Author Institution: Department of Physiological Chemistry, Ohio State Universit

Matthews, Richard H.

Sardovia-Iyer, Marlinda R.

The Ohio Journal of Science

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Copyright © 1980 Ohio Acad. Sci. 0030-0950/80/0005-0220 $1.00/0BRIEF NOTEAMINO ACID LEVELS IN SARCOMA 37 ASCITES TUMOR CELLSCOMPARED WITH ASCITIC FLUID AND NORMAL MURINE TISSUES1MARLINDA R. SARDOVIA-IYER and RICHARD H. MATTHEWS, Department of Physio-logical Chemistry, Ohio State University, 1645 Neil Avenue, Columbus, OH 43210OHIO J. SCI. 80(5): 220, 1980The functional characteristics of 2amino acid transport systems known asSystems A and L have been studied asthey occur in Sarcoma 37, (S37), murineascites tumor cells (Matthews et al 1970,Matthews 1972, Matthews et al 1975,Matthews et al 1977, Matthews and Zand1977). In order to examine the physio-logical significance of these active trans-port systems, we have measured levels ofindividual endogenous amino acids inS37 cells in vivo and in the surroundingcell-free ascitic fluid. For comparativepurposes, we also measured levels of in-dividual amino acids in blood plasma andin several normal murine tissues.Analyses were performed on pooledsamples from 5 S37 tumor-bearing mice.Solid tissues were freed of fat and con-nective tissues, and at least 1 g of solidtissue was used in each case. Freshlydrawn blood, or ascites fluid (10 ml), wasused for analysis. Picric acid was usedfor deproteinization and was subse-quently removed using Dowex 2-X8 resin.Amino acids were eluted from the resinusing 0.02 N HC1. The eluate was con-centrated by lyophilization and sampleswere neutralized with NaOH. After 4hours at 25 °C to convert cysteine tocystine, samples were adjusted to pH 2,transferred to a volumetric flask, and di-luted to 5 ml using pH 2.2 acetate buffer.Amino acid analyses were carried outusing a Beckman model 120-C aminoacid analyzer. Cystine and valine, glu-tamine and asparagine, as well as thebasic amino acids, were not well resolvedby the methods employed and are not in-xManuscript received 21 May 1979 and in re-vised form 17 October 1979 (#79-31).eluded. The results suggest that most ofthe amino acids of S37 cells have beenaccounted for in that the total aminoacids measured for the S37 cell in thepresent study (32.3 mM) exceeded slightlythe level of the total intracellular aminoacid pool measured in another prepara-tion of S37 cells by a non-discriminatingmethod.Earlier measurements showed aminoacid levels were high in rapidly-growingtissues and in cancers in particular (Hol-den 1962, Ryan and Lorincz 1964). Inthe present study, total and neutralamino acids were higher for S37 cells thanfor any normal tissues assayed (tables 1and 2). Acidic amino acids were higher inbrain than in S37 cells due to the glutamicacid, which has a central metabolic rolein brain (Neame 1968). High totalamino acid and neutral amino acid levelswere maintained in S37 cells despite thefact that the ascites fluid possesses lowertotal and neutral amino acid levels thandoes blood plasma. This finding thatascites fluid is poorer in amino acids thanblood plasma is in accordance with thestudy by Drewes and McKee (1967) us-ing Ehrlich tumors, and it suggests thatdiffusion of amino acids from plasma toascites fluid is followed by active trans-port into tumor cells. We attribute themoderately high level of glutamic acid inS37 cells to metabolism rather than trans-port since S37 cells transport acidicamino acids relatively ineffectively. Glu-tamic acid is formed in metabolism bytransamination of the amino group fromother amino acids to a-ketoglutarate de-rived from the Krebs tricarboxylic acidcycle.If we sum essential amino acids and220Ohio J. Sci. AMINO ACIDS IN TUMOR CELLS 221TABLE 1Amino Acid Levels in Murine Tissues and Fluids.*AminoAcidPheTyrLeulieMetAlaGlvProSerThrGluAspAscitesFluid0.130.050.340.140.061.010.630.370.090.390.280.23BloodPlasma0.340.250.900.580.212.331.86trace0.490.741.260.19S37Cells0.410.120.810.440.329.005.823.640.653.447.300.35Liver0.400.321.160.620.614.472.51trace0.470.621.840.50Muscle0.290.200.760.460.293.733.401.030.750.800.790.35Braintracetrace0.460.180.161.962.63none0.543.5811.000.21*Units are ^moles/g for solid tissues and /xmoles/ml for fluids.nonessential amino acids and computethe ratio (E/N) we note that it is mar-ginally lower in ascites fluid than inplasma and markedly lower in S37 cellsthan in normal tissues (table 2). Thelow E/N ratio in S37 cells may reflect 2aspects of cancer cell metabolism: first, acontinuing protein synthesis and minimalprotein breakdown associated withgrowth. The tendency of tumor cells togrow to the limits of essential amino acidsavailable was suggested by tissue cultureexperiments in which it was shown thatnormal cells arrest in culture due to limi-tations in peptide growth