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[CANCER RESEARCH 30, 768-772, March 1970] Inhibitory Effect of L-Asparaginase in Lymphocyte Transformation Induced by Phytohemagglutinin Moriji Miura, Masami Mirano, Kumi Kakizawa, Akimitsu Merita, Tadaaki Uetani, and Kazumasa Yamada First Department of internal Medicine, Nagoya University School of Medicine, Nagoya, Japan SUMMARY The effect of Escherichia coli L-asparaginase on phytohemagglutinin (PHA)-induced transformation of human lymphocytes was studied. Transformation was remarkably suppressed by the enzyme when it was added to the cul ture not later than 24 hr after its initiation. The effective concentration was more than 0.2 i.u./tube. Guinea pig serum and L-0-aspartohydroxamic acid, an L-asparagine analog, have a suppressive effect on lymphocyte transfor mation in this system. PHA-treated lymphocytes became sensitive to L-asparaginase 12 hr after the culture was started, while no L-asparagine dependency was detected at any stage of the culture. An addition of L-asparagine or L-glutamine to the culture system treated with both PHA and L-asparaginase at zero time caused the partial re covery of thymidine-'H uptake. Thus, it is concluded that one of the mechanisms in hibiting PHA lymphocyte transformation by L-asparag inase is due to the deprivation of exogenous L-asparagine and i.-glutamine from the tissue culture medium. INTRODUCTION Many investigators have reported that L-asparaginase inhibits tumor growth in the mouse (4-7, 15), rat (16), dog (23), and human (13, 22), through the inhibition or dele tion of tumor-specific metabolism relating to L-asparagine. Becker and Broome (3) found, however, that L-asparag inase affects nonneoplastic tissue such as regenerating rat liver. The biochemical similarities of PHA'-transformed lym phocytes to regenerating rat liver subsequent to partial hepatectomy regarding a state of the activated nucleic acids (9, 12, 26) and protein synthesis (2, 14, 24, 25) lead to a question of whether or not L-asparaginase can affect the PHA-transformed lymphocytes. Astaldi et al. (1) re ported that L-asparaginase could intensively inhibit biastic transformation of human peripheral lymphocytes in the PHA culture system in vitro. McElwain et al. (17) re ported that the lymphocytes from the L-asparaginasetreated patients showed no transformation by PHA. The stimulation of lymphocytes by PHA to grow and divide in ' The abbreviations used are: PHA, phytohemagglutinin: L-0-aspartohydroxamic acid. Received June 2. 1969; accepted August 6, 1969. 768 LAHA, vitro provides an adequate experimental system for the study of growth control mechanism or nutritional require ment of the cell. This study was undertaken to investigate the influence of L-asparaginase in the course of lymphocyte transforma tion induced by PHA and, furthermore, to clarify the mechanisms involved. MATERIALS AND METHODS Leukocyte Preparation. Ten ml blood from healthy sub jects with blood type O were obtained, with citric acid-trisodium citrate-dextrose solution as an anticoagulant. The blood was mixed with 25% of its volume of 6% dextranphosphate-buffered saline (-) (Dextran: Meito Co. Ltd., Nagoya, Japan; M. W. 270,000, pyrogen free), and the red cells were allowed to sediment at 37°for 40 to 50 min. The supernatant leukocyte-rich plasma was drawn with a sterile pipet and subsequently left to stand at 37° for 4 hr to eliminate as many neutrophils and monocytes as possible. The supernatant was obtained which con tained more than 50% lymphocytes. The lymphocyte-rich plasma was then centrifuged for 10 min at 1200 rpm to re move platelets. The cells remaining were suspended in warm culture medium, and the concentration was ad justed to 10 X 106/ml. Culture Medium. Eagle's HeLa Medium (Grand Island Biological Company, Grand Island, N. Y.) supplemented with 10% inactivated fetal calf serum (Grand Island Bio logical Company, Grand Island, N. Y.) was used. PHA. One vial PHA-P (Difco Laboratories, Inc., De troit, Mich.) was dissolved in 5 ml hemagglutination buf fer (Difco). Before use, it was diluted 100-fold with Eagle's HeLa medium, and 0.1 ml of this PHA solution was added to each culture tube. L-Asparagine and L-Glutamine. L-Asparagine (Daiichi Pure Chemical Co., Ltd., Tokyo, Japan) and L-glutamine (Kyowa Hakko Co., Ltd., Tokyo, Japan) were dissolved