The Effects of L-Asparaginase on the Amino Acid Incorporation of Mouse Lymphoid Tumors1

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[CANCER RESEARCH 28, 961-967, May 1968] The Effects of L-Asparaginase on the Amino Acid Incorporation of Mouse Lymphoid Tumors1

L. T. Mashburn and C. S. Gordon Research Institute Â¡orSkeletomuscular Diseases of the Hospital Â¡orJoint Diseases and Medical Center, New York, New York 10035

SUMMARY strain was treated in a similar manner, indicating that the re duction in microsomal incorporation was relevant to the corti- The effects of in vivo L-asparaginase treatment on the in coid-induced lymphocytolysis. vitro amino acid incorporation into four lymphoid tumors We have investigated the effects of in vivo EC-2 treatment (P1798 CS, P1798 CR, EARAD1 and 6C3HED) have been on the in vitro incorporation of amino acids into the micro measured. Normal mouse liver supernatant was used when somal fraction isolated from several different asparaginase- amino acid incorporation into the tumor microsomal fraction sensitive and -resistant tumors, as well as other mouse tissues. was determined. Eighteen hours after treatment, amino acid The supernatant fraction of each of the tissues was also studied incorporation into the microsomal fraction of three (P1798 CS, following EC-2 treatment. It was found that EC-2 has a pro P1798 CR, and EARAD1) of the four tumors was inhibited found inhibitory effect on the amino acid incorporation into (40-90%). There was no inhibition in the incorporation during the microsomal fraction of three of the four asparaginase- the first four hours following treatment. The microsomal frac sensitive tumors tested, while treatment stimulates the ability tions of the asparaginase-resistant strains of P1798 CS and of the supernatant fraction to mediate in vitro asparagine in P1798 CR showed no inhibition of amino acid incorporation corporation into normal liver microsomes. A preliminary report eighteen hours after treatment. Both the asparaginase-sensitive of this work has been published (11). and -resistant strains of 6C3HED had increased incorporation into the microsomal fraction following eighteen-hour treatment. MATERIALS AND METHODS Among the tissues studied from tumor-bearing and normal BALB/c mice, only the spleen microsomal fraction had a de Asparagine-14C (uniformly labeled, specific activity 42 \ia/ creased amino acid incorporation eighteen hours after treat /Â¿mole)and aspartic acid-3H (generally labeled, specific activity ment. The supernatant fraction from each of the tumors, as 185 /tc/Vmole) were purchased from Nuclear Chicago Corp., well as the other mouse tissues used (except the brain), had a Des Plaines, Illinois. Leucine-14C (uniformly labeled, 223 Â¿ic/ stimulatory effect on the incorporation of asparagine into the /Â«mole)wasobtained from New England Nuclear Corp., Boston, microsomal fraction of normal mouse liver. A similar effect was Mass. BBOT2 was purchased from Packard Instrument Co. not demonstrable on the incorporation of leucine into the same Inc., Downers Grove, Illinois and naphthylene was from Baker microsomal fractions. Chemical Co., Phillipsburg, N. J. All of the amino acids used were of the L-form and were purchased from Calbiochem, Los INTRODUCTION Angeles, California. Distillation Products Industries, Rochester, N. Y. was the source of 2-mercaptoethanol. The nucleotides Purified EC-2 (L-asparaginase; L-asparagine amidohydro- ATP and GTP were purchased from Schwarz BioResearch, lase, EC.3.5.1.1) isolated from Escherichia coli has been shown Inc., Orangeburg, N. Y. Phosphoenolpyruvate and phospho- to be a potent antilymphoma agent against several tumors cnolpyruvate kinase were products of Sigma Chemical Co., St. when tested by the delayed method (4, 10, 14). Sobin and Louis, Mo. Formalated cellulose (Cellex-XF-1) was obtained Kidd (21) have reported a rapid decrease in the in vivo from LKB Instrument*, Rockville, Md., and the hydroxylapa- incorporation of amino acids into ascites cells of 6C3HED tite used was Bio-Gel HT from Bio-Rad Laboratories, Rich lymphosarcoma following intraperitoneal injection of guinea mond, California. pig serum. Guinea pig serum has been shown to contain an Preparation of L-Asparaginase (EC-2) from E. coli. Lyo- asparaginase with antilymphoma properties (1, 13, 22). philyzed E. coli B cells, ECB 6402 or ECB 6506 (Worthing- A decrease in amino acid incorporation into isolated micro- ton Biochemical Corp., Freehold, New Jersey), were suspended somes of the corticoid-sensitive strain of P1798 lymphosarcoma in cold Tris buffer (0.05 M, pH 8.6) to give a 10% suspension. following treatment with 9<*-fluoroprednisolone has been re All further operations were carried out at 0-3Â°C. The suspen ported (6). No inhibition was seen when the corticoid-resistant sion was subjected to sonication for 30 minutes and centri-

