On the Mechanism of the Nucleoside Stimulation of Amino Acid Incorporation Into Protein of Ehrlich Ascites Tumor Cells in Vitro{

[CANCER RESEARCH 27 Part 1, 1073-1083, June 1967]

On the Mechanism of the Nucleoside Stimulation of Amino Acid Incorporation into Protein of Ehrlich Ascites Tumor Cells in Vitro{

M. L. BELKHODE,- A. M. GOTTO,3 AND O. TOUSTER

Departments of Molecular Hiology and liiochcmistry, Vunderbill University, Nashville, Tennessee 37203

SUMMARY tain congenital hemolytic anemias (55, 56). Possible mechanisms which have been suggested to account for these effects are in The action of the nucleoside stimulators of amino acid in creased formation of ATP (7-9, 19, 24, 44, 45), glutamine (20, corporation into proteins of Ehrlich ascites tumor cells, as well 21), or pentose phosphate (17, 62). as the lesser stimulation by glucose, is blocked by inhibitors of protein synthesis, of energy production, and of glutamine syn- We have reix>rted in preliminary form that certain purine and pyrimidine nucleosides stimulate the incorporation of amino thetase, but not by actinomycin D. Preincubation of cells was acid into the protein of Ehrlich ascites tumor cells (10, 11, 13, required to obtain the maximal stimulation by nucleosides. 15). We have also found an enhancement by inosine of uracil Effects on the uptake of amino acids, on pH, or on cell viability and 5-fluorouracil incorporation into nucleic acids (12) and could not be correlated with the rate of incorporation, nor was there a correlation with the intracellular levels of adenosine tri- have shown that these cells contain a purine nucleoside phosphorylase, which readily catalyzes the conversion of nucleoside phosphate or adenine nucleotides. Although a possible role of to ribose-l-phosphate and free base (14, 41, 47). The aim of glutamine was suggested by several observations, a large part this paper is to present evidence concerning the mechanism of of the nucleoside effect does not seem to involve enhanced syn nucleoside stimulation of amino acid incorporation into protein thesis of glutamine. Preliminary cleavage of the nucleoside to ribose-1-phosphate of Ehrlich ascites tumor cells. appears to be required for stimulation, since only ribonucleosides which are substrates for tumor cell purine and pyrimidine nucleo MATERIALS AND METHODS side phosphorylases were found to enhance amino acid incor Materials. Amino acids were purchased from Mann Research poration. Laboratories, New York, New York. Uracil-2-14C and a-aminoisobutyric acid-l-14C were purchased from Calbiochem, Los INTRODUCTION Angeles, California; all other labeled amino acids were obtained Nucleosides and glucose have been reported to exert a number from New England Nuclear Corporation, Hoston, Massachu of stimulatory effects in ascites tumor cells and erythrocytes, setts. Methionine sulfoximine was obtained from Nutritional e.g., in ascites cells, stimulation of the rate of purine biosynthesis Biochemicals Corporation, Cleveland, Ohio. We are grateful to in vitro (63), of adenine and uracil transport and incorporation other investigators for several chemicals: for DON, R. W. into acid soluble nucleotide (62), and of formate incorporation Brockman, Southern Research Institute, Birmingham, Alabama, into purines (17), nucleic acid, serine, and protein (20, 21). and John Dice, Parke Davis and Company, Detroit, Michigan; In erythrocytes the presence of nucleosides or glucose prolongs for puromycin, N. Bohonas, Lederle Laboratories, Pearl River, the physiologic viability of the cells, leads to an increased syn New York; for the enzymes myokinase, (Â»tato apyrase, and thesis of ATP4 (7-9, 24, 44), and reverses autohemolysis in cer- 5'-adenylic acid deaminase, B. Pogell, Albany Union Medical College, Albany, New York. Other chemicals were of the highest 1This study was supported in part by grants from the National grade commercially available. The late H. B. Goldie, Meharry Science Foundation (G-25126),from the National Cancer Institute Medical College, Nashville, Tennessee, generously provided our of the USPHS (CA-07489),and from the Institutional Grant from culture of Ehrlich ascites tumor cells. the American Cancer Society. Culture and Incubation of Tumor Cells. Ehrlich ascites 2Present address: McGill University Cancer Research Unit, tumor cells were grown intraperitoneally in Swiss Webster mice Montreal, Canada. with weekly transfers. The cells from several mice were [Â»oled, 3Aided by a grant for a Postdoctoral Research Scholarship from susjÃ¬ended,and thrice washed with 0.9% saline at 4Â°C.For the American Cancer Society. Present address: Department of in vitro experiments the washed cells were suspended in 1 volume Medicine, School of Medicine, Harvard University, Boston, Mas of 0.9% saline. To conical flasks (25 ml) were added 0.3 to 0.4 sachusetts. 4The abbreviations used are: ATP, adenosine triphosphate; ml of cell susjiension, a mixture of 20 L-amino acids at a final DON, 6-diazo-5-oxo-norleucine; AMP, adenosine monophosphate; concentration of 1 nui each, salt mixture (49), and (XÃ¬tassium ADP, adenosine diphosphate; U, uniformly labeled; and PÂ¡,in phosphate buffer (pH 7.4) to 5 mM final concentration, in a organic phosphate. total volume of 5 ml. The mixtures were shaken for 2 hr at 37Â°C Received August 1, 1906; accepted January 31, 1967. in a Dubnoff water bath. After this 2-hr period of preincubation,

