Alteration in Spleen Lymphoid Populations Associated with Specific Amino Acid Depletion During L-Asparaginase Treatment1

Home , Germinal center

[CANCER RESEARCH 42, 252-258, January 1982] 0008-5472/82/0042-0000$02.00 Alteration in Spleen Lymphoid Populations Associated with Specific Amino Acid Depletion during L-Asparaginase Treatment1

John A. Oistasio,2 Donald L. Durden, Ronald D. Paul, and Mehrdad Nadji

Departments of Microbiology (D4-4) [J.A.D., D.L.D., P.O.P.] and Pathology (R-1), [M.N.] University of Miami School of Medicine, Miami, Florida 33/0)

ABSTRACT thought to represent a unique prototype of selective therapy in which treatment could be directed specifically against aspara- Treatment with antileukemic asparaginases isolated from gine-dependent cells. The low levels of asparaginase present Escherichia coli and Erwinia carotovora is accompanied by in guinea pig serum made it necessary to seek a more practical pronounced toxicity and immunosuppression. The capability of source of antineoplastic enzyme. Asparaginases isolated from these enzymes to hydrolyze L-glutamine and to limit serum Escherichia coli and Erwinia carotovora were subsequently levels of this amino acid as well as asparagine has led investi shown to be effective in the treatment of acute lymphoblastic gators to hypothesize that glutaminase activity may be respon leukemia (4, 31). Unlike the guinea pig enzyme (14), clinical sible for the side effects observed during treatment. We have use of the microbial asparaginases is associated with pro isolated a glutaminase-free asparaginase with potent antilym- nounced toxicity including liver and pancreas dysfunction and phoma activity from Vibrio succinogenes. With the use of this immunosuppression (11, 20, 24). enzyme, we have demonstrated that asparagine depletion One difference between the E. coli and E. carotovora aspar alone does not result in suppression of humoral or cell-medi aginases and the guinea pig enzyme is the nonspecific ami- ated responses after immunization with sheep red blood cells. dohydrolase activity associated with the microbial enzymes (7, In contrast, treatment with E. coli asparaginase abolishes these 22). As a result, patients treated with the microbial enzymes immune responses. In the present study, we investigated the demonstrate a reduction in serum levels of glutamine as well effects of specific amino acid depletion resulting from asparag as asparagine (11, 28). Some investigators have established a inase treatment on spleen histology and lymphocyte popula correlation between glutamine deprivation and toxicity (21, 27, tions. Administration by i.p. injection of 50 IU of E. coli aspa 29). The importance of glutamine in mammalian intermediary raginase per day for 4 days resulted in a marked reduction in metabolism, e.g., the amide donor in important transamination the size of lymphoid follicles within the spleen concomitant with reactions and in nucleotide biosynthesis, suggests that gluta reduced germinal centers. There appeared to be a significant mine deprivation may be deleterious to important cellular pro change in reactivity of the germinal centers, evidenced by a cesses. Lymphoid tissues have been shown to have relatively decrease in immunoblasts (B-cells). These findings were con low levels of glutamine synthetase activity, suggesting that firmed by a sensitive peroxidase-antiperoxidase staining pro these tissues may be particularly sensitive to the depletion of cedure for immunoglobulin-containing cells. Examination of glutamine (17). In contrast, some investigators have proposed lymphocyte subpopulations within the spleen revealed that that asparagine depletion alone may be responsible for aspa- there was nearly a 40% decrease in the percentage of Slg- raginase-induced immunosuppression (3, 9, 25). bearing cells accompanied by an increase in the ratio of Thy We have isolated a glutaminase-free asparaginase with po 1.2-bearing cells. The ratio of Lyt-2 to Lyt-1 cells was un tent antilymphoma activity from Vibrio succinogenes (12, 13). changed as compared to control animals. The data support the The use of this enzyme has allowed us to establish clearly the hypothesis that glutamine or glutamine combined with aspara first direct evidence that asparagine depletion alone cannot be gine depletion resulting from the treatment of E. coli asparagi responsible for the immunosuppressive effects of E. coli as nase causes a marked decrease in spleen lymphocytes of the paraginase (15, 16). Treatment of mice with doses of E. coli B-cell lineage. In contrast, asparagine deprivation alone, asparaginase, which are capable of depleting serum levels of caused by the administration of the glutaminase-free asparag both L-asparagine and L-glutamine, results in a marked sup inase from V. succinogenes, does not affect spleen histology pression of antibody response and specific cell-mediated cy- or lymphocyte marker distribution. Thus, the asparaginase from totoxicity against the T-dependent antigen, SRBC.3 In contrast, V. succinogenes should serve as an effective antileukemic treatment of animals with equivalent doses of V. succinogenes agent without causing deleterious effects on the immune sys asparaginase, which depletes serum levels of L-asparagine and tem. lacks glutaminase activity, does not cause suppression of the immune response. INTRODUCTION While many studies have documented the immunosuppres sive effects of asparaginases with glutaminase activity, little Asparaginase isolated from guinea pig serum was first shown information exists concerning the underlying mechanism(s) (1, by Broome (6) to be inhibitory to certain animal tumors. Since 5, 9, 24). Our analysis of the specific cytotoxic response after L-asparagine is a nonessential amino acid, asparaginase was SRBC immunization revealed that it requires both the synthesis of antibody (IgG specific for the immunizing antigen) and a ' This work was supported by Grant F79UM-1 from the Florida Division of the American Cancer Society (Jeffrey Frohman Research Grant), Grant RR 05363- 17 from the NIH, and grants from the United Way and the Women's Cancer 3 The abbreviations used are: SRBC, sheep red blood cells; ADCC, antibody- Association of the University of Miami. dependent cell-mediated cytotoxicity; PFC, plaque-forming cells; FITC, fluores- 2 To whom requests for reprints should be addressed. cein isothiocyanate conjugated; PAP. peroxidase-antiperoxidase immune com Received July 6, 1981; accepted September 28, 1981. plexes.