!This work was supported by USPHS Grants CA 08765 and - Abbreviations used in this paper are : GTP, guanosine triphos- CA 10064. phate; TCA, trichloroacetic acid; RNase, ribonuclease; BBOT, Received August 28, 1967; accepted January 16, 1968. 2,5-bis[2-(5t-butylbenzoxazolyl)]thiophene.

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fuged to remove the cellular debris as well as any unbroken measurement of 3 diameters. The rate of growth or regression cells. The clear supernatant was treated with 0.05 volume of 1 was calculated as the change in average tumor diameter each M MnCl2 and the resultant precipitate was removed by centrif- day. The asparaginase-resistant strain of P1798 CS was devel ugation. The supernatant was stored in the refrigerator over oped in this laboratory by frequent, increasingly large injec night and again clarified by centrifugation before being brought tions of EC-2 through several transplant generations. This to 50% saturation (2 M) with solid ammonium sulfate. A pH strain, designated P1798 CS/AH, was treated with repeated greater than six was maintained by the addition of 1 ml of 1 injections of 9Â«-fluoroprednisolone by a method previously used N NaOH to each 10 gm of ammonium sulfate. The precipitate to develop a corticoid-resistant strain (8). The resulting double was removed by centrifugation and discarded. Solid ammonium resistant strain was designated P1798 CR/AU. Both of these sulfate was added to the supernatant until the solution was sat asparaginase-resistant strains have been regularly transplanted urated (4 M). The precipitate was collected by centrifugation, subcutaneously for more than two years. The resistant strain suspended in glass-distilled water, and dialyzed against half- of 6C3HED was obtained from Dr. John Broome, New York strength electrophoresis buffer (see below) until it was negative University, and has been regularly transplanted in our labora to Nessler's solution. The dialyzed enzyme solution was applied tory for three years. Mice with subcutaneous transplants of to a large Porath electrophoresis column (LKB Instruments, EARAD1 leukemia (3) were a generous gift from Dr. E. A. Inc., Hockville, Maryland), which had been packed with 1.5 kg Boyce of Sloan-Kettering Laboratory. of formulated cellulose and equilibrated with 0.2 M Tris-borate Determination of Amino Acid Incorporation. Tumor-bearing buffer, 0.0084 M ethylenediaminetetraacetic acid, pH 8.8. The mice were injected intraperitoneally with 0.1 ml of an EC-2 electrophoresis was carried out for 17 hours at 700 volts and preparation containing 1.2-1.4 U or with 0.1 ml of saline. 300-310 milliamperes. The material was eluted with the same The animals were sacrificed by dislocation of the cervical ver buffer; fractions containing asparaginase were pooled and tebrae 2, 4, or 18 hours following the injection. The methods concentrated at 37Â°Cin a rotary evaporator under vacuum. of Ochoa and Weinstein (16) were used for the preparation Foaming was prevented by the addition of n-octyl alcohol. of the microsomal fraction and supernatants, as well as for the The concentrated enzyme solution was dialyzed against 0.05 measurement of amino acid incorporation. The 122,000 X g M Na2HP04 (pH 8.6) and applied to a hydroxylapatite col pellet is referred to as the microsomal fraction and the 122,000 umn which had been equilibrated with the same buffer. The X g supernatant is referred to as the supernatant. The micro column was washed with 0.1 M Na2HP04 and 0.05 M K2HP04 somal incorporation was measured in the presence of the super before the enzyme was eluted with 0.1 M K2HPO4 (4). Frac natant from liver of normal BALB/c mice. The effects of the tions containing asparaginase activity were pooled and con supernatants of the tumors as well as of other tissues were centrated as before in the rotary evaporator. The concentrated studied on the microsomal fraction of normal BALB/c mouse enzyme was dialyzed against 0.01 M Na2HP04 in 0.9% NaCl, liver. The microsomal fraction (0.3-0.4 mg protein) and the pH 8.0, sterilized by Millipore filtration, and stored at â€”20Â°C supernatant (0.4-0.5 mg protein) were incubated in a final until used. The EC-2 samples used in experiments reported volume of 0.4 ml for 15 minutes at 37 Â°C.The incubation mix here showed 1-3 faint protein bands in addition to the very ture was the following: 0.01 M Tris-HCl buffer, pH 7.4, 5.0 strong asparaginase band on disc electrophoresis (Canalco HIM magnesium acetate, 0.06 M potassium chloride, 0.25 mM Co.) and had a specific activity of 23-67 U/mg protein. GTP, 0.625 HIM ATP, 3.12 HIM phosphoenolpyruvate, 6.0 HIM Assay of L-Asparaginase Activity. An aliquot of enzyme was 2-mercaptoethanol, 0.051 mM arginine, 0.0241 mM cystine, diluted to 0.1 ml with Tris-HCl buffer (0.05 M, pH 8.6) and 0.0229 mM hist Â¡dine,0.0983 HIM isoleucine, 0.0985 HIM leucine, mixed with 1.4 ml of the same buffer. Immediately before the 0.0098 IÃ•IMlysine, 0.0251 mM methionine, 0.0489 HIM phenyl- start of the incubation, 0.5 ml of asparagine (0.04 M in the alanine, 0.108 mM threonine, 0.0098 mM tryptophan, 0.051 Tris buffer) was added. After 30 minutes at 37Â°C,the reaction mM tyrosine, and 0.11 niM valine containing 30 jug/ml phos was stopped by the addition of 0.2 ml of 1.5 M TCA. If any phoenolpyruvate kinase and 0.25 microcurie/ml of the radio precipitate formed upon the addition of the acid, the mixture active amino acid (asparagine-14C, leucine-14C, or aspartic was diluted 1:2 with glass-distilled water and centrifuged. The acid-3H). When incorporation of leucine was measured, the supernatant was brought to a volume of 9.5 ml, and 0.5 ml unlabeled leucine was omitted from the incubation mixture. of Nessler's solution was added. The orange color was measured The reaction was stopped by addition of 3.0 ml of 5% TCA at 500 mfi between 5-15 minutes after the addition of the containing 1% unlabeled amino acid corresponding to the radio Nessler's solution. One international unit (U) is that amount active one. The resulting suspension was heated in boiling water of enzyme which liberates one micromole of ammonia per for 30 minutes, cooled to room temperature, and washed with minute under the assay conditions. 3.0 ml fresh TCA-amino acid solution. The washed precipitate Determination of Asparaginase Sensitivity in Vivo. Tumors was dispersed on a Millipore filter and again washed with the were transplanted by trocar subcutaneously in the right flank TCA-amino acid solution (3 X 2 ml) followed by 2 X 2 ml of the host female mice. The 6C3HED tumors were trans of 70% ethyl alcohol. The filters were dissolved in a 1:1 mix planted into C3H/HeJ mice from Jackson Laboratories, Bar ture of dioxane-ether on aluminum planchÃ©isand, after drying, Harbor, Maine and the P1798 tumors were transplanted into were counted in a low background gas-flow counter (Nuclear BALB/c mice from Simonsen Laboratories, Gilroy, California. Chicago). When aspartic acid-3H was used in the incubation After 10-14 days, intraperitoneal injections of EC-2 were given mixture, the Millipore filters were put in glass vials, dissolved in equal daily doses for 4 days immediately following tumor in 12 ml of the scintillation fluid (300 ml methyl cellosolve,