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. M. L. Belkhode, A. AI. Gotto, and 0. Touster l pe of labeled amino acid (4.2 mamÃ³les) and various comjÃ¬ounds TABLE 1 to be tested were added. Incubation at 37CC was continued for 2 Effect of Nucleosides on the Incorporation of Isoleucine-Â¡ÃCinto hr. Maximum specific activity was observed when 0.20 ml of Protein by Ehrlich Ascites Tumor Cells in Vitro cells was used per 5 ml of reaction medium. Cells were preincubated for 2 hr as described in Materials and Methods. After preincubation, 1 pc (4.17 mn moles) of isoleucine- U-UC and nucleosides (at a final concentration of 5 mM) were \n;ih tir Methods added. Incubation was continued for 2 hr. For details see Mate rials and Methods. U, uniformly labeled. Incorporation of Labeled Amino Acids into Protein. For this purpose, aliquota of 1.0 ml were transferred at timed AdditionsNoneInosineUridineGuanosineCytidineAdenosineThymidineSpecificactivity (cpm/mgprotein)9102992294017741631910720 intervals into 1.0-ml portions of 10% trichloracetic acid. The precipitates were thoroughly dried and then dissolved in 0.50 ml of 0.5 N NaOH. Aliquots of 0.10 ml were placed on discs of Whatman Xo. 3.M.M pa[>cr, 2.3 cm in diameter. The paper discs were washed by the procedure of Mans and Novelli (37). Radio activity was measured in a Packard Tri-Carb scintillation counter. Protein content of the precipitate was determined by analysis of another aliquot by the method of Lowry el al. (36). Measurement of Uptake of Amino Acids. In order to de termine the effect of various compounds on the uptake of labeled of labeled amino acid into protein except that no amino acid amino acids, the cells were incubated in 5 ml of the medium mixture was added. After preincubation, various additions described above. At timed intervals 2.0-ml samples were re were made and the cells were incubated in a total volume of moved, pipetted into 10 ml of salt mixture (49) buffered with 5 5.0 ml for a further 2 hr, at which time samples were removed, mM phosphate (pH 7.4) at 0Â°C,and centrifuged. After centrif heated for 5 min in a boiling water bath, and centrifuged at 2000 ugador, the cells were washed once with the medium and rpm for 10 min. A sample (0.2 ml) of the sujx;rnatant was added extracted for 30 min with 3 ml of 95% ethanol (4, 28). The radio to 1.8 ml of 0.2 M sodium citrate buffer (pH 2.2) and analyzed activity in the ethanol extract was measured in a Packard Tri- with specific interest in the separation of glutamic acid, glycine, Carb scintillation counter. The scintillation liquid (2) was pre and serine plus glutamine, by the procedure of Moore, Spack- pared by dissolving 2.15 gm of PPO (2,5-diphenyloxazole), man, and Stein (42). 17.5 mg of POPOP (1 ,4-di-[2-(5-phenyloxazoly))]-benzene), Measurement of Cell Viability. Cell viability was deter and 22.0 gm of naphthalene in 250 ml of p-dioxane and 50 ml of mined by the uptake of eosin stain by nonviable cells (6). Count xylene. ing of cells was [>erformed in a whole cell counting chamber. Incorporation of L'racil-2-14C into Nucleic Acids. For this purix)se, cells were incubated in the medium described for RESULTS amino acid incorporation except that uracil-2-14C (1 Â¿uc,33.3 Effects of Nucleosides oil Amino Acid Incorporation into m/umoles) was substituted for the radioactive amino acid. Sam Protein: Specificity, Concentration, and Sensitivity to ples (2 ml) from the incubation medium were pipetted into 1 ml I'IIIOIHM Â¡nand p-Eliioropheiiylalanine of 10% trichloracetic acid. The precipitate was washed twice with 1-ml portions of 5% trichloracetic acid at 5Â°C,once with The presence of certain nucleosides enhanced the incori>oration 3 ml of hot ethanol Â¡ether (3:1), and once with 3 ml of ether. of amino acids into the protein of Ehrlich ascites tumor cells by The precipitate was dried at room tem|)erature. Nucleic acids as much as threefold (Table 1). were extracted from the precipitate with 1.5 ml of 0.5 M per This effect was shown with the following amino acids which chloric acid at 80Â°Cfor 30 min (60). Radioactivity of the extract were tested: L-isoleucine, L-leucine, L-glutamine, and glycine. was measured in a Packard Tri-Carb scintillation counter with Of the purine and pyrimidine nucleosides tested, inosine and the p-dioxane-xylene medium described above. undine were the most active, followed by guanosine and then Estimation of Adenine Nucleotides. For the determination eytidine. Adenosine, thymidine, nucleotides, and deoxynucleo- of AMP, ADP, and ATP, cells were incubated in the usual me sides did not stimulate. Deoxyinosine did not stimulate, although dium except that the total volume was increased to 20 ml by it was converted to deoxyribose-phosphate by the cells (A. M. proportionate increases in cells and all components. Aliquots Gotto, M. L. Belkhode, and O. Touster, manuscript in prepa (5 ml) were removed at 0 hr, 1 hr, and 2 hr and centrifuged. To ration). the packed cells were added 2 ml of 5% perchloric acid. The That the enhanced incorporation of isoleucine involved protein tubes were shaken, centrifuged, and decanted. The su]>ernatant synthesis is indicated by the observation that it is abolished by was neutralized with 4 N KOH. After removal of potassium the addition of puromycin at a final concentration of 50 MS/ml l>erchlorate by centrifugal ion, the solutions were analyzed for of medium (specific activity: control, 765; inosine, 4312; puro AMP, ADP, and ATP by the method of M unch-Pe tersen and mycin, 47; inosine plus puromycin, 74). Further evidence for the Kalckar (43). ATP in the solutions was also determined by the involvement of protein synthesis was provided by the fact that luciferase reaction (61). ATP concentrations by the two methods p-fluorophenylalanine (20 nui) blocked the stimulation by ino- were in agreement. sine of isoleucine incorporation (specific activity: control, 1530; Estimation of (iln lumie Acid. Ascites cells were preincu- inosine, 3785; p-fiiioroplienylahmiiie, 275; inosine plus p-fluoro- bated as described for experiments measuring the incorporation phenylalanine, 938). Similar results were obtained when glu-

