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'-diphosphate 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 phosphoribosyltransferase, 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 37Â° 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 Â¿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 4Â°.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 /Â¿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.

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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

DECEMBER 1977 4383

L5I78Y 1.65 Â»IO7 CELLS , CONTROL

0.06 0.03

O.O4 0.02 >

S < 0.02

1.75 x IO7 CELLS 6-azaUR, 5 pM, 2 hrs O.O6 0.03 ATP

ADP S" o 0.04 O 02

I 6TP 0.02 UTP 0.01 GDP

M, 4O 60 BO R E TENTION TIME (mln) Chart 2. High-pressure liquid chromatograms of acid-soluble extracts of L5178Y cells. Top, untreated cells; bottom, an extract of cells exposed to 5 , 6-aza-UR for 2 hr. UDPG, UDP-glucose; XMP, xanthosine monophosphate. identical with that of IMP, but it has not been positively Table 1 identified at this time. Concentrations of major nucleotides Nucleotide concentration of L5178Y cells estimated from this and similar chromatograms are pre NucleotideATP cells"1.06x 109 sented in Table 1. Â±0.46" Shown in Chart 2, bottom, is the nucleotide profile of ADPGTP 0.14 Â±0.090.29 cells grown for 2 hr in the presence of 5 /Â¿M6-aza-UR.The most obvious differences between this profile and that of Â±0.11 the control cells are the marked decreases in the sizes of GDPUTP 0.02 Â±0.020.44 all pyrimidine nucleotide peaks (UTP, UDP, CTP, CDP, and Â±0.20 UDP-glucose), increases in ATP and ADP peaks, and the UDPCTP 0.06 Â±0.040.21 appearance of 2 new peaks in the nucleoside monophosphate region. Nelson (22) also observed similar peaks and Â±0.06 tentatively assigned the larger of the 2 peaks to orotidine. CDPUDP-glucose'/Â¿moles/1.5 0.08 Â±0.060.15 These 2 new peaks have strong absorption at 290 nm and Â±0.10 pH 4.5. The first peak was identified as orotidine by super- " Average of 8 experiments. One ml of packed cells equals imposition of an authentic sample. The second of the 2, approximately 1.5 x 109 cells. with a much higher A2,,,/A.2.-,.iratio,was identified as orotic " Mean Â±S.D. acid by the method to be described below. r A maximum value, since other minor peaks, e.g., xanthosine For positive identification of the orotic acid peak, an monophosphate, are usually poorly resolved. enzymic peak shift technique was applied, as described in "Materials and Methods", and the results are shown in peak in the control extract (marked as orotic acid in Chart Chart 3. Under the experimental conditions, decarboxyla- 3, fop) disappeared, and a new peak with a retention time tion of OMP to UMP was largely blocked by 6-aza-UMP. identical with that of OMP appeared, (Chart 3, bottom). Upon this enzymic treatment, the major 290-nm absorbing The 290-nm absorbing peak, which eluted before the orotic

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L5I78Y I.SnlO7 CELLS. 6-azoUR