962 CANCER RESEARCH VOL. 28

450 ml toluene, 60 gm naphthylene, and 3 gm BBOT), and sensitivity. The asparaginasc-resistant strain (P1798 CS/AR) counted in a liquid scintillation spectrometer (Packard Instru did not regress, although the host mice were treated with more ments Co.). Protein was determined by the method of Lowry than 10 times the dose of EC-2 required to cause rapid regres et al. (9), using bovine serum albumin as a standard. The data sion of the sensitive strain (P1798 CS/AS). Similarly, the are calculated for incorporation of labeled amino acid per resistant strain of Gardner lymphosarcoma 6C3HED continued milligram of microsomal protein and are expressed as percent to grow when the host mice were treated with twice the amount of control in Tables 2, 3, and 6. of EC-2 necessary for complete regression of the sensitive The nucleic acid levels of the microsomal fractions were strain (Chart 1). estimated by measuring the optical density at 260 m/i and at All of the components in the incubation medium are neces 280 m/x and using the nomograph by E. Adams, which is dis sary for maximum incorporation of asparagine into the micro tributed by Calbiochem, Los Angeles, California. The data are somal fraction of the various tissues (Table 1). It appears, calculated as micrograms of nucleic acid per milligram protein however, that the amino acid mixture does not enhance the and expressed as percent of control in Table 6. The microsomal incorporation activity of the P1798 CS/AS fraction before or fraction from one group of tumors (P1798 CS/AS) was resus- after EC-2 treatment. The increased dependence of the micro pended in water instead of the sucrose-containing buffer, so somal fraction of treated P1798 CS/AS on supernatant and that a pentose determination could be carried out by the orcinol on GTP may reflect an added requirement of the treated method of Khym and Doherty (7). The results of that pro microsomal fraction which has not previously been detected. cedure agreed with the values reported in Table 6. The requirements of the system of the P1798 CR/AS seem to be similar to those of the liver in all respects. Mercaptoethanol RESULTS is more necessary in the P1798 CS/AS system than in that of the liver or the P1798 CR/AS. This difference, therefore, Asparaginase sensitivity of the tumors used in these experi seems to be related to the corticoid sensitivity rather than to ments is shown in Chart 1. The rapid regression following EC-2 the asparaginase sensitivity of the tumors. The microsomal treatment of the two strains of P1798 (CS and CR), which fraction of each of the tumors is more dependent on magnÃ©sium were developed by Lampkin antl Potter (8), indicates that ions than is the liver microsomal fraction. there is no correlation between corticoid and asparaginase The effects of EC-2 on the incorporation of amino acids into

PI798 CS PI798 CR 6 3 16.3 0 â€¢3 6

3 4

6C5HED EARADI 6 6 UJ o 3 3 O O o _ 3 â€¢3 -6 â€¢6

I DAYS OF TREATMENT

Chart l : Sensitivity of various lymphoid tumors to purified L-asparaginase. Tumor-bearing mice (asparaginase-resistant strain (C C). asparaginase-sensitive strain â€¢ â€¢)) were injected with equal daily doses of asparaginase (EC-2), i.p., for 4 days immediately following tumor measurement of 3 diameters. Control asparaginase-sensitive tumor-bearing mice (Q O) were in jected with buffered saline instead of EC-2. Data shown for EARADI are from a previous publication (3). The numbers on the curves represent the total amount (U) of EC-2 given each mouse during the test period. Each point is the average of 4 mice.

