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The Competitive Effect of Canavanine on Utilization of Arginine in Growth of Walker Carcinosarcoma 256 Cells in Vitro* PAULF. KRUSE,JR., ANDTHOMASA. McCov (Biomédical Division, The Samuel Roberts Noble Foundation, Inc., Ardmore, Okla.) aseptically; solutions were sterilized by passage through Selas filters. Each treatment was made in triplicate, for a duration of 72 hours. In the canav anine experiments it was necessary to replenish amino acids differ structurally only in the replace the media at the end of 48 hours, but this was not necessary in the ornithine or citrulline studies. ment of a méthylène group by oxygen: After 72 hours' incubation, growth response was NH NH2 determined by cell counts. I Arginine<->H2N—C—NH—CH2—CH2—CHr-CH—COOH L-Canavanine • H2S042 was added to produce canavanine/arginine molar ratios between 0.5 and NH NH2 10.0, and these ratios were studied at three differ II I Canavanine<-*H2N—C—NH—O—CH2—CH2—CH—COOH ent arginine concentrations (0.2, 0.6, and 1.0 HIM). In the ornithine-citrulline studies, arginine was Accordingly, it was of interest to study arginine- replaced by 0.02-10.0 mM concentrations of either canavanine relationships in actively multiplying L-ornithine-HCl or L-citrulline. Later, 0.02 mM tumor cells. Arginine has been shown to be essen L-arginine-HCl (one-tenth the usual concentra tial for the growth of the Walker carcinosarcoma tion in Medium 3) was included to insure the 256 in vitro (8) ; and, with the recent developments maintenance of approximately two-thirds of the in tissue culture (9, 11) in this laboratory, it was initial inoculum during the 72-hour incubation possible to study the effects of canavanine on argi period. Thus, sufficient cells were present to yield nine metabolism during tumor cell growth. The a response, if any, to the supplements. Further present report describes this investigation as well tests with citrulline included the addition of vary as related growth experiments with ornithine and ing amounts of aspartic acid or ammonium chlo citrulline. ride, with and without adenosine triphosphate (ATP). MATERIALS AND METHODS C14 experiments.—Twelve replicate cultures Tissue cultures.—The experimental technics of were made. After incubation for 48 hours, two of tissue culture of the Walker tumor in T-15 tissue the cultures were counted; the medium in the culture flasks have been described (9). Except for other ten was replaced with fresh medium con the changes in concentration of arginine and the taining 1.0 HIML-arginine-U-C14-HCl, ca. 350,000 additions of canavanine, the amino acid composi counts/min/mg.3 The medium in five of the flasks tion of the medium was composed of only the four also contained 1.0 mM L-canavanine'HjSO.!. At teen essential amino acids of Medium 3 (8), 1.0 the end of 24 hours of incubation, the cultures were mM DL-serine, and 0.5 mM glycine. The concentra tion of vitamins, antibiotics, salts, glucose, and chilled in a refrigerator for several hours to free the dialyzed human serum was the same as that re cells from the glass. Cell counts showed that the ported for Medium 2 (11), except that inositol was cultures had progressed uniformly within each raised to 9 fig/ml.1 All operations were performed group, and their contents were combined. The cells in each group were then centrifuged, washed * Presented in part at the 132d National Meeting of the Some striking examples of antagonism between amino acids in microbiological systems have been those dealing with the interference of arginine by canavanine (4, 17-22). These naturally occurring American Chemical Society, New York, N.Y., September 8-13, 1957. 1R. E. Neuman, this laboratory, unpublished data. Received for publication September 30, 1957. 2Obtained from California Foundation for Biochemical Research. 'Purity established by radioscan of paper strip chromatogram. 