252 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Effects of Asparaginase on Lymphoid Populations population of nonspecific effector cell(s) (ADCC) (16). Exami lymphocytes (immunoblasts) within each germinal center. All the pa nation of spleen cells from E. coli asparaginase-treated mice rameters were semiquantitatively graded from 1 to 3 + . revealed that the decreased specific ADCC results from de Spleen Cell Preparation for Marker Analysis. Spleens were re moved aseptically and homogenized in 2 ml Hanks' balanced salt creased synthesis of specific IgG. Spleen cells isolated from treated animals do not show a reduced ability to serve as solution in a loosely fitting homogenizer. The resulting cell suspension effector cells in this cytotoxic response. From our previous was centrifuged at 300 x g for 20 min, and the cell pellet was subjected to hypotonie shock to remove RBC. work, it is clear that the immunosuppressive effects of com The levels of B- and T-lymphocytes, as well as T-cell subsets, in the bined L-asparagine and L-glutamine depletion cause a de spleens of mice were determined by flow cytometric analysis of fluo creased number of IgM and IgG RFC in animals immunized with rescence using a Beckton-Dickinson FACS III fluorescence-activated SRBC (15). Although Â¡mmunoglobulin production is a B-cell cell sorter. Briefly, appropriately stained cells in a fluid stream are function, it has become clear that regulatory cells play a excited by an argon ion laser beam (wavelength, 488 nm). The emitted primary role in the expression of immunoglobulin synthesis. fluorescence is integrated by a series of filters and photomultiplier Both helper and suppressor T-cells are involved in the regula tubes and converted into an electrical signal which is stored in a tion of antibody production (10, 19). Therefore, in the present multichanneled pulse height analyzer. Data are presented as percent study, we have examined the effects of specific amino acid age of fluorescent cells and displayed as histograms in which the depletion on spleen lymphocyte populations (Slg, Thy 1.2, Lyt- oridinate represents the number of cells; the abscissa represents fluorescent intensity. In each sample, 1 x 10s cells were analyzed. 1, Lyt-2) and spleen histology in an effort to further characterize FITC rabbit anti-mouse immunoglobulin was generously donated by the mechanism of E. coli asparaginase-induced immuno- the Division of Biological Carcinogenesis Branch, National Cancer suppression. Institute, from stocks supplied by the Frederick Cancer Center. FITC monoclonal anti-Thy 1.2 and monoclonal biotin-conjugated Lyt-1 and MATERIALS AND METHODS Lyt-2 were purchased from Beckton-Dickinson Monoclonal Antibody Center, Mountain View, Calif. The biotin-conjugated Lyt-1 and Lyt-2 Animals. The animals used in these studies were 9- to 12-week-old were each used in an individual immunofluorescent assay with FITC BALB/cCrgl mice, originally obtained from the Cancer Research Ge avidin also supplied by Beckton-Dickinson Monoclonal Antibody Cen netics Laboratory, University of California, and maintained in our lab ter. The dilutions of antisera:avidin used were those recommended by oratory by brother-sister matings. Beckton-Dickinson for use with the fluorescence-activated cell sorter. L-Asparaglnase Preparations. L-Asparaginase from v succino- In these experiments, individual samples containing 1 x 106 spleen genes was purified to homogeneity as described previously (13). The cells were stained according to the procedure of Rabellino et al. (26). L-asparaginase, EC-2 (Lot 1028A) from E. coli was obtained from Staining for Cytoplasmic Immunoglobulin in Cross-Sections of Merck Sharp and Dohme, West Point, Pa. Further purification of E. coli Spleen. The peroxidase-antiperoxidase procedure for immunoglobulin was carried out on Bouin's-fixed, paraffin-embedded splenic tissue, asparaginase by gel filtration (23) on Ultrogel AcA44 (34) (LKB Instru ments, Inc., Rockville, Md.) yielded a homogeneous preparation, as