(CANCERRESEARCH38,2768-2772,September1978] 0008-5472/78/0038-0000$02.00 Deoxycytidine Kinase and Cytosine Nucleoside Deaminase Activities in Synchronized Cultures of Normal Rat Kidney Cells1 CharnWingWan2andTak W. Mak Ontario Cancer Institute and Department of Medical Biophysics, university of Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9 ABSTRACT increased levels of cytidmnedeaminase in HeLa cell treated with ara-C. Other studies (1, 10, 23, 24, 34, 38, 40), however, Previouswork has suggestedthat 1-fi-D-arabinofurano have shown that the levels of kinase were important in sylcytosine5'-triphosphateis the active metaboliteof 1- determining the sensitivity of cells to ara-C treatment. On @-o-arabinofuranosylcytosine.Theamountof 1-/3-o-arabi the other hand it has been suggested that the nofuranosylcytosine5'-trlphosphate formed in tissues kinase:deaminase ratio could be indicative of the response has been shownto be influencedby the relativelevels of to treatment with ara-C (16, 18). These different observa deoxycytidine kinase and cytosine deaminase. In this tions are probably due to the wide variations in the kinases studywe have measuredthe intracellularlevels of deox and deaminase activities, which are known to exist among ycytidine kinase and cytosine deaminase activities in various normal and malignant tissues or cells (4, 8, 11, 18, synchronizedcultures of normal rat kidney cells. The 33). deoxycytidinekinase activftywas found to be cell cycle Since the action of ara-C is to inhibit DNA synthesis, it is relatedwith a minorpeak of activityin early G1phaseand believed to be a cell cycle-dependent drug (20, 22, 42). a major peak of activity in middleand late S phase. The Karon and Shirakawa (20, 23) and Young and Fischer (42) cytosinedeaminase activity was also found to be cycle have also determined that the action of ara-C is specifically dependent with a peak of activity at G1phase and another in the S phase of the cell cycle. at S phaseof the cell cycle. Similarresultswere obtained The purpose of this study was to measure the intracellular when cytosinedeaminase activitieswere measuredwith levels of both deoxycytidmne kinases and cytosine nucleo cytidine, deoxycytidine, or 1-$-D-arabinofuranosylcyto side deaminases in synchronized cultures of NRK cells and sine as substrate.Presentstudiesalso confirmedearlier to determine the sensitivity of these synchronized cells to studies by other workers that the main effect of 1-,3-D- ara-C. arabinofuranosylcytosineisin the late S phaseof the cell cycle. MATERIALSAND METHODS Materials. [‘4C]ara-C(NSC63878; specific activity, 56 INTRODUCTION mCi/mmol) was supplied by the Drug Development Branch, ara-C3 is a potent inhibitor of cellular proliferation and Cancer Chemotherapy National Services Center, National DNA synthesis in mammalian cells (6, 7, 14, 19, 25, 32, 42). Cancer Institute. The sample was diluted with unlabeled It has also been demonstrated to be effective in the treat ara-C to give the final specific activity of 0.53 mCi/mmol. [2- ment of various cancers and to be a potent agent in the I @C]Deoxycytidmne (specific activity, 29.7 mCi/mmol) was therapy of acute myeloblastic leukemia (5, 17, 27). purchased from New England Nuclear, Boston, Mass. The The mechanism of action of ara-C has been well studied radioactive compound was diluted with unlabeled deoxy and is currently believed to be one or a combination of the cytidmne to give a final specific activity of 1 mCi/mmol. following mechanisms: (a) inhibition of cellular DNA polym [“CjCytidine(specific activity, 500 mCi/mmol) was pur erases by ara-CTP (13, 15, 26, 30); and (b) incorporation chased from Amersham/Seanle Corp., Chicago, III. and was into cellular DNA (15, 31). diluted to give a final specific activity of 0.5 mCi/mmol. The active compound ara-CTP is formed intracellularly by Pyruvate kinase, unlabeled ara-C, deoxycytidine, cytidine, sequential action of nucleoside and nucleotide kinases (34, and phosphoenolpyruvate were purchased from Sigma 35). However, the effective concentration of the active Chemical Co., St. Louis, Mo. compound ara-CTP is also affected by the level of intracel Cells and Culture Methods. NRK cells were obtained lular cytidine deaminase, which converts ana-C to the inert from Dr. A. Bernstein (Ontario Cancer Institute, Toronto, metabolite 1-/3-D-arabinofuranosyluracil (6, 9). Canada). Cells were grown in culture in a-MEM with aspar Stewart and Burke (37) found higher levels of cytidmne agine, 50 @g/ml,and supplemented with 10% FCS (Flow deaminase in leukocytes from leukemic patients whose Laboratories, Rockville, Md.) in 75-sq cm Falcon tissue disease did not respond to ara-C than in cells from patients culture flasks. The cultures were maintained at 37°in sensitive to the agent, and Meyers et a!. (29) have described humidified atmosphere supplemented with 5% CO2. Cell SynchronizationProcedure. NRK cells were syn chronized by a modified serum deprivation method previ I This work is supported by a grant from the Medical Research Council of ously described by Farmilo and Stanners (12). In short 2.5 Canada. 