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Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells' [CANCER RESEARCH 33, 94-103, January 1973] Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells' E. M. Scholar and P. Calabresi Department of Medicine, The Roger Williams General Hospital, Providence, Rhode Island 02908, and The Division of Biological and Medical Sciences,Brown University, Providence,Rhode Island 02912 SUMMARY monocytes, and lymphocytes, it is difficult to know in what particular fraction an enzyme activity is present. It would therefore be advantageous to separate out the different Extracts of lymphocytes from normal donors and from components of the leukocyte fraction before investigating patients with chronic lymphocytic leukemia (CLL) and acute their enzymatic activities. All previously reported work on the lymphoblastic leukemia were examined for a variety of enzymes of purine nucleotide metabolism was done at best in enzymes with activity for purine nucleotide biosynthesis, the whole white blood cell fraction. Those enzymes found to interconversion, and catabolism as well as for a selected be present included adenine and guanine phosphoribosyltrans number of enzymes involved in pyrimidine nucleotide ferase (2, 32), PNPase2 (7), deoxyadenosine deaminase (7), metabolism. Lymphocytes from all three donor types (normal, ATPase (4), and inosine kinase (21). CLL, acute lymphocytic leukemia) contained the following A detailed knowledge of the enzymatic pathways of purine enzymatic activities: adenine and guanine phosphoribosyl and pyrimidine nucleotide metabolism in normal lymphocytes transferase , adenosine kinase , nucieoside diphosphate kinase, and leukemia cells is important in elucidating any biochemical adenylate kinase, guanylate kinase, cytidylate kinase, uridylate differences that may exist. Such differences may be exploited kinase, adenosine deaminase, purine nucleoside phosphorylase, in chemotherapy and in gaining and understanding of the and adenylate deaminase (with ATP). In contrast, no adenine metabolic basis for the development of resistance to certain deaminase, guanine deaminase, or xanthine oxidase activity antileukemic drugs. An investigation of the enzymes of purine could be demonstrated. Deoxycytidine deaminase was found nucleotide biosynthesis, interconversion, and catabolism has in the lymphocytes from all the CLL patients but was not been carried out in sonic extracts of purified lymphocytes detected in the lymphocytes from the normal donors. The from the peripheral blood of normal donors and from patients enzymes necessary for the de novo synthesis of purines were with CLL and acute lymphoblastic leukemia. A few of the absent in lymphocytes from both normal donors and patients enzymes concerned with pyrimidine nucleotide metabolism with CLL. The activity of all enzymes were quantitatively the were also studied. In addition, we examined the murine same for the normal and CLL cells but severalfold higher leukemic lymphoblastic lines Ll2 10 and L5 178Y and the activities were found for enzymes present in lymphoblasts from patients with acute lymphocytic leukemia. A related study involving the murine leukemic cells L5 l78Y 2The abbreviations used are: APRT, adenine phosphoribosyl and Ll 2 10 and the murine ascites Sarcoma 180 cells showed transferase (adenylate :pyrophosphate phosphoribosyltransferase) (EC similar enzymatic patterns but with excessively elevated 2.4.2.7); PNPase,purine nucleoside phosphorylase (purine nucleoside: activities in comparison to lymphocytes from normal donors orthophosphate ribosyltransferase) (EC 2.4.2.1); CLL, chronic lymphocytic leukemia; MMPR, methylmercaptopurine ribonucle or from patients with CLL or acute lymphoblastic leukemia. oside; NDPK, nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase) (EC 2.7.4.6); AMPK, adenylate kinase (ATP:AMP phosphotransferase) (EC 2.7.4.4); GMPK, guanylate kinase (ATP:GMP phosphotransferase) (EC 2.7.4.8); INTRODUCTION IMPK, inosinate kinase; UMPK, uridylate kinase (ATP:UMP phosphotransferase)(EC 2.7.4); CMPK, cytidylate kinase (ATP:CMP phosphotransferase) (EC 2.7.4); HGPRT, hypoxanthine-guanine The enzymes of purine nucieotide biosynthesis and phosphoribo syltransferase (inosinate:guanylate:pyrophosphate interconversion present in leukocytes obtained from normal phosphoribosyltransferase) (EC 2.4.2.8). Trivial names used are: and leukemic subjects have been only partially studied, unlike deoxycytidine deaminase, cytidine deaminase, pyrimidine nucleoside those of pyrimidine nucleotide metabolism, which have deaminase (cytidine aminohydrolase) (EC 3.5.4.5); xanthine oxidase been extensively investigated (6, 15, 23). Since leukocytes are (xanthine:O3 oxidoreductase) (EC 1.2.3.2); adenosine kinase (ATP:adenosine 5'-phosphotransferase) (EC 2.7.2.20); adenosine a heterogeneous mixture composed of granulocytes, deaminase(adenosine aminohydrolase) (EC 3.5.4.4); guanase,guanine deaminase (guanine aminohydrolase) (EC 3.5.4.3); adenase,adenine deaminase (adenine aminohydrolase) (EC 3.5.4.2); AMP deaminase I A preliminary report of this work has appeared (28). This work was (5'-AMP aminohydrolase) (EC 4.3.5.6); thymidine kinase supported by USPHSGrant GM 16538-03. (ATP:thymidine 5'-phosphotransferase) (EC 2.7.2.21); uridine kinase ReceivedAugust 16, 1972; aceeptedOctober 4, 1972. (ATP:uridine 5'-phosphotransferase)(EC 2.7.2.48). 