with Earle's solution at concentrations of 5 and 30 mg/ml, respectively. These were sterilized by passage through a Millipore filter with a pore size of 0.45 ¿¿. Guinea Pig Serum. Blood was aseptically obtained by heart puncture, and then fresh serum was absorbed with washed human blood cells at 4°for 30 min to eliminate the natural heteroantibodies. This was repeated twice. The absorbed sera were stored at —80°. CANCER RESEARCH VOL. 30 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research. L-Asparaginase and PHA-indiiced Lymphocyte Transformation LAMA. LAHA (HONHCOCH2CHNH2COOH) (Tanabe Farm Co., Ltd., Osaka, Japan), one of the struc tural analogs of L-asparagine (21) which possesses antineoplastic activity in certain rodent tumors (20), was sub jected to the experiment on inhibition of PHA-induced lymphocyte transformation. This compound was dissolved with Earle's solution at a concentration of 10 mg/ml. E. coli L-Asparaginase. One vial L-asparaginase (Kyowa Hakko Co., Ltd., KW-020-1, 10,000 i.u./vial, Lot No. 1047, specific activity 184 i.u./mg protein) was dissolved with 5 ml Earle's solution and kept at —80° as a stock so lution. Before use, it was diluted to a desired concentra tion with Earle's solution. Leukocyte Culture. One ml culture medium, 0.1 ml leukocyte suspension, and 0.1 ml PHA-P solution were incubated in a rubber-stoppered tube at 37°for 72 hr. Procedures for L-AsparaginaseSensitivity Test and L-Asparagine Dependency Test. A triplicate of leukocyte cul tures at various time intervals after the initiation served as a set for these tests. To the first leukocyte culture tube was added 0.1 ml Earle's solution; to the second tube 2.0 i.u. L-asparaginase in 0.1 ml Earle's solutions were added; to the third tube 0.05 mg or 0.005 mg L-asparagine in 0.1 ml Earle's solution was added. Then, uridine-5-' H or thymidine-o-'H (Daiichi) was added to a final concentration of 2 ^Ci/tube. At the end of the 12 hr incubation, the cells were harvested, and the acid-insoluble residue was obtained on a filter paper disc (Toyo Roshi Co., Ltd., Tokyo, Japan, No. 5 B) by successive treatment of the cells with 5% cold trichloroacetic acid. The acid-insoluble residue on the filter paper disc was dried. Each disc was placed in a standard 20-ml low-potassium-counting vial, and then 8 ml liquid scintillator A (PPO and POPOP in toluene) were added. The samples were counted for 1 min with a Model TEN, GSL-163 (Kobe Kogyo, Kobe, Japan) liquid scintillation spectrometer. A formula for the determination of L-asparaginase sen sitivity is as follows. cpm control culture —cpm L-asparaginase-treated culture X 100 cpm control culture where in control culture, Earle's solution was added to the culture instead of L-asparaginase. The values above 40% represent L-asparaginase sensitivity and the values at 40% or less represents L-asparaginase insensitivity. A formula for the determination of L-asparagine de pendency is as follows. Blastic Transformation Rate. Blastic transformation rate was calculated as follows. Smear preparations of the cul tures were made after 72 hr incubation, and stained with May-Griinwald-Giemsa solutions. Five hundred cells of the lymphoid population per culture were then examined and the percentage of blast-like cells was taken as the blastic transformation rate. RESULTS Table 1 shows the effect of L-asparaginase on lympho cyte transformation when incubated with the enzyme and PHA for 72 hr. A remarkable suppression of lymphocyte transformation was observed at concentrations more than 0.2 i.u./tube L-asparaginase. There was no inhibitory ef fect of L-asparaginase at concentrations less than 0.02 i.u./tube. The lymphocytes left untransformed in the cul tures were morphologically intact. In the next experiment, inhibitory effect of L-aspara ginase in relation to preincubation time with PHA was studied as shown in Table 2. A remarkable suppression was observed in cultures preincubated with PHA for less than 24 hr. In parallel to the above experiments, the effect of guinea pig serum and LAHA, an L-asparagine analogy, was studied (Table 3). Leukocytes were cultured with guinea pig serum or LAHA in the presence of PHA for Table 1 Inhibitory effect of L-asparaginase on human lymphocyte transformation by PHA (%°)L-Asparaginase Experi (i.u./tube)200i.u.0NTNTNT20I.U.00002.0i.u.00000.2i.u.NT*5.012.0 