1074 CANCER RESEARCH VOL. 27

TABLE 2 Effect of the Simultaneous Presence of Inosine and Glucose on 8 Amino Acid Incorporation by Ehrlich Ascites Tumor cells in Vitro 1200 The experimental conditions were as described for Table 1. E Ã–L o Addition Specific activity (cpm/mg protein) t 800 Experiment 1 None 565 0.2 mM glucose 1242 o 400 1 mm glucose 2715 25 mM glucose 1134 0.2 mM inosine 1109 ÃÃ¡ o. 0.2 mM inosine + 0.2 mM glucose 1742 V) 0.2 HIMinosine + 1 mM glucose 2842 8 12 16 20 0.2 mM inosine + 25 mM glucose 1144 Experiment 2 INOSINE (mM) None 596 CHART1.Effect of inosine concentration on the incorporation of 0.2 mM inosine 1311 isoleucine-14C into protein by Ehrlich ascites tumor cells in vitro. 1 mM inosine 3432 Cells were preincubated for 2 hr as described in Materials and 5 mM inosine 4887 Methods, following which time 1 /zc of isoleucine-U-14C (4.17 mju- 0.2 mM glucose 1149 moles) and inosine at varied concentrations were added. Incuba 1 mM glucose 3788 tion was continued for 2 hr. For details see the Materials and 0.2 mM inosine + 0.2 mM glucose 2395 Methods section. U, uniformly labeled. 1 mM inosine + 1 ITIMglucose 4258 5 HIMinosine + 1 mM glucose 4685 6000 Effects of Glucose on Amino Acid Incorporation into Protein: Relationship to the Nucleoside-mediated Stimu Â£ 5000 lation e o. The addition of u-glucose resulted in a .stimulation of amino acid incorporation which was similar in several respects to the f nucleoside-dependent effect. Maximal incorporation occurred 4000 E at a glucose concentration of 1 to 2 HIM.However, in contrast d. u EXPT. 2 to the results obtained with inosine, the further addition of glucose beyond a concentration of 2 HIMresulted in reversal of 3000 the enhancement of incorporation (Chart 2). When glucose and inosine were added simultaneously at relatively low concentra I- tions (less than 1 HIM),their separate effects were approximately o < 2000H additive (Table 2). However, if either glucose or inosine were present at a concentration which gave maximal stimulation, / tAKEXPT. I. I the further addition of the other component did not result in an o additional increase (Table 2). IODO] The stimulation by inosine was abolished at high concentra tions (25 mia) of glucose. A decrease in the pH of the reaction medium of 1 to 2 units was observed in the presence of high glucose concentration. The magnitude of the change in pH was dependent upon the concentration of glucose and of cells. At a 10 15 20 25 concentration of 1 mia or less, incubation with glucose did not GLUCOSE (mM) decrease the pH. Inosine at 5 mM concentration decreases the CHART2.Effect of concentration of D-glucose on the incorpora pH 0.1 to 0.3 of a pH unit. tion of isoleucine-HC by Ehrlich ascites tumor cells in vitro. The In order to rule out the possibility that the effects of nuclco- conditions were as described for Table 1, except that glucose was sides and glucose on the incorporation of labeled amino acid were added instead of inosine. exerted through an influence on pH, the experiments described in the following section were performed. cose (1 ITIM)was substituted for inosine (specific activity: glucose, 2875; glucose plus p-fluorophenylalanine, 941). Effect of pH 011 the Nucleoside- and Glucose-dependent As the concentration of inosine was raised, there was increased Incorporation of Amino Acids incorporation of isoleucine-I4C until a maximum was reached In order to avoid any change of pH during the incubation at about 2 mia (Chart 1). period due to the metabolism of the substrate, the use of a

JUNE 1967 1075

substance for protein synthesis which is depleted during pre incubation, the following experiment was carried out. Cells ~ 2000 were preincubated with phosphate buffer, salts, and amino acids (see Materials and Methods section) in the presence or absence INOSINE of 5 HIMinosine. After preincubation for 2 hr, the cells were rapidly centrifuged at 4Â°Cand transferred to fresh medium in the E 1500 presence or absence of 5 niM inosine. Preincubation with inosine o. had little effect on the incorporation of isoleucine-I4C, since pre o vious exposure to this nucleoside did not lead to increased in corporation when cells were transferred to a medium devoid of > 1000 inosine. Thus, if inosine replenishes a substance which is de pleted during preincubation, this substance was extremely o labile or did not accumulate during the conditions of this ex periment. C 500 Other possible explanations for the preincubation effect, for O example, disappearance of a substance which would otherwise Lu NO INOSINE inhibit the inosine effect or formation of a substance which is O. cn required for the inosine effect, would agree with the experimental 5.0 5.5 6.0 6.5 7.0 7.5 8.0 observations, but there is no direct evidence to support them. PH Effect of Inosiiie oil Cell Viability CHART3. Effect of pH on the inosiiie-stimulated amino acid incorporation. The conditions were the same as for Chart 1, ex Incubation with inosine protected cells against loss of viabil cept that the pH of the incubation mixture was varied as indi ity as estimated by staining ability with 1% eosin (6). There was cated. little loss of viability (17 and 29% in 2 experiments) during the initial 2-hr incubation period, although a significant loss was ob stronger buffer system, viz. 20 IHMtris(hydroxymethyl)amino- served during a further 2 hr in the absence of inosine. The mag nitude of the cell death was insufficient to account for the inosine methane, was employed. Under this condition, there was no stimulation of amino acid incorporation, if one assumes that observed change of pH during the incubation period. Although nonviable cells are unable to incorporate amino acid. stimulation by inosine occurred with the stronger buffer, there was more incorporation of amino acid into protein in the pres ence of the weak buffer. The use of the stronger buffer had little Effect of Inosine on Amino Acid Uptake influence on the stimulation by glucose. These findings indicated The nucleoside-de]>endent stimulation of amino acid incor that the nucleoside and glucose effects are not exerted through a poration could be exerted upon the transport of amino acid into change of the pH of the reaction medium. When tumor cells the cell or upon a subsequent reaction involved in protein syn were incubated in the presence of 5 miÂ«inosine at different pH thesis. An experimental approach to the difficult problem of values, it was evident that pH had very little effect upon the separation of these two processes was made in two ways. First, action of inosine between pH 6.0 and 7.5 (Chart 3). Outside this the transport of a-aminoisobutyric acid was studied. This range there was a decrease in the magnitude of stimulation by compound has been employed to separate the phenomena of inosine. amino acid transport and incorporation (3), owing to the fact that it is actively trans]X>rted into Ehrlich ascites cells but is Effect of Preiiu'iibation on the Nucleoside-dependent not further metabolized. Inosine did not enhance the uptake by Stimulation of Amino Acids the cells of a-aminoisobutyric acid-l-14C under our ex]>erimental conditions. As a second approach to the possible involvement of Nucleosides gave maximal stimulation only if the cells were amino acid transport, the effect of inosine on the labeling of free preincubated in the reaction medium prior to addition of the intracellular isoleucine compared with cell protein was studied. radioactive amino acid. The observation that preincubation also The free intracellular isoleucine-14C was estimated by measuring lowers the specific activity of the control flask suggests a the radioactivity extractable with 9590 ethanol. Although ino- depletion of a substance or substances essential for protein syn sine increased isoleucine incorporation into protein by threefold, thesis. It would then follow that inosine can reverse this deple it was without effect on the intracellular level of free isoleucine- tion, or in fact give abnormally high levels of the essential fac- 14C.These results are suggestive evidence that increased amino tor(s). A similar role has been suggested for purine nucleosides acid transport is not the mechanism of inosine stimulation, but in preserving the physiologic viability of erythrocytes, in which they do not absolutely rule out this possibility. case ATP synthesis was thought to be the critical factor (7-9,

