[CANCER RESEARCH 29,807—811,April1969]

Stimulatory Effects of Inosine and Deoxyinosine on the Incorporation @ of@ 4C, ‘IC,and ‘ICinto Nucleic Acids by Ehrlich Ascites Tumor Cells in Vitro1

A. M. Gotto,2 M. L. Belkhode,3 and 0. Touster

Department ofMolecular Biology, Vanderbilt University, Nashville, Tennessee 37203

SUMMARY been demonstrated. The transfer of pentose from nude osides to bases is the subject of this communica The incorporation of -2-14C into the nucleic acids of tion. Evidence is presented for the stimulation by inosine of Ehrlich ascites tumor cells is increased 10- to 50-fold by the the incorporation of uracil-2-14C into nucleic acids by Ehrlich addition of inosine. Maximum stimulation is obtained at an ascites cells. This phenomenon may be utilized to investigate inosine concentration of 1 mM . Actinomycin D (50 j.Lg/ml) purine and pyrimidine interconversions. The study, abolishes incorporation. The effect of inosine seems unlikely which has obvious therapeutic implications, was extended to to be mediated by an enhancement of synthesis of ATP, measure the incorporation into nucleic acids of the halo 5-phosphoribosyl-1-pyrophosphate, or nucleic acids by de genated , 5-fluorouracil and 5-bromouracil, and to novo pathways. Unlike uracil incorporation, the labeling of compare the effects of inosine with those of glucose, deoxy by -2-14C is extensive in the absence of inosine, and 6-mercaptopurine as potential inosine. The incorporation of 5-fluorouracil-2-14 C into RNA stimulators of pyrimidine base incorporation. A preliminary also is greatly increased by inosine. Inosine has little effect on account of this work has been presented (5). the incorporation of 5-bromouracil-2-14 C into nucleic acids, whereas deoxyinosine is a potent stimulator. Deoxyinosine, on MATERIALS AND METhODS the other hand, is a relatively weak enhancer of uracil incorpo ration. These results suggest that -1-phosphate and Materials deoxyribose-1-phosphate may be rate limiting for the incorpo ration of uracil (or 5-fluorouracil) and 5-bromouracil into The isotopically labeled and bases were pur RNA and DNA respectively. Inosine and deoxyinosine serve as chased from Schwarz Bioresearch, Inc., Orangeburg, New precursors of ribose- 1-phosphate and deoxyribose- 1-phos York, and Calbiochem, Los Angeles, California. The specific phate. These interconversions involve purine and pyrimidine activities of the isotopes employed were as follows: uracil-2- nucleoside phosphorylases. The possible application of these 14C, 30 pc/pmole; 5-fluorouracil-2-14C, 20 pc4tmole; 5- findings in is discussed. bromouracil-2-14 C, 8.8 j.td/j.zmole; 14C, 25 pc/pmole. All other chemicals were of the highest purity commercially INTRODUCTION available.

The ribose moiety of pyrimidine may be Methods transferred by intact Ehrlich ascites tumor cells to externally supplied to form free nucleosides (20) or to Incorporation of Uradil-2-'4C, 5-Fluorouradil-2-'4 C, and form the purine of nucleic acids (9, 10). With both 5-Bromouradil-2-14 C into Nucleic Acids. Ehrlich ascites carci whole cell (20) and cell-free preparations (24) of this tumor, noma cells were grown for 6 to 7 days in the peritoneal cavi the reciprocal process, i.e., the transfer of purine ribonucleo ties of Swiss Webster mice, removed aseptically, and washed 3 side ribose (inosine and ) to pyrimidine bases, has times at 4°Cwith 0.9% NaCl by centrifugation for 5 min at 500 X g. The washed cells were suspended in 1 volume of 0.9% saline. To conical flasks (25 ml) was added 0.3 to 0.4 ml of 1This study was supported in part by grants from the National cell suspension, potassium phosphatebuffer (pH 7.4) to a final Science Foundation (G-25126), the National Cancer Institute of the concentration of5 mM, a mixture of salts (23), and a mixture USPHS (CA-07489), and an Institutional Grant from the American of the usual 20 L-amino acids found in most proteins at a final Cancer Society. concentration of 1 mM each. The incubation mixture, in a 2j@jded by a grant for a Postdoctoral Research Scholarship from the total volume of 5 ml, was shaken for 2 hr at 37°C in a Dub American Cancer Society. Present address: Molecular Disease Branch, National Heart Institute, NIH, Bethesda, Maryland. noff water bath. After this 2-hr period of preincubation, 1 pc 3Present address: Radiation Protection Division, Department of Na of the appropriate radioactive nucleoside or base and various tional Health and Welfare, Ottawa, Canada. compounds to be tested were added. At timed intervals, ali Received June 10, 1968; accepted November 22, 1968. quots of 1.0 ml or 2.0 ml were removed and pipetted into an

