for 2 h inhibits de novo pyrimidine biosynthesis by 70%. Similarly, treatment of L1210 cells with 10 MM uridine for 1 h inhibits de novo pyrimidine biosynthesis by 70% (27). PALA and uridine, both of which inhibit de novo pyrimidine biosynthesis, had little effect on PRPP levels (33, 34, 36). To our knowledge, the only report of increases in PRPP due to PALA is in MCF-7 cells (37). Therefore, in many tumor cell lines, inhibition de novo pyrimidine biosynthesis does not necessarily coincide with increased intracellular PRPP. Our results suggest that an inhibition of de novo pyrimidine biosynthesis due to uridine, without any corresponding accumulation of PRPP, is still suffi cient to allow for increased anabolism of FUra by OPRTase. Effect of Uridine on UTP Levels. In S-49 cells treated for 6 h with 10 MMuridine there was a trend (which was not statistically significant) toward increased UTP levels. Nevertheless, it is prob able that the levels of UTP which were seen with 10 MMuridine accounted for the inhibition (via carbamoyl phosphate synthetase II) of de novo pyrimidine biosynthesis. In L1210 cells a 50% expansion of the uridine nucleotide pool inhibits de novo pyrimi dine biosynthesis by 50% (27). By comparison, an apparent increase of UTP levels in S-49 cells of 46% (which, however, was not statistically significant; Table 1), was associated with a 70% inhibition of de novo pyrimidine biosynthesis (Chart 5). Furthermore, the increase in UTP seen in cells treated with 10 MMuridine appeared to compete with FUTP for RNA polymerase, since the incorporation of FUra into RNA was more closely related to the FUTP: UTP ratio than to FUTP levels (Table 1). It is of interest that the combination of PALA plus FUra, which increases the FUTP:UTP ratio by decreasing UTP levels, also increases FUra incorporation into RNA as compared to FUra alone (38). Effect of Uridine on Macromolecular Synthesis. Uridine (10 MM)did not increase the amount of FdUMP which was bound to thymidylate synthetase. In addition, free thymidylate synthetase was apparently still present in cells treated with 1.0 MM FUra, since treatment with 1.7 MMFUra resulted in increased amounts of the thymidylate synthetase:FdUMP:A/5,A/10-methylenetetrahydrofolate complex (Chart 4). This indicated that uridine did not increase the inhibition of thymidylate synthetase by FUra. The incorporation of [3H]deoxyguanosine into DMA, which was used as a measure of DNA synthesis (Chart 6), is also an indirect measure of thymidylate synthetase activity. Incubation of S-49 cells with either 1.0 MM FUra plus 10 MMuridine or 1.0 MM FUra alone did not affect DNA synthesis even after 24 h. In contrast, in cells treated with 2.4 MM FUra, DNA synthesis was inhibited by 50% at 24 h. The differential effect on the inhibition of DNA CANCER RESEARCH VOL. OF FUra ANABOLISM synthesis at 24 h by 1.0 MMFUra plus 10 MMuridine as compared to 2.4 MM FUra suggested that there may be differences in the mechanism of cell kill due to the two treatments. Despite the increase in FUra ribonucleotide formation by uri dine, the enhanced cell kill due to the combination of uridine plus FUra could not be completely accounted for by increased FUra anabolism. At most it could account for approximately 50% of the increased cytotoxicity of FUra plus uridine. Furthermore, uridine did not appear to increase the inhibition of DNA synthesis by FUra. In studies to be reported elsewhere6 we have observed a thymidine-preventable component of the cytotoxicity of 1.0 MM FUra plus 10 MMuridine which can also account for approximately 50% of the cytotoxicity of this combination. These studies,6 together with the data presented here, indicate that uridine enhanced FUra cytotoxicity by increasing both the anabolism of FUra to ribonucleotides and a DNA-directed component of FUra action. REFERENCES 1. Myers, C. E. The pharmacology of the fluoropyrimidines. Pharmacol. Rev., 33. 1-15, 1981. 2. Cheng, Y. C., and Nakayama, K. Effect of 5-fluoro-2'-deoxyuridine on DNA metabolism in HELA cells. Mol. Pharmacol., 23: 171-174, 1983 3. Danenberg, P. V., Heidelberger. C., Mulkins, M A . and Peterson, A. R. The incorporation of 5-fluoro-2'-deoxyuridine into DNA of mammalian tumor cells. Biochem. Biophys. Res. Commun., 102: 654-658. 1981. 4. Ingraham, H. A.. 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Pharmacol., 30: 2045-2049, 1981. 45 SEPTEMBER 1985 4256 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research. Enhancement by Uridine of the Anabolism of 5-Fluorouracil in Mouse T-Lymphoma (S-49) Cells William B. Parker and Philip Klubes Cancer Res 1985;45:4249-4256. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/9/4249 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.