24, 44), and for pyrimidine nucleosides in enhancing formate Effects of Nucleosides and Glucose on the Levels of Ade- incorporation into purines and proteins by Ehrlich ascites tumor iiiiic Nuclcotides and Relationship lo Energy Metabolism cells, in which case glutamine synthesis was pro]Â»sed as the mechanism of action (20, 21). The ]x>ssibleroles of inosine, uridine, and glucose as substrates To test the hypothesis that inosine replenished a critical for energy production were studied by measuring the intraeellu-

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TABLE 3 TABLE 4 Effects of Nucleosides and Glucose on the Content of AMP, ADP, Effect of Glucose on the Content of ATP and the Incorporation of and A TP and the Incorporation of Iso1eucine-liC into Isoleucine-]iC into Protein by Ehrlich Ascites Tumor Protein by Ehrlich Ascites Tumor Cells in Vitro Cells in Vitro" The cells were incubated as described under Materials and Methods, except that the volume of the reaction mixture was Glucose(mM)01250125Length of activity (Â»uÃ±Ã³lesper100 incubation(hr)000222Specific(cpm/mgprotein)75926301471ATPmg cellprotein)162.692.453.3128.4124.7184.4 increased to 20 ml without altering concentrations of the sub stances added. For experimental details see Materials and Meth ods. The adenine nucleotides were all determined by the enzymic procedure of Muneh-Petersen and Kalckar (43). The final con centrations were: inosine, 5 DIM; uridine, 5 mia; and glucose, 25 niM.

of nucleotide per 100mg of activity proteinAMP"0.340.230.020.240.130.230.150.080.160.020.140.050.050.060.0ADP0.280.170.060.210.080.230.140.060.220.020.130.110.110.020.0ATP0.650.940.490.960.350.700.951.051.421.510.670.911.201.831.%Total1.271.340.571.400.561.161.201.191.801.550.941.071.361.911.96cell AdditionsNoneUridineGlucoseInosineInosineincubation (cpm/mg 0 The cells were incubated as described under Materials and (hr)0000iiiii22222Specificprotein)341247113362488222311624784172841163158(Â¿moles Methods, except that the volume of the reaction mixture was increased to 20 ml without altering concentrations of the sub stances added. ATP was estimated by the enzymic procedure of Munch-Petersen and Kalckar (43). ATP, adenosine triphosphate.