2 Recipient of a studentship from the National Cancer Institute of Canada. x 10@cells in 25 ml of a-MEM supplemented with 0.75% 3 The abbreviations used are: ara-C, 1-$-D-arabinofuranosylcytosine; ara FCS were seeded in a 150-sq cm tissue culture flask. After CTP,1-@-D-arabinofuranosylcytosine5'-triphosphate;NRK,normalrat kid nay; a-MEM, minimal essential medium (Eagle's); FCS, fetal calf serum. 3 days at this low serum concentration, the medium was Received November 29, 1977; accepted June 5, 1978. replaced by fresh a-medium containing 10% FCS. The 2768 CANCER RESEARCHVOL. 38 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. Kinaseand Deaminasein Synchronized NRK Cells addition of the serum stimulates entry of the cells into the formed after 8 days of incubation at 37°in a humidified G, phaseofthecellcycle(39). atmosphere with 5% CO2. DNA Synthesis.The rate of DNA synthesisafterserum stimulation was determined by incorporation of RESULTS [3H]thymidmneinto acid-insoluble material. For pulsing a 20- l.Ll aliquot of [3H]thymidine (47 Ci/mmol; Amersham/Searle) SynchronizationofNRK Cells. NRKcellswere synchro was addedto 1.6 ml of medium(30-mmx 10-mm Falcon nized by serum deprivation as described in “Materialsand Petni dish) for 30 mm. The cells were then washed 3 times Methods.―The degree of synchrony of these cells was with 5 ml cold phosphate-buffered saline (0.02 M measured by determining the rates of DNA synthesis with Na@HPO4:0.14MNaCI, pH 7.2), trypsinized, and precipitated [3H]thymidmne at various times after addition of serum. As with 10% cold tnichloroacetic acid. The precipitate was can be seen in Chart 1, the addition of serum resulted in a collected on Millipore membrane filters and washed 3 times peak of DNA synthesis 9 to 20 hr later. The number of cells with 30 ml of 10% trichloroacetic acid, washed once with 10 per plate was also determined to assess the synchrony of ml of cold ethanol (95%), and dried. Radioactivity was the cultures. As illustrated in Chart 1, the number of cells monitored in 10-mi Econofluor (New England Nuclear, per plate remained fairly constant for the first 14 hr, after Boston, Mass.) in a Beckman LS-230 scintillation spectnom which it increased gradually to approximately double the eter. initial cell number by 25 hr. The DNA synthesis and the cell PreparationofCell Extracts.Extractsofcellsuspensions doubling profiles are highly reproducible from experiment for enzymatic analysis were prepared as described by Ho to experiment. A lag time of 13 to 15 hr of DNA synthesis (18). In short, cells were trypsmnized and washed twice with and doubling time of 24 to 25 hr were seen in 8 independent ice-cold 0.9% NaCI solution. The cells were then resus experiments. pended at 2 x 10. cells/mI in cold sucrose (0.25 M) and Deoxycytidine Kinase Activity in Synchronized NRK sonically disrupted for 30 sec in a MSE sonic oscillator. The Cells. The level of deoxycytidmne kinase as a function of extract was then centrifuged for 1 hr at 130,000 x g in a 50 time after serum stimulation was measured. At various Ti rotor at 4°,and the supennatant was removed for enzy times after the addition of serum, cells were trypsinized, matic analysis of the kinase and deaminase. Protein con extracts were prepared, and the deoxycytidmne kinase activ centrations were determined by the method of Lowry et a!. ities were assayed . The results of 3 independent expeni (28). ments are illustrated in Chart 2. The enzymatic activity is DeaminaseEnzymeAssays.Cytidinenucleosidedeami cell cycle related with a reproducible minor peak of activity nase enzyme activities were measured by a modification of in early G, (about 5 hr after the addition of serum) and a the method described by Ho (18). The reaction mixture major peak of activity in middle and late S (between 15 and contained Tnis buffer, pH 8.0, 40 @.&mol;[‘4C]deoxycytidmne, 20 hr). [‘@C]cytidine,or[1@C]ara-C,60 nmol; and cell-free superna Cytosine Nucleoside Deaminase Activity in Synchro tant to give a total volume of 100 @I.Thereaction mixture nized NRK Cells. The intracellularconcentrationof the was incubated at 37°for 60 mm, and the reaction was active compound ara-CTP found inside a cell is known to terminated by putting the mixture into boiling water for 2 be affected by the cellular cytosine nucleoside deaminase, mm. The precipitate was removed by centnifugation at 1000 x g for 10 mm. Fifty @Iofthesupernatant fluid were spotted on Whatman No. 1 filter paper and subjected to ascending chromatography for 18 hr with a solvent system consisting of isopropyl alcohol:water:ethyl acetate (22.5:12.5:65).