60 120

L5I78Y I 5 x IO' CELLS , 6-ozoUR OPRTose, PRPP, 6-oiollMP t O 03

ADP 00Â« ,002 g

< 0.02 GTP_ ODI CDP UTP GDP

20 40 60 80 100 120 RETENTION TIME (mm ) Chart 3. Identification of orotidine and orotic acid peaks in a high-pressure liquid chromatogram by an enzyme peak shift technique with the use of orotate phosphoribosyltransferase (OPTRase). Top, the nucleotide profile of an acid-soluble extract of L5178Y cells exposed to 5 nM 6-aza-UR for 2 hr; bottom, the same extract after treatment with orotate phosphoribosyltransferase in the presence of PRPP. MgCls. and 6-aza-UMP. XMP. xanthosine monophosphate; UDPG, UDP-glucose. acid peak, remained unchanged, while the peak of added 6-aza-UMP appeared. These findings confirm the identities of the orotic acid and orotidine peaks shown in Chart 1. Chart 4 summarizes the time sequence of changes in the concentrations of various nucleotides after addition of 6- . 100 aza-UR. The pyrimidine nucleotides decreased markedly, as expected. On the other hand, the ATP concentration increased steadily, while the GTP concentration decreased for a few hr and then returned to the normal level. Orotic acid and orotidine, which are undetectable in the control by these methods, also accumulated. Orotic acid tends to accumulate before orotidine, but evenutally orotidine ex ceeds orotic acid. Since 6-aza-UR is believed to exert its action after conver sion to 6-aza-UMP by blocking decarboxylation of OMP, one might expect to see OMP accumulation; however, OMP does not accumulate in detectable amounts. As will TIME Ihr) be discussed later, this suggests the presence of an active Chart 4. Time course of changes in nucleotide levels in L5178Y cells phosphatase, possibly 1 of the 5'-nucleoside monophos- during exposure to 5 ^M 6-aza-UR. phatases, possessing a low Kn,for OMP. To find explanations for the accumulation of orotidine of 0.01 mw 6-aza-UMP, however, 65% of the original radio and orotic acid but not of OMP, we carried out the following activity was converted to orotidine, but no significant experiments. As may be seen in Table 2, [7-14C]OMP was amount was converted to orotic acid. It appears that 6-aza- completely metabolized during a 2-hr incubation at 37C UMP did not block the decarboxylation completely, and with an aliquot of the low-speed supernatant of the homog- the radioactivity that was not accounted for as orotidine enate dialyzed in Tris buffer. In the absence of 6-aza-UMP, plus OMP was lost as CO2. Similar experiments were also OMP was almost completely decarboxylated to UMP, and carried out in the presence of 6-aza-UMP for various periods the radioactivity was lost as CO2.Only a small amount (7%) of time, and the results are shown in Chart 5, left. In a of the radioactivity was present as orotidine. In the presence similar experiment, in which the phosphate buffer was