+glucoseNoneUridineGlucoseInosineInosine ent, it was possible to dissociate completely the enhancement of amino acid incorporation and of ATP concentration, for the presence of 1 HIMglucose stimulated amino acid incorporation but did not increase the ATP levels (Table 4). When oxidative phosphorylation was uncoupled by 2 min +glucoseNoneUridineGlucoseInosineInosine 2,4-dinitrophenol (58, 59) or when the high energy phosphate pool was depleted by 10 mM 2-deoxyglucose (31-33, 38, 63), the stimulation of amino acid incorporation by inosine (5 HIM) was totally abolished. Stimulation by glucose also was abolished + glucoseLength by these inhibitors. These results indicated that although energy was required to demonstrate the nucleoside and glucose effects, " AMP, adenosine monophosphate; ADP, adenosine diphos- the mechanism of the enhancement cannot be explained by an pluite; ATP, adenosine triphosphate. 6 The fact that there has been a brief time for reaction in the increased formation of ATP or adenine nucleotide. 0-hr samples is discussed in the text. Relationship of Nucleoside Effect to Glutamine Metalw- lisin lar levels of AMP, ADP, and ATP in the presence and absence of these compounds. Although uridine gave slightly more stimula Herscovics and Johnstone (21), from a study of the stimulatory tion of amino acid incorporation than did inosine, it was, by effects of glucose, uridine, and cytidine on the incorporation of comparison, relatively ineffective in raising the ATP level (Table formate-14C into tumor cell protein, obtained evidence that the 3). Estimation of ATP by the luciferase method gave similar three stimulators acted by increasing glutamate levels, thereby values. It will be noted that the presence of inosine led to a net increasing the supply of the presumably rate-limiting amino increase in the total level of adenine nucleotides compared with acid glutamine. The hypothesis that inosine increases amino the initial values. Incubation with both inosine and 25 HIM acid incorporation by increasing the intracellular concentration glucose produced a significant decrease in the inosine-dependent of glutamine was tested in five ways: (a) suppressibility of the stimulation after 2 hr but did not depress the ATP level. effect by methionine sulfoximine, an analog of glutamic acid There are several ]x>ints of interest concerning the action of which inhibits glutamine synthetase (57); (6) effect of inosine glucose. The presence of glucose invariably resulted in a depres on the intracellular level of glutamic acid in the presence and sion of the ATP, ADP, and AMP levels at zero time. Since the absence of methionine sulfoximine; (c) suppressibility by the samples were obtained by chilling with ice and centrifugation glutamine antagonist DON (34); (d) effect of preincubation at 4Â°Cfor5 min, the cells were briefly exixxsed to glucose and the with glutamine on the magnitude of inosine stimulation; and initial samples do not represent a true zero time. Since the (e) studies on the incorporation of glutamine-MC into protein. samples were obtained by chilling with ice and centrifugation at a) Effect of Methionine Sulfoximine on Inosine Stimula 4Â°Cfor 5 min, the cells were briefly exposed to glucose and the tion. In several experiments, methionine sulfoximine was found initial samples do not represent a true zero time. Temporary to block the inosine stimulation of protein synthesis, although it initial depression of ATP levels on the addition of glucose has did not appreciably decrease isoleucine-14C incorporation in previously been observed in several laboratories (22, 23, 25, 26, controls (Table 5). These results suggest that the action of ino 29, 32, 35). During the subsequent incubation with glucose, the sine is dependent on the production of glutamine from glutamic level of ATP increased but the concentrations of AMP and ADP acid. However, although glutamine itself increased incorporation, remained low. When varied concentrations of glucose were prÃ¨s- its action was always additive with that of inosine. Addition of

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TABLE .5 Inhibition of Inosine and Glucose Stimulation by Melhionine â€¢ GLUTAMINE PRESENT DURING PREINCUBATION; Sulfoximine" NO INOSINE ADDED 0 GLUTAMINE PRESENT DURING PREINCUBATION; Specific activity INOSINE (5mM) ADDED AFTER PREINCUBATION Additions (cptn/mg - 6000 protein) o a Experiment 1 o- None 120:> 4800 Inosine 4993 Glutamine 3102 Sulfoximine 1011 Inosine + sulfoximine 1456 3600 Inosine + glutamine 7240 Inosine + sulfoximine + glutamine 7276 Experiment 2 None 2916 2400 Glucose 4093 Sulfoximine 2773 O 2.-)00 ÃœJ Glucose + sulfoximine O_ (/) 1200 " Experimental conditions were as described for Table 1 and under Materials and Methods. Methionine sulfoximine (10 IHM), glutamine (5 HIM), inosine (5 mM), and glucose (1 mM) were pres ent as indicated. Methionine sulfoximine was added at zero time. 0.0 1.0 5.0 25.0 GLUTAMINE (mM) glutamine in the presence of inosine and methionine sulfoximine CHART4. Effect of preincubation of cells with glut amine and its not only reversed the inhibition but also restored incorix>ration effect on the magnitude of inosine stimulation of isoleucine-"C to the level seen with glutamine and inosine together. This incorporation into protein. The conditions are the same as de would suggest that the addition of glutamine not only provided scribed under Materials and Methods except that glutamine was the cell with a substance which is stimulatory to protein syn present during preincubation at the concentrations indicated. thesis but also overcame the blocked inosine effect and per Inosine at a final concentration of 5 mM and isoleucine (1 Â¿ic)were mitted the nucleoside to exert its full stimulatory action. Pos added after 2 hr of preincubation. sibly, glutamine actively competed with methionine sulfoximine for an enzyme site. Hypotheses based on experiments with the sulfoximine depend, of course, on the assumption that the only of the tumor cells with 2 mM DON, whereas the glutamine (5 site of action of the analog is the enzyme glutamine synthetase. mM) enhancement was essentially abolished (specific activity The glucose stimulation of amino acid incorporation similarly after 2-hr incorporation period: control, 641; inosine, 2974; was inhibited by methionine sulfoximine (Table 5). glutamine, 2363; DON, 385; DON plus inosine, 1317; DON b) Kflect of Inosine on the Intracellular Level of Glu- plus glutamine, 382). These experiments indicate that the nucleo lamic Acid ami Glulamine. Since it has been reported that side effect is not mediated entirely through the production of free glutamine cannot be detected in Ehrlich ascites tumor cells glutamine. (30, 50), it was decided to determine whether the addition of d) Kflect of Preincubation -with Glutamine 011 the Mag both inosine and methionine sulfoximine leads to an increase in nitude of the Inosine Stimulation. When Ehrlich ascites glutamic acid (see Materials and Methods). It was observed cells were not preincubated with glutamine, i.e., if glutamine was that both the nucleoside and sulfoximine were required for an omitted from the incubation mixture, the basal level of incor- increase in glutamic acid to occur (Â¿imolesglutamic acid i>er ml |K>ration was increased 5- to 10-fold by the addition of 5 mM of packed cells: control, 1.35; 5 mM inosine, 1.71; 10 HIMmethio inosine (Chart 4). Preincubation with increasing quantities of nine sulfoximine, 1.44; 5 mM inosine plus 10 mM methionine glutamine led to progressive increase in the basal level of in sulfoximine, 4.14). Glycine did not accumulate in increased corporation. This resulted in a diminished calculated percent amounts under these conditions, and the serine plus glutamine of stimulation by inosine. At a 5 IÃ•IMconcentration of glutamine fraction showed a comparatively small increase. These results (which gave optimal stimulation), the addition of inosine in are in close agreement with those of Herscovics and Jolmstone creased incorporation 2-fold above the control value. These (21), who found with other technics that glucose, uridine, or exi>eriments indicate that a significant stimulation by inosine cytidine increased the level of glutamate in ascites tumor cells. occurs even when the cells are saturated with glutamine. <â€¢)Kflect of DON on Inosinc Stimulation. DON is a po Â«â€¢)Incorporation of Glutamine-U-14C into Protein. tent competitor of glutamine and is capable of causing irreversible Glutamine-U-14C was readily incorporated into the protein of inactivation of a glutamine-linked enzyme by an alkylation reac Ehrlich ascites cells. As with other amino acids, the addition of tion (35). The inosine (5 IHM) enhancement of amino acid incor inosine increased the incorporation of this amino acid (specific poration was only moderately reduced by a 2-hr preincubation activity: control, 7205, 6825; inosine, 12958, 11647).