94 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1973 American Association for Cancer Research. Nucleotide Metabolism in Human Lymphocytes murine Sarcoma 180 ascitic cells for the presence of the same WHOLE BLOOD enzymes that were studied in the human cells. PLASMAGEL MATERIALS AND METHODS (REMOVAL OF RBC) Materials. The sodium salts of AMP, ATP, GMP, IMP, UMP, CMP, dTDP, the magnesium salt of 5-phosphoribosyl PLASMAGEL SUPERNATANT 1-pyrophosphate; and adenosine, adenine, guanine, hypoxanthine, and deoxycytidine were purchased from P-L Biochemicals (Milwaukee, Wis.). The sodium salts of I COTTONWOOLCOLUMN phosphoenolpyruvic acid and NADH were purchased from (REMOVAL OF GRANULOCYTES) Sigma Chemical Co. (St. Louis, Mo.). MMPR-methyl-' 4C was synthesized by Dr. Shth-Hsi Chu of this laboratory. Guanine-8-' 4C (50 mCi/mmole), adenine-8-' 4C (58 LYMPHOCYTES + RED BLOOD CELLS mCi/mmole), thymidine-methyl-3 H (6 Ci/mole), uridine-5-3 H (26 Ci/mole), and Tris base (enzyme and buffer grade) were LOW SPEED CENTRIFUGATION obtained from Schwarz/Mann (Orangeburg, N. Y.). Uniformly labeled glycine-' 4C (83 mCi/mmole) was from New England (REMOVAL OF PLATELETS) Nuclear (Boston, Mass.). Plasmagel was purchased from Roger Bellon Laboratories (Neuilly, France). Hepariized plastic bags were from Abbott Laboratories (N. Chicago, Ill.). Fischer's LYMPHOCYTES + RED BLOOD CELLS medium for leukemic cells and horse serum were obtained from Grand Island Biological Co. (Grand Island, N. Y.). Wright's stain was purchased from New England Reagent HYPOTONIC LYSIS Laboratories (Riverside, R. I.). Xanthine oxidase was (REMOVAL OF RBC) purchased from Worthington Biochemical Corp. (Freehold, N. J.). Pyruvate kinase (rabbit skeletal muscle, 150 units/mg), lactic acid dehydrogenase (rabbit muscle, 360 units/mg), and PURE LYMPHOCYTES glutamate dehydrogenase (bovine liver, 45 units/mg, 50% (90.95%) solution in glycerol) were from Boehringer Mannheim Corp. Chart 1. Schematic diagram for the purification of lymphocytes (New York, N. Y.). PPO and dimethyl-POPOP were purchased from human peripheral blood. Seetext for discussion. from Packard Instruments Co., Inc. (Downers Grove, Ill.). Saponin solution was obtained from Coulter Diagnostics not put through cotton wool columns. The eluate coming off (Hialeah, Fla.). Whatman DE8 1 filter paper discs were from H. the column contained both lymphocytes and contamiliating Reeve Angel (Clifton, N. J.), and Eastman cellulose erythrocytes as well as some platelets. The platelets were Chromagram sheets were purchased from Eastman Kodak Co. removed by low-speed centrifugation at 100 X g for 20 mm in ( Rochester, N. Y.). All other chemicals used were of the an International PR-2 centrifuge. The remainder of the highest purity available and obtained from Fisher Scientific erythrocytes were removed as follows (5). Lymphocytes and Co. Boston, Mass. erythrocytes were centrifuged at 300 X g and the supernatant Blood. Blood from normal donors was collected in Abbott was removed. Cold 0.85% NaCl solution was used to suspend heparinized plastic bags (Pliapak-Pan Hepann). Leukemic the cell pellet, and 3 volumes of ice-cold distilled water were blood was collected in plastic syringes containing heparin as rapidly added. The preparation was mixed for 20 sec by gentle the anticoagulant. inversion and restored to isotonicity by the addition of 1 Isolation of Lymphocytes. The procedure used for the volume of 3.5% NaCl followed by centrifugation at 160 X g purification of human lymphocytes is shown in Chart 1. The for 10 mm. The clean lymphocyte pellet was resuspended in initial step in lymphocyte isolation involves sedimentation of 0.85% NaCl solution and washed twice more. The entire erythrocytes and removal of the leukocyte-plasma purification procedure took approximately 5 hr. Smears of the supernatant. Whole blood was mixed with 0.2 volume of an final lymphocyte suspension were examined microscopically isotonic solution of gelatin (Plasmagel) and allowed to settle after addition of Wright's stain and were found to be 90 to for approximately 40 mm at 37°.This procedure allowed for 95%purewithnoredbloodcellspresent.Asjudgedby
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