according to the method of Taylor and Chir (32). Briefly, 3 /ÃŒMsections determined by polyacrylamide disc gel electrophoresis in the presence were incubated at 58Â°for 18 hr, deparaffinized in xylene, and carried of sodium dodecyl sulfate (33). through decreasing grades of ethanol. After blocking the endogenous The antilymphoma activity of homogeneous preparations of V. suc- peroxidase by a solution of hydrogen peroxide:methanol, the slides cinogenes and E. coli asparaginases used in these studies was deter were sequentially treated with normal swine serum, rabbit anti-mouse mined routinely using the 6C3HED lymphosarcoma in C3H mice as IgG, excess swine anti-rabbit immunoglobulin, and PAP (consisting of described previously (12). rabbit anti-horseradish peroxidase and horseradish peroxidase anti Assay for L-Asparaginase Activity. Enzyme activity was measured gen). The final reaction was visualized by adding aminoethylcarbazol by determining the amount of ammonia produced upon hydrolysis of in the presence of hydrogen peroxide. The dilutions of antisera used L-asparagine as described previously (13) except that 0.01 M sodium were as follows: rabbit anti-mouse IgG, 1:200; swine anti-rabbit im phosphate buffer (pH 7.0) was used in the reaction mixture. Enzyme munoglobulin, 1:10; and rabbit PAP, 1:500. The control for each assay and substrate blanks were included in all assays along with a standard consisted of an adjacent section in which the primary antiserum was curve prepared with ammonium sulfate. Enzyme activity is expressed substituted by nonimmune normal rabbit serum. All reactions were as ID (the amount of enzyme catalyzing the formation of 1 fimol carried out at room temperature. All washes and dilutions were done ammonia per min under the conditions of the assay). In all experiments, with phosphate-buffered saline (pH 7.4). Rabbit anti-mouse IgG, swine the enzyme activity of V. succinogenes and E. coli asparaginases was anti-rabbit immunoglobulin, rabbit PAP, and normal swine and rabbit determined as the average from triplicate assays of each enzyme sera were commercially obtained (Dako Corp., Santa Barbara, Calif.). Analysis of Data. Student's t test was utilized to evaluate the preparation. The activity of the 2 enzymes was always determined simultaneously and less than 24 hr before injection. observed differences in lymphocyte markers between E. coli and V. Asparaginase Treatment. Mice between 9 and 12 weeks of age succinogenes asparaginase-treated animals and controls. were treated with equivalent dosages of E. coli or V. succinogenes asparaginase. One i.p. injection of 0.2 ml was administered daily for 4 days. The dosage of enzyme was 50 ID/injection. Control animals RESULTS received 4 injections of 0.01 M phosphate buffer (pH 7.0). Preparation of Spleens for Histology. Mice were exsanguinated by Examination of cross-sections of spleen tissue from mice retroorbital bleeding in order that serum samples could be obtained for treated for 4 days with 50 IU of E. coli asparaginase per day other studies. Spleens were removed and immediately immersed in revealed a reduction in the size of lymphoid follicles accom 10% buffered formaldehyde solution. After conventional tissue proc panied by a reduction in the size of germinal centers (Fig. 1; essing, 7 Â¡imsections were taken from the paraffin blocks and stained with hematoxylin and eosin. The sections were then evaluated for the Table 1) as compared to spleen tissue of control animals. The general splenic structure, number of lymphoid follicles, size of follicles, number of lymphoid follicles appeared unchanged. The most reactivity of the follicles, and status of the intervening lymphocytes not striking difference between the spleens of E. coli asparaginase- associated with follicles. The reactivity of the follicles was estimated by treated mice and the control group was observed in reactivity the relative size of the germinal centers and the number of reactive of the germinal centers, which was demonstrated by a pro-

