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Effects of 6-Azauridine on Nucleotides, Orotic Acid, and Orotidine in L5178Y Mouse Lymphoma Cells in Wfro1

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Effects of 6-Azauridine on Nucleotides, Orotic Acid, and Orotidine in L5178Y Mouse Lymphoma Cells in Wfro1 [CANCER RESEARCH 37, 4382-4388, December 1977] Effects of 6-Azauridine on Nucleotides, Orotic Acid, and Orotidine in L5178Y Mouse Lymphoma Cells in Wfro1 Claude M. Janeway and Sungman Cha Division ot Biology and Medicine, Brown University, Providence, Rhode Island 02912 SUMMARY In addition to drastic changes in pyrimidine nucleotides and their precursors, significant effects have been observed Murine lymphoma L5178Y cells in culture were exposed in the purine nucleotide concentrations. A working hypoth to 5 fj.M 6-azauridine (6-aza-UR) for up to 8 hr. The nucleo- esis is offered to explain the mechanism of accumulation tides in the acid-soluble extracts were determined by high- of orotic acid and orotidine. Plausible explanations are pressure liquid chromatography. The concentrations of also offered for previously observed but unexplained find uracil and cytosine nucleotides decreased rapidly in the ings, i.e., the potentiation of the effects of ara-C by 6-aza- treated cells. Adenosine 5'-triphosphate and adenosine 5'- UR when 6-aza-UR is given prior to ara-C (2). diphosphate increased steadily, whereas guanosine 5'-di- phosphate decreased for a few hr before returning to the normal level. Orotic acid and orotidine, but no detectable MATERIALS AND METHODS amount of orotidine monophosphate (OMP), accumulated Chemicals. Fischer's medium, horse serum, and antibiot in treated cells. In homogenates of the cells harvested from ascitic fluids of mice, the presence of a membrane-bound ics necessary for the culture of L5178Y cells were obtained phosphatase with activity for OMP was demonstrated, from Grand Island Biological Co., Grand Island, N. Y. 6- which may be responsible for the lack of OMP accumula Aza-UR, 6-aza-UMP, orotidine, and OMP were purchased tion. When incubated with the homogenate, [7-'"C]OMP from Calbiochem, Los Angeles, Calif. [7-14C]OMP (39.3 was converted to [7-'4C]orotidine in the absence of inor mCi/mmole) was obtained from New England Nuclear, ganic phosphate, but it was converted to [7-14C]orotic acid Boston, Mass. OMP pyrophosphorylase (orotate phospho- ribosyltransferase, EC 2.4.2.10) from baker's yeast and 5- in the presence of phosphate, suggesting the occurrence of an orotidine phosphorylase. The increase in adenosine fluoroorotate were purchased from P-L Biochemicals, Inc., 5'-triphosphate levels and the rapid decrease in the pyrimi- Milwaukee, Wis. The enzyme was purified further, approxi dine nucleotides observed after treatment with 6-aza-UR mately 10-fold, by the use of a Sephadex G-200 column may be responsible for the observation (Brenckman ef al., eluted with a 0.2 M Tris-CI buffer containing 0.05 M MgCI2 Biochem. Biophys. Res. Commun., 52: 1368-1373, 1973) at pH 8.O. Even after purification, the preparation had that 6-aza-UR potentiates the activity of 1-/3-D-arabinofu- considerable OMP decarboxylase (EC 4.1.1.23) activity. The ranosylcytosine only when 6-aza-UR is added prior to 1-/3- enzyme was assayed spectrophotometrically by the method D-arabinofuranosylcytosine. of Dahl ef al. (8) with 5-fluoroorotate as substrate. All other nucleotide standards used for identification of peaks on high-pressure liquid chromatography were pur INTRODUCTION chased from Sigma Chemical Co., St. Louis, Mo. Cell Culture and Extraction Conditions. The L5178Y cells Various aspects of the clinically useful pyrimidine anti- were obtained from Dr. M-Y. Chu of Brown University. The metabolite, 6-aza-UR,2 have been extensively reviewed by cells were grown in culture in 500-ml bottles at 37° in several investigators (16, 19, 28). Taking advantage of the Fischer's medium supplemented with 10% horse serum, as recent improvements (e.g., dual wavelength monitoring) in described by Fischer and Sartorelli (12). Cell numbers were high-pressure liquid chromatography, we have undertaken counted with a Model B Coulter counter (Coulter Electron a study of the effects of 6-aza-UR on the nucleotide pools ics, Inc., Hialeah, Fla.). Exponentially growing cultures of of mouse lymphoma L5178Y cells in culture. The simplicity L5178Y cells were exposed to 6-aza-UR (final concentra and versatility of this Chromatographie technique allowed tion, 5 ¿UM)forvarying periods of time. Cells were collected us to determine quantitatively many nucleotides and to by centrifugation and washed twice with cold 0.9% NaCI follow the time course of changes in metabolic concentra solution. The packed cells (approximately 5 x 107 cells) tions, particularly those of orotic acid, orotidine, and OMP. were extracted twice with 0.5 ml of trichloroacetic acid at 4°.The extracts were combined, and trichloroacetic acid 1 Supported by USPHS Grants CA 12531 and CA 13943. 