DECEMBER 1977 4385

used throughout the homogenization, dialysis, and incuba previous observations. Nevertheless, the depletion of the tion, more orotic acid was formed than orotidine (Chart 5, pyrimidine nucleotides is very rapid and is almost complete right) suggesting that orotidine was converted to orotic in less than 1 hr. acid by a phosphorylase. Chart 6 shows the results of Since 6-aza-UMP inhibits OMP decarboxylase (14, 15, experiments in which Tris-CI buffer was used throughout 24), it is natural to expect an accumulation of OMP. As the procedure. The putative phosphatase present in the discussed previously, however, no detectable amounts of homogenate (as indicated in Chart 6, left) was removed by OMP accumulated at any time after 6-aza-UR treatment. centrifugation at a high speed (100,000 x g, 5 hr) (Chart 6, Instead, orotic acid and orotidine accumulated. Accumula right). tion of orotic acid and orotidine has been observed after 6- aza-UR treatment in bacterial culture media (29), experi mental tumors of mice (24), and human urine (5, 11). The DISCUSSION simplest explanation for the accumulation of these com pounds, but not of OMP, may be that they are the degrada 6-Aza-UR exerts its effects after phosphorylation to 6- tion products of OMP that accumulate after 6-aza-UR treat aza-UMP, which in turn inhibits OMP decarboxylation, thus ment. The accumulation of OMP was demonstrated in the preventing the de novo biosynthesis of pyrimidine nucleo- particle-free supernatant fraction of L5178Y cells (24) and tides (14, 15, 24). The drastic decrease in the concentra of rat liver (1). However, to the best of our knowledge, tions of the pyrimidine nucleotides observed above in detectable accumulation of OMP has never been demon 6-aza-UR-treated cells are, therefore, consistent with these strated in any intact cell. Therefore, it seems appropriate to raise questions as to how orotic acid and orotidine are Table 2 formed. OMP phosphatase activity ot L5178Y cell homogenate The results presented in Table 2 and Charts 5 and 6 cpm demonstrate that orotidine can be formed rapidly from OMP in homogenates of L5178Y cells and suggest the Addition OMP Orotic acid Orotidine existence of a membrane-bound phosphatase. This is con ,490"50 NoneHomogenate Â±150 Â±60610 Â±40950 sistent with the observations of other investigators cited Â±350 Â±330 Â± previously. Homogenate + 250 Â±1 50190 330 Â±130200 7,700 Â±380 azaUMP12,750 For the mechanism of accumulation of orotidine and orotic acid in the 6-aza-UR-treated cells, we propose the Mean Â±S.D. following working hypothesis on the basis of available ioo< evidence, as illustrated in Chart 7. First, de novo synthesis of orotic acid increases as the consequence of a series of events. 6-aza-UMP, the active metabolite of 6-aza-UR, inhibits OMP decarboxylation. UTP and CTP concentrations decrease. The first and rate-limit ing step of pyrimidine biosynthesis, the aspartate carbamyl- transferase section, is stimulated by the removal of feed back inhibition by UTP and CTP (3), as well as by the possible increase in the amount of the enzyme, as shown in Sarcoma 180 cells by Ennis and Lubin (10). I HOURS 2 Second, the OMP pyrophosphorylase reaction may be Chart 5. Metabolism of [7-'4C]OMP by low-speed (800 x g, 5 min) super inhibited as a result of the inhibition of OMP decarboxylase natant fluid of homogenate in 0.05 M Tris buffer (pH 7.4) (left) and whole homogenate in 0.05 M phosphate buffer (pH 7.4). (right) In both experiments. by 6-aza-UMP. There is evidence that these 2 enzymes 10 mM MgClj and 10 ^M 6-aza-UMP were present OMP, orotidine. and normally exist as a complex (26) and that OMP formed orotic acid were separated by polyethyleneimine cellulose thin-layer chro- from orotic acid by the pyrophosphorylase reaction is matography directly channeled to the carboxylase without a rapid ex change with exogenous free OMP in the medium (31). If this is so, upon inhibition of the decarboxylase the endog- HOMOGENATE TRIS

OROTIDINE ... 6-Azo.UMP-x

OROTIDINE â€¢OROTATE--^-E- MP â€¢UMP

OROTIC ACID

OROTIDINE- OMP OROTIC ACID

05 HOURS 2 0.5 HOURS 2 Chart 7. A hypothetical scheme accounting for the accumulation ot orotic Chart 6. Metabolism of [7-"C]OMP. Experimental conditions were similar acid and orotidine and the lack of OMP accumulation after treatment with 6- to those of Chart 5. except that different enzyme preparations were used. aza-UR. The large arrow pointing to orotate signifies the increased de novo Left, homogenate in 0.1 M Tris-CI buffer (pH 7.4); right, cytosol (supernatant synthesis of orotate: R-1-P, ribose 1-phosphate; E-OMP, enzyme-bound fluid of homogenate after centrifugation at 100.000 x g for 5 hr). OMP