mentNo.910Test control(%)83.583.095.093.0PHA cellNo.1234PHA ' %, blastic transformation rate. ' NT, not tested. Table 2 Inhibitory effect of L-asparaginase on human lymphocyte transformation by PHA: relationship i.u./tube)Test cpm L-asparagine-treated culture —cpm control culture X 100 cpm control culture where in control culture, Earle's solution was added in stead of L-asparagine. The values above 40% represent L-asparagine dependency, and values at 40% or less rep resent L-asparagine independency. MARCH with PHA preincubation time L-Asparaginase (20 cellNo.234PHAcontrol83.095.093.01201.4.Preincubationhr with36 hr1.05 24 hr22.349.046.5PHA48 hr30.078.075.1 12.50 13.0time 1%, blastic transformation rate. 1970 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research. 769 Miura, Mirano, Kakizawa, Marita, Uetani, and Yamada 72 hr, and blastic transformation rate was calculated. Both materials inhibited the lymphocyte transformation; 0.1 ml the serum/tube or 0.1 mg LAHA/tube lowered the blastic transformation rate to less than 50%. L-Asparagine dependency and L-asparaginase sensitivity were examined at various time intervals of culture of the transforming lymphocytes (Table 4). Acquisition of Lasparaginase sensitivity was noted at 12 hr after leuko- cytes were exposed to PHA. In contrast, L-asparagine dependency was not observed at any stage of culture ex amined. Restorative effect of L-asparagine and L-glutamine on the inhibition of PHA lymphocyte transformation by Lasparaginase was studied (Table 5). Leukocyte cultures incubated with PHA, L-asparaginase, and one of the fol lowing. L-glutamine, and L-asparagine plus L-glutamine. After 48 hr incubation, thymidine-'H was added. DNA synthesis in these groups was compared with controls con taining PHA alone or PHA plus L-asparaginase. The up take of thymidine-'H appeared augumented in these 3 groups (PHA, L-asparaginase plus L-asparagine; PHA, (1 : 100) plus GPS L-asparaginase plus L-glutamine; and PHA, L-asparagi (ml/tube)0.125.022.539.0 nase, L-asparagine plus L-glutamine) as compared to that of control treated with PHA plus L-asparaginase. The up take of thymidine-'H in these 3 groups did not reach the level of the PHA-treated control. Table 3 Effect of CPS°and LAH A on the lymphocyte transformation induced b\ PHA (%')PÃŽI Experi ment No.GPS A(1:100)0.285.5 15.5 17.0 20.0 NT NTPHA 50.0100) 12Experi 89.0 90.0 Table 5 Influence of L-asparagine and L-glutamine on the L-asparaginase inhibition of PHA -induced lymphocyte transformation (%'0.05NT46.573.5')(1:100) (1:GPS plus LAHA (mg/tube)1.0 LAHA ment No.LAHA 0.164.0 L-as Test paraginase, ginase, L- ginase, L- L-asparagine, cellNo.123PHA(cpm)23,83527,52539,314PHA,L-aspara ginase asparagine glutamine L-glutamine, (cpm)3921,742717PHA,L-aspara (cpm)4381,7711,157PHA,L-aspara (cpm)6952,4551,630PHA, (cpm)5953,8252,906 0.00140.0 1 2PHA 0 2.0 89.0 NT 14.5 48.0 90.0 NT 18.00.01 53.5 ° GPS, guinea pig serum; NT, not tested. ' %, blastic transformation rate. 52.5 NT NT0.000165.0NT NT At time zero, L-asparaginase, L-asparagine, or L-glutamine was added to each culture tube at a concentration of 2.0 i.u./tube, 0.05 mg/tube and 3.0 mg/tube, respectively. Forty-eight hr after the start of culture, 2 ltd thymidine-'H were added to each culture tube and uptake of thymidine-3H was measured 12 hr thereafter. Table 4 L-Asparagine dependency and L-asparaginase sensitivity of PHA-induced transforming lymphocytes PretreatmentNo (cpm)Earle's uptake of preof L-as of L-as cell incubation paragine de paraginase sen No.565656565656Uridine-'H (hr)00122448Test pendency13.5-21.225.5-2,25.47.034.7-8.86.64.3-1 sitivity3.320.631.420.553.0 solution3705315326351,6351,5445,0285,10211,7769,333L-Asparagine (0.05mg/tube)42041866S6211,7241.6636,7734,65112,5549,7399,706°11,934°L-Asparagi (2.0i.u./tube)3584223655057716252,1181,4964,9575,177Values treatmentPHAPHAPHAPHATime ' L-Asparagine content was 0.005 mg/tube. 770 CANCER RESEARCH VOL. 30 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research. L-Asparaginase and PHA-induced Lvmphocyte Transformation DISCUSSION E. coli L-asparaginase inhibited the lymphocyte trans formation by PHA. The following mechanisms are con sidered the inhibitory effect of this enzyme. Acquisition of L:Asparaginase Sensitivity by Transform ing Lymphocytes. The fact that the lymphocyte transfor mation by