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Home , Adenosine deaminase, AMP deaminase, Cytidine deaminase, Cytidylate kinase, Guanine, Guanine deaminase

[CANCER RESEARCH 33, 94-103, January 1973] Identification of the Enzymatic Pathways of in Human 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 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 of nucleotide metabolism was done at best in enzymes with activity for purine nucleotide , the whole white blood cell fraction. Those enzymes found to interconversion, and catabolism as well as for a selected be present included and phosphoribosyltrans number of enzymes involved in nucleotide ferase (2, 32), PNPase2 (7), deaminase (7), metabolism. Lymphocytes from all three donor types (normal, ATPase (4), and (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 , kinase , nucieoside diphosphate kinase, and leukemia cells is important in elucidating any biochemical , , cytidylate kinase, uridylate differences that may exist. Such differences may be exploited kinase, , purine phosphorylase, in 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, , or oxidase activity antileukemic drugs. An investigation of the enzymes of purine could be demonstrated. 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 of 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 ) (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, -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 , deaminase, pyrimidine nucleoside those of pyrimidine nucleotide metabolism, which have deaminase (cytidine ) (EC 3.5.4.5); been extensively investigated (6, 15, 23). Since leukocytes are (xanthine:O3 ) (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 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); kinase supported by USPHSGrant GM 16538-03. (ATP:thymidine 5'-phosphotransferase) (EC 2.7.2.21); kinase ReceivedAugust 16, 1972; aceeptedOctober 4, 1972. (ATP:uridine 5'-phosphotransferase)(EC 2.7.2.48).

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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 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 (enzyme and buffer grade) were LOW SPEED CENTRIFUGATION obtained from Schwarz/Mann (Orangeburg, N. Y.). Uniformly labeled -' 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.). (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 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 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 the sedimentation of most of the red blood cells. fluorochromasia, the lymphocytes were over 90% viable (25). For removal of granulocytes and monocytes from this white Cell Counts. All lymphocyte counts were made in a Coulter cell-rich plasma, the leukocyte-plasma suspension was placed Model B counter (Coulter Electronics, Hialeah, Fla.). In the on a cotton wool column for 30 mm at 37° (14). impure lymphocyte fractions, 3 drops of saponin solution Lymphocytes were washed off with Fischer's medium plus were added to the counting vials to lyse any erythrocytes 10% horse serum while granulocytes and monocytes adhered present. to the cotton wool. Blood from patients with acute Isolation of Animal Tumor Cells. Sarcoma 180, L5 178Y, or lymphoblastic leukemia and some of the blood of patients Ll210 cells, harvested from mice 7 days after i.p. tumor with CLL contained over 90% lymphocytes and therefore was inoculation of either 1 or 2.5 X 106 cells, were washed with