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. A. M. Gotto, M. L. Belkhode, and 0. Touster equal volume of 10% trichloroacetic acid. The total length of Table 1 incubation was 2 hr. The precipitate was removed by cen activity trifugation and washed twice with 1.0-rnl volumes of 5% ReactionUnits)1A. flask AdditionSpecific (cpm(hr/10 A2@ trichloroacetic acid at 5°C,once with 3.0 ml of hot : IncorporationC1 of uracil-'4 ether (3: 1), and then once with 3.0 ml of ether at room tem None1572 perature. Inosine90253 After the precipitate was dried at room temperature, nucleic Glucose19084 D25 Actinomycin acids were extracted with 1.5 ml of 0.5 N perchloric acid at Inosine41lB. Actinomycin D + 80°C for 30 mm. Radioactivity was measured in a Packard Tri-Carb scintillation counter. An aliquot (0.5 ml) of the cx IncorporationC6 of uridine-'4 None38327 tract was placed in 5.0 ml of scintillation liquid (2) and count Inosine9181 ed for 10 mm or until 1,000 total counts were recorded. Specific activity was calculated on the basis of the absorbancy Incorporation of uracil-2-'4C and uridine-2-14C into nucleic acids by Ehrlich ascites cells. Cells were preincubated for 2 hr as described in Mate at 260 m,.L of the perchloric acid extract and was expressed as rials and Methods. After preincubation, 1 zc of uracil-2-'4C (1A) or 1 @Lcof cpm incorporated/hr/b A260 units. A specific activity of uridine-2-14C (1B) and inosine (5 mM), glucose (25 mM), or actinomycin D 4,500 cpm/hr/10 A260 units corresponded approximately to (50!Jg/ml)were added as indicated. Incubation was continued for 2 hr. an incorporation of 0.07 mj.zmole of uraci/hr/mg protein cell suspension. In the presence of inosine, the specific activity 151 !.LgATP/100 mg protein; plus inosine, 165 @gATP/100 mg varied from 0.03 to 0.15 [email protected] of uracil incorporated/hr/mg protein. Experiment 2: control, 133 pg ATP/100 mg protein; protein. The usual range of values in the presence of inosine plus inosine, 140 jig ATP/100 mg protein). Glucose (25 mM) was 0.05 to 0.10. Duplicate samples varied less than 5% in a was a less effective stimulator than inosine of uracil-2-'4 C single experiment. However, there was considerable variation incorporation (Table 1). between different batches of cells. The variability between batches of cells was less when inosine was present (usually 2- to 3-fold but rarely as much as 5-fold variation) than when it was omitted (5- to 10-fold variation). Also, it was observed that a 2-hr period of preincubation decreased the variability in different experiments. In all experiments, however, with or without preincubation and despite variability of incorporation, (I, inosine exerted similar effects on the incorporation of uracil I— and 5-fluorouracil. Omission of the 2-hr period of preincuba z tion did not change the results qualitatively with either uracil 0 to or 5-fluorouracil although the magnitude of the stimulatory c@J effects was decreased (Table 5). A similar preincubation effect 0 has been described for the inosine-mediated stimulation of incorporation by Ehrlich cells (1). The mechanisms 0. involved have not been clarified. 0 Counting efficiency was 65 to 70%. The total quantity of >- radioactivity incorporated into nucleic acids did not exceed I-. 10% of the counts added as tracer in any experiment. > Measurement of Intracellular Al? Levels. ATP was assayed F- by the luciferase reaction (27) or by the method of Munch 0 Petersen and Kalckar (16). A description of the aliquots used and of the experimental conditions has been given previously 0 LL (1). w0 RESULTS U)