279 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research. Cancer Research 280 with ice-cold Earle's balanced salt solution, ex tracted with 80 per cent alcohol, alcohol-ether (1:1), cold 10 per cent trichloroacetic acid (TCA), and hot 5 per cent TCA, according to the method of Schneider¿(14).The protein residues were hydrolyzed in 6 N HC1 for 22 hours and evaporated in vacuo to dryness. The evaporation was repeat ed after addition of water, and the residues were dissolved in 10 ml. of water. Arginine radioassays were performed by pre cipitant-carrier methods (7). All assays were made in a windowless gas-flow sealer; glycine was dis solved in aliquots of the cell extracts taken for L-ARGININE-HCI • -0.2 rtiM x —0.6 mM °— I.OmM Vol. 18, April, 1958 the inhibition of growth of the tumor cells by canavanine was of a competitive nature with arginine. The essentiality of arginine in the tissue culture medium (8) was emphasized in the present study by the failure of either ornithine or citrulline to replace arginine, as shown in Table 1 (although some alleviation of cell death was apparent at the higher concentrations of citrulline). Analysis of the citrulline by ion exchange and paper chromatography failed to reveal any arginine impurity. These analytical methods were designed to detect as little as 0.1 per cent arginine impurity. Therefore, the slight response of citrulline was unexplained, since a higher concentration of arginine impurity would be necessary to yield the same alleviation of cell death (8). For practical purposes, however, the cells were incapable of utilizing either of the two common precursors of arginine. The same results were obtained when, in addition to citrulline, TABLE 1 GROWTHRESPONSESTUDIESOF WALKERCAR CINOSARCOMA 256 CELLSCUL TUREDin Vitro* Argininewas replacedby ornithine or citrulline. SUPPLEMENT CONCEKTHAT10N (mu) 0 o ^ RATIO; L-CANAVANINE-H?S04/ L-ARGININE-HCI (mM) ' CHAKT1.—Growth response studies with arginine and canavanine of Walker carcinosarcoma 256 cells cultured in vitro. For culture conditions see text. assay purposes in order to make all counts in the 3.4-4.3 mg/sq cm weight range (7). Assays of the TCA extracts were made by first evaporating ali quots to dryness with a few drops of l N HC1 in glass counting containers. RESULTS In the 0.2-, 0.6-, and 1.0-mM arginine control cultures with no canavanine, the cells increased 5.9-, 3.7-, and 4.3-fold, respectively. Chart 1 illus trates the subsequent inhibitions found when ca navanine was added. It can be seen that the degree of inhibition was due primarily to the molar ratio of antimetabolite to metabolite and was inde pendent of the absolute concentrations of the amino acids. The inhibition indices, selected as the ratios which permitted only 50 per cent of the in crease in cells in the controls, were found to be 1.2, 1.2, and 1.1. The constancy of these indicated that PlR CENT DECREASE IN CELL COUNTsf L-Ormthine-HCl L-Citrulline 87 87 0.02 82 73 0.05 (ÃŒ7 80 0.2 fiS 78 1.0 7i 65 5.0 74 43 10.0 74 19 * For culture conditions, see text, t The decrease in cells divided by the initial inocu lum, 500,000 cells/T-15 flask. varying amounts of aspartic acid or NH4C1 and ATP were included in the medium. Subsequent studies of the mechanism of growth inhibition by canavanine were confined solely to the interference of the utilization of arginine by the cells. These were performed with the use of L-arginine-C14 with and without an equimolar amount of L-canavanine. Table 2 shows that canavanine inhibited the utilization of arginine quite uniformly throughout the cell extracts and in the cell protein. In both experiments, the total of the extracts and protein radioactivities was 25 per cent of the amount of arginine utilized, and the activity of each protein hydrolysate was found to be due only to incorporated arginine. There was a close correlation between the decrease in utiliza tion of arginine (29 per cent) and the decrease in the protein-bound arginine (25 per cent). Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research. KRUSE AND McCoY—Effect of Canavanine DISCUSSION The severe toxicity of canavanine on tumor cell growth was particularly interesting, since canava nine is a naturally occurring amino acid. For the purposes of discussion, however, comparisons will be made with microbiological systems, since the authors have been unable to find any studies con cerning canavanine and its effect on tumor growth in vitro. Horowitz and Srb studied the effect of canavanine on three wild strains of Neurospora (4). While the sensitivity and/or resistance to canavanine appeared to be genetically determined, an inhibition quotient of 1.2 (canavanine/arginine) was