Effects of Threonine Deaminase on Growth and Viability of Mammalian Cells in Tissue Culture and Its Selective Cytotoxicity Toward Leukemia Cells1

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[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 â€œnonessentialâ€• 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, â€œessentialâ€•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)COOHâ€”@CH3CH2COCOOH+NH3 (A)

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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 37Â°.This enzyme preparation had a specific activity of 4.5 vated enzyme at concentrations equivalent to 1.3 and 0.26 units/mg protein. The enzyme was stored frozen at 0Â°in 50 units/mi, maintained more than 95% viability throughout mM potassium phosphate buffer, pH 7.2. The enzyme was the 48-hr period. sterilized by filtration through a sintered glass filter before Chart lB describes the growth of RADA1 cells in this use in tissue cultures. experiment. There was no increase in cell number after the Effects of Threonine Deaminase on Cell Growth and addition of enzyme to the cultures. Cells placed in thmeo Viability. EARAD1 and RADA1 cells were inoculated at mdi nine-free media also stopped dividing. The inactivated en cated densities in a medium to which threonine deaminase zyme appears to have a stimulatory effect on the growth of was added . At the indicated times, aliquots were removed, RADA1 cells. The reason for this is not yet clean. the cells were counted, and viability was determined. Thre Similar results were observed when EARAD1 cells were onine-free medium was supplemented with fetal calf serum treated with threonine deaminase (Chart 2, A and B). dialyzed for 24 hr against 0.15 M NaCl to remove traces of EARAD1 cells lost 98% viability after 24 hr of exposure to the threonine. enzyme at a concentration of 0.26 unit/mI. Cell division was Mouse L-cells were grown in plastic culture plates con also rapidly inhibited by the enzyme (Chart 2B). A compani taming wells 35 x 10 mm. Each well was initially plated with son of the rate of cell death between RADA1 and EAAAD1

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and 24 hr. After addition of the enzyme to EARAD1 cultures, aliquots of cells were removed at 0, 2, 4, 6, 8, 12, and 24 hr. These cells were washed twice with media, resuspended in

BIB @1 complete media, and analyzed for cell growth over a 48-hr III period. Cell division was observed for those aliquots me U moved between 0 and 12 hm,while those removed at 24 hr MI -@ (when more than 97% were dead, as shown by thetrypan 4 blue test) showed no cell growth. Thus, cultures containing > 21 an appreciable proportion of cells that were viable accord ing to the trypan blue test could be rescued by transferring the cells to complete media, while cultures in which almost all cells were stained with trypan blue could not be made to grow. This experiment thus qualitatively confirms the valid 11 ity of the trypan blue exclusion test in oursystem. In another control experiment, EARAD1 cells were grown in media containing amounts of a-ketobutynate and ammonia that would have been present if all the threonine in the media had been deaminated. Theme was no loss of cell viability and no inhibition of cell growth under these conditions. Effects of Enzyme Concentration and Threonine Con 0 centration on Cytotoxicity. The correlation between the x cytotoxic effect of threonine deaminase and threonine con centration on EARAD1 cells is shown in Chart 3A. Enzyme E concentrations of 0.26 and 0.13 unit/mI killed 90 to 95% of BIB -I -I MI U

VI .4 -I IN U IN -a 4

HOURS Chart 1. A, cytotoxic effects of threonine deaminase on RADA1 cells in vitro. RADAI leukemia cells were grown in media that contained the follow HOURS ing additions: a and b, threonine deaminase (0.26 and i .3 units/mI, re spectively) inactivated with sodium borohydride; c, no additions; d, no additions, threonine-free medium; e and f, threonine deaminase (0.26 and i .3 units/mI, respectively). At the indicated times, aliquots were removed, cells were counted, and viability was determined by trypan blue exclusion. 6 B, inhibition of RADA1 cell division by threonine deaminase. Curves a to I give the cell numbers in the corresponding experiments described in A. 5 MB 24 cells indicates that EAAAD1 cells are slightly more sensitive N to enzyme treatment than are RADA1 cells. When cell viability was measured during the 1st 5 hn, VI there was an initial lag period of approximately 4 to 5 hr before both EARAD1 and RADA1 cells began to lose viabil V ity. Following addition of the enzyme (0.26 unit/mI), amino acid analyses of the media showed threonine levels to be undetectable after 2 hr. Thus, in the absence of extracellu Ianthneonine, these cells apparently have sufficient intracel lular threonine to maintain viability for about 3 hr. Theneaf HOURS ten, presumably as a result of exhaustion of intracellular Chart 2. A, cytotoxic effect ofthreonine deaminase on EARAD1 leukemia cells in vitro. EARAD1 leukemia cells were grown in media to which the threonine, the cells begin to lose viability. following additions were made: â€¢,threonine deaminase (0.26 unit/mI); 0, The following experiment was performed to determine same enzyme inactivated with sodium borohydride. B, inhibition of EARAD1 whether the trypan blue exclusion test was a reliable indica cell division by threonine deaminase. EARAD1 growth was determined in the experiments described in A. Symbols refer to the corresponding experi tion of cell viability during the cytotoxic period between 4 ments.