1078 CANCER RESEARCH VOL. 27

2500-"*NXXX\XXX>_ii-ISOLEUCINE-I4C^Â¿/^|Â¿//XÂ¿Â¿X/Â¿^^/!sLU

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ytZ

tUÃ®^ LU _l : Z Z Z? Ã¶0 Ãœu Ã i Ã• S2 0â€”J~a10,000HCO7r///>^///i^gÃ¬1- i S i 00 Ã› J- (_ 2< Z 0 0 < < Â« CHAUT5. Effect of actinomycin 1) on thÃ¨inosine enhancement of isoleucine-l4C incorporation into protein and of uracil-HC incor poration into nucleic acid. The conditions were as described in Materials and Methods.

Role of Rilx>se-phosphate Formation and of RNA Synthe That inosine may serve as a major source of ribose-1-phosphate sis in Inosine Stimulation was clearly shown by its ability to stimulate the incorporation of uracil-I4C into nucleic acid (Chart 5). The sj)ecific activity of the Ehrlich ascites tumor cells contain a nucleoside phosphorylase acid-insoluble nucleic acid fraction was increased from 157 in which catalyzes the following reaction (14, 41): the control to 9025 in the presence of inosine. There was virtu 1'urine nucleoside + PÂ¡^ ribose-1-phosphate + purine base ally no incorporation in the presence of actinomycin D (50 Mg/ml). While actinomycin D completely abolished the inosine- The partially purified enzyme acts u[xm inosine and guanosine. stimulated incorporation of uracil-HC into nucleic acid, it pro The occurrence of a uridine phosphorylase in these cells had been duced only about a 25% decrease of the enhanced incorporation demonstrated previously by Reichard and SkÃ¶ld(53), who pre of amino acid in the same experiment. sented evidence for the involvement of the enzyme in the initial Direct evidence for the ribose-phosphate hy|x>thcsi.s could not step of a pathway for polynucleotide synthesis from free uracil. be obtained. Ribose, ribose-1-phosphate, and ribose-5-phosphate On the assumption that the supply of ribose-1-phosphate is rate did not stimulate amino acid incorporation. Permeability bar limiting, we tested the ability of inosine to serve as a precursor riers may have prevented entry of the phosphorylated deriva for this substance in in vitro experiments. This approach took tives into the cell. Free ribose is poorly metabolized by these advantage of the two phosphorylase reactions involving ribose-1- cells (17). phosphate, i.e., purine nucleoside and uridine phosphorylases, and the subsequent incorjwration of uridine into nucleic acid. DISCUSSION Thus, a coupling of the following reactions was measured by the ability of inosine to enhance the incorporation of uracil into Possible Mechanisms of the Nucleoside Stimulation: nucleic acids: Effect of Preincuhatioii Inosine + Pi^Â±ribose-1-phosphate -f- hypoxanthine The stimulatory actions of micleosides and glucose either share Ribose-1-phosphate + uracil ;=Â±uridineâ€”>â€”>â€”>nucleicacid a common pathway or are exerted via the same mechanism, for