Effect of Concentration of Exogenously Added Inosine on Intracellular Level of AlP and on Uradil-2-'4C Incorporation into Nucleic Acids. The incorporation of uracil-2-14C into the nucleic acids of Ehrlich ascites cells was increased as much as 0 0.2 0.4 0.6 0.8 1.0 5.0 50-fold by the addition of 5 mM inosine (Table 1). Actino mycin D (50 j.tg/ml) inhibited incorporation in the presence or CONC. OF INOSINE(mM) absence of inosine. Maximal stimulation was obtained at an Chart 1. Effect of inosine concentration on the incorporation of inosine concentration of 1 mM (Chart 1). Reproducible stimu uracil-2-14C into nucleic acids by Ehrlich ascites tumor cells in vitro. lations were observed at inosine concentrations of 0.1 to 0.2 For experimental conditions, see Table 1 and Methods. Each flask con mM . At a concentration of 0. 2 mM , the intracellular level of tamed 1pc ofuracil-2-14C. Specific activity is based on the radioactivity ATP was little affected after 1 hour (Experiment 1: control, incorporated over a period of 1 hour.

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Incorporation of Uridine-2-'4 C into Nucleic Acids. Uridine 3Incubation Table 2-14C labeled nucleic acids much more extensively than did units)Experiment systemSpecificactivity (cpm/hr/10 A2@ uracil-2-14C in the absence of inosine (Table 1). Compared to A its effect with uracil, inosine was a relatively weak stimulator 1. No incubation of the incorporation of uridine-2-14 C (Table 1). However, it 2. Tris buffer 19,946 should be noted that uracil-2-14C and uridine-2-14C produced 3. Tris buffer + DNase 21,000 similar labeling of nucleic acids in the presence of inosine. 4. Tris buffer + RNase 11,600 Effects of 6-Mercaptopurine and its Ribonucleoside on 10,980Experiment5. 1.0 N perchloric acid at 4°C22,874 Uracil-2-'4 C Incorporation into Nucleic Acids. The addition B of 6-mercaptopurine did not interfere with the stimulation by 1. Noincubation 4,561Experiment2. 1.0 N perchioric acid at 4°C6,100 inosine (Table 2). It was of interest that the ribonucleoside of 6-mercaptopurine was as effective as inosine in increasing the C labeling of nucleic acids by uracil (Table 2). 1. No incubation Release of Radioactivity from Labeled Nucleic Acids by 2. 1.0 N perchloric acid at 4°C4,237 3,444 RNase and Cold Perchioric Acid Extraction. Ehrlich ascites Effects of RNase, DNase, and cold perchioric acid extraction on the cells were incubated with uradil-2-14C and inosine (5 mM) as 14C-labeled nucleic acids of Ehrlich ascites cells. Experimental conditions are as described in Methods and Ref. 1, except that 5 i.tcof uracil-2-14C described in Methods. The nucleic acid fraction was isolated in were used in Experiment A and the entire reaction mixture incubated 2 hr. the usual way (see Methods). Treatment of the nucleic acid Inosine was present at a concentration of 5 mM. Aliquots were removed and fraction with RNase for 15 mm released approximately 5CY@of taken through the following steps: 1 . Precipitation and washing of nucleic the incorporated radioactivity so that it was not precipitated acids with 10% trichloroacetic acid at 4°C.2. Washing with hot ethanol:ether (3: 1). 3. Washingwith ether. The precipitates from Step 3 were treated in by 10% trichloroacetic acid (Table 3). The limited release of various ways. In Incubation System 1 of Experiments A, B, and C the radioactivity was possibly due to the brief exposure to RNase. precipitate was extracted directly with 0.5 N perchloric acid for 30 mm at Extraction for 18 hr with 1 N perchloric acid at 4°Cextracted 80°Candused for counting. Whereindicated, the precipitate was incubated 50 to 80% of the incorporated radioactivity (Table 3). This 15 mm at 37°Cin 100 mM Ti-is buffer, pH 7.2, or with Tm buffer contain procedure has been shown to extract RNA predominantly, ing 1 mg DNase or RNase. After the period of incubation (Experiment A, Incubation Systems 2—4), nucleic acid was again precipitated and washed but, as in the present experiments, the extraction is not usu with 10% tricloroacetic acid at 4°C.The precipitates were extracted for 30 ally quantitative (17, 18). Incubation with DNase, on the miii at 80°C with 0.5 N perchloric acid, and the extracts thus obtained were other hand, did not release a significant quantity of acid used for counting and determination of specific activity. Finally, a precipi soluble radioactivity. These results indicate that, under our tate from Step 3 above was incubated 18 hr at 4°Cwith 5 ml of 1.0 N experimental conditions, most of the measured incorporation perchloric acid. After centrifugation, an aliquot of the supernatant was used for counting. It should be noted that, in Systems 2—4ofExperiment A, from uracil-2-14C is in RNA. specific activities are based on the radioactivity that is still precipitable (i.e., Incorporation of 5-Fluorouracil-2-14C into Nucleic Acids. not hydrolyzed or acid soluble) with 10% trichloroacetic acid after the van As observed with uracil, the incorporation of 5-fluorouracil-2- ous incubations. In System 5 from Experiment A and System 2 from Exper 14 C into nucleic acids is markedly enhanced in the presence of iments B and C, activity was based on the radioactivity and absorbancy at 5 mM inosine and to a lesser extent in the presence of 25 mM 260 nip of the material which was solubilized with 1.0 N perchloric acid at 4°C. glucose (Table 4). The magnitude of the labeling is greater when a period of preincubation is employed. This effect may reflect a depletion of intracellular uracil or of an intermediate 4AdditionSpecific Table compound in the incorporation of the tracer uracil into units)5A activity (cpm/hr/10 A260 nucleic acid. The incorporation is blocked by actinomycin D .PreincubatedNone22Glucose2154Inosine4096SB. and is progressively diminished as the ratio of uracil to 5- fluorouracil is increased (Table 5). Effects of Inosine and Deoxyinosine on the Incorporation of Uradil-2-'4 C and S-Bromouracil-2-'4 C into Nucleic Acids. NotpreincubatedNone78Glucose904Inosine2779 Inosine increases the incorporation of uracil-2-14 C much more