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the cells in 24 hr. At 0.065 unit/mI, there was a lag period of Thneonine concentrations of the media were measured 30 hr in which EARAD1 cells remained viable. However, throughout the experiment and are shown in Chart 3B. By over the next 42 hr, almost 100% of the cells were killed. 12 hn, threonine levels were undetectable at enzyme con Lower concentrations of enzyme showed no cytotoxic ef centrations of 0.26 and 0.13 unit/mi. At 0.065 unit/mI, thre fects in 72 hr. onine levels decreased to 31 @Mby24 hr and were unde tectable by 50 hn, at which time the cells were losing viabil ity. The lower enzyme concentrations did not reduce threo ninelevelsmuch below 100 @Mby 50 hn,and no lossof viabilitywasobserved. In another experiment, threonine was added at several concentrations to threonine-free media, and the viability of EARAD1 cells was measured over 48 hr. it was found that media containing 10 @Mthreonineor less could not support cell growth on viability. At threonine concentrations of 26 j.@Mor above, cells remained viable and were able to multi ply. This result correlates very well with the observations made intheexperimentdescribedinChart3,A and B. At a concentration of 0.065 unit/mI, the enzyme reduced threo nine levelsbelow20 j.@Matapproximately40hr and, at about this time, the cells started to die. We conclude that EAAAD1 cells require greater than 20 @Mthreonineconcen trations to grow and remain viable. Effect of Threonine Deaminase on Macromoiecular Syn thesis. In order to understand more fully the mechanism of HOURS the cytotoxic effects observed with threonine deaminase, we looked at macromolecular synthesis during the expo sure of RADA1 cells to the enzyme. The effects of threonine deaminase on protein, ANA, and DNA synthesis are shown in Table 1. [3H]Leucine incorporation into tnichloroacetic E acid-precipitable material was not significantly different in z treated and control cells oven the 1st 5 hn, but essentially z stopped thereafter in enzyme-treated cells. Similarly, RNA 0 synthesis measured by [3H]unidine incorporation stopped 5 I hr after threonine deaminase was added to the cultures. The rate of DNA synthesis was about normal for 5 hr and gradu ally decreased to zero by 24 hr. Loss of viability also started 12 24 36 48 about 5 hr after enzyme addition. HOURS Effects of Threonine Deaminase on Mouse L-CeIls. To Chart 3. Correlation between cytotoxic effect of threonine deaminase on compare the results described above for leukemia cells with EARAD1 cells and threonine level in the medium. A, decrease in cell viability the response of other cell types to threonine deprivation, at different enzyme concentrations. Additions: â€¢,threoninedeaminase, 0.26 unit/mI; A, 0.13 unit/mI; 0, 0.065 unit/mI; x, 0.032 unit/mI; 0, 0.013 unit! mouse L-cells were also treated with thneonine deaminase. @ ml; 0.26 unit/mI, inactivated with sodium borohydride. Other condi As shown in Chart 4, the growth of L-cells was inhibited tions are as in Chart 1. B, decrease in threonine level of medium at different enzyme concentrations. Threonine levels in the experiments described in A after addition of the enzyme to the cultures and remained so were determined on a Durrum D500 amino acid analyzer. up to 96 hr. At this time, the number of cells had declined

Table 1 Inhibition of protein, RNA, and DNA synthesis by TDA6 in RADA1 leukemia cells The final concentration of TDA was 0.26 unit/mI. In the controls, the same amount of enzyme inactivated with sodium borohydride was used.