2 The abbreviations used are: 6-aza-UR, 6-azauridine; OMP, orotidine 5'- was removed by extracting 3 times with water-saturated monophosphate; ara-C, 1-/3-D-arabinofuranosylcytosine; 6-aza-UMP, 6- diethyl ether. The aqueous phase (pH 6.0) was lyophilized, azauridine 5'-monophosphate; PRPP, 5-phospho-a-D-ribosylpyrophosphate; ara-CMP, 1-fl-D-arabinofuranosylcytosine 5'-monophosphate; ara-CTP, 1-/3- and the solids were redissolved in 100 /¿Iof water and kept o-arabinofuranosylcytosine 5'-triphosphate. frozen until analysis by high-pressure liquid chromatogra Received June 22. 1977; accepted August 31. 1977. phy. 4382 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. 6-aza-UR Effects in L5178Y Chromatographie Procedures for Nucleotides. Nucleo- Aliquots (50 or 100 /xl) of these preparations (equivalent tide levels in the acid-soluble extracts were determined by to approximately 5 mg of cells) were incubated with 10 high-pressure liquid chromatography with a Varian Aero nmoles of [7-1<C]OMP (1540 cpm/nmole) in 0.05 M Tris-CI graph LCS-1000 instrument equipped with a Partisil PXS buffer or 0.05 M potassium phosphate/0.01 M MgCIj buffer 10/25 SAX column (Whatman, Inc., Clifton, N. J.). The (pH 7.3) in a total volume of 1 ml in the presence of 10 nucleotides (usually 30 to 50 /J of the 100-,ul extract) were nmoles of 6-aza-UMP. At various times, aliquots of the eluted from the column with a potassium phosphate buffer reaction mixture were removed, and reactions were termi (phosphoric acid adjusted to pH 4.5 with KOH) under the nated by addition of perchloric acid (final concentration, following conditions: low concentrate was 2 HIM and high 5%). After the precipitate was removed by centrifugation, concentrate was 0.5 M; starting volume in the mixing the supernatant fluid was neutralized with KOH. and the chamber was 25 ml (low concentrate). The elution scheme precipitate was removed by centrifugation. The radioactive used in these studies consisted of elution with low-concen materials in these extracts were separated by chromatog trate buffer (2 HIM) for 30 min at a flow rate of 25 ml/hr raphy with the use of polyethyleneimine cellulose thin-layer through the column (pump settings were 25 and 0 ml/hr sheets (Cel 300 PEI, Brinkmann Instruments. Inc., West- for the column and gradient pumps, respectively) followed bury, N. Y.). After the samples were applied, the sheets by a convex gradient from 2 mw to 0.5 M buffer at a flow were washed with methanol/water (1/1) to remove the rate of 40 ml/hr through the column (both column and salts. The chromatograms were developed in 0.5 M LÃŒGIfor gradient pump flow rates were 40 ml/hr). Elutions were 1 hr. The Rt values for OMP. orotic acid, and orotidine carried out at a constant pH value of 4.5. During the were 0.07, 0.64, and 0.77, respectively. Appropriate spots gradient delay, the volume of buffer in the mixing chamber were cut out, and radioactivity was counted in a toluene decreased from 25 to 12.5 ml and was maintained at this solution of 10% Bio-Solv (Beckman Instruments. Fullerton, latter value throughout the remainder of the elution. UV Calif.) and 0.7% PPO (Packard Instrument Co.. Inc., Down absorption of the eluate was monitored at 2 wavelengths. ers Grove. III.) with a Packard Tri-Carb Model 3320 Liquid 254 and 290 nm. The A29(,/A2S4absorption ratios greatly scintillation spectrophotometer. facilitated identification of the peaks. At pH 4.5, the cyto- sine nucleotides and orotic acid absorb strongly at 290 nm. The extinction coefficient of orotic acid (8800) at 290 nm RESULTS and pH 4.5 is much greater than those of orotidine (1900) The effects of 6-aza-UR on the growth of L5178Y cells in and OMP (2300). Some nucleotide peaks were identified by Fischer's medium are shown in Chart 1. On the basis of comparison of retention time and by superimposition with these results, 5 ¿¿Mwaschosen as the concentration of the known standards. The nucleotide concentrations were de termined by comparison of the peak areas with those of a drug for studies of its effects on various nucleotides. A high-pressure liquid chromatography profile of an acid- series of standard nucleotides by the use of a digitized planimeter (Model EGC; Numonics Corp., North Wales, Pa). soluble extract of cells in exponential growth phase is Identification of Orotic Acid by an Enzyme Peak Shift. shown in Chart 2, fop. All but a few peaks have been An acid extract of cells treated with 6-aza-UR, containing identified by the methods described previously. Contrary to 17 nmoles of orotic acid and 11 nmoles of orotidine. was results from other laboratories (4. 22, 30), UDP eluted incubated at room temperature for 5 hr with 0.01 unit of before CDP, and UTP eluted before CTP. while UMP eluted OMP pyrophosphorylase, 25 ¿¿molesofTris-CI (pH 7.9), after CMP.
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