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. 6-aza-UR Effects in L5178Y enous OMP presumably bound to the enzyme complex pyrimidine nucleotide metabolism beyond UMP and the would increase to a saturating level before the concentra consequential cessation of nucleic acid synthesis. The fact tion of the free OMP in the medium increases significantly. that ATP increases relatively more than ADP is also consist Then, this enzyme-bound OMP might serve as a product ent with this possibility of an ATP-sparing effect. inhibitor of the pyrophosphorylase. At the same time, 6- Regardless of the mechanism of the ATP increase, this aza-UMP might also serve as an alternative product inhibi phenomenon, together with the decrease in pyrimidine tor. In addition to this product inhibition, it is conceivable nucleotides, may explain an interesting observation made that the inhibition of the decarboxylase moiety of the by Brenckman ef al. (2). These investigators reported that enzyme complex might cause simultaneous inhibition of 6-aza-UR and ara-C exhibited a potentiation when L5178Y pyrophosphorylase by a conformational change. The valid cells were exposed to 6-aza-UR before ara-C, an additive ity of these speculations, however, must be tested by future effect if ara-C preceded 6-aza-UR, and a less-than-additive experimentation. The pyrophosphatase reaction is readily effect if the 2 drugs were administered simultaneously. It is reversible with Â«,,â€žof0.12 in the direction of OMP formation generally acepted that ara-C is converted to ara-CMP by (13). Nevertheless, the notion that orotic acid may accumu deoxycytidine kinase and then to ara-CTP by various nu late simply due to the increased OMP concentration and by cleotide kinases (6, 7). Therefore, depletion of deoxycyti the reversal of the pyrophosphatase reaction appears to be dine compounds as a consequence of the decrease in CTP untenable, because there is no evidence that OMP actually may result in stimulation of ara-CTP formation by decreas accumulates to a significant degree in treated cells and ing the levels of substrates that compete for various en because the concentration of PPÂ¡isvery low in most cells. zymes in the pathway. Since the conversion of ara-C to Third, free OMP is rapidly converted to orotidine. As the ara-CTP requires ATP, increases in ATP may also facilitate endogenous OMP reaches a saturating level, it will disso this conversion. The K,,,value of ATP for ara-CMP formation ciate from the enzyme complex and then be rapidly dephos- by calf thymus deoxycytidine kinase was reported to be 2 phorylated to orotidine by a membrane-bound phospha- x 10~4 M (9, 21), and that for dCDP formation by rabbit tase. Thus, the concentration of free OMP is kept low. muscle deoxycytidylate kinase was reported to be 3.1 x Finally, orotidine and orotate are interconverted by a 10 5 M (20). Although the Kmvalues of these enzymes from phosphorylase. This putative phosphorylase may or may L5178Y cells for ara-C or its derivatives as a second sub not be the same enzyme as the uridine phosphorylase strate are not known, the ATP concentration of 1 ITIMin the described by Pontis ef al. (25). control cells is 5-fold greater than the Kâ€ž,valueof ATP for The changes in the concentrations of the purine nucleo- the calf thymus deoxycytidine kinase cited above. There tides were unexpected findings. ATP and ADP increased fore, a 2-fold increase in the ATP concentration could steadily for at least 8 hr, while guanine nucleotides (GTP significantly