Incorporation (cpm)No. of ce (cells! of ml x 10@) ce (%) incuba â€” tionTDATimeTDAControlTDA53823192,162 (hr)[3H]Leucine[3H]Uridine[3H]ThymidineControlTDAControl Control15b TDAViableControllIsb 8324744432302459796185288,946 1 1,9551,9565774502459737243,0934281 9323,4559026582559720304,0313893,8648659232389840,055 1

a TDA, threonine deaminase.

b Values represent the average of 2 experiments.

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ity.Protein and ANA synthesis ceased 5 hr after enzyme addition, which coincided with the initiation of cell death. DNA synthesis gradually decreased between 5 and 24 hr. The data are consistent with the hypothesis that the growth inhibitory and cytotoxic properties of thneonine deaminase are due to its ability to deplete threonine in the media. 0 However, the possibility that the enzyme also affects cells in N VI other ways is not excluded. Concentrations of threonine in the media were also come MI U lated with cell death. When threonine levels were reduced 0 below 31 @tMby the enzyme, the cytotoxic effects were N MI observed. In the absence of threonine deaminase, it was found that threonine concentrations below about 20 @M z were unable to support the growth on viability of EAAAD1 cells. Thus, it appears that in order to achieve a therapeutic response in vivo, threonine deaminase must be adminis tened in doses that will reduce plasma and extracellulan fluid

HOURS Chart 4. Growth inhibition of mouse L-cells by threonine deaminase. The following additions were made to cultures of L-cells: â€¢,threoninedeaminase (0.26 unit/mI); 0, same enzyme inactivated with sodium borohydride. Arrows, time when cells were washed twice and placed in fresh, complete media with no enzyme additions. slightly, probably because of sloughing off of dead cells. However, those cells adhering to the culture plates looked normal under microscopic examination and were viable (>95%). At 96 hr, the cells were washed twice and placed in HOUIS fresh medium.Thistneatmentinitiated cell multiplication. Chart 5. Growth inhibition of GM 38 fibroblasts by threonine deaminase: Thus, the response of L-cells to threonine deprivation is ., threonine deaminase 0.26 unit/mI; 0, inactivated threonine deami quite different from that of AADA1 and EARAD1 leukemia nasa. Arrow, time when media were replaced by fresh complete media containing no threonine deaminase. (Cells treated with inactive enzyme had cells. The L-cells seem to enter into a â€œnestingstateâ€•in reachedconfluenceat i32 hr.) which they do not undergo cell division but maintain their viability for a relatively long period of time. Addition of Table 2 threonine allows the cells to move out of this â€œrestingstateâ€• Stimulatory effect of trypsinization on GM 38 fibroblasts inhibited and reenter their normal cell cycle. by threonine deaminase Effect of Threonine Deaminase on Human Fibroblasts. GM 38 cells were plated in Petri dishes to which threonine The g@owthof human GM 38 fibroblasts was also inhibited deaminase (0.26 unit/mI) or an equivalent amount of inactivated by threonine deaminase after addition of the enzyme to the enzyme was added. Controls in which there were no additions were cultures (Chart 5). The fibroblasts were able to remain un also set up. The cells were incubated and counted at the indicated den these conditions up to 5 days without loss of viability. times. After 72 hr, the cells were trypsinized and replated with fresh media. Enzyme-treated cells were replated at one-half the 72-hr However, unlike L-cells, they could not be induced to divide density, and controls were replated at one-fourth of their 72-hr by placing them in fresh media. Cell division could be densities. Cell growth was thentimes.No. determined at the indicated initiated by trypsinizing the cells and replating them with dish6Time of cells/Petri fresh media (Table 2). (hr) EnzymeadditionsAfter Inactive enzyme No additions0 DISCUSSION 144,300 144,300 144,300 24 198,100 231,200 251,000 Threonine deaminase was shown to be highly growth 48 253,100 477,500 518,700 inhibitory and cytotoxic toward RADA1 and EAAAD1 leuke 72 245,600608,700After 441,200 mias in vitro. The cytotoxic effects were correlated with trypsinization48 reduction of threonine levels in the culture media. Follow 128,700 187,500 199,100 ing the addition of threonine deaminase to cell cultures, 96 543,100 336,800 416,200 thneonine levels were undetectable in the medium after 2 hr 881,200a240 931 ,100 622,500 and, beginning at 5 hn, there was a rapid loss of cell viabil Average of 2 Petri dishes.