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0.85% NaCl solution. Red blood cells were removed by lysis After incubation for 20 mm at 37°, the reaction was with hypotonic NaCl solution, and the tonicity was terminated by the addition of 50 @ilof 4 N . An completely restored by addition of 3.5% NaC1 solution. After 3 aliquot (10 p1) of the reaction mixture was spotted on additional washes, the cells were counted and then sonically Eastman Kodak cellulose thin layers on Mylar sheets that had disrupted as described below. been spotted previously with carrier amounts of the Enzyme Preparation. After being washed, the purified appropriate nonradioactively labeled nucleotide or base. The lymphocytes or leukemic cells were suspended in 0. 1 M nucleotide products were separated from the by Tris-acetate buffer, pH 7.4, and disrupted sonically for 75 sec development in 5% Na2 HPO4 for 45 mm. Following with a sonifier cell disruptor (Heat Systems-Ultrasonics, Inc, development, the appropriate nucleotide area as seen by UV Plainview, N. Y.) set at a 10-watt output. The animal tumor (254 nm) was cut out and counted in a toluene-based liquid cells were sonically disrupted for 120 sec and like the human scintillation counting system (5 g PPO and 0.5 g cells were kept at 00 during the process. The resulting dimethyl-POPOP per liter of toluene). Nucleotide formation suspensions were centrifuged for 60 mm at 39,000 X g. The showed a linear relationship to incubation time and supernatant was used for the enzyme assays. The enzyme concentration under the conditions used. activities in the pellet were not determined, but previous Adenosine kinase activity was assayed by a radiochemical studies have shown that very little, if any, activity was present method as modified from that of Schnebli et al. (27). The there. incubation mixture contained, in a final volume of 200 ,il, Enzyme Assays. All the enzymes were assayed at room 200 mM Tris-acetate, pH 7.0; 1 mM ATP; 6 mM phosphoenol temperature unless otherwise indicated. PNPase was assayed pyruvate; 0.2 mg pyruvate kinase; 100 mM KC1; and 10 mM by the coupled xanthine oxidase method of Kalckar (1 1). The MgCl2. MMPR-methyl-' “Cwas present as the substrate at a assay conditions and definition of enzyme units for PNPase are fmal concentration of 0.50 mM . The reaction was started by described by Kim et al. (1 3). Xanthine oxidase was assayed as addition ofMMPR and, after incubation for 20 mm at 25°,the described in the Boehringer Mannheim Corp. catalog except a reaction was stopped by immersing the reaction tubes in a 1.0-mi volume was used. NDPK was assayed by the coupled boiling water bath for 90 sec. The reaction mixtures were then pyruvate kinase-lactic dehydrogenase method as described by centrifuged for 30 mm at (39,100 X g). An aliquot (10 jil) of Mourad and Parks (20) except that the substrate was 0.4 mM the supernatant with the appropriate carriers was spotted on dTDP instead of dGDP. AMPK activity was measured Eastman Kodak cellulose thin layers, and the MMPR spectrophotometrically by a coupled pyruvate kinase-lactic 5'-phosphate was separated from MMPR by develop dehydrogenase system identical to that used for NDPK ment in 5% Na2 HPO4 for 45 min. The area of the thin layer except that the substrate was 0.1 mM AMP. GMPK activity containing the radioactive MMPR-5'-phosphate was located by was measured spectrophotometrically, as with the NDPK inspection under UV (254 nm) and then cut out and counted assay, except that the substrate was 0.1 mM GMP, by in a toluene-based liquid scintillation counting system as determining the rate of formation of ADP and GDP in the previously described. Nucleotide formation showed a linear presence of nonrate-limiting concentrations of relationthip to incubation time and protein concentration phosphoenolpyruvate, NADH, Mg2 @,pyruvate kinase, and under the conditions used. lactic dehydrogenase. IMPK activity was assayed similarly Adenosine deaminase, guanase, adenase, deoxycytidine except that 0.1 mM IMP was the substrate. In most deaminase, and AMP deaminase were assayed experiments the reactions were started by the addition of the spectrophotometrically by coupling the production of substrate; however, blanks were taken without addition of with the glutamic acid dehydrogenase reaction (8). enzyme. Appropriate blanks were also included to detect The reaction mixture in 1 ml consisted of: 100 mM potassium background activity resulting from the presence of phosphate buffer, pH 7.4; 1 mM EDTA; 17 mM a-ketoglutaric contaminants such as phosphatases. The phosphorylation of 1 acid; 0.3 mM NADH; and 100 j.zl of glutamic acid j.tmole of AMP or GMP in the presence of AlP ultimately dehydrogenase (in glycerol, ammonia free). In addition, one of results in the oxidation of 2 j.zmoles of NADH because both the following substrates were used: 1 mM adenosine, 0. 1 mM ADP and GDP serve as substrates for pyruvate kinase (1 , 18). adenine, 0.1 mM guanine, 1 mM deoxycytidine, and 1 mM UMPK and CMPK activities were also measured by a coupled AMP with or without 8 mM AlP. The reaction was started by pyruvate kinase-lactic dehydrogenase system identical to that the addition of substrate after taking a background with used for NDPK except that the substrates were 0. 1 mM UMP enzyme plus cofactors. Measurements were made by following and 0.1 mM CMP. In this case the phosphorylation of 1 j.zmole the decrease in absorbance of NADH at 340 nm with a Gilford of UMP or CMP in the presence of AlP results in the recording spectrophotometer. oxidation of only 1 pmole of NADH, since both UMP and and uridine kinase were assayed by a CDP were found to be poor substrates for pyruvate kinase modification of the method of Ives et al. (10). The final (34). reaction mixture (200 p1) contained: Tris-HC1, pH 7.4, 50 HGPRT and APRT activities were assayed by the mM ; ATP, 5 mM ; MgCl2 , 5 mM ; KC1, 50 mM; radiochemical method of Kelley et aL (12). The final reaction phosphoenolpyruvate, 0.6 mM ; pyruvate kinase, 0.1 mg; and mixture (200 j.z1) contained 50 mM Tris buffer, pH 7.4; 5 mM thymidine-methyl-3H, 0.10 mM or uridine-5-3H, 0.10 mM. MgC12; 1 mM 5-phosphoribosyl 1-pyrophosphate; extract; and The reaction was carried out for 15 mm at 37°and was either 0.1 mM adenine-8-' 4C (specific activity, 10 j.zCi/j.zmole) terminated by immersing the reaction tubes in a boiling water or 0.1 mM guanine-8-1 4C (specific activity, 12 jzCi/.tmole). bath for 90 sec. The resultant reaction mixtures were