MAY 1968 963

Table 1 eighteen hours appears to consistently inhibit the incorporation systemP1798 Percent of complete of leucine (Table 3). CS/AS CS/AS CR/AS Eighteen hours after EC-2 treatment, the corticoid-resistant, controlNoneMicrosomesSupernatant6Mg+Omission treated"100038407352289639P1798control10002865746947775Liver10003681707987460asparaginase-sensitive strain of P1798 has almost complete inhibition of amino acid incorporation into the microsomal fraction, while the supernatant from these tumors strongly enhances the incorporation of asparagine, but not of leucine, +K+GTPATP, into liver microsomal fraction (Table 3). Mice bearing E AR ADI (3) were obtained from Dr. E. A. Boyse to ascertain if EC-2 treatment of these mice would affect the ability of the micro PEPkinPEP, and aseAmino somal fraction to incorporate asparagine. On the basis of that acidmixtureMercaptoethanol100073577276189135P1798 one experiment, it appears that the microsomal amino acid incorporation of EAR ADI is impaired similarly to that of P1798 CS/AS (Table 3). The increased incorporation into the The effect of incomplete incubation systems on asparagine in corporation into the microsomal fraction of tumors. GTP, guano- microsomal fraction of EC-2 treated 6C3HED/AS (Table 3) clearly separates it from the other asparaginase-sensitive sine triphosphate ; PEP, phosphoenolpyruvate. a Eighteen hours after asparaginase treatment. tumors tested. After this result was obtained the in vivo sensi 6Supernatant from livers of normal BALB/c mice was used in tivity of the tumors to asparaginase was reaffirmed and the each experiment. resulting curve is shown in Chart 1. Other tissues of tumor-bearing mice (P1798 CS/AS) were assayed for changes in the microsomal fraction and in the the tumor microsomal fraction of the asparaginase-sensitive supernatant in order to determine the specificity of the effects and the asparaginase-resistant strains of P1798 CS are given seen in the tumor fractions. Of the tissues studied only the in Table 2. Although there is no significant change in the as spleen, another lymphoid tissue, showed a substantial reduction paragine incorporation during the first four hours after treat of asparagine incorporation into the microsomal fraction ment, there is a remarkable, reproducible inhibition of amino (Table 4). However, the microsomal fraction of kidneys from acid incorporation into the microsomal fraction of the sensitive treated mice had greatly increased incorporation similar to tumors eighteen hours after treatment. Similar inhibition is that seen in the 6C3HED/AS following EC-2 treatment. To noted for the incorporation of leucine and aspartic acid after eliminate any relationship of the tumor to the other affected 18 hours, but, again, no reduction in incorporation is observed tissues, the asparagine incorporation of the microsomal frac at the earlier times studied. No inhibition is demonstratable in the microsomal fraction of the asparaginase-resistant strain tions of those tissues (i.e., spleen and kidney) from normal BALB/c mice was measured. The treatment affected the tissues of the tumor at any of the times studied. The effect of supernatant of tumors from EC-2 treated from the two groups of mice similarly (Table 4). On the basis animals on the asparagine incorporation into the microsomal of these two single experiments, it is difficult to appraise the fraction of normal liver is striking (Table 2). Two hours after difference in magnitude of the values obtained for the effect on treatment, the supernatant from both sensitive and resistant the fraction of normal kidneys and that of kidneys from the strains enhanced the incorporation of asparagine but gave no tumor-bearing mice. However, we feel that the incorporation stimulation of leucine incorporation. This stimulation seems into the microsomal fraction of the spleens in both cases can to diminish after an initial peak, although it is still present in be considered to be inhibited similarly to the lymphoid tumors. most samples 18 hours after treatment (Tables 3, 5). On the Asparagine incorporation into the microsomal fraction of other hand, the tumor supernatant from animals treated liver is stimulated by the supernatant of all tissues (except the