AUGUST 1977 2527

threonine levels below 20 @Movenan extended period of cells may have lost this control. Thus, when deprived of an time. In contrast, rapid cell death during threonine depniva essential nutrient, normal cells enter a quiescent state of tion was not observed in mouse L-cell on in human GM 38 low metabolic activity and are able to survive for long pe fibnoblast cultures. These findings indicate that leukemic nods, while cells which have lost this â€œrestrictionpointâ€• cells might be selectively killed during treatment with threo control, located in the G, phase, continue to other phases of ninedeaminaseinvivo. the cell cycle in which they die under the adverse condi L-cells and fibroblasts apparently can survive thneonine tions. Our results are consistent with this hypothesis. deficiency by entering a nesting state, while the leukemic It would be of value to test other tumors and cell lines for cells cannot. The-L-cells can easily reenter the normal cell their response to essential amino acid deprivation. The data cycle after thneonine in the medium is replenished. Fibno presented above suggest that an asparaginase-nesistant tu blasts can be brought back into their normal cell cycle mom may be sensitive to threonine deaminase treatment. following trypsinization and replenishment of the medium Thus it may be possible to increase the effectiveness of with thneonine. aspanaginase therapy by combining it with another enzyme When we looked at fibroblast intracellular amino acid such as threonine deaminase. Such combined enzyme then levels after the addition of threonine deaminase, we found apy may help prevent development of aspanaginase resist that the levels of most of the amino acids were near normal ance by killing resistant cells that appear during treatment. except for those of threonine (R. S. Greenfield and D. Well ner, unpublished results). Threonine was present in only trace amounts after 24 hr. It would appear that these cells ACKNOWLEDGMENTS make extremely efficient use of intracellular threonine de nived from protein turnover in order to survive for long Wewishto thank Dr. LloydJ. Oldfor his helpand interestin this work. periods of time in threonine-deficient environments. Similar observations on the effects of amino acid depniva tion were made by Tobey and Ley (20) when L-cells and REFERENCES Chinese hamster ovary cells were grown in isoleucine-free media. These cells were found to be arrested in the G, i . Abell, C. W., Hodgins, D. 5., and Stith, W. J. An in Vivo Evaluation of the Chemotherapeutic Potency of Phenylalanine Ammonia-Lyase. Cancer phase of the cell cycle, and the cells could be maintained up Res., 33: 2529-2532, i973. to 5 days in isoleucine-free media with no loss of viability. 2. AbeIl,C. W., Stith, W. J., and Hodgins,D. S. The Effectsof Phenylala Everhart and Prescott (5) were also able to arrest Chinese nine Ammonia-Lyase on Leukemic Lymphocytes in vitro. Cancer Res., 32: 285-290, 1972. hamster ovary cells in G by partial leucine deprivation. It 3. Boyse, E. A., and Old, L. J. Suppression of Murine Leukemias by L may be that the L-cells and fibroblasts used in this work Asparaginase. J. Exptl. Med., 125: 17-31, 1967. were also arrested in the G1 phase by threonine deprivation. 4. Boyse, E. A., Old, L. J., and Stockert, E. Inhibitory Effect of Guinea Pig Serum on a Numberof New Leukemias in Mice. Nature, 198: 800, 1963. We also tested RADA1 cells for growth and viability in iso 5. Everhart, L. P., and Prescott, 0. M. Reversible Arrest of Chinese Hamster leucine-deficient media. It was found that, in the absence of Cells in Gi by Partial Deprivation of Leucine. Exptl. Cell Res., 75: 170â€” 174, i972. isoleucine, the cells did not divide and rapidly lost viability 6. Greenfield, R. S., and Wellner, 0. Isozymes of Sheep Liver Threonine (A. S. Greenfield and D. Wellnen, unpublished results). Deaminase. In: C. Markert (ed), Isozymes, Vol. 2, pp. 60i-608. New Thus, the response of RADA1 cells to isoleucine deficiency York: Academic Press, Inc., 1975. 