reported for the complete growth inhibi tion of the most sensitive strain. In the present study, an inhibition ratio of 1.2, which permitted only 50 per cent of maximum growth, was also re markably small, since Woolley has pointed out that inhibition or antibacterial indices for most competitive compounds have been calculated to range from 100 to 10,000 (24). Some interesting comparisons were made by Volcani and Snell (21) of the effect of canavanine on eight strains of bacteria differing in their ar ginine requirements. In two strains which could utilize only preformed arginine, L. mesenteroides and S. faecalis, canavanine was without effect. This was shown also by Suzuki, Muraoka, and Konobu (18, 19). Other bacteria which could syn thesize arginine from inorganic substances, ornithine, or citrulline were all inhibited by canava nine. The situation in the present system is some what the reverse. While the Walker 256 cells re quired preformed arginine for growth, they were markedly inhibited by canavanine. The cause of resistance in S. faecalis has been shown to be due to one major (5) and one minor (6) canavanine-destroying enzyme. The former catalyzed the reductive cleavage of canavanine to guanidine and homoserine. Apparently, this en zyme is not present in the Walker 256 cells. Fur thermore, the present experimental data indicated the absence of arginosuccinase (2), since the cells could not utilize citrulline. There is no doubt that the cause of canavanine inhibition of the present system rested only with its interference of the utilization (and not the synthesis) of arginine. The experiments with arginine-C14 indicated that cell growth, arginine utilization, and arginine incorporation were in hibited to approximately the same extent as the result of supplementation with canavanine. These results, as well as the radioactivities noted in the various cell extracts, inferred that canavanine ex hibited a general inhibition of arginine metabolism on Walker 256 281 and did not affect any specific area of its utiliza tion. In addition, the C14 studies have revealed an other interesting facet of tumor metabolism. Only 25 per cent of the arginine utilized could be ac counted for in the cellular components. This, coupled with the fact that the total radioactivity in the protein hydrolysates was due only to argi nine, indicated that the carbons from arginine were extensively converted to some substance(s) other than protein-bound amino acids. TABLE2 DISTRIBUTION OFRADIOACTIVITY IN CELLFRACTIONS OFWALKER CARCINOSARCOMA 256 The cells were grown in vitro* in the presence of L-arginineC14with and without L-canavanine. At C-At Total countfl/min Total counta/min Medium: Initial arginine radioactivity 1,000,000 1,000,000 Final arginine radioactivity 790,000 850,000 Arginine utilized 210,000 150,000 Cells: 80 per cent alcohol extract 2,600 1,700 Alcohol-ether (1:1) extract 1,000 50 Cold 10 per cent trichloroacetic 900 700 acid extract Hot 5 per cent trichloroacetic 1,900 1,100 acid extract Residual protein (as hydroly45,700 34,100 sate) Growth: Number of cells at 72 hours 12,900,000 11,700,000 Number of cells at 48 hours 6,200,000 6,200,000 Increase in number of cells 6,700,000 5,500,000 * For culture conditions see text; the cells were cultured on unlabeled medium for 48 hours, followed by 24 hours in medi um containing 1.0 millimolar L-arginine-C"-HCl, 350,000 counts/min/mg. 11.0 millimolar L-arginine-C'4-HCl. 11.0 millimolar L-arginine-C"-HCl and 1.0 millimolar L-ca navanine • BUSO4. While canavanine has been found to inhibit the multiplication of certain viruses (12, 13), negative tumor regression data were reported with the use of in vivo systems (3, 15, 16). These latter results might be explained by known enzymes in the mammalian liver and kidney, which catalyze urea production (1) and transamidination to glycocyamine (10) and arginine (23) from canavanine. SUMMARY Canavanine was found to be a competitive an tagonist of the utilization of arginine by Walker carcinosarcoma 256 under tissue culture condi tions, with an inhibition index of 1.2 for 50 per cent of maximum growth. The tumor cells failed to utilize either ornithine or citrulline in place of Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research. 