Table 2 microsomal fraction with normal supernatant with normal liver control)Asparagineaverage98liver supernatant (% of control)Asparagineaverage188149135206ib(4)(3)(6)(2)(2)(2)range117-293130-17697-216171-24187-26261-227Leucineaverage80microsomal fraction (% of after Tumor treatment acidaverage90 Aspartic acidaverage strainP1798 (hours)24182418Tumor range85-19139-48average range100 CS/ASP1798CS/ARTime (3)Â«107 (1)138 (1)72 (1)78 (3)52 (3)44 (3)50 (7)90 (2)93 53-5595-102Tumor54 (2) (2)97 (2) 73-83 (2)117 (1)99 (1)85 (2)119 (2)125 (2)60 (2)range79-13364-13745-6580-99109-12583-154Leucine(1)range (1)range62-7948-5170-100Aspartic Effects of asparaginase (EC-2) treatment on the amino acid incorporating system of P1798 CS lymphosarcoma. "â€¢Parenthesesindicate the number of experiments. Each experiment contained two separate groups of control and treated samples which were processed simultaneously. 6 Since the two experiments gave distinctly different results, the values were not averaged and the approximate value is still considered unknown.

964 CANCER RESEARCH VOL. 28

Tables Tumor microeomal fraction Tumor supernatant with normal with normal liver supernatant liver microaomal fraction (% of control) (% of control)

TumorP1798 range52 range44 range135 range50 CS/ASP1798 (7)Â«45-65119 (2)39-48125 (6)97-216b170 (2)48-5160 CS/ARP1798 (2)83-15420 (1)8 (1)36 CR/ASP1798 (3)9-37131 (1)130 (3)148-187260 (1)84 CR/AREARAD16C3HED/AS6C3HED/ARAsparagineaverage(1)66 (1)120 (1)224 (1)50 (1)188 (1)203 (3)148-225170 (1)Asparagineaverage(3)156-243255 (1) (1)Leucineaverage (1)Leucineaverage The amino acid incorporating system of tumors eighteen hours after treatment with asparaginase (EC-2). " Parentheses indicate the number of experiments. Each experiment contained two separate groups of control and treated samples which were processed simultaneously. 6Two experiments gave distinctly different results (61, 227). The values were not averaged and the approximate value is still considered unknown. brain) studied 18 hours after EC-2 treatment (Tables 2, 3, 10,800-122,000 X g and may be of a different composition from and 5). that of the control samples. However, the presence of the The change in nucleic acid content of the microsomal frac microsomal fraction is necessary for any amino acid incorpora tions of the tumors 18 hours after treatment is shown in tion to take place in the system of the treated tumors as well Table 6. There is little agreement in the degree of reduction as that of the control tumors (Table 1). Further, the change of nucleic acid with the inhibition of amino acid incorporation. in the microsomal fraction which seems to be responsible for With a 25% reduction in nucleic acid, P1798 CS/AS had con the marked decrease in amino acid incorporation does not siderable inhibition of amino acid incorporation, while the appear to be closely related to the decrease in nucleic acid incorporation into the microsomal fraction of 6C3HED/AS was (Table 6). Although both DNA and RNA are measured by stimulated. Further, a significant reduction in amino acid the procedure used, the change is likely to be the result of the incorporation in EARAD1, but a large stimulation in the in increased RNase which has been found in 6C3HED (15) and in corporation into the microsomal fraction of 6C3HED/AS was P1798 (12) following treatment. However, DNA should be associated with no change in nucleic acid. The two experiments lower in all regressing tissues. If the increased RNase were with P1798 CR/AS show a large reduction in both amino acid acting on messenger or ribosomal RNA, there should be the incorporation and nucleic acid. same definite decrease in incorporation of amino acids into the microsomal fraction of 6C3HED/AS as is seen in that of DISCUSSION the P1798 CS/AS, since both tumors have elevated RNase 18 hours after treatment. Because of the lack of agreement A dramatic fall in microsomal amino acid incorporation is in the degree of reduction of the nucleic acid to the inhibition seen in the two asparaginase-sensitive strains of P1798 and in of amino acid incorporation, we plan more detailed analyses an asparaginase-sensitive strain of EARAD1 18 hours after of the microsomal fractions in order to ascertain the exact treatment while there is no decline in incorporation in any of nature of the change. the asparaginase-resistant tumor strains. Sobin and Kidd (22) have reported a rapid decrease in pro The fall in amino acid incorporation appears to represent a tein synthesis in ascites 6C3HED following intraperitoneal in definite change in the microsomal fraction. This fraction con jection of heated guinea pig serum into tumor-bearing mice. tains material which is deposited by centrifugation between Incorporation of valine-14C into the cell during a five-minute