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centrifuged at 12,100 X g for 30 mm, and an aliquot (20 p1) Table 1 of the supernatant was spotted on DEAE-impregnated Enzymesofpurine and pyrimidine metabolism in lymphocytes from Whatman DE81 paper discs (20 mm in diameter). Unreacted the peripheral blood ofnormal donors thymidine or uridine was removed from the disc by incubation Enzymes were assayed in sonic extracts of the lymphocytes as in 0.001 M formate (10). The radioactive described in “MaterialsandMethods.― remaining on the disc were counted as previously Units/lO' ° described, and the same linear relationships were found as in proteinAnabolicN0.° lymphsUnits/mg the previous assays. In almostallcases,enzymeactivitieswereassayedonthe enzymesGMPK day the extracts were prepared. In a few early studies, the 0.06AMPK 9 3.0 ±1•5b0.09 ± extracts had been stored for up to 2 months at —20°or—80° 0.3UMPK 9 56 ±281.5 ± before being assayed. In order to evaluate the effects of 0.05CMPK 5 9.7 ±2.60.25 ± different storage times, conditions, or temperatures, extracts 0.25IMPKC 4 10.6 ±6.20.30 ± were stored at 4°,—20°(withbovine plasma albumin added to 0.240.02Adenosine 2 0.43 ± 0.070.01Thymidinekinase 5 0.25 ± a concentration of I mg/ml), and —80°.Whenthe extracts 0.01TraceUridinekinase 1 were stored at 4°,most of the enzymes lost significant activity TraceTraceAPRTkinase 1 within a thort time, while with those stored at —20°or—80° 1.90.10±0.03HGPRT 6 3.8± activity was retained over a fairly long time. Adenosine kinase 0.01NDPK 6 1.5 ±0.20.04 ± and thymidine kinase, however, lost activity rapidly and were 1.6Catabolic9 188 ±514.8 ± assayed immediately upon preparation of the extracts. enzymesPNPase Enzyme activities were expressed as micromolar units/i X 101 0 cells and, in some cases in micromolar units/mg of 9.8Adenosinedeaminase 9 19.4 ± protein. With all enzymes, product formation was linear with 0.12Adenase 8 9.9 ±5.60.23 ± 00Guanase 5 respect to time and protein concentration. A micromolar unit 00Deoxycytidine 9 was the amount of enzyme that would convert 1 pmole of 00AMP deaminase 8 substrate per mm. Protein was measured by the method of 00AMPdeaminase (—ATP) 5 Lowryetal. (16). 0.01Xanthinedeaminase (+ ATP) 3 0.05 ±0.300.02 ± 1k Nowo Pathway of Purine Biosynthesis. Purified oxidase 9 00 lymphocytes (3 X l0@) obtained from normal donors or from leukemic patients or L5 l78Y murine lymphoblasts (3 X l0@) a No. of donors whose blood was assayed for that particular enzyme. were incubated in 2 ml of Fischer's medium with 10% horse b Mean ±S.D. serum containing 5 pCi of uniformly labeled glycine-' 4C C Measured by an indirect assay and thus may not refer strictly to (specific activity, 100 pCi/pmole). The incubation was carried IMP kinase (see “Results―). out for 90 or 240 mm at 37°in tightly stoppered flasks. The pH was maintained at 7.4 by addition, if necessary, of 0.1 N some cases, as units/mg of protein. It has been determined that HC1. Microscopic examination indicated that the cells there are approximately 0.2 to 0.3 X 10' °lymphocytes per remained in good physiological condition during the ml of packed cells. incubation. After termination of the reaction by dropwise Both APRT and HGPRT were found to be present in the addition of the cell suspension (0.5 ml) to 1 ml of a rapidly lymphocytes. Hypoxanthine phosphoribosyltransferase prob agitated 12% solution of trichloroacetic acid, the mixture was ably is also present since it is believed that the same protein centrifuged at 12,100 X g for 20 mm. Aliquots (50 p1) of the catalyzes IMP formation from hypoxanthine and GMP neutralized supematant fractions (pH 7.0 to 7.5 by addition of formation from guanine. The fact that adenosine kinase 2 M Tris) were spotted on Whatman No. 3M paper. The paper activity was also detected indicates that human lymphocytes was developed by ascending chromatography in boric possess 2 fundamentally different pathways for the synthesis acid--H2O-NH@OH (1.5 g, 158 ml, 67 ml, 25 ml) of AMP, one utilizing adenine as substrate and the other according to the technique of Miech and Santos (19) followed utilizing adenosine. by location of known standards by IN (254 nm). The At least 2 purine nucleoside monophosphate , AMPK radioactivity of the separated compounds was determined by and GMPK, were also present. AMPK had more than 10-fold cutting the chromatographs into 1-cm strips and counting the the GMPK activity. In addition, a small amount of IMP kinase strips in a toluene-based scintillation solution. activity was detected, but it is not known whether this activity is due to a separate IMPK enzyme or whether IMP is an RESULTS alternative substrate for another enzyme. Partially purified GMPK from RBC has been shown to have a small amount of The activities of selected enzymes of purine and pyrimidine activity with IMP (1). Another kinase, NDPK, which is metabolism present in purified lymphocytes from the necessary in the formation of nucleoside triphosphates and peripheral blood of healthy male and female donors ranging in nucleic acids, was extremely active in normal human age from 20 to 60 years are shown in Table 1. As far as could lymphocytes. Two pyrimidine nucleoside monophosphate be determined, none of the donors were taking medication. kinases, CMPK and UMPK, were also detected in lymphocytes. Enzyme activities were expressed as units/ 1010 cells or, in The activities of these enzymes were relatively high, being