JUNE 1967 1079

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. M. L. Belkhode, A. M. Gotto, and 0. Touster when one com[xmnd was present at a saturating level, the addi Glutamiiie Metabolism tion of the other caused no further stimulation. Their separate Recently, Herscovics and Johnstone (21) have proposed that effects were additive below saturating concentrations. Also, the action of each was blocked by the presence of methionine sul- increased glutamine synthesis is the mechanism of action of glucose and pyrimidine nucleosides in stimulating formate in- foximine. coi'i>oration into the protein and purine nucleotides of Ehrlich Although preincubation was required to demonstrate maximal ascites cells. Glutamine has been shown to be required for pro stimulation by nucleosicles, appreciable stimulation was regularly tein synthesis in these cells (52) and for the growth of tumor cells observed without preincubation. We could not obtain evidence in tissue culture (5). We found that methionine sulfoximine, an that inosine leads to the formation of a metabolically stable inhibitor of glutamine synthetase (57), interfered with the ino- intermediate essential for protein synthesis. The presence of sine stimulation. Its action was readily reversed by adding gluta inosine during preincubation, but not during the incorporation mine. Furthermore, the addition of inosine and methionine ])eriod, did not produce stimulation. Alterations in cell viability sulfoximine led to an increased content of free glutamic acid or pH of the medium could not be correlated with the rate of inside the cell. No such increase occurred if the compounds were incorporation of amino acid into protein. added separately. Similar increases in the intracellular concen tration of glutamate have been reported when Ehrlich cells ATI* and Adeiiine Â¡Vueleotide Metabolism were incubated with glucose, cytidine, or uridine (21). Herscovics and Johnstone (21) suggest that there is a conversion of nucleo- The mechanism of enhancement by glucose and nucleosides side to glutamic acid, since the presence of unlabeled uridine of formate and purine incorporation into nucleic acid has been a lowers the specific activity of glutamic acid formed from glucose- subject of disagreement for several years. Henderson and Le- 14C. Studies in our laboratory indicate that guanosine-14C is indeed converted into glutamate-uC. This work will be the sub Page (19) concluded that the principal role of glucose is to supply energy. The importance of glucose as an energy source for Ehr ject of a separate communication. lich ascites cells is well documented (27, 48, 51). The ex]>eri- In spite of these findings, we have collected sufficient evidence to conclude that the glutamine hypothesis cannot account for mental data of this paper clearly demonstrated that glucose the nucleoside effect on amino acid incorporation. This con and inosine can lead to an increased adenine nuclcotide synthesis clusion is based on the inability of glutamine to abolish the in these cells. These results are in close agreement with those of inosine effect, on the ability of DON to block completely stimu Yushok et al. (65) but would seem to differ from those of Thomp lation by glutamine but not by inosine, and, most important, on son et al. (62), who found that glucose did not increase the ATP the stimulation of glutamine-14C incorporation into protein by content. However, since the length of incubation was only 45 inosine. If the function of inosine were merely to increase the min in these experiments (62), there is no real discrepancy be intracellular level of glutamine, it would be expected to dilute tween these results and those of the present paper. The fact that out the incorporation, rather than enhance it. Thus, although uridine, or glucose at 1 mm concentration, stimulates amino acid the action of nucleoside apparently is dependent upon the con incoriÂ»ration without increasing the ATP levels indicates a version of glutamate to glutamine, and although nucleoside mechanism other than energy production under these ex[>eri- can increase the level of glutamate and, by inference, of gluta mine and can overcome the effect of an apparent depletion of mental conditions. glutamine, the glutamine hypothesis cannot account for the A comment should be made concerning the very low levels of stimulation of amino acid incorporation into protein by nucleo ATP, ADP, and AMP found in the early time samples in solu sides. tions to which glucose was added. After the initial loss of adenine nucleotides, the presence of glucose subsequently led to an in Amino Acid Transport creased intracellular content of ATP but not of ADP or AMP. Our studies revealed that inosine did not enhance the trans The failure of ADP to increase may be caused by a high rate of port of a-aminoisobutyric acid, a compound which is actively ATP generation or by a relative block in the utilization of ATP. trans]K>rted but not incorporated into protein by Ehrlich ascites High levels of ATP with concomitant low levels of ADP have cells (3). Furthermore, the presence of inosine did not alter the also been described in the erythrocytes of premature infants intracellular concentration of isoleucine but simultaneously in (16). In a subsequent study in this laboratory (A. W. Meikle, creased its incorjKiration into protein threefold. It has been re]x>rted by Riggs and Walker (54) that amino acid transport A. M. Gotto, and O. Touster (40), it was found that during the is much more rapid in Ehrlich ascites cells than amino acid period of ATP depletion there was excreted into the medium a incorporation and that incorporation could be stimulated by material with an absorption sj)ectrum similar to that of inosine. increasing the intracellular concentration of free amino acid only This phenomenon apj>ears to be similar to ones described by until the incorporation system was saturated. Our data indicate Letnansky (33) and McComb and Yushok (39) in Ehrlich and that altÃ©rationin amino acid transport does not account for the Krebs ascites cells in the presence of 2-deoxyglucose and by Wu inosine effect. and Racker (64) in Ehrlich ascites cells in the presence of glucose and iodoacetate. However, the depletion induced by glucose alone Ribosr Phosphate 1'rodurtioii is subsequently reversed because of the further metabolism of When Harrington found that the action of glucose on formate glucose-6-phosphate incorporation into purines could be duplicated by certain pyrimi-