JANUARY 1982 253

Table 1 Slg Thy 1.2 Changes in spleen histology after treatment with asparaginase Mice were given i.p. injections of 50 IU of asparaginase in 0.01 M phosphate buffer, pH 7.0, or buffer alone (controls) on Days 0. +1, + 2, and +3. Spleens were removed on Day +4 For other details, see "Materials and Methods".

LymphocytesLymphoid centerSize+3-1-2+ folliclesTreatmentUntreatedÂ£. associated(No.)+3+1+3UJ

^untreated 3-/ u -EC coli asparagi 2/ +3+ +1+3not o: naseV. Lu unstained succinogenesasparaginaseSize-1-3+2-1-3No.+2X+3+2/ + 3Germinal2/ + 3Reactivity+ 0_ Lyt-1 Lyt-2 (fÃ

LU nounced decrease in the number of immunoblasts. The density U of lymphocytes not associated with follicles also appeared decreased in treated animals. None of these changes was or _Ec observed in cross-sections of spleen tissue from mice treated LU m for 4 days with 50 IU of V. succinogenes asparaginase per day. Spleen cells from asparaginase-treated and untreated ani mals were assayed for Slg, Thy 1.2, Lyt-1, and Lyt-2 (Chart 1;

Table 2). Lymphoid cell preparations from mice given i.p. unstained injections of 50 IU of E. coli asparaginase daily for 4 days contained 19.5% Slg-bearing cells. This represented a marked â€” FLUORESCENT INTENSITY => reduction as compared to the spleens of control mice which Chart 1. Effect of asparaginase treatment on spleen lymphoid populations. contained 32.6% Slg-bearing cells. There was an apparent Mice were given i.p. injections of 50 IU of asparaginase (control animals received shift in lymphoid populations of the spleens from enzyme- buffer) per day on DaysO, +1, +2, and +3. On Day +4, spleens were removed, and single-cell suspensions were prepared and treated with an appropriate treated mice since the drop in Slg-bearing cells was accom antibody marker as described in "Materials and Methods." Cell populations were panied by a 9.2% increase in Thy 1.2-positive cells as com examined with a FACS III cell sorter. Results are shown as fluorescent histograms of Slg-, Thy 1.2-, Lyt-1-, or Lyt-2-bearing cells. EC, spleen cells from E. coli pared to controls. Spleens from the V. succinogenes asparag asparaginase-treated animals; Vs. spleen cells from v succinogenes asparagi inase-treated animals contained control numbers of Slg- and nase-treated animals. Thy 1.2-positive cells. When spleens from E. coli asparaginase- treated animals were examined for subpopulations of T-cells, it was shown that there was a significant increase in the Table 2 percentage of cells with Lyt-1 and Lyt-2 on their surfaces as Marker distribution of spleen cells from animals treated with asparaginase Mice were given i.p. injections of 50 IU of asparaginase in 0.01 M phosphate compared to control animasi (P < 0.05). The increase was not buffer, pH 7.0, or buffer alone (controls) on Days 0, +1, +2. and +3. Spleens as marked, however, as that observed in the percentage of Thy were removed on Day +4. For other details, see "Materials and Methods." 1.2-positive cells. No change was seen in the Lyt-1 or Lyt-2 coli cell populations of V. succinogenes asparaginase-treated ani asparaginase succinogenes mals. We observed no significant change in the Lyt-2:Lyt-1 cell (%)32.6 (%)19.5 asparaginase33.2 Â±1.90a Â±1.336 Slg Â±3.08 ratio in any of the asparaginase-treated animals (p > 0.05). 54.2 Â±1.216 Thy 1.2 45.2 Â±1.26 44.3 Â±0.65 44.4 Â±0.96C In order to determine whether the preferential decrease in Lyt-1 41.4 Â±0.44 40.9 Â±1.08 21.1 Â±0.60Â° Slg-bearing cells within the spleens of Â£.coli asparaginase- Lyt-2 18.1 Â±0.49 16.9 Â±0.56 Lyt-2:Lyt-1Untreated 43.8 Â±0.92E. 47.4 Â±1.47V. 40.4 Â±1.38 treated mice represented a specific depletion of cells within the germinal centers, we assayed for cytoplasmic immunoglobulin Mean Â±S.E. of total spleen cells. The data for each experimental group were compared to the data for untreated controls. using the PAP-staining procedure. There was an elimination of " Significant difference at p < 0.001. immunoglobulin-containing cells localized within the germinal c Significant difference at p < 0.05. centers and only a partial decrease in the immunoglobulin- positive cells not associated with lymphoid follicles (Fig. 2). survival time of skin grafts, promoted allogeneic tumor take, Moreover, there was no apparent modification in immunoglob and increased the numbers of experimental mÃ©tastases(5,18). ulin-containing cells in the spleens of V. succinogenes aspa An investigation of the effects of asparaginase on the immune raginase-treated animals. system of leukemia patients revealed that the development of delayed hypersensitivity to primary antigens and antibody pro DISCUSSION duction was extremely suppressed. In contrast, established delayed hypersensitivity was left unchanged (24). The immunosuppressive effects of Â£.coli and E. carotovora In an attempt to determine the mechanism of asparaginase- asparaginases have been well documented in both animal and induced immunosuppression, we investigated the effects of human systems. In animal studies, asparaginase was shown to enzyme treatment on the immune response to the T-dependent inhibit antibody and lymphokine production, blastogenesis, and antigen, SRBC. Our previous experiments have shown that the number of PFC produced in response to specific antigenic mice treated with E. coli asparaginase display a pronounced stimulation (1, 9). In addition, treatment prolonged the median reduction in the number of specific IgM and IgG PFC to SRBC