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1973 American Association for Cancer Research. E. M. Scholar and P. Calabresi greater than GMPK but only about one-third that of AMPK. had a peripheral WBC count of 202,000 with 93% The pyrimidine nucleoside kinases, uridine kinase and lymphoblasts. He had been treated with , thymidine kinase, were also present in normal lymphocytes p r e d n i sone , , arabinosylcy to sine, although their activities were relatively low. 6-, and 6-thioguanine over a 2-year period and Among the catabolic enzymes, adenosine deaminase and presently was not responsive to any of these compounds. Most purine nucleoside phosphorylase were present in the human of the selected enzymes assayed in these lymphoblasts had lymphocytes whereas guanase , adenase, deoxycytidine activities severalfold higher than those in normal or CLL deaminase, and xanthine oxidase could not be detected. AMP lymphocytes. In contrast, HGPRT and APRT had about the deaminase activity was found only in the presence of same activity and PNPase had about one-half the activity exogenous ATP, which is a known activator of this enzyme in found in normal lymphocytes. The 2nd patient with acute mammalian tissue (31). lymphoblastic leukemia(D. P.), a 7-year-old male who over the Tables 2 to 4 shows the activities of these same enzymes in past 2 years had been treated with various chemotherapeutic lymphocytes from the blood of patients with CLL. Enzyme agents, was in relapse with a WBC of 40,000 and 90% activities were divided into 1 of 3 groups depending on the lymphoblasts. After the patient received arabinosylcytosine, a WBC count of the patients from whom the lymphocytes were specimen of blood was obtained for study and showed a obtained. These groups were: Table 2, WBC counts below differential count of 90% lymphocytes and about 7% blasts. 25,000; Table 3, WBC between 25,000 and 50,000; and Table Most of the enzyme activities determined were lower than 4, WBC above 50,000. None of the patients with CLL received those found in normal lymphocytes. It is not known whether medication during the study period except R. L. and A. B. this unexpected finding was a result of the chemotherapy that (Table 2), who were taking both prednisone and . the patient received or was due to the smaller percentage of Apart from significant deoxycytidine deaminase activity lymphoblasts present in his blood. It is also possible that the found only in lymphocytes from CLL patients, there were no lymphocytes present are normal by light microscopy. More differences in which enzymes were present and their activities patients wifi have to be studied before this question can be between normal and CLL lymphocytes in either treated or answered satisfactorily. Guanase, adenase , deoxycytidine untreated patients. In addition, the WBC count in the patients' deaminase, or xanthine oxidase could not be detected in cells peripheral blood did not seem to influence the level of from either of the 2 acute leukemic patients. enzymatic activity. As part of a continuing study, the enzymes The activities of the enzymes of nucleotide metabolism in the lymphocytes from 1 patient in each group were obtained from 3 murine ascitic cell lines. Sarcoma 180, L1210, reassayed from 1 to 3 months later; no significant changes in and L5178Y are found in Tables 6 to 8. Chart 2 presents in activities were found. comparative form the values for the 3 animal tumor lines. The Table 5 shows the enzyme activities in the lymphocytes of base line of activity is arbitrarily set at the values for normal peripheral blood from 2 patients who had acute lymphoblastic human lymphocytes. In comparison to normal human leukemia. Patient S. M. (6-year-old male) was in relapse. He lymphocytes, large increases were found in the activities of