influence the rate of conversion of ara-C to and GDP) decreased for a few hr before returning to control ara-CMP. levels. These findings are superficially analogous to those of Nelson and Parks (23), which showed that DTP increased in Sarcoma 180 cells after treatment with 6-methylmercap- ACKNOWLEDGMENTS topurine ribonucleoside. Nelson and Parks attributed this The authors gratefully acknowledge Dr. M-Y. Chu for her invaluable to the increased availability of PRPP for pyrimidine biosyn contributions throughout these studies. Dr. Woo Ik Hwang for carrying out thesis due to the inhibition of purine nucleotide biosyn the initial phase of this work, Dr. G. A. Fischer and Dr. G. W. Crabtree for thesis by the 5'-monophosphate derivative of 6-methylmer- valuable advice, and Arpie Shiragian for her assistance in the high-pressure liquid chromatography. captopurine ribonucleoside. However, in this situation one would expect to find a decrease of PRPP rather than an increase, because the increased concentration of orotic REFERENCES acid may cause increased consumption of PRPP by the 1 Blair, D. G. R., Stone, J. E., and Potter. V. R Formation of Orotidine 5'- OMP pyrophosphorylase reaction. Furthermore, if the in Phosphate by Enzymes from Rat Liver. J. Biol. Chem., 235. 2379-2386. creases in adenine nucleotides were due to the stimulation 1960. 2. Brenckman, W. D., Jr., Chu, M. Y., and Fischer, G. A. Schedule of purine biosynthesis by an increase in PRPP, one would Dependence of the Lethal Effects of 6-Azauridine and Cytosine Arabino- expect a concurrent increase in GTP, which is contrary to side on Murine Leukemia Cells (L5178Y) in Culture. Biochem. Biophys. the previous observation. Indeed, our preliminary experi Res. Commun., 52; 1368-1373, 1973. 3. Bresnick. E. Inhibition by Pyrimidines of Aspartase Transcarbamylase ments to determine PRPP concentrations by the method of Partially Purified from Rat Liver. Biochim. Biophys. Acta. 67. 425-434. Henderson and Khoo (17) in the extracts prepared by the 1963 method of Hisata (18) revealed no indication of an increase 4 Brown. P. R. The Rapid Separation of Nucleotides in Cell Extracts Using High-Pressure Liquid Chromatography. J. Chromatog.. 52: 257- in the concentrations (approximately 75 /J.M)of PRPP after 272. 1970. 6-aza-UR treatment. Since the above data are expressed on 5 Buttoo. A. S.. Israels. M. C. G., and Wilkinson. J. F. Hypercholesterolae- mia and Orotic Aciduria during Treatment with 6-Azauridine. Brit Med. a per cell basis, it is conceivable that the ATP increase J., 1: 552-554, 1965. merely reflects increases in cell size following 6-aza-UR 6. Chu. M. Y., and Fischer. G. A. Comparative Studies of Leukemic Cells treatment, whereas the ATP concentration per se remained Sensitive and Resistant to Cytosine Arabinoside. Biochem. Pharmacol., constant. While the answer to this question must await cell 14: 333-341. 1965. 7. Creasey. W. A Arabinosylcytosine. In: A. C. Sartorelli and D. G. Johns size distribution studies, the slower increase in ADP and (eds.), Antineoplastic and Immuno-suppressive Agents II, pp. 232-256. the decrease in GTP do not support this explanation. A Berlin: Springer Verlag. 1976. 8 Dahl, J. L., Way. J. L., and Parks. R. E., Jr. The Enzymatic Synthesis of more plausible explanation for the increase of ATP is that 5-Fluorouridme 5'-Phosphate. J. Biol. Chem.,234: 2998-3002, 1959. the consumption of ATP may be spared due to the lack of 9. Durham, J. P.. and IvÃ©s.D.H. Deoxycytidine Kinase 1. Distribution in