254 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Effects of Asparaginase on Lymphoid Populations when the data are expressed as both the number of PFC/106 asparaginase treatment, we hope to elucidate the effects of the spleen cells and the number of PFC/total spleen (15). These enzyme on thymocyte differentiation. data suggest that the suppression of the humoral response to Cellular cooperation in immunogenic responses to T-depend- SRBC may be due to a specific depletion of lymphoid cells ent antigens has been extensively characterized in the mouse within the spleen. This is in agreement with earlier work that system. It is well established that T-helper (Lyt-1+) and T- showed that treatment of mice with Â£.coli asparaginase pref suppressor (Lyt-2+, 3+) cell populations cooperate to control erentially affected antibody-forming precursor cells resident in and regulate specific humoral responsiveness (10, 19). In the bone marrow (9). Other evidence for this concept exists in addition, macrophages are known to function in antigen proc that suppression of the specific ADCC response to SRBC in essing and presentation. The present study suggests that the mice treated with E. coli asparaginase appears to be the result numbers of T-cells within lymphocyte subpopulations of the of a decrease in the synthesis of specific immunoglobulin (16) spleen are unaltered by asparaginase treatment. Future studies and not due to the depletion of effector cells. in our laboratory will concentrate on determining the effects of The data presented here support the hypothesis that aspa- E. coli asparaginase treatment on the function of these acces raginase-induced immunosuppression is the result of prefer sory cells in an effort to further define the role of specific amino ential depletion of lymphocytes of the B-cell lineage. Treatment acid depletion during asparaginase-induced immunosuppres of mice with asparaginase from E. coli resulted in a reduction sion. in the size of lymphoid follicles within the spleen concomitant Our comparison of the effects of E. coli and V. succinogenes with reduced germinal centers (Fig. 1; Table 1). There was a asparaginases on the spleen cell surface marker distribution significant change in the reactivity of germinal centers that was and on spleen histology suggests that specific depletion of L- evidenced by a decrease in immunoblasts (B-cells). PAP stain asparagine is not deleterious to the immune system. Thus, we ing of immunoglobulin-containing cells confirmed these find propose that the glutaminase-free asparaginase from V. suc ings (Fig. 2). In earlier studies by Astaldi et al. (2), it was cinogenes will serve as an effective antileukemic agent without reported that E. coli asparaginase treatment resulted in a causing immunosuppression. decrease in size and cellular density of the collar surrounding the germinal center of the spleen. Other cells within the ger ACKNOWLEDGMENTS minal center appeared unaffected. The apparent discrepancy between their findings and ours may be due to a difference in The authors wish to thank Harry S. Kurchner who worked on this project as an undergraduate research assistant; Mantley Dorsey, Jr., for his excellent the route of asparaginase injection or to the use of a different technical assistance; and Dr. Jocelyne Ziegels-Weissman for her technical advice animal model. Astaldi et al. used rabbits for their experiments and expertise in the preparation of histological specimens. and administered enzyme by i.v. injection. More importantly, they injected the enzyme only once at a dosage of 1000 to REFERENCES 3000 ID/kg body weight. In our experiments, mice were given i.p. injections of 50 IU (approximately 2000 ID/kg body weight) 1. Ashworth, L. A. E., and MacLennan, M. P. Comparison of L-asparaginases from Eschehchia coli and Erwinia carotovora as immunosuppressants. Can asparaginase daily for 4 days. Our examination of lymphocyte cer Res.. 34: 1353-1359, 1974. populations within the spleen demonstrated that E. coli aspa 2. Astaldi. G., Micu, D., Astaldi, A., Burgio, G.