2SystemSpecificactivityTable Incorporation of 5-fluorouracil-2-'4C into nucleic acids by Ehrlich ascites cells. Each reaction flask contained 1 @icof5-fluorouracil-2-'4 C. The van units)Control236Inosine45066-MP1686-MP (cpmfhr/10 A2@ ous additions, as indicated above, were inosine (5 mM) and glucose (25 mM). In 5A, as described in Methods, the cells were preincubated in the medium for 2 hr at 37°Cprior to addition of the isotope and of the compound to be tested. In 5B, the preincubation period was omitted, and riboside52926-MP all additions were made, therefore, at zero time. For experimental condi +inosine44986-MP tions, see Table 1 and Methods. riboside + inosine4696

Effects of inosine, 6-mercaptopurine (6-MP), and 6-MP riboside on the effectively than does deoxyinosine (Table 6A), corroborating incorporation of uracil-2-14C into nucleic acids by Ehrlich ascites tumor cells. The concentration of inosine, 6-MP,and 6-MPriboside was 5 mM. The the evidence cited previously that uracil labels primarily RNA. conditions were as described in Table 1 and Methods. Each flask contained However, when 5-bromouracil-2-14 C is substituted for uracil I @Lcofuracil-2-14C. 2-14C, deoxyinosine becomes a potent stimulator while mo