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Normal and Neoplastic Tissues and Interrelationships of Deoxycytidine 22. Nelson, J. A. Some Clinical and Pharmacological Applications of High- and 1-ÃŸ-Arabinofuranosylcytosine Phosphorylation. Mol. Pharmacol., 5: Speed Liquid Chromatography. Advan. Chromatography. 15: 273-305. 358-375. 1969. 1977. 10. Ennis, H. L., and Lubin, M. Capacity for Synthesis of a Pyrimidine 23. Nelson, J. A., and Parks, R. E., Jr. Biochemical Mechanisms for the Biosynthetic Enzyme in Mammalian Cells. Biochim. Biophys. Acta. 68: Synergism between 6-Thioguanine and 6-(Methylmercapto)purine Ri- 78-83, 1963. bonucleoside in Sarcoma 180 Cells. Cancer Res., 32. 2034-2041, 1972. 11. FallÃ³n,H. J.. Frei, E.. III. Block, J., and Seegmiller, J. E. The Uricosuria 24. Pasternak, C. A., and Handschumacher, R. E The Biochemical Activity and Orotic Aciduria Induced by 6-Azauridine. J. Clin. Invest.. 40: 1906- of 6-Azauridine: Interference with Pyrimidine Metabolism in Transplant- 1914, 1961. able Mouse Tumors. J. Biol. Chem., 234: 2992-2997, 1959. 12. Fischer, G. A., and Sartorelli, A. C. Development, Maintenance and 25. Pontis, H., Degerstedt, G., and Reichard, P. Uridine and Deoxyuridine Assay of Drug Resistance. Methods Med. Res., 10: 247-262, 1964. Phosphorylases from Ehrlich Ascites Tumor. Biochim. Biophys. Acta, 13. Flask, J. G. Orotidine 5'-Phosphate Pyrophosphorylase. Methods En- 5): 138-147, 1961. zymol., 6: 148-152, 1963. 26. Prabhakararao, K., and Jones, M. E. Radioassay of Orotic Acid Phospho- 14. Habermann, V., and Sorm, F. Mechanism of the Cancerostatic Action of ribosyltransferase and Orotidylate Decarboxylase Utilizing a High-Volt 6-Azauracil and Its Riboside. Collection Czech. Chem. Commun., 23: age Paper Electrophoresis Technique or an Improved "CCvRelease 2201-2206. 1958. Method. Anal. Biochem., 69: 451-467. 1975. 15. Handschumacher, R. E. Orotidylic Acid Decarboxylase: Inhibition Stud- 27. Scholar. E. M., and Calabresi, P. Identification of the Enzymatic Path ies with Azauridine 5'-Phosphate. J. Biol. Chem.. 235: 2917-2919, 1960. ways of Nucleotide Metabolism in Human Lymphocytes and Leukemia 16. Handschumacher, R. E., Calabresi, P., Welch, A. D., Bono, V., FallÃ³n, Cells. Cancer Res., 33. 94-103, 1973. H., and Frei, E., III. Summary of Current Information on 6-Azauridine. 28. Skoda, J. Azapyrimidine Nucleosides. In: A. C. Sartorelli and D. G. Cancer Chemotherapy Rept., 21: 1-18, 1962. Johns (eds), Antineoplastic and Immuno-Suppressive Agents II. pp. 17. Henderson, J. F., and Khoo, M. K. Y. Synthesis of 5-Phosphoribosyl 1- 348-372. Berlin: Springer Verlag, 1976. Pyrophosphate from Glucose in Ehrlich Ascites Tumor Cells in Vitro. J. 29. Skoda, J., and Sorm, F. The Accumulation of Orotic Acid, Uracil and Biol. Chem..240: 2349-2357, 1965. Hypoxanthine by Escherichia coli in the Presence of 6-Azauracil and the 18. Hisata, T. An Accurate Method for Estimating 5-Phosphoribosyl 1-Pyro- Biosynthesis of 6-AzaUridylic Acid. Collection Czech. Chem. Commun.. phosphate in Animal Tissues with the Use of Acid Extraction. Anal. 24: 1331-1337, 1959. Biochem., 68: 448-457, 1975. 30. Snyder, F. F., Henderson, J. F., Kim, S. C., Paterson, A. R. P., and 19. Jones, M. E. Regulation of Pyrimidine and Arginine Biosynthesis in Brox, L. W. Furine Nucleotide Metabolism and Nucleotide Pool Sizes in Mammals. Advan. Enzyme Regulation, 9: 19-49, 1970. Synchronized Lymphoma L5178Y Cells. Cancer Res., 33: 2425-2430, 20. Mendicino, J. Phosphorylation of Cylidine Monophosphate in Animal 1973. Tissues. J. Biol. Chem., 237. 165-169, 1962. 31. Traut, T. W., and Jones, M. E. Substrate to Product Channeling by a 21. Momparler, R. L., and Fischer, G. A. Mammalian Deoxynucleoside Multi-Enzyme Complex in Pyrimidine Biosynthesis: Orotate Phosphori- Kinases.' I. Deoxycytidine Kinase: Purification, Properties, and Kinetic bosyltransferase-Orotidine-5'-Phosphate Decarboxylase Federation Studies with Cytosine Arabinoside. J. Biol. Chem., 243: 4298-4304, Proc.,36: 876, 1977. 1968.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. Effects of 6-Azauridine on Nucleotides, Orotic Acid, and Orotidine in L5178Y Mouse Lymphoma Cells in Vitro

Claude M. Janeway and Sungman Cha

Cancer Res 1977;37:4382-4388.

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