10NO CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. Nudeoside Stimulation of Amino Acid Incorporation cline nucleosides, she postulated that ribose phosphate formation amino acid analyses and to Mrs. Vera Coleman for the cell viabil was the mechanism of action (17, 18). Thompson et al. (62) pre ity measurements. sented evidence to support the ribose-phosphate hypothesis and failed to substantiate the increased ATP formation suggested by REFERENCES Henderson and LePage (19). If the explanation for inosine stimu 1. Abrams, R., Edmonds, M., and Libenson, L. Deoxyribosyl lation were the provision of ribose phosphate for nucleotide and Exchange Activity Associated with Nucleoside Phosphorylase. nucleic acid synthesis, then actinomycin D might be expected Biochem. Biophys. Res. Commun., 20: 310-314, 196.5. to interfere. The absence of such interference means that general 2. Bray, G. A. A Simple Efficient Liquid Scintillator for Counting DXA-directed RNA synthesis is not involved. It is conceivable Aqueous Solutions in a Liquid Scintillation Counter. Anal. that some particular nucleic acid fraction, the synthesis of which Biochem., /: 279-285, 1960. was insensitive to actinomycin, was being formed, although there 3. Christensen, H. N. Active Transport with Special Reference to the Amino Acids. Perspectives Biol. Med., a: 228-242, 1959. is no evidence to support this explanation for the effects reported 4. Coles, N. W., and Johnstone, R. M. Glutamine Metabolism in herein. Although a general stimulation of nucleic acid synthesis Ehrlich Ascites-Carcinoma Cells. Biochem. J., 83: 284-291, does not appear to be the basis of the inosine effect, there is 1962. strong evidence that inosine can provide ribose-1-phosphate by 5. Eagle, R. Nutrition Needs of Mammalian Cells in Tissue Cul means of the purine nucleoside phosphorylase demonstrated to ture. Science, Igg; 501-504, 1955. occur in Ehrlich ascites cells (14, 41). This purine nucleoside 6. Eaton, M. D., Scala, A. 11., and Jewell, M. Methods for Meas phosphorylase, acting in conjunction with uridine phosphorylase uring Viability of Ascites Cells: Dye Exclusion and Respira (53), can account for the large increase of uracil incorporation tion as Affected by Depletion, Poisons, and Viruses. Cancer into nucleic acid when inosine was added. Thus, the provision Res., 19: 945-953, 1959. by nucleoside of ribose-1-phosphate is clearly shown. There is 7. Gabrio, B. W., Donohue, D. M., and Finch, C. A. Erythrocyte strong evidence that this conversion is essential for the nucleo Preservation. V. Relationship between Chemical Changes and Viability of Stored Blood Treated with Adenosine. J. Clin. side effect on protein synthesis. Deoxyinosine failed to stimulate Invest., 34: 1509-1512, 1955. incorporation, although it is split by the cells. Those ribonucleo- 8. Gabrio, B. W., Finch, C. A., and Huennekens, F. M. Erythro sides (inosine, guanosine, uridine) which are substrates of cyte Preservation: A Topic in Molecular Biochemistry. Blood. nucleoside phosphorylases present in tumor cells (14, 41, 53) 11: 103-113, 1956. show the stimulatory effect on amino acid incorporation. Cyti- 9. Gabrio, B. W., and Huennekens, F. M. The Role of Nucleoside dine, which has recently been found in our laboratory to be a Phosphorylase in Erythrocyte Preservation. Biochim. Bio phosphorylase substrate, stimulates moderately, while thymi- phys. Acta, 18: 585-586, 1955. dine is neither a substrate nor a stimulator. Only the behavior of 10. Gotto, A. M., Belkhode, M. L., Hester, R. W., Siler, R., and adenosine requires additional comment. It was generally inactive Touster, O. Protein Synthesis in Ascites Tumor Cells: En hancement by Nucleotides and Nucleosides. Biochim. Bio in stimulating incorporation, although occasionally a small phys. Acta, 80: 163-165, 1964. stimulation was observed. Partially purified purine nucleoside 11. Gotto, A. M., Belkhode, M. L., and Touster, O. Aspects of phosphorylase shows considerable activity toward adenosine, Nucleoside and Glutamine Metabolism in Ehrlich Ascites presumably as a result of its prior deamination to inosine (14). Tumor Cells. Federation Proc., 24: 597, 1965. It seems likely that the rate of ribose-1-phosphate formation 12. Gotto, A. M., Belkhode, M. L., and Touster, O. The Enhance from adenosine by whole cells is too slow to enhance amino acid ment by Inosine of the Incorporation of C14 Uracil and C14 incorporation. Fluorouracil into Nucleic Acids of Ascites Tumor Cells. Proc. It is possible that the explanation for the nucleoside effect Am. Assoc. Cancer Res., 5: 22, 1964. resides in a quantitatively minor transformation in the cell which 13. Gotto, A. M., Belkhode, M. L., and Touster, O. Mechanism of is highly significant in a particular subcellular compartment. Nucleoside Enhancement of Amino Acid and Uracil Incorpora tion by Ascites Tumor Cells. In: Proceedings of the 6th In Present evidence suggests that study of the further metabolism ternational Congress of Biochemistry, New York, IUB Vol. of ribose-l-phosphate will be required for an understanding of 32, 1-64, p. 56, 1964. the sjiecific mechanism of the nucleoside stimulation. The prob 14. Gotto, A. M., Meikle, A. W., and Touster, O. Nucleoside lem has been made more difficult by the fact that the nucleoside Metabolism in Ehrlich Ascites Tumor Cells: Phosphorolysis of stimulation is not demonstrable in cell-free preparations. More Purine Nucleosides. Biochim. Biophys. Acta, 80: 552-561, over, recent work (1, 47) has shown that the purine nucleoside 1964. phosphorylase of Ehrlich ascites tumor cells has both phos- 15. Gotto, A. M., Touster, O., Belkhode, M. L., Hester, R. W., and phorolytic and ribosyl transfer activity and that these two Siler, R. Effects of Nucleosides and Nucleoside Phosphates on types of catalytic activity are interconvertible (47). Perhaps the Incorporation of Amino Acids into Proteins of Ascites Carcinoma Cells. Federation Proc., 22: 237, 1963. such potentially important alterations in enzyme activity, 16. Gross, R. T., Schroeder, E. A. R., and Brounstein, S. A. En governed by intracellular conditions, influence the cellular re ergy Metabolism in the Erythrocytes of Premature Infants sponse to nucleosides. Compared to Full Term Newborn Infants and Adults. Blood, gl: 755-763, 1963. ACKNOWLEDGMENTS 17. Harrington, H. Effect of Glucose and Various Nucleosides on Discussions with Dr. H. M. Johnstone and the technical assist Purine Synthesis by Ehrlich Ascites Tumor Cells In Vitro. ance of Mr. Gordon Page are gratefully acknowledged. We are J. Biol. Chem., Â«33:1190-1193, 1958. indebted to Dr. L. W. Cunningham and Mr. J. 1). Ford for the 18. Harrington, H. Effect of Cytidine on Purine Nucleotide For-

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Mechanism of Action of of Purine Compounds in Ehrlich Ascites Tumor Cells: Evi Glucose and Pyrimidine Nucleosides on I4C Formate Utiliza dence for a Salvage Pathway of Inosine Metabolism. Biochim. tion by Ehrlich Ascites Cells. Biochim. Biophys. Acta, 91: Biophys. Acta, 1S8: 445-451, 1967. 365-373, 1964. 41. Meikle, A. W., Gotto, A. M., and Touster, O. Purine Nucleo- 22. Hess, B., and Chance, B. Metabolic Control Mechanisms. VI. side Phosphorylase in Ehrlich Ascites Tumor Cells: Charac Chemical Events After Glucose Addition to Ascites Tumor terization and Possible Metabolic Role. Federation Proc., 23: Cells. J. Biol. Chem., 236: 239-246, 1961. 168, 1964. 23. Hess, B., and Chance, B. ÃœberZellulare Regulationsmecha- 42. Moore, S., Spackman, D. H., and Stein, W. II. Chromatography nismen und ihr Mathematisches Modell. Naturwissenschaften, of Amino Acids on Sulfonated Polystyrene Resins. Anal. 46: 248-257, 1959. Chem., 30: 1185-1190, 1958. 24. Huennekens, F. M. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. On the Mechanism of the Nucleoside Stimulation of Amino Acid Incorporation into Protein of Ehrlich Ascites Tumor Cells in Vitro

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

Cancer Res 1967;27:1073-1083.

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