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Table 5Specific presence of 5-phosphonibosyl-1-pyrophosphate (PRPP)] to activitySystemUracil/5.fluorouracil(cpm/hr/10 inosine monophosphate (15). Whole cell preparations of units)Control063Inosine09740Inosine A260 Ehrlich cells incorporated inosine into nucleic acids (15). The strong stimulatory action of 6-mercaptopunine niboside and the relatively weak activity of deoxyinosine suggested the +uracil204125Inosine importance of the nibose moiety to the inosine effect. A gen +uracil502200Inosine+uracil100820Inosine eral stimulation of the de novo synthesis of nucleic acids was made unlikely by the fact that 6-mercaptopurine riboside en +uracil500526Inosine +uracil1000257Inosine hances the labeling of nucleic acids just as effectively as does + actinomycin076 inosine. Although a small loss of cell viability, as judged by staining Effect of uracil on the incorporation of 5-fluorouracil-2-14C into nucleic acids of Ehrlich ascites tumor cells. Each reaction flask contained 1 pc of 5- with eosin, occurred during the 2-hr period of preincubation, fluorouracil-2-14C (50 mpmoles). The molar ratio of uracil to 5-fluoroura and there was a small decrease in intracellular concentrations cil is indicated above. The concentration of actinomycin D was 50 @.sg/ml of ATP and , the cells were nonetheless able when present. For experimental conditions, see Table 1 and Methods. to perform active transport of amino acids, to incorporate amino acids into protein, and to restore the intracellular level sine has much less effect (Table 6B). In separate experiments it of ATP if inosine or glucose was added (1). A plausible ex was found that the small stimulation by inosine is blocked by planation for the effect of inosine on uracil and 5-fluorouracil the addition of 5 mM uracil. Glucose (25 mM ) also is ineffec incorporation is that it replaced a rate-limiting substance tive in stimulating the incorporation of 5-bromouracil-2-14C. which became partially or completely depleted during the period of preincubation. Omission of inosine or of preincuba DISCUSSION tion thus introduced a greater degree of variability into the experiments. Three such substances which might have been The labeling of RNA by uradil-2-14C in these experiments rate limiting are ATP, PRPP, and ribose-1-phosphate. Although was indicated by the inhibition by actinomycin D, the release inosine increased the intracellular levels of ATP (1), it stimu of radioactivity from the nucleic acid fraction by RNase or lated the incorporation of uracil by 3- to 4-fold at a concentra cold perchloric acid, the failure of DNase to release radioactiv tion (0.2 mM) which did not affect the level of ATP. The ity, and the 6-fold greater potency of inosine than deoxyinose provision of PRPP seemed unlikely to explain the inosine ef in promoting incorporation. While these data do not permit an fect since inosine is a more effective stimulator of uradil incor absolute measurement of the radioactivity in RNA, 5-fluoro poration than is glucose (Table 1), but glucose is a better uracil-2-14C and uracil-2-14C were incorporated to similar precursor of PRPP (12). The incorporation ofsmall quantities extents in the presence of inosine. It has been shown pre of uracil should not be dependent on the provision of PRPP, viously that 5-fluorouridine is incorporated into RNA but not since PRPP synthesis occurs at an appreciable rate as measured into DNA (8). by synthesis from purine bases (1 1). Further The mechanism of the inosine-mediated stimulation of uracil more, we are unaware of any report on the detection of un and 5-fluorouracil incorporation is probably related to the dine monophosphate pyrophosphorylase in mammalian cells. metabolic fate of this nucleoside. Ehrlich ascites cells have The possibility that ribose-1-phosphate is rate limiting for been shown to possess enzymic activities for the phospho uracil incorporation was suggested by the extensive labeling of rolysis of inosine to hypoxanthine and nibose-1-phosphate (6, nucleic acids by uridine-2-14C in the absence ofinosine (Table 19), for the phosphorylation of inosine to inosine monophos 1). The marked increase of uracil incorporation into nucleic phate (21, 22) and for the conversion of hypoxanthine [in the acids which inosine produced could be effected by purine nucleotide phosphorylase (5, 19), unidine phosphorylase (25), and uridine kinase (26), acting in conjunction with the estab 6System Table lished reactions for nucleic acid synthesis.