factors, whereastransformed cells arrest due to limitationsin glucose and some essential amino acids(Moses et al 1978). Second, stimulatedgrowth has been associated with in-creased activity of transport system A,which favors several nonessential aminoacids as substrates (Tramacere, Borghettiand Guidotti 1977). This stimulation ofsystem A is reflected in the present studyin the concentrating of several typical Asystem substrates (ala, gly, pro, ser andthr) by the S37 cell (table 1). Thus, the2 factors of use of essential amino acidsin ongoing protein synthesis and a highactivity of a transport system favoringmany nonessential amino acids leads tothe observed low E/N ratio in the S37cell.Elevated amino acid levels may con-tribute to the unregulated growth char-acteristic of tumor cells in ways apartfrom supplying material for protein syn-thesis. Increased levels of amino acidsappear to stimulate protein synthesis(Clemens and Korner 1971). Further-TABLE 2Totals of Various Amino Acid Groups.*GroupNeutralAcidicTotalEssentialNonessentialE/N Ratio**AscitesFluid3.210.513.721.062.660.40BloodPlasma7.701.459.152.776.380.43S37Cells24.657.6532.305.4226.880.20Liver11.182.3413.523.4110.110.34Muscle11.711.1412.852.6010.250.25Brain9.5111.2120.724.3816.340.27*Units are ^moles/g for solid tissues and yumoles/ml for fluids.**Essential to nonessential amino acid ratio.222 M. R. SARDOVIA-IYER AND R. H. MATTHEWS Vol.more, glycine and glutamic acid arecomponents of glutathione, which is syn-thesized and consumed in the 7-glutamylcycle in a manner that may supportamino acid transport. Some evidence forthe operation of the 7-glutamyl cycle insupport of transport systems L and Ahas been presented (Matthews and Sar-dovia 1975). 7-Glutamyl-cysteine syn-thetase has a Km value of 1.6 mM forglutamic acid (Orlowski and Meister1971), and glutathione synthetase has aKm of 2 mM for glycine (Mooz andMeister 1967). The levels of glycine andglutamic acid in the S37 cell (table 1) farexceed these Km values, so that thesereactions would be catalyzed at maximumvelocity in the S37 cell. This raises thepossiblity of a positive feedback mech-anism contributing to metabolic instabil-ity, since the 7-glutamyl cycle's opera-tion at maximal velocity could work toincrease amino acid transport, and in-creased amino acid transport could becontributing to the operation of the 7-glutamyl cycle at maximal velocity.LITERATURE CITEDClemens, M. J. and A. Korner 1971 Activa-tion of the protein synthetic capacity of ratliver cell sap by amino-acids. Nature, NewBiol. 232: 252-253.Drewes, P. A. and R. W. McKee 1967 Amino-acid composition of ascitic fluid and bloodplasma from mice bearing Ehrlich-Lettretumour. Nature 213: 411-412.Holden, J. T. 1962 Amino Acid Pools. El-sevier, New York. 815 pp.Matthews, R. H. 1972 Characteristics of atransport system serving for the transfer ofhistidine into S37 ascites tumor cells. Bio-chim. Biophys. Acta 282: 374-382., C. A. Leslie and P. G. Scholerleld 1970Histidine uptake and exchange in S37 ascitestumor cells. Biochim. Biophys. Acta 203:457-463.and 1975 The energetic basis ofamino acid transport into S37 ascites tumorcells. Biophys. J. 15: 311a.-, , N. J. Lewis and R. Zand 1975Biphasic kinetic plots and specific analogsdistinguishing and describing amino acidtransport sites in S37 ascites tumor cells.Biochim. Biophys. Acta 394: 182-192.f .G.E.Milo and N.J.Lewis 1977Effects of cysteine upon tumor cells. CancerBiochem. Biophys. 2: 65-69.and R. Zand 1977 Basis for substratepreference of amino acid transport system Lover amino acid transport system A. Bio-chemistry 16: 3820-3824.Mooz, E. D. and A. Meister 1967 Tripeptide(glutathione) synthetase. Purification, prop-erties, and mechanism of action. Biochem-istry 6: 1722-1734.Moses, H. L., J. A. Proper, M. E. Volkenant,D. J. Wells and M. J. Getz 1978 Mechanismof growth arrest of chemically transformedcells in culture. Cancer Res. 38: 2807-2812.Neame, K. D. 1968 Transport, metabolismand pharmacology of amino acids in brain.In: A. N. Davison and J. Dobbing (eds.), Ap-plied Neurochemistry. F. A. Davis, Phila-delphia. 119-177 pp.Orlowski, M. and A. Meister 1971 Isolationof highly-purified •y-glutamylcysteine syn-thetase from rat kidney. Biochemistrv 10:372-380.Ryan, W. L. and A. B. Lorincz 1964 Freeamino acids of human breast cancer. Ne-braska State Med. J. 49: 321-325.Tramacere, M., A. F. Borghetti and G. G.Guidotti 1977 Serum-mediated regulationof amino acid transport in cultured chick em-bryo fibroblasts. J. Cell. Physiol. 93: 425-434.

Amino Acid Levels in Sarcoma 37 Ascites Tumore Cells Compared With Ascitic Fluid and Normal Murine Tissues'

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Amino Acid Levels in Sarcoma 37 Ascites Tumore Cells Compared With Ascitic Fluid and Normal Murine Tissues'

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