7. Horowitz, B., Madras, B. K., Meister, A., Old, L. J., Boyse, E. A., and was similar to the response of these cells to threonine Stockert, E. Asparagine Synthetase Activity in Mouse Leukemias. Sci deficiency. This supports the hypothesis that the effect is ence, 160: 533-535, 1968. due to an inhibition of protein synthesis. Also RADA1, an 8. Kreis, W., and Hession, C. Biological Effects of Enzymatic Deprivation of L-Methionine in Cell Culture and an Experimental Tumor. Cancer Res., aspanaginase-nesistant line, and EARAD1, an aspanaginase 33: i866-i869, 1973. sensitive line, are both sensitive to threonine deprivation. 9. Mashbum, L. T., and Wriston, J. C. Tumor Inhibitory Effect of L-Aspara ginase. Biochem. Biophys. Res. Commun., 12: 50-55, 1963. The experimental evidence suggests that deprivation of an 10. Meadows, G. G., DiGiovanni, J., Minor, L., and Elmer, G. W. Some amino acid essential for growth can evoke 1 of the 2 Biological Properties and an in Vivo Evaluation of Tyrosine Phenol-Lyase responses in different cell lines: (a) entering into a resting on Growth of B-i6 Melanoma. Cancer Res., 36: 167-17i, 1976. ii. Oettgen, H. F., Old, L. J., Boyse, E. A., Campbell, H. A., Philips, F. S., state or (b) rapid cell death. RADA1 cells are resistant to Clarkson, B. D., Tallal, L., Leaper, R. D., Schwartz, M. K., and Kim, J. H. aspanaginase because they have high levels of aspanagine Inhibition of Leukemias in Man by L-Asparaginase. Cancer Res., 27: 2619-263i, 1967. synthetase . Thus, they do not require extracellular aspara 12. Old, L. J., Boyse,E.A., andStockert,E.AntigenicPropertiesofExperi gine for growth. However, they fall into the 2nd category, as mental Leukemias. I. Serological Studies in vitro with Spontaneous and do EARAD1 cells, since they rapidly die when deprived of an Radiation Induced Leukemias. J. NatI. Cancer Inst., 31: 977-986, 1963. 13. Pardee, A. B. A Restriction Point for Control of Normal Animal Cell essential amino acid such as threonine on isoleucine. Proliferation. Proc. NatI. Aced. Sci. U. S., 71: i286â€”1290,i974. The ability of some cells to enter into a nesting phase 14. Pardee, A. B., and James, L. T. Selective Killing of Transformed Baby while others die during amino acid deprivation cannot be Hamster Kidney (BHK) Cells. Proc. Natl. Acad. Sci. U. S., 72: 4994-4998, 1975. fully explained at this time. However, this difference may be 15. Prager, M. 0., and Bachynsky, N. Asparagine Synthetase in Asparagi related to the effects of intracellular amino acid concentra nase Resistant and Suceptible Mouse Lymphomas. Biochem. Biophys. Res. Commun., 31: 43-47, i968. tions on protein synthesis and control of the cell cycle. 16. 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18. Sugimura, T., Bimbaum, S. M., Winitz, M., and Greenstein, J. P. Quanti Female C57BL Mice and Their Implanted BW10232 Adenocarcinomas. J. tative Nutritional Studies with Water Soluble, Chemically Defined Diets. Nutr., 101: 223â€”232,1971. VIII. The Forced Feeding of Diets Lacking in One Essential Amino Acid. 20. Tobey,A.A.,andLay,K.D.Isoleucine-mediatedRegulationofGenome Arch. Biochem. Biophys., 81: 448-455, 1959. Replication in Various Mammalian Cell Lines. Cancer Res., 31: 46-51, 19. Theuer, A. C. Effect of Essential Amino Acid Restriction on the Growth of 1971.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1977 American Association for Cancer Research. Effects of Threonine Deaminase on Growth and Viability of Mammalian Cells in Tissue Culture and Its Selective Cytotoxicity toward Leukemia Cells

Robert S. Greenfield and Daniel Wellner

Cancer Res 1977;37:2523-2529.

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