Table 4 mice mice (A/Â«noles/mgprotein)%of protein)Control113105714.612325.54TreatedÂ»11560.7413.061.192.98%(/x/imoles/mg of TissueBrainKidneyLiverSpleenTumor Controlcontrol1.48 Treated" control100274895154

14814.610.992.19

0.66 67Tumor-bearing (P1798 CS/AS)Normal Effect of asparaginase treatment on asparagine-14C incorporation into the microsomal frac tion of various tissues. a Eighteen hours after asparaginase (EC-2) treatment.

MAY 1968 965

Table 5 and that the resultant tumor regresses when further EC-2 Asparagine-t4C incorporation treatment is given. Similar results have been obtained with protein)TissueBrainKidneyLiverSpleenTumor (/i/imoles/mg EARAD1 (3). (c) Increased alkaline RNase activity has been control114181154155216observed two hours after treatment with EC-2 in 6C3HED/AS (15), while no significant increase occurred within four hours in P1798 CS/AS (12). Since early reports on clinical trials with EC-2 have shown that large quantities of enzyme are necessary for remission of some lymphoid tumors in man (P1798CS/ AS)Control7.443.499.504.404.48TreatedÂ«8.4763014.656.839.68%of(17, 18), we feel that 6C3HED is not an ideal test system to The effect of supernatant from tissues of tumor-bearing mice on use in studying possible mechanisms of the antilymphoma action of EC-2. Agreement from several other systems may amino acid incorporation into normal liver microsomes. a Eighteen hours after asparaginase (EC-2) treatment. be more meaningful. The evidence that EC-2 affects the microsomal fraction of the spleen of the tumor-bearing and of normal BALB/c mice pulse following an earlier injection of guinea pig serum was significantly reduced. The reduction could be caused by an (Table 4) to approximately the same extent as those of the increase in amino acids in the ascites fluid due to tumor break sensitive tumors (Table 3) indicates that the effect of aspara down or by lack of asparagine as they observed in vitro (20). ginase on the microsomal fraction is not specifically limited to Reduction in incorporation could not be demonstrated here certain tumors, but acts on normal lymphoid tissue as well. in the separated parts of the protein synthesizing system of This, together with the lack of any significant change in amino 6C3HED, although a significant decrease was observed in the acid incorporation into the microsomal fraction four hours other three asparaginase-sensitive tumors tested (Table 3). after treatment with EC-2, suggests to us that the change in The decrease in incorporation of leucine and aspartic acid into the microsomal fraction is not the primary site of the antitumor the microsomal fraction of treated P1798 asparaginase-sensitive action of asparaginase, although it probably is an important tumors could not be due to a lack of asparagine, as has been secondary effect. suggested for 6C3HED cells (2, 20). Here, the asparagine con The striking increase in the asparagine incorporation into centration is the same in treated and control incubations since liver microsomal fractions which are incubated with super the same liver supernatant is used. The decrease in leucine and natant from treated tissues is probably due to a reduced level aspartic acid, as well as in asparagine incorporation, is, there of unlabeled asparagine in the supernatant. Since the entire fore, strong evidence of a change in the microsomal fraction. asparagine pool of the mouse should be decreased following The stimulation of amino acid incorporation into the micro systemic treatment with asparaginase, all of the tissues should somal fraction of 6C3HED/AS following EC-2 treatment is give the same response. Tables 2, 3, and 5 show that the super puzzling, but it should be pointed out that this tumor is dif natant of each of the tissues tested except that of the brain ferent from the other three in several respects, (a) Permanent did, in fact, give a significant increase in asparagine incorpora tion into the liver microsomal fraction following EC-2 treat remission of subcutaneous 6C3HED has been achieved often in this laboratory as well as in others (22) following treatment ment. The reason for the lack of response in the brain may be with only 1.0 U of asparaginase while the others require 40-80 that the asparaginase does not easily cross the blood-brain U of EC-2 for permanent remission. (6) After EC-2 induced barrier. Dolowy et al. (5) found it necessary to treat mice remission of 6C3HED of all the host mice which we have bearing intracerebral 6C3HED with substantially more enzyme tested became immune to reinoculation with the same tumor. (6.7 U) than is necessary to treat mice with subcutaneous A high percentage of immunity of host mice followed regres 6C3HED (1.0 U) to achieve permanent remission. The varia sion with guinea pig serum (22) or EC-2 on the ascites form tion in the effect of treatment on the asparagine incorporation of the tumor (19). We have found that P1798-bearing mice in the presence of the supernatant of the P1798 CS/AR are susceptible to reinoculation following complete remission (Table 2) cannot be explained, but the constancy of the effect on all of the other systems makes the low values suspect. Table6 Any hydrolysis of asparagine to aspartic acid in the incuba tion mixture would also increase the apparent incorporation acid incorporation acid of asparagine, since both labeled asparagine and labeled aspar Tumor6C3HED/AS6C3HED/ASP1798 (% ofcontrol)1481806255173766Nucleic(% ofcontrol)957464756359106tic acid would be incorporated. It is unlikely that such a phenomenon is taking place here, since in an experiment using asparagine-14C and aspartic acid-3H with supernatant of CS/ASP1798 P1798 CS/AS after two hours of treatment, the carbon in CS/ASP1798 corporation was increased (293% of control), while the tritium incorporation was not changed. If there were both aspartic CR/ASP1798 acid-14C and aspartic acid-3H in the incubation of the super CR/ASEARAD1Amino natant of the treated tumor and only aspartic acid-3H in the incubation of the supernatant of the control tumor, a decrease Change in nucleic acid in the microsomal fraction of tumors in tritium incorporation should accompany any increase in following asparaginase (EC-2) treatment. carbon incorporation if the source of the increase is as-