units)6A. Specific activity (cpmlhr/10 A260 The possibility of enhancing the antitumor activity of 5- fluorouracil by the addition of inosine was suggested by our uracil-2-14CControl90Inosine3211Deoxyinosine5046B.Incorporation of results. In this regard it is of interest that glucose, which also increases uracil and 5-fluorouracil incorporation into nucleic acids, has been found to potentiate the inhibition by 5- fluorouracil of Flexuer-Jobling carcinoma (14). In a recent CControl184Inosine250Deoxyinosine3007Incorporation of 5-Bromouracil-2-14 preliminary report on human and mouse leukemia cells, inef fective doses of 5-fluorouracil were therapeutic in combination with inosine (7). Inosine and glucose increased the formation Effects of inosine and deoxyinosine on the incorporation of uracil-2-14 C of nucleotides, and it was suggested that the enhanced anti and 5-bromouracil-2-14C into nucleic acids by Ehrlich ascites tumor cells. tumor activity was related to the formation of 5-fluorouracil The concentration of inosine and deoxyinosine was 5 mM. In 6A, each flask riboside. contained 1 pc of uracil-2-14 C. In 6B, each flask contained 2 pc of 5-bromo The labeling of nucleic acids by 5-bromouracil-2-14C in the uracil-2-14C. Where indicated, inosine and deoxyinosine were present at a concentration of 5 mM . For experimental conditions, see Table 1 and presence of deoxyinosine (Table 6), the lack of incorporation Methods. of 5-bromouracil into mammalian DNA (see Ref. 13 for re