Table 2 Enzymesofpurine and pyrimidine metabolism in lymphocytesfrom patients with CLL (<25,000 WBC) Enzymeswere assayedin sonic extracts of the lymphocytes as described in “MaterialsandMethods.―

enzymes(units/lO' °lymphs)Adenosine

HGPRT1.PatientWBCLymphs (%) GMPKAnabolicAMPK UMPK CMPK IMPKa NDPK kinaseAPRT

C. B. 1.3 26.7 25.3 0.50 277 0.60 1.3 2.H.W. 25,000 81 4.0 70.8 20.0 13.8 262 3.2 1.5 H.W.b 23,000 81 5.0 72.8 26.5 31.8 285 3.1 1.6 3. R. L. 19,000 84 3.4 68.5 18.6 14.3 293 2.7 1.3 4.A.B. 22,00084 90 2.1 34.3 Mean±S.D.12,000 3.2 ±1.538.757.0 ±18.90.1Catabolic 22.9 ±4.3 21.3 ±8.8 0.50 266 ±30 0.602.3 2.8 ±0.4 1.4 ±

lymphs)PatientPNPaseAdenosine enzymes(units/lO' °

Xanthine AMP deaminase deaminase ATP)l.C.B. deaminaseDeoxycytidineGuanase Adenase deaminase oxidase (—ATP)AMP (+

0 2.3 0 0 2.H.W. 8.5 4.3 0 0 3.5 0 0 H.W.b 8.5 4.6 0 0 0 0 3. R. L. 4.3 4.6 0 0 1.2 4.A.B. 4.6 4.6 0 Mean±S.D.10.36.6 ±3.02.7 4.2 ±0.90 0 0 2.3 ±1.2 0 00.66 0.66

a Measured by an indirect assay and thus may not refer strictly to IMP kinase (see “Results―). b Enzymes were assayed 2.5 months after the initial study.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1973 American Association for Cancer Research. Nucleotide Metabolism in Human Lymphocytes Table 3 Enzymesofpurine and pyrimidine metabolism in lymphocytes from patients with CLL (25,000 to 50,000 WBC) Enzymes were assayed in sonic extracts of the lymphocytes as described in “MaterialsandMethods.―

lymphs)Lymphs Anabolic enzymes(units/lO' °

Adenosine HGPRT1.D.P.Patient WBC (%) GMPK AMPK UMPK CMPK NDPK kinase APRT

32,500 85 2.9 42.4 20.0 15.2 233 0.19 2.4 1.2 2.P.H. 40,000 81 1.1 18.8 139 P. H.@ 46,000 70 3.3 23.7 12.1 9.2 232 0.14 3.5 Mean±S.D. 2.5 ±1.3 27.9 ±12.81.2Catabolic 16.0 ±5.6 1.2 ±0.4 201 ±54 0.16 ±0.04 2.9 ±0.8

lymphs)Adenosine enzymes(units/lO' °

Deoxycytidine Xanthine AMP deaminase AMP deaminase ATP)1.Patient PNPase deaminase Guanase Adenase deaminase oxidase (—ATP) (+

D. P. 5.2 2.7 0 0 0.6 0 0 0.19 2.P.H. 8.3 2.5 0 0 0 P.H.@ 9.6 1.9 0 0 0 Mean±S.D.0.19a 7.7±2.2 2.4±0.4 0 0 0.6 0 0

Enzymes were assayed 1 month after initial study.Table

4 Enzymesofpurine and pyrimidine metabolism in lymphocytes from patients with CLL (>50,000 WBC) Enzymes were assayedin sonic extracts of the lymphocytes as describedMethods.―Anabolic in “Materialsand