PHA was inhibited not only by E. coli Lasparaginase and guinea pig serum but also by LAHA suggests the involvement of the interference with L-asparagine metabolism as a possible mechanism. The transforming lymphocytes showed L-asparaginase sensitivity 12 hr after the initiation of culture, but they did not require L-asparagine for their metabolism. This might be due to the presence of L-asparagine in culture medium. There are possibly 2 sources of L-asparagine in tissue culture system. One is the fetal calf serum used in this experiment which contained 7.80 ^g/ml L-asparagine and the other is either red blood cells, platelets, or leuko cytes. This discrepancy between the results on L-aspara gine dependency and L-asparaginase sensitivity may imply a mechanism other than specific disturbance of L-aspara gine metabolism. The Involvement of Glutaminase Activity in i.-Asparaginase in the Inhibition of PHA Lymphocyte Transforma tion. Campbell et al. (8) reported that L-asparaginase from E. coli had small but definite glutaminase activity. Miller et al. (19) confirmed the presence of glutaminase activity in this enzyme and found that the sera of the leukemic patients during L-asparaginase treatment contained neither L-asparagine nor L-glutamine. The glutaminase activity of our particular sample KW-020-1, Lot No. 1047, was measured as about 4% of the L-asparaginase activity. L-Glutamine as well as L-asparagine partially restored lym phocyte transformation from the suppression with L-as paraginase. Therefore, L-glutamine depletion in the tis sue culture medium induced by L-asparaginase might be a cause of the suppression. PHA-stimulated lymphocytes in a metabolically acti vated state synthesize proteins (2, 14, 24, 25) and nucleic acids (9, 12, 26). For synthesizing of these materials, many amino acids that are not essential for normal lymphocytes may become essential for PHA-stimulated ones. The re duced amount of exogenous L-asparagine and L-glutamine in a tissue culture medium after treatment with L-aspara ginase might become critically low for the process essen tial for lymphocyte transformation, although adequate for normal lymphocytes. Other Possible Mechanisms of L-Asparaginase Inhibition of PHA Lymphocyte Transformation. Another question is whether L-asparaginase acts on the exogenous L-aspara gine alone or on both exogenous and endogenous L-aspar agine. Several unexplainable side effects of L-asparagi nase treatment in patients with leukemia, such as hypofibrinogenemia (10, 11), hypocholesterolemia (11), liver damage (11, 18), and myelosuppression (11) have been re ported, although the highly purified, almost crystallized MARCH enzyme was used for the treatment. It seems difficult to explain all of these side effects by the deprivation of exog enous L-asparagine and L-glutamine by this enzyme. The interference with the metabolism of endogenous L-aspar agine and L-glutamine might be involved in these phe nomena. REFERENCES 1. Astaldi, C., Burgio, G. R., Krc, J., Genova, R., and Astaldi, A. A. L-Asparaginase and Blastogenesis. Lancet. 759: 423, 1969. 2. Bach, F., and Hirschhorn, K. -pGlobulin Production by Human Lymphocytes in Vitro. Exptl. Cell Res., 32: 592-595, 1963. 3. Becker, F. F., and Broome. J. D. i.-Asparaginase: Inhibition of Early Mitosis in Regenerating Rat Liver. Science, 756: 1602 1603. 1967. 4. Boyse. E. A., Old, L. 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Genetic Control of LáclateDehydrogenase and Malate Dehydrogenase Isozymes in Cultures of Lym phocytes and Granulocytes: Effect of Addition of Phytohemagglutinin, Actinomycin D and Puromycin. Biochim. Biophys. Acta, 139: 264, 1967. Rabinowitz, Y., Lubrano, T., Whilhite, B. A., and Dietz, A. A. Lac tic Dehydrogenase of Cultured Lymphocytes: Response to Environ mental Conditions. Exptl. Cell Res., 48: 675-678, 1967. Tormey, D. C., Kamin, R., AND Fudenberg, H. H. Quantitative Studies of Phytohemagglutinin Induced DNA and RNA Synthesis in Normal and Agammaglobulinemic Leukocytes. J. Exptl. Med. 125: 863-872, 1967. CANCER RESEARCH VOL. 30 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research. Inhibitory Effect of l-Asparaginase in Lymphocyte Transformation Induced by Phytohemagglutinin Moriji Miura, Masami Hirano, Kumi Kakizawa, et al. Cancer Res 1970;30:768-772. 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