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 phos- phorylase, 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-amino- isobutyric 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, JUNE 1967 1073 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.

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