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

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The Effects of L-Asparaginase on the Amino Acid Incorporation of Mouse Lymphoid Tumors1 [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 ¡orSkeletomuscular Diseases of the Hospital ¡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 /¿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 ¿ic/ stimulatory effect on the incorporation of asparagine into the /«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 0-3°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. MAY 1968 961 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. L. T. Mashburn and C. S. Gordon 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 9«-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 37°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 —20°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 °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 ¡dine,0.0983 HIM isoleucine, 0.0985 HIM leucine, mixed with 1.4 ml of the same buffer.
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