lymphs)Lymphs enzymes(units/lO' °

Adenosine HGPRT1.J.G.Patient WBC (%) GMPK AMPK UMPK CMPK IMPK NDPK kinase APRT

55,000 91 4.0 82.0 21.6 12.8 o.16a 360 0.34 3.3 1.2 2.A.W. 85,000 90 2.1 26.2 11.4 267 1.6 0.8 A.W.b 97,000 99 2.6 40.0 13.0 20.4 196 2.4 0.8 3. S. P. 55,000 60 3.8 94.7 16.7 30.7 434 0.45 6.0 4. P. B. 480,000 95 1.2 14.5 0.6 7.5 0.49 131 0.09 5.4 1.5 Mean±S.D. 2.7 ±1.2 51.5 ±35.10.3Catabolic 13.8 ±5.7 17.8 ±10.0 0.33 ±0.20 277 ±122 0.29 ±0.19 3.7 ±1.9 1.0 ±

lymphs)Adenosine enzymes(units/lO' °

Deoxycytidine Xanthine AMP deaminase AMP deaminase ATP)1.J.G.Patient PNPase deaminase Guanase Adenase deaminase oxidase (—ATP) (+

8.0 2.4 0 0 0.5 0 0 0.32 2.A.W. 7.1 2.7 0 1.4 A.W.@' 4.4 1.8 0 0 1.5 0 0 3.S.P. 10.7 1.7 0 0 1.1 0 0 4.P.B. 2.9 2.6 0 0 0.2 0 0 0.12 Mean±S.D. 6.6±3.0 2.2±0.1 0 0 0.9±0.6 0 0 0.22±0.10

a Measured by an indirect assay and thus may not refer strictly to IMPK (see “Results―). b Enzymes were assayed 3 months after initial study.

GMPK, AMPK, UMPK, CMPK, NDPK, adenosine kinase, and biosynthesis, we incubated purified human lymphocytes from adenosine deaminase. APRT was high only in Sarcoma 180 normal donors and from patients with CLL with ascites cells, and PNPase was elevated in Sarcoma 180 and the high-specific-activity glycine-' 4C for periods up to 4 hr. The murine lymphoblast line L12l0 but was in the normal range in components of the acid extracts of the washed cells were L5178Y cells. Guanine deaminase activity was detected in all 3 separated by paper chromatography as described in “Materials murine cell lines (Tables 6 to 8) but not in normal or leukemic and Methods.―The distribution of the radioactivity is seen in human lymphocytes or lymphoblasts. No deoxycytidine Chart 3. Only a small percentage of the initial radioactivity deaminase, adenase, xanthine oxidase, or AMP deaminase was found in the acid extracts, and with normal and CLL activity could be found in any of the murine cell lines. lymphocytes chromatography showed this to be present as In order to determine whether human lymphocytes like glycine and not as nucleotides. Conversely, in the L5 178Y L5 178Y cells contained a de novo pathway for purine lymphoblasts which possess a de novo pathway for purine

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Table 5 Enzymesofpurine and pyrimidine metabolism in acute lymphoblastic leukemia cells Enzymes were assayedin sonic extracts of the cells asdescribed in “MaterialsandMethods.―

Patient S. M. % of D. P. of (202,000 WBC) “normal (42,000 WBC) “normal (units/lO' °cells)activity@@aAnabolic activity@@aPatient(units/lO' °cells)%

enzymesGMPK

313 AMPK 124 221 7.3 13 UMPK 20.8 214 1.1 11 CMPK 29.8 274 1.3 12 IMPK Trace Trace NDPK 645 343 91 48 Adenosinekinase 0.11 44 APRT 2.0 NC 2.1 NC HGPRT9.4 2.2NCCatabolic NC1.1 2.037

enzymesPNPase

65 Adenosinedeaminase 73.2 737 10.0 NC Guanase 0 NC 0 NC Adenase 0 NC 0 NC Deoxycytidinedeaminase 0 NC Xanthine oxidase 0 NC 0 NC AMP deaminase (- ATP) 0 NC3.5 0 NC AMP deaminase (+ ATP)12.7 0.0618 NC

a Activities expressei as a percentage of the activity in normal lymphocytes. NC, no change. See Table 1 for normal activities.

Table 6 Table 7 Enzymesofpurine and pyrimidine metabolism in Enzymesofpurine and pyrimidine @netabolismin murine Sarcoma180 ascitic cells murine L51 78Ylymphoblasts Enzymes were assayedin sonic extracts of the cells as described in Enzymes were assayedin sonic extracts of the cells as described in “MaterialsandMethods.― “MaterialsandMethods.―

No. of Units/lO' ° No. of Units/lO' ° proteinAnabolicanimals cellsUnits/mg proteinAnabolicanimals cellsUnits/mg enzymesGMPK5 enzymesGMPK

