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Pyrimidine Nucleoside Monophosphate Kinase from Human Leukemic Blast Cells1

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Pyrimidine Nucleoside Monophosphate Kinase from Human Leukemic Blast Cells1 [CANCER RESEARCH 38, 579-585, March 1978] Pyrimidine Nucleoside Monophosphate Kinase from Human Leukemic Blast Cells1 Kenneth R. Hände and Bruce A. Chabner Clinical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20014 ABSTRACT inhibition of DMA polymerase (10). Conversion of ara-C to this active compound is carried out through a series of Pyrimidine nucleoside monophosphate kinase (EC phosphorylating enzymes (Chart 1), while deamination of 2.7.4.14) catalyzes the phosphorylation of various pyrimi- ara-C or its nucleotides to arabinofuranosyluracil or arabi- dine nucleoside monophosphates to their corresponding nofuranosyluracil nucleotides results in drug inactivation. diphosphates. In addition to its role in the synthesis of Several workers have attempted to correlate clinical re nucleic acid precursors, this enzyme is essential for the sponse to chemotherapy with the ability of leukemic cells conversion of the antileukemic agent 1-/3-o-arabinofu- to activate or degrade ara-C. Steuart and Burke (28) have ranosylcytosine to its active metabolite 1-/3-o-arabino- reported that elevated levels of the initial degrading enzyme furanosyl 5 -triphosphate. Pyrimidine nucleoside mono- (cytidine deaminase) were associated with a decreased phosphate kinase has been purified 520-fold from human response to ara-C. However, other investigators (15, 31) leukemic blast cells. Substrate specificity and kinetics for have concluded that levels of the initial activating enzyme, various pyrimidine nucleoside monophosphates and ana deoxycytidine kinase, are more important than is deami logs have been investigated and compared with previous nase concentration in the determination of therapeutic studies of this kinase isolated from other sources. A responsiveness. These reports would suggest that this single enzyme appears responsible for the phosphoryla initial phosphorylating step through deoxycytidine kinase tion of cytidine 5 -monophosphate, deoxycytidine 5 - is rate controlling in the activation of ara-C. monophosphate, uridine 5 -monophosphate, and deoxy- Previous investigations (2, 5) in this laboratory have uridine 5 -monophosphate as well as the pharmacological concerned the characterization of deoxycytidine kinase and substrates 1-0-D-arabinofuranosylcytosine 5 -monophos- cytidine deaminase from human leukemic blast cells. The phate, 5-fluorouridine 5 -monophosphate, 5-fluorodeoxy- subsequent phosphorylation and degradation steps in the uridine 5 -monophosphate, and 1-ß-o-arabinofuranosyl- metabolic pathway of ara-C have not been investigated in uracil 5 -monophosphate. Enzyme levels of pyrimidine detail. In this work, the second phosphorylating enzyme in nucleoside monophosphate kinase have been deter the ara-C activation pathway, pyrimidine nucleoside mono- mined in leukocyte blast cells isolated from normal human phosphate kinase (also called dCMP kinase, EC 2.7.4.14), donors (1.16 units/mg protein) and from patients with has been partially purified and characterized. Its levels in acute lymphocytic leukemia (1.96 units/mg protein), acute leukemic cells have been determined, and its affinities for myelogenous leukemia (1.99 units/mg protein), and physiological and pharmacological substrates have been chronic myelogenous leukemia (1.88 units/mg protein). established. These levels are 100-fold higher than those of deoxy cytidine kinase, the enzyme responsible for the initial MATERIALS AND METHODS phosphorylation of 1-ß-oarabmofuranosylcytosine. How ever, the affinity of deoxycytidine kinase for 1-/l-o-arabino- Materials furanosylcytosine (K,„,2.56 x 10 M) is greater than that of the monophosphate kinase for 1-/i-o-arabinofuranosyl- Unless otherwise mentioned, all non radioactive nucleo cytosine 5 -monophosphate (Kni, 6.8 x 10"* M). tides and enzymes were purchased from Sigma Chemical Company, St. Louis, Mo. ara-UMP and FUMP were kindly INTRODUCTION supplied by Dr. Robert Engle, Drug Research and Develop ment, National Cancer Institute, Bethesda, Md. All radioac- ara-C3 is a pyrimidine nucleoside analog widely used for tively labeled nucleotides with the exception of ara-CMP treatment of acute myelogenous leukemia (7). The active were purchased as 14C-labeled compounds from either New metabolite of the drug appears to be cytosine arabinoside England Nuclear, Boston, Mass., or Schwartz/Mann, triphosphate ara-CTP, which causes cytotoxicity through Orangeburg, N. Y. 3H-Labeled ara-CMP (specific activity, 15 Ci/mmole) was custom synthesized by Amersham/Searle 1 Presented in part at the Annual Meeting of the American Association Corp., Arlington Heights, III. All enzymes used in the en For Cancer Research, May 8, 1976 (14). 