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. Incorporation of Uracils into Nucleic Acids view), and the incorporation of 5-bromouracil deoxyriboside 12. Henderson, J. F., and Khoo, M. K. Y. Synthesis of 5-Phospho by mammalian cells into DNA (4) suggested that provision of ribosyl l-Pyrophosphate from Ribonucleosides in Ehrlich Ascites deoxynibose-1-phosphate is rate limiting for the conversion of Tumor Cells in Vitro. J. Biol. Chem., 240: 2363—2366, 1965. this base analog to nucleic acid. Except for one strain of yeast 13. Karnofsky, D. A., and Clarkson, B. D. Cellular Effects of Antican (seeRef.3 for review),5-bromouracilanditsribosidehavenot cer Drugs. Ann. Rev. Pharmacol., 3: 357—427, 1963. been found to label RNA. 14. Kung, S., Goldberg, N. D., Dahl, J. L., Park, R. E., and Kline, B. E. Potentiation of 5-Fluorouracil Inhibition of Flexner-Jobling Carci noma by Glucose. Science, 141: 627—628, 1963. ACKNOWLEDGMENTS 15. Meikle, A. W., Gotto, A. M., and Touster, 0. The of The technical assistance of Mr. Gordon Page is gratefully acknowl Purine Compounds in Ehrlich Ascites Tumor Cells: Evidence for a edged. Salvage Pathway of Inosine Metabolism. Biochim. Biophys. Acta, 138: 445—451,1967. REFERENCES 16. Munch-Petersen, A., and Kalckar, H. M. Determination ofATP and ADP in Tissue Filtrates. In: S. P. Colowick and N. 0. Kaplan (eds.), 1. Belkhode, M. L., Gotto, A. M., and Touster, 0. On the Mecha Methods in Enzymology, Vol. 3, pp. 869—871. New York: Aca nism of the Nucleoside Stimulation of Amino Acid Incorporation demic Press, Inc., 1957. into Protein of Ehrlich Ascites Tumor Cells in Vitro. Cancer Res., 17. Munro, H. N., and Fleck, A. The Determination of Nucleic Acids, 27: 1073—1083, 1967. Methods ofBiochemical Analysis, 14: 113—176,1966. 2. Bray, G. A. A Simple Efficient Liquid Scintillation for Counting 18. Ogur, M., and Rosen, G. The Nucleic Acids ofPlant Tissue. I. The Aqueous Solutions in a Liquid Scintillation Counter. Anal. Bio Extraction and Estimation of Desoxypentose Nucleic Acid and chem., 1: 279—285, 1960. Pentose Nucleic Acid. Arch. Biochem. Biophys., 25: 262—276, 3. Brockman, R. W., and Anderson, E. P. Biochemistry of Cancer 1950. (Metabolic Aspects). Ann. Rev. Biochem., 32: 463—512, 1963. 19. Paterson, A. R. P., and Sutherland, A. Metabolism of 6-Mercapto 4. Cheong, L., Rich, M. A., and Eidinoff, M. L. Introduction of the punine Ribonucleoside by Ehrlich Ascites Carcinoma Cells. Can. J. 5-Halogenated Uracil Moiety into Deoxyribonucleic Acid of Mam Biochem. Physiol.,42:1415—1423,1964. malian Cells in Culture. J. Biol. Chem., 235: 1441—1447,1960. 20. Paterson, A. R. P. The Synthesis of Extracellular Ribonucleosides 5. Gotto, A. M., Belkhode, M. L., and Touster, 0. The Enhancement by Ascites Tumor Cells in Vitro. Can. J. Biochem. Physiol., 43: by Inosine of the Incorporation of C14-Uracil and C14-Fluoro 257—269, 1965. uracil into Nucleic Acids of Ascites Tumor Cells. Proc. Am. Assoc. 21. Pierre, K. J., Kimball, A. P., and LePage, G. A. The Effect of Cancer Res., 5: 22, 1964. Structure on Nucleoside Kinase Activity. Can. J. Biochem. 6. Gotto, A. M., Meikie, A. W., and Touster, 0. Nucleoside Metabo Physiol., 45: 1619—1632, 1967. lism in Ehrlich Ascites Tumor Cells: Phosphorolysis of Purine 22. Pierre, K. J., and LePage, G. A. Formation of Inosine-5'-Monophos Nucleosides. Biochim. Biophys. Acta, 80: 552—561, 1964. phate by a Kinase in Cell-Free Extracts of Ehnlich Ascites Cells in 7. Hall, T. C., Kessel, D., Goodsill, A., and Roberts, D. Unidine Vitro. Proc. Soc. Exptl. Biol. Med., 127: 432—440, 1968. Phosphorylation, An Overlooked Pathway?; 5-Fluorouridine, Ne 23. Rabinovitz, M., Olson, M. E., and Greenberg, D. M. Independent glected Drug? Proc. Am. Assoc. Cancer Res., 9: 27, 1968. Antagonism of Amino Acid Incorporation into Protein. J. BioL 8. Harbers, E., Chaudhuri, N. K., and Heidelberger, C. Studies of Chem., 210: 837—849,1954. Fluorinated Pyrimidines. VIII. Further Biochemical and Metabolic 24. Reichard, P., and Sköld, 0. Formation of Uridine Phosphate from Investigations. J. Biol. Chem., 234: 1255—1261, 1959. Uracil in Extracts of Ehrlich Ascites Cells. Acta Chem. Scand., 11: 9. Harbers, E., and Heidelberger, C. Studies on Nucleic Acid Bio 17—23, 1957. synthesis in Ehrlich Ascites Cells Suspended in a Medium Per 25. Reichard, P., and Sköld, 0. of Uracil Metabolism in mitting Growth. J. Biol. Chem., 234: 1249—1254, 1959. Ehrlich Ascites Tumor and Mammalian . Biochim. Biophys. 10. Harrington, H. Effect of on Purine Nucleotide Formation Acta, 28: 376—385, 1958. in Ascites Tumor Cells in Vitro. Biochim. Biophys. Acta, 68: 26. Sköld, 0. Unidine Kinase from Ehrlich Ascites Tumor: Purification 509—518, 1963. and Properties. J. Biol. Chem., 235: 3273—3274, 1960. 11. Henderson, J. F., and Khoo, M. K. Y. Availability of 5-Phospho 27. Strehler, B. L., and McElroy, W. D. Assay of Tn ribosyl 1-Pyrophosphate for Ribonucleotide Synthesis in Ehrlich phosphate In: S. P. Colowick and N. 0. Kaplan (eds.), Methods in Ascites Tumor Cells in Vitro. J. Biol. Chem., 240: 2358—2362, Enzymology, Vol. 3, pp. 871—873. New York: Academic Press, 1965. 1957.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. Stimulatory Effects of Inosine and Deoxyinosine on the Incorporation of Uracil-2- 14C, 5-Fluorouracil-2-14C, and 5-Bromouracil-2- 14C into Nucleic Acids by Ehrlich Ascites Tumor Cells in Vitro

A. M. Gotto, M. L. Belkhode and O. Touster

Cancer Res 1969;29:807-811.

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