966 CANCER RESEARCH VOL. 28

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. Tumor Amino Acid Incorporation partic-14C. The lack of a decrease in the tritium incorporation Development of Cortisone Resistance in a Cortisone-sensitive also indicates that the aspartic acid pool in the supernatant is Lymphosarcoma of the Mouse. J. Nati. Cancer Inst., 20: 1091- not significantly changed two hours after treatment. Therefore, 1112, 1958. the supernatant of the untreated tumor probably has a suffi 9. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. cient amount of aspartic acid so that subsequent treatment Chem., 193: 265-275, 1951. with asparaginase and, thus, hydrolysis of the asparagine in 10. Mashburn, L. T., Boyse, E. A., Old, L. J., and Campbell, H. A. the tumor does not significantly alter it. A Comparison of Concurrent and Delayed Tests for Antitumor The inhibition of leucine incorporation by supernatants from Activity of L-Asparaginase. Proc. Soc. Exptl. Biol. Med., 124'- treated tumors can probably be explained by a change in pool 568-571, 1967. size. Due to breakdown of the tumor protein, the amino acid 11. Mashburn, L. T., and Gordon, C. S. Effect of Asparaginase on pool would increase. This increase would be reflected in the Protein Synthesizing System of P1798 Lymphosarcoma. Fed cell supernatant fraction and would dilute the amount of labeled eration Proc., 26: 807. 1967. amino acid incorporated into microsomes which are incubated 12. Mashburn, L. T., and Landin, L. M. Changes in Ribonuclease with that supernatant. Alternatively, the reduction in incor Activities in P1798 Lymphosarcoma Following Asparaginase poration of leucine could be the result of a reduced amount of Treatment. Arch. Biochem. Biophys., in press. asparagine in the supernatant. 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MAY 1968 967

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. The Effects of l-Asparaginase on the Amino Acid Incorporation of Mouse Lymphoid Tumors

L. T. Mashburn and C. S. Gordon

Cancer Res 1968;28:961-967.

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