[CANCER RESEARCH 37, 2523-2529, August 1977] Effects of Threonine Deaminase on Growth and Viability of Mammalian Cells in Tissue Culture and Its Selective Cytotoxicity toward Leukemia Cells1 Robert S. Greenfield2 and Daniel Wellner3 Department of Biochemistry, Cornell University Medical College, New York, New York 10021 SUMMARY These observations imply that some neoplasms may not have the ability to synthesize adequate amounts of certain The purpose of this work was to investigate the effects of “nonessential― amino acids when exposed to an environ threonine deprivation on the growth and viability of malig ment deficient in these amino acids. Deprivation of nones nant and nonmalignant cells in vitro. Threonine deaminase sential amino acids, however, is difficult to achieve, since was added to the culture media of two mouse leukemias the host organism synthesizes large amounts of these (RADA1 and EARAD1), mouse L-cells, and human GM 38 amino acids. In addition, the tumors may acquire the ability fibroblasts. This enzyme irreversibly deaminates threonine to synthesize the missing amino acids under stressful con to a-ketobutynate and ammonia, products that cannot be ditions. Such a phenomenon is observed when asparagi utilized by the cell for the mesynthesisof threonine. Growth nase-sensitive leukemias become resistant to enzyme treat inhibition was observed in all cases, but only the two leuke ment. This was found to be associated with an increase in mia cell lines were killed in 24 to 48 hr after treatment. In the aspamagine synthetase activity in the tumors (7, 15). threonine-deficient medium, L-cells and GM 38 fibroblasts These problems can be circumvented by manipulation of showed little on no loss of viability after 3 and 5 days, “essential―aminoacid levels. A major requirement for this respectively. The inhibition of cell division and loss of viabil approach is that the cancer cells should be more sensitive ity were related to the ability of threonine deaminase to to the amino acid deprivation than normal cells. Dietary reduce threonine levels in the medium. However, addition deprivation of an essential amino acid may not be very of thneonine deaminase to the cultures was more effective effective, since the tumor may be supplied through the than simply using a threonine-free medium. Protein and blood with amino acids originating from the breakdown of RNA synthesis in RADA1 cells ceased 5 hr after treatment protein from tissues such as muscle. On the other hand, with the enzyme, while DNA synthesis gradually decreased injection of enzymes that degrade essential amino acids between 5 and 24 hr. When the threonine-depleted medium reduces substrate levels in the blood and may allow mainte was replaced by complete medium, inhibited L-cells me nance of a sufficiently low concentration of amino acid in sumed cell division, while fibnoblasts did not. When the the environment of the tumor for therapeutic effectiveness. fibnoblasts were trypsinized and replated in fresh medium, Several studies on enzymes that degrade essential amino the cells resumed normal growth. These findings demon acids have been carried out. Phenylalanine ammonia-lyase strate that certain leukemia cells are more sensitive to thne isolated from yeast, when added to tissue cultures of human onine deprivation than some nonmalignant cells and sug leukemic cells and L5178Y lymphoblasts, markedly in gest the possibility that threonine deaminase may have hibited the growth of these cells (2). When this enzyme was therapeutic uses in the treatment of cancer. tested in vivo, plasma phenylalanine levels rapidly de creased and the average life-span of mice beaning L5178Y tumors increased (1). Meadowsetal. (10) recently reported INTRODUCTION that administration of tyrosine phenol-lyase from Erwinia herbicola significantly inhibited the growth of established Recently, great interest has been shown in control of B-16 melanoma tumors. Kneis and Hession (8) found that tumor growth by dietary restriction of amino acid intake or methioninase, a methionine-degnading enzyme isolated by use of enzymes that degrade amino acids. The require from Clostridium SporogenS, caused inhibition of growth of ments of several mouse sarcomas (9), mouse leukemias (3), P815 and L1210 cells in vitro. Walker 256 carcinoma-bear and several human leukemias (11) for aspanagine explain ing rats showed significant tumor growth inhibition when the tumor regression observed following the administration treated with this enzyme. of the enzyme aspanaginase. Tumor regressions were also These observations prompted us to study the effects of observed when glutamine deficiency was induced in vivo threonine deprivation on tumors and normal cells in vitro. by administration of the enzyme glutaminase (16, 17). Threonine-free diets used in previous studies (18, 19) were shown to be relatively effective in inhibiting tumor growth. I This work was supported in part by NIH Grant CA-i 3259. Threonine deaminase nonoxidatively deaminates threon me 2 Present address: Department of Pathology, Tufts University School of Medicine, Boston, Mass. 