-R., and Krc, I. Further investi gation of L-asparaginase and immune reactions. Colloq. Int. Cent. Nati. raginase treatment resulted in nearly a 40% decrease in the Rech. Sci., 197: 205-219, 1971. number of Slg-bearing cells (B-cells), accompanied by an 3. Baechtel, F. S., Gregg, D. E., and Prager, M. D. The influence of glutamine, increase in the ratio of Thy 1.2-bearing cells (T-cells). The Lyt- its decomposition products, and glutaminase on the transformation of human 2:Lyt-1 cell ratio was unchanged. The data support the concept and mouse lymphocytes. Biochim. Biophys. Acta, 427. 33-43, 1976. 4. Beard, M. E. J., Crowther, D., Galton, D. A. G., Guyer, R. J., Hamilton that glutamine or glutamine combined with asparagine deple Fairley, G., Kaz, H. E. M., Knapton, P. J., Malpas, J. S., and Bodley Scott, tion results in a profound decrease in the B-cell population of R. L-Asparaginase in treatment of acute leukemia and lymphosarcoma. Br. Med. J., 9; 191-195, 1970. the spleen. In contrast, asparagine deprivation alone caused 5. Brambilla. G., Parodi, S., Cavanna, M., Caracemo, C. E., and Baldini, L. The by the administration of asparaginase from V. succinogenes Â¡mmunodepressive activity of Escherichia co/i L-asparaginase in some trans plantation systems. Cancer Res., 30: 2665-2670, 1970. did not affect spleen histology or lymphocyte marker distribu 6. Broome, J. D. Evidence that the L-asparaginase activity of guinea pig serum tion. is responsible for its antilymphoma effects. Nature (Lond.), 191: 1114- Preliminary studies in our laboratory indicate that E. coli 1115, 1961. 7. Campbell, H. A., Mashburn, L. T., Boyse, E. A., and Old, L. J. Two L- asparaginase treatment in mice results in a significant reduction asparaginases from Escherichia coli B. Their separation, purification, and in thymic weight, which was associated with a greater than antitumor activity. Biochemistry, 6: 721 -730, 1967. 40% decrease in the total thymocyte population.4 It is well 8. Cantor, H., and Weissman, I. Development and function of subpopulations of thymocytes and T lymphocytes. Prog. Allergy, 20: 1-64, 1976. established that the thymus plays an essential role in differen 9. Chakrabarty, A. K., and Friedman. H. L-Asparaginase-induced immuno tiation and functional maturation of bone marrow-derived pre suppression: effects on antibody forming cells and serum titers. Science cursors (8, 30). Since we have demonstrated that T-cells (Thy (Wash. D. C.), 167: 869-879, 1970. 10. Claman, H. A., Chaperon, E. A., and Triplett, R. F. Thymus-marrow cell 1.2, Lyt-1, Lyt-2) within the spleen are unaffected by specific combinations. Synergism in antibody production. Proc. Soc. Exp. Biol. Med., amino acid depletion (Table 2), it is possible that early stages Ã•22: 1167-1171, 1966. 11. Cooney, D. A., and Handschumacher, R. E. L-Asparaginase and L-aspara- of stem cell differentiation within the thymus may be preferen gine metabolism. Annu. Rev. Pharmacol., 10: 421-440, 1970. tially affected by E. coli asparaginase treatment. By monitoring 12. Distasio. J. A., Niederman, R. A., and Kafkewitz, D. Antilymphoma activity of the phenotypic expression of specific cell surface antigens a glutaminase-free L-asparaginase of microbial origin. Proc. Soc. Exp. Biol. (Thy 1.2, TL, Lyt-1, Lyt-2) of thymic cell populations during Med., )55: 528-531, 1977. 13. Distasio, J. A., Niederman, R. A., Kafkewitz, D., and Goodman, D. Purifica tion and characterization of L-asparaginase with antilymphoma activity from Vibrio succinogenes. J. Biol. Chem.. 25): 6929-6933. 1976. 14. Dolowy, W. C., Henson, D., Cornet, J. C., and Sellin, H. Toxic and antineo- 4 D. L. Durden, and J. A. Distasio. unpublished observations. plastic effects of L-asparaginase. Cancer (Phila.), 19: 1813-1819, 1966.