282 Cancer Research arginine. Carbon-14 studies have indicated that the inhibition rests in a general interference with the utilization of arginine. Some postulates have been made concerning the absence of certain en zyme activities in the Walker 256 cells. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Merle Maxwell, Henry Carter, Sarah Seldin, and Nancy Hubbell. REFERENCES 1. DAMODARAN, M., and NARAYANAN, K. G. A. A Compara tive Study of Arginase and Canavanase. Biochem. J., 34: 1449-59, 1940. 2. FINCHAM,J. R. S., and BOYLENJ. B. Neurospora crossa Mutants Lacking Arginosuccinase. J. Gen. Microbio!., 16:438-48, 1957. 3. FREEDLANDEH, B. L. Chemotherapy Studies on Trans planted Mouse Tumors. Cancer Research (Suppl.), 2:59, 1955. 4. HOROWITZ,N. H., and SRB, A. M. Growth Inhibition of Neurospora by Canavanine and Its Reversal. J. Biol. Chera., 174:371-78, 1948. 5. KIHARA,H.; PRESCOTT,J. M.; and SNELL,E. E. The Bac terial Cleavage of Canavanine to Homoserine and Guanidine. J. Biol. Chem., 217:497-503, 1955. 6. KIHARA,H., and SNELL,E. E. The Enzymatic Cleavage of Canavanine to O-Ureidohomoserine and Ammonia. J. Biol. Chem., 226:485-95, 1957. 7. KRUSE, P. F., JR.; PATTERSON,M. K., JR.; and McCoT, T. A. The Isolation of Glycine, Alanine, Leucine, Arginine, and Glutamic Acid for Radiometrie Assay in Tracer Ex periments. Arch Biochem. & Biophys., 66:146-54, 1957. 8. McCoT, T. A.; MAXWELL,M.; and NEDMAN,R. E. The Amino Acid Requirements of the Walker Carcinosarcoma 266 in Vitro. Cancer Research, 16:979-84, 1956. 9. McCoy, T. A., and NEUMAN,R. E. The Cultivation of Walker Carcinosarcoma 256 in Vitro from Cell Suspen sions. J. Nat. Cancer Inst, 16:1221-29, 1956. 10. NAKATSU,S. Enzymatic Transamidination from Canava nine to Glycine by Hog Kidney Extracts. J. Biochem. (Japan), 43:675-81, 1956. Vol. 18, April, 1958 11. NEDMAN,R. E., and McCoT, T. A. Dual Requirement of Walker Carcinosarcoma 256 in Vitro for Asparagine and Glutamine. Science, 124:124-25, 1956. 12. PEARSON,H. E.; LAOERBOHO, D. L.; and WINZLER,R. J. Effect of Certain Amino Acids and Related Compounds on Propagation of Mouse Encephalomyelitis Virus. Proc. Soc. Exper. Biol. & Med., 79:409-11, 1952. 13. PILCHER,K. S.; SOIKE,K. F.; SMITH,V. H.; TROSPER,F.; and FOLSTON,B. Inhibition of Multiplication of Lee Influ enza Virus by Canavanine. Proc. Soc. Exper. Biol. & Med., 88:79-86, 1955. 14. SCHNEIDER,W. C. Phosphorus Compounds in Annual Tis sues. I. Extraction and Estimation of Desoxypentose Nu cleic Acid and of Pentose Nucleic Acid. J. Biol. Chem., 161:293-303, 1945. 15. SKIPPER,II. E., and THOMSON,J. R. Test of Compounds against Certain Mouse Tumors and Leukemias. Cancer Research (Suppl.), 2:172, 1955. 16. STOCK,C. C.; CLARKE,D. A.; PHILIPS, F. S.; and BAR CLAY,R. K. Sarcoma 180 Inhibition Screening Data. Can cer Research (Suppl.), 2:219, 1955. 17. SUZUKI,T., and MUBAOKA,S. Antimetabolites of Amino Acids. II. Antagonism of Canavanine to Arginine with Lactobacillus Fermenti. J. Pharm. Soc. Japan, 74:534-37; 1954; "Abstr.," Chem. Abstr., 48: 10116, 1954. 18. SUZUKI,T.; MUHAOKA,S.; and KONUBU,K. Antimetabo lites of Amino Acids. III. Antagonism of Canavanine and Canaline to Arginine by Lactobacillus arabinosus. J. Pharm. Soc. Japan, 74:537-40, 1954. 19. . Antimetabolites of Amino Acids. IV. Relation of Canavanine Inhibition to Arginine Requirement of Micro organisms. Ibid., pp. 540-43. 20. TEAS, H. J. Effect of Canavanine on Mutants of Neuro spora and Bacillus subtilis. J. Biol. Chem., 190:369-75, 1951. 21. VOLCANI,B. E., and SNELL,E. E. The Effects of Canav anine, Arginine, and Related Compounds on the Growth of Bacteria. J. Biol. Chem., 174:893-902, 1948. 22. WALKER,J. lì.Canavanine and Homoarginine as Antimetabolites of Arginine and Lysine in Yeast and Algae. J. Biol. Chem., 212:207-15, 1955. 23. — . Biosynthesis of Arginine from Canavanine and Ornitbine in Kidney. Ibid., 218:549-56, 1956. 24. WOOLLEY,D. W. A Study of Antimetabolites, p. 67. New York: John Wiley & Sons, Inc., 1952. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research. The Competitive Effect of Canavanine on Utilization of Arginine in Growth of Walker Carcinosarcoma 256 Cells in Vitro Paul F. Kruse, Jr. and Thomas A. McCoy Cancer Res 1958;18:279-282. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/18/3/279 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.