02111. to a-ketobutynate and ammonia, according to Equation A. 3 To whom requests for reprints should be addressed. Received December 6, 1976; accepted April 27, 1977. CH3CH(OH)CH(NH2)COOH—@CH3CH2COCOOH+NH3 (A) AUGUST1977 2523 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1977 American Association for Cancer Research. R. S. Greenfield and D. Wellner This enzyme has been highly purified in this laboratory from 180,000 cells in 1 ml of complete minimal essential medium sheep liver (6). An important feature of this reaction is that and incubated for 24 hr. Another 1.5 ml of medium were the products cannot be neutilized for the synthesis of threo added to each well, followed by the addition of threonine nine in mammalian cells. deaminase. Cells were trypsinized, stained with trypan blue, The purpose of this study was to determine the effects of and counted at the indicated times. threonine deaminase in malignant and nonmalignant cells Human fibroblasts GM 38, generously provided by Dr. J. in tissue culture. It was found that, following the addition of Lombardi of this department, were treated in the same thneonine deaminase to cell cultures, threonine concentra fashion as mouse L-ceils. tions were lowered to undetectable levels. In the presence Determination of Protein, RNA, and DNA Synthesis. The of the enzyme, RADA1 and EARAD1 leukemia cells were effect of threonine deaminase on protein, RNA, and DNA rapidly killed. Protein, RNA, and DNA synthesis stopped synthesis in RADA1 cells was tested by measuring the incon between 5 and 24 hr after addition of the enzyme. Human poration of [3H]Ieucine, [3H}unidmne,and [3H]thymidine into fibroblasts and mouse L-cells stopped growing in the pres tnichlonoacetic acid-precipitable material. In separate ex ence of the enzyme but maintained their viability for at least peniments, each radioactive substrate was added to the 3 and 5 days, respectively, in the threonine-depleted media. tissue culture medium at 0.2 pCi [3H]leucine per ml, and 0.1 Mouse L-cell growth was reinitiated by placing them in pCi [3Hjunidmneor [3H]thymidine per ml. For studies of RNA complete media while the growth of the human fibroblasts and DNA synthesis, unlabeled unidine (1 @g/ml)and unla was reinitiated after trypsinization and neplating. beled thymidine (1 @g/ml),respectively, were also added to the cell cultures. At the indicated times, 1-mI aliquots were removed and added to 1 ml of 10% tnichlonoacetic acid . This MATERIALS AND METHODS mixture was filtered through Whatman glass fiber filters and washed with 5% tnichloroacetic acid. The filters were dried Materials. [methy!-3H]Thymidine, specific activity, 2 Cu and counted in 7 ml of Packard Permablend scintillation mmole; [5-3H]unidine, specific activity, 25 Ci/mmole; and L fluid on a Packard Tni-Carb Model 3380 scintillation [4,5-3H]leucine, specific activity, 5 Ci/mmole, were ob counter. tamed from New England Nuclear, Boston, Mass. All media Sodium Borohydride Inactivation of Threonine Deami and supplements were obtained from Grand Island Biologi nase. Threonine deaminase was inactivated by NaBH4and cal Co., Grand Island, N. Y. Unlabeled thymidine and un used as a control in several experiments. Inactivation was dine were from Sigma Chemical Co. , St. Louis, Mo. performedbytheadditionof0.1mlofa 1-mg/mIsolutionof Cell Cultures. EARAD1 and RADA1 leukemias used in NaBH4 to 0.45 mg of enzyme in 1 ml of 0.1 M potassium these experiments originated from X-irnadiation of female phosphate buffer, pH 7.2. The inactivated enzyme was then C57BL/6 x A F, hybrids (4) and Strain A female mice (12), dialyzed against 50 mM potassium phosphate buffer, pH respectively. The leukemia cells were withdrawn from as 7.2, to remove sodium bonohydnide. cites fluid and washed 2 times with minimal essential me dium with Earle's salts supplemented with nonessential amino acids, glutamine, penicillin (50 units/mI), streptomy RESULTS cm (50 j.@g/ml), and 10% dialyzed fetal calf serum. The cells were maintained in culture by passage every 3rd or 4th day Cytotoxic Effects of Threonine Deaminase on RADA1 with an initial inoculum of approximately 200,000/mI. (All and EARAD1 Cell Cultures. The cytotoxic effects of threo cell counts were done in a hemocytometen.) Viability was nine deaminase on RADA1 leukemia cells in vitro are shown determined by the trypan blue exclusion test. in Chart 1A. At enzyme concentrations of 1.3 units and 0.26 Preparation of Threonine Deaminase. Threonine deami unit/mI, more than 95% of the cells lost viability by 48 hr, as nase from sheep liven was prepared by the method of measured by trypan blue exclusion. Similar results were Greenfield and Wellnen (6) through the DEAE-cellulose observed when RADA1 cells were grown in threonine-free chromatography procedure. One unit of enzyme is that medium. The controls, RADA1 cells grown in complete me amount which produced 1 @moleof a-ketobutynate pen mm dia with no additions or in media containing NaBH4-inacti at 37°.This enzyme preparation had a specific activity of 4.5 vated enzyme at concentrations equivalent to 1.3 and 0.26 units/mg protein.