JANUARY 1982 255

15. Durden, D. L., and Distasio, J. A. Comparison of the Â¡mmunosuppressive Cancer Res., 30: 1605-1611, 1970. effects of asparaginases from Escherichia coli and Vibrio succinogenes. 25. Prager, M. D., and Mehta, J. M. Enzymes as Â¡mmunosuppressants: basic Cancer Res., 40: 1125-1129, 1980. considerations. Transplant. Proc., 5: 1171-1175, 1973. 16. Durden, D. L., and Distasio, J. A. Characterization of the effects of asparag- 26. Rabellino, E., Colon, S., Grey, H. M., and Unanue, R. R. Immunoglobulins inase from Escherichia coli and a glutaminase-free asparaginase from Vibrio on the surface of lymphocytes. I. Distribution and qualification. J. Exp. Med., succinogenes on specific cell-mediated cytotoxicity. Int. J. Cancer, 27. 59- Ã•33: 156-167, 1971. 65, 1981. 27. Schmid, F. A., and Roberts, J. Antineoplastic and toxic effects of Acineto 17. EI-Asmar, F. A., and Greenberg, D. H. Studies on the mechanisms of bacter and Pseudomonas glutaminase-asparaginases. Cancer Chemother. inhibition of tumor growth by the enzyme glutaminase. Cancer Res., 26. Rep., 58 829-840, 1974. 116-122, 1966. 28. Schrek, R.. Batra, K. V., Holcenberg, J. S., Roberts. J., and Dolowy, W. L. 18. Fidler, I. J. Duration of in vivo effects of L-asparaginase on experimental Effect of L-glutaminase on transformation and DMA synthesis of normal metastasis. Nature (Lond.), 229. 564, 1971. lymphocytes. Acta Haematol. (Paris), 48: 12-15, 1972. 19. Gershon, R. K., Cohen, P., Hencin, R., and Liebhaber, S. Suppressor T 29. Spiers, A. S. D., and Wade, H. E. Achromobacter L-glutaminase-L-asparag- cells. J. Immunol., 108: 586-590, 1972. inase: human pharmacology, toxicology, and activity in acute leukemias. 20. Haskell, C. M., Canellos, G. P., Leventhal, B. G., Carbone, P. P., Block, J. Cancer Treat. Rep., 63: 1019-1024, 1979. B., Serpick, A. A., and Selawry, O. S. L-Asparaginase; therapeutic and toxic 30. Stutman, O. Intrathymic and extrathymic T cell maturation. Immunol. Rev., effects in patients with neoplastic disease. N. Engl. J. Med., 287: 1028- 42: 138-184, 1978. 1034, 1969. 31. Tallal, L., Tan, C., Oettgen, H., Wollner, N., McCarthy, M., Helson, L., 21. Holcenberg, J. S., Tang, E., and Dolowy, W. D. Effects of Acinetobacter Burchenal, J., Karnovsky, D., and Murphy, M. L. E. coli L-asparaginase in glutaminase-asparaginase treatment on free amino acids in mouse tissues. the treatment of leukemia and solid tumors in 131 children. Cancer (Phila.), Cancer Res., 35: 1320-1325, 1975. 25:306-320, 1970. 22. Howard, J. B., and Carpenter, R. H. L-Asparaginase from Erwinia carotovora: 32. Taylor, C. R., and Chir, B. Immunoperoxidase techniques: practical and substrate specificity and enzymatic properties. J. Biol. Chem., 247: 1020- theoretical aspects. Arch. Pathol. Lab. Med.. 102: 113-121, 1978. 1030, 1972. 33. Weber, K., and Osborn, M. The reliability of molecular weight determinations 23. Loos, M., Vadlamudi, S., Meltzer, M., Shifrin, J., Borsos, T., and Golding, A. by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244: Detection of endotoxin in commercial L-asparaginase preparations by com 4406-4412, 1969. plement fixation and separation by chromatography. Cancer Res., 32: 34. Wu, M-c., Arimura, G. K., and Yunis, A. A. Mechanism of sensitivity of 2292-2296, 1972. cultured pancreatic carcinoma to asparaginase. Int. J. Cancer, 22: 728- 24. Ohno, R., and Hersh, E. M. Immunosuppressive effects of L-asparaginase. 733, 1978.

Fig. 1. Effect of asparaginase treatment on spleen histology. Mice were given i.p. injections of 50 IU of asparaginase (control animals received buffer) per day on Days 0, +1. -1-2. and +3. On Day -1-4, spleens were removed, and cross-sections were prepared and stained with hematoxylin and eosin. For other details, see "Materials and Methods." A, cross-section of spleen from control mice; B, cross-section of spleen from mice treated with E. coli asparaginase; C, cross-section of spleen from mice treated with V. succinogenes asparaginase. X 32. Fig. 2. Effect of asparaginase treatment on immunoglobulin-containing cells within the spleen. Mice were given i.p. injections of 50 IU of asparaginase (control animals received buffer) per day on Days 0, +1, +2, and +3. On Day +4, spleens were removed, and cross-sections were prepared and stained for immunoglobulin- containing cells. For other details, see "Materials and Methods." A, cross-section of spleen from control mice; ÃŸ,cross-section of spleen from mice treated with E. coli asparaginase; C. cross-section of spleen from mice treated with V. succinogenes asparaginase. x 320.

256 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. 257 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. 7

* *>Ãª "* f* * Â£â€¢â€¢

â€¢2C

258 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Alteration in Spleen Lymphoid Populations Associated with Specific Amino Acid Depletion during L-Asparaginase Treatment

John A. Distasio, Donald L. Durden, Ronald D. Paul, et al.

Cancer Res 1982;42:252-258.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/1/252

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/42/1/252. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.