0.10AMPK5 32.5 ±25.2a0.17 ± 15.6±7.4a 0.20UMPK3 336 ±1361.8 ± AMPK 4 231 ±119 3.8 ±1.4 0.13CMPK3 66.0 ±64.30.27 ± UMPK 1 55 0.75 0.22IMPK1 138 ±1100.55 ± CMPK 1 64 0.88 1•9bAdenosine IMPK 1 057b o.oi 1.40.01APRT3kinase3 2.4 ± Adenosinekinase 1 0.75 0.01 0.04HGPRT5 31.8 ±20.10.14 ± APRT 4 2.23 ±0.14 0.04 ±0.01 0.01NDPK5 7.1 ±3.60.05 ± HGPRT 4 4.21 ±1.61 0.08 ±0.04 11.8Catabolic 7760±480240.3± NDPK4 2.0Catabolic4 2543 ±6510.25±0.1043.4 ± enzymesPNPase5 enzymesPNPase

0.03Adenosine 4.4±290.20 ± 9.7 ±5.8 ±0.10 6.50.10Adenase2deaminase6 20.0 ± Adenosinedeaminase 4 57 ±15 1.1 ±0.1 00Guanase3 Adenase 2 0 0 0.50.01Deoxycytidine 1.0 ± Guanase 3 1.0 ±0.2 0.02 00AMP deaminase1 Deoxycytidine deaminase 2 0 0 00AMPdeaminase (—ATP)2 AMP deaminase (—ATP) 3 0 0 00Xanthinedeaminase (+ ATP)1 AMP deaminase(+ ATP) 1 0 0 oxidase2 00 y ‘nthine oxidase3 3 00.20 0

a Mean ± S.D. a Mean ±S.D. b Measured by an indirect assay and thus may not refer strictly to b Measured by an indirect assay and thus may not refer strictly to IMP kinase (see “Results―). IMP kinase (see “Results―).

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Micleotide Metabolism in Human Lymphocytes

Table 8 Enzymesofpurine and pyrimidine metabolism in murine L1210 lymphoblasts 0 L5178V @ Enzymes were assayedin sonic extracts of the cells asdescribed in L1210 “MaterialsandMethods. I- > 0 S-IsO I-. No. of Units/lO' ° U 4 proteinAnabolicanimals cellsUnits/mg z w enzymesGMPK5 (I) w4 SOC l.Oa0.24AMPK5 11.7± U 0.9UMPK3 177±603.6 ± z I- 60( 0.03CMPK3 17.0 ±5.80.36 ± z

0.19IMPK1 25 ±130.52 ± U 4O( 026bo.oiAdenosine ‘U 0.170.02APRT4kinase3 0.74 ± 0. 0.01HGPRT5 2.3 ±0.70.05 ± 3.5±0.90.72±0.21NDPK5 2.4Catabolic 3095±14776.0 ± ILi ‘U tUtU 0. 0. 0. 0. 0. 0. ZU) @@@ enzymesPNPaseS 2 2 0 ‘flu) 0. 0. a 0 4 D U Z 04 4 (a 0. Zz x z 0. W4 0.69Adenosine 45 ±390.89 ± 4 OW 0.3Adenase3deaminase5 72±181.5 ± 00Guanase4 Chart 2. Increasein activity of enzymesfrom L5178Y, L1210, and 0.170.01Deoxycytidine 0.60 ± Sarcoma 180 cells compared to normal human lymphocytes. Baseline, 00AMP deaminase3 normal activity (seeTables 6, 7, and 8); bars, percentageof increasein 00AMPdeaminase (—ATP)3 activity compared to normal human lymphocytes. 00Xanthinedeaminase (-I-ATP)1 oxidase3 00

a Mean ±S.D. b Measured by an indirect assay and thus may not refer strictly to IO@ IMP kinase (see “Results―).

biosynthesis, considerable radioactivity was detected in the nucleotide region. These results with isolated lymphocytes are in agreement with those of others who have shown that the whole WBC fraction lacks at least the initial enzymes necessary IO@ for de novo purine biosynthesis (29, 30).