2 To whom requests for reprints should be addressed, at Building 10, zyme assay reaction as well as phosphoenolpyruvate, DTT, Room 6N119; NIH, Bethesda, Md. 20014. bovine serum albumin, and pHMB were purchased from 3 The abbreviations used are: ara-C, 1-0-D-arabinofuranosylcytosine; ara- CTP. 1-/3-o-arabinofuranosylcytosine 5'-triphosphate; ara-UMP, 1-,3-D-arabi- Sigma. RNase A, chymotrypsinogen, and ovalbumin, as nofuranosyluracil 5'-monophosphate; FUMP, 5-fluorouridine 5'-monophos- well as Sephadex G-150 resin, were purchased from Phar phate; ara-CMP, 1-/5-D-arabinofuranosylcytosine 5'-monophosphate; FdUMP, macia Fine Chemicals, Inc., Piscataway, N. J. Human trans- 5-fluorodeoxyuridine 5'-monophosphate; DTT, dithiothreitol; pHMB, p-hy- droxymercuribenzoate. ferrin was purchased from Behring Diagnostics, Woodbury, Received June 7. 1977; accepted November 16, 1977. N. Y. DEAE-cellulose resin was purchased from Whatman MARCH 1978 579 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1978 American Association for Cancer Research. K. R. Händeand B. A. Chabner . deoxycvtidine kinase with 4 M formic acid, with 1 mM ammonium formate as mpnophospnale kinase diphosphate kinase solvent. cvtidine deoxvcyridine deaminase monophosphate For kinetic determinations in which FdUMP, FUMP, ara- deaminase UMP. cytosine 2'- or 3'-monophosphate, and 5-methyl- or Aia-U Ata-UMP dATP 5-bromo-dCMP were used as substrate, the spectrophoto- inactive) 'inactive! dGTP TTP DNA metric assay system of Maley and Ochoa (19) was used. In dCTP polymer äse this assay, pyruvate kinase was coupled with láclate dehy- Chart 1. Metabolie pathway of ara-C. drogenase to measure ADP formation. The disappearance of NADH as measured by decreasing absorbance at 340 nm Biochemicals Ltd., Reeve Angel, Clifton, N. J. AG 1-X10 was used as a measure of monophosphate kinase activity. resin (200 to 400 mesh, chloride form) and hydroxylapatite Standard conditions for the spectrophotometric assay (Bio Gel HTP) were purchased from Bio-Rad Laboratories, were: monophosphate substrate; 0.05 M Tris-CI, pH 7.5; 8 Richmond, Calif. Aquasol scintillation counting fluid was mM ATP: 8 mM MgCI; láclatedehydrogenase, 3 units/assay; purchased from New England Nuclear. 3 /¿Mphosphoenolpyruvale; 0.133 /J.MNADH; pyruvale ki nase, 20 unils/assay; and enzyme and waler lo a lolal Enzyme Assay for Pyrimidine Nucleoside Monophosphate volume of 1.00 ml. Reaclions were carried out at 37°. Kinase Because of the strict dependence of the pyrimidine nu Two different assays for pyrimidine nucleoside mono- cleoside monophosphate kinase on sulfhydryl reducing agents for activity, all assays were carried out after a 15- phosphate kinase were used in these studies. The assay used for the majority of studies was based on ion-exchange min preincubation with 50 HIM DTT unless otherwise speci Chromatographie separation of isotopically labeled sub fied. One unit of enzyme was defined as thai amounl of strate and product. In this assay, an excess of nucleoside enzyme required to convert 1 /MimóleofdCMP to dCDP per diphosphate kinase was added to the reaction mixture to hr. convert all diphosphate formed to triphosphate as previ Enzyme Preparation ously described by Cheng and Prusoff (3). Unreacted mono- phosphates were then separated from triphosphates by For studies of enzyme purification and substrale specific selective elution from an AG 1 ion-exchange column with ity, pyrimidine nucleoside monophosphate kinase was ob NH,CI solutions of increasing molarity, in a manner similar tained from CEM cells, a human acute lymphoblastic cell to that described by Lindsey ef a/. (17) for the separation of line (9), grown in spinner culture in Roswell Park Memorial uridine nucleotides. Inslitute Medium 1640 wilh 10% heal-inaclivaled fetal calf Pyrimidine nucleoside monophosphate kinase assays serum supplemented with glulamine. Cells were harvesled were performed in a total volume of 0.25 ml. The reaction by cenlrifugalion, washed Iwice wilh 0.05 M sodium phos- mixture contained: 8 rriM ATP; an ATP-regenerating system phale buffer, pH 7.5-0.8% NaCI, and pyrimidine nucleoside consisting of 1.0 unit creatine phosphokinase and 3 rriM monophosphale kinase exlracled from Ihe cells by freeze- phosphocreatine; 8 HIM MgCI; 1% albumin; 0.05 M Tris-CI, thawing in 0.02 MTris-CI, pH 7.5, with 50 mM DTT, followed pH 7.5; 0.2 unit nucleoside diphosphate kinase, and a 3 by Dounce homogenizalion. Cell debris was removed by mM concentration of the appropriate monophosphate cenlrifugalion al 40,000 x g for 30 min, and Ihe superna- (UMP, dCMP, CMP. or ara-CMP) (0.03 to 0.06 Ci/mole). lanl fluid was used for furlher sludies. The reaction was initiated by the addition of 10 to 100 /il of Pyrimidine nucleoside monophosphale kinase was also enzyme and incubated
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