0. DISCUSSION C)

The activities of a selected number of the enzymes 02 responsible for purine and pyrimidine nucleotide biosynthesis, interconversion and catabolism have been studied in purified lymphocytes obtained from the peripheral blood of normal donors, from patients with CLL, and from lymphocytes and lymphoblasts of patients with acute lymphocytic leukemia. Previously, only a limited number of enzymes present in the whole white blood cell fraction had been studied. Since the white blood cell fraction of the peripheral blood is a variable heterogeneous mixture of cells composed of granulocytes, lymphocytes, and monocytes, interpretation of the previous 0 0 20 30 results is somewhat difficult. Granulocytes account for 60 to cm. FROM ORIGIN 80% of the WBC fraction in normal blood, but in different Chart 3. Chromatographic localization of ‘4Cfollowing incubation leukemias the percentage can vary from almost 0 to 90% of of normal lymphocytes, CLL lymphocytes, or L5178Y cells with the WBC fraction. A low enzyme activity found in the total glycine-' 4C. Five @Ciofuniformly labeledglycine-' 4C were incubated for 4 hr with purified lymphocytes from either normal donors or from WBC fraction could be a result of an extreme dilution of a patients with chronic lymphocytic leukemia or L5178Y murine high cellular activity or of a low overall activity. In extreme lymphoblasts as described in “MaterialsandMethods.―Aliquots of cases, a relatively low activity in a cell group that is present as neutralized trichloroacetic acid extracts were then developed on paper a small percentage of the total fraction could be entirely chromatograms.1, the areawhere AMP, ADP, and ATP arelocated; 2, missed. location of adenosine;3, location of adenineand glycine.

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While present studies in purified lymphocytes showed the of action and development of resistance to the antileukemic lack of de novo purine synthesis, there were found enzymes drugs. It is important to know both the qualitative and capable of synthesizing purine nucleotides by the salvage quantitative differences in enzymatic activity that may exist pathways. Of these enzymes, APRT, HGPRT, adenosine between normal and leukemic cells so that they may be kinase, and purine nucleoside phosphorylase were found. In exploited in chemotherapy. Systematic investigations of addition, several kinase enzymes important in purine and enzymatic activity as were carried out in the present study pyrimidine nucleotide interconversion also were present. may provide a more rational use of certain antineoplastic NDPK, GMPK, AMPK, CMPK, and UMPK were among these. agents as was the case with L- (9, 22). They may It was of considerable interest to find a lack of the catabolic also be of considerable utility in monitoring and predicting the enzymes xanthine oxidase, guanase, and adenase in normal and development of resistance or toxicity during and/or following leukemic lymphbcytes. This deficiency may be important treatment of leukemia with (2, 26). when considering the toxicity of certain antimetabolites such as 6-mercaptopurine and 6-thioguanine to normal and leukemic lymphocytes, because these compounds could not be ACKNOWLEDGMENTS catabolized without the presence of these enzymes. Marchesi The authors acknowledge the technical assistanceof Mr. William and Sartorelli (17) have demonstrated that, while Henry. We would also like to thank Mrs. Archetto and the staff of the 6-thioguanine was quite toxic to rabbit bone marrow which Roger Williams General Hospital Blood Bank for drawing blood from was devoid of guanase, it was nontoxic to the intestinal normal donors and to thank the following people for obtaining blood mucosa, in which there was an active guanine deaminase, from leukemic patients: Dr. F. Cummings and Dr. M. Hoovis of the thereby catabolizing a large fraction of the 6-thioguanine. Department of Medicine, Roger Williams General Hospital; Dr. M. Another catabolic enzyme studied, deoxycytidine Albala, Rhode Island Hospital, Providence, R. I.; Dr. L. Stolbach, deaminase, could be detected only in lymphocytes isolated Pondville Hospital, Norfolk, Mass.; and Dr. D. Traggis, Children's from the blood of patients with CLL. It is possible that Cancer ResearchFoundation, Boston, Mass.We also wish to thank Dr. lymphoblasts contain the enzyme as the only patient with K. C. Agarwal, Dr. M. Hoovis, Dr. R. P. Miech, and Dr. K. Palmer for their adviceduring the preparation of this manuscript. acute lymphoblastic leukemia examined for deoxycytidine deaminase had a high percentage of lymphocytes in the blood. The reason for its presence only in this cell type is not clear at REFERENCES this time, but deoxycytidine deaminase (also known as ) activity has been correlated with the 1. Agarwal, R. P., Scholar, E. M., Agarwal, K. C., and Parks, R. E., Jr. susceptibility of leukemic cells to arabinosylcytosine. This Identification and Isolation on a Large Scaleof Guanylate Kinase enzyme deaminates arabinosylcytosine to arabinosyluridine from Erythrocytes. Effects of Monophosphate Nucleotides of which has no therapeutic activity (33). Elevated deoxycytidine Purine Analogs.Biochem. Pharmacol.,20: 1341—1354,1971. deaminase has also been found in the spleen of mice infected 2. Davidson, J. D., and Winter, T. S. 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