DOCSLIB.ORG

Deoxycytidine Kinase and Cytosine Nucleoside Deaminase Activities in Synchronized Cultures of Normal Rat Kidney Cells1 Charnwingwan2andtak W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Deoxycytidine Kinase and Cytosine Nucleoside Deaminase Activities in Synchronized Cultures of Normal Rat Kidney Cells1 Charnwingwan2andtak W (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).
Load more

Recommended publications
  • Nucleoside Phosphate-Conjugates Come of Age: Catalytic Transformation, Polymerase Recognition and Antiviral Properties.# Elisabetta Groaz* and Piet Herdewijn
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Lirias Send Orders for Reprints to [email protected] Current Medicinal Chemistry, Year, Volume 1 Nucleoside Phosphate-Conjugates Come of Age: Catalytic Transformation, Polymerase Recognition and Antiviral Properties.# Elisabetta Groaz* and Piet Herdewijn Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium Abstract: Over the past few decades, different types of nucleoside phosphate-conjugates have been under extensive investigation due to their favorable molecular lability with interesting catalytic hydrolysis mechanisms, recognition as polymerase substrates, and especially for their development as antiviral/anticancer protide therapeutics. The antiviral conjugates such as nucleoside phosphoesters and phosphoramidates that were discovered and developed in the initial years have been well reviewed by the pioneers in the field. In the present review, we will discuss the basic chemical and biological principles behind consideration of some representative structural classes. We will also summarize the chemical and biological properties of some of the more recent analogues that were synthesized and evaluated in our laboratory and by others. This includes new principles for their application as direct substrates of polymerases, nucleobase-dependent catalytic and antiviral activity, and a plausible ‘prodrug of a prodrug’ strategy for tissue/organ-specific targeted drug delivery. Keywords: Prodrug – Delivery – Stability – Polymerase - Phosphoramidates INTRODUCTION profile of these molecules through the “tuning” of the nature of the existing ligands and the pursuit of new protecting The ability of unnatural nucleoside-based therapeutics to moieties relying on chemical rather than enzymatic induce inhibition of uncontrolled cell proliferation or viral dependant activation mechanisms.
    [Show full text]
  • Depression of Thymidylate Synthetase Activity in Response to Cytosine Arabinoside1
    [CANCER RESEARCH 32, 1160-1169, June 1972] Depression of Thymidylate Synthetase Activity in Response To Cytosine Arabinoside1 DeWayne Roberts and Ellen V. Loehr St. Jude Children's Research Hospital, Memphis, Tennessee 38101 ¡D.R., E. V. L.J and Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tennessee 38101 [D. R.J SUMMARY Cytosine arabinoside induces remission of acute granulocytic leukemia (11). After the administration of Depression of thymidylate synthetase activity by cytosine cytosine arabinoside to patients, changes in the enzyme arabinoside in CCRF-CEM cells was correlated with a blocking activity pattern of leukemic leukocytes occur (32). After drug of precursor incorporation into RNA and with cell lysis. With administration, a decrease in thymidylate synthetase activity is increasing concentrations of cytosine arabinoside in the observed as early as 1 hr and persists for 24 hr in some culture media, a proportional decrease of uridine patients. A concentration-dependent decrease in thymidylate incorporation into RNA and decrease in thymidylate synthetase activity also follows the addition of cytosine synthetase activity occurred and was accompanied by lysis of arabinoside to CCRF-CEM cultures (33). the cells. These effects continued to develop with drug Methotrexate elevates thymidylate synthetase activity in concentrations in excess of that required to block thymidine leukocytes from patients with acute leukemia (32), in rat liver incorporation into DNA. The addition of deoxycytidine at and transplantable tumors (27), and in cells of LI210 or concentrations that failed to reverse inhibition by cytosine CCRF-CEM cultures (32, 33). The simultaneous addition of arabinoside of thymidine incorporation into DNA reversed the cytosine arabinoside and methotrexate to CCRF-CEM cultures drug effects on uridine incorporation, thymidylate synthetase modulated the effect of each drug on thymidylate synthetase activity, and cell lysis.
    [Show full text]
  • 2'-Deoxyguanosine Toxicity for B and Mature T Lymphoid Cell Lines Is Mediated by Guanine Ribonucleotide Accumulation
    2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation. Y Sidi, B S Mitchell J Clin Invest. 1984;74(5):1640-1648. https://doi.org/10.1172/JCI111580. Research Article Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8- aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'- deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine.
    [Show full text]
  • Hyperhomocysteinemia: Focus on Endothelial Damage As a Cause of Erectile Dysfunction
    International Journal of Molecular Sciences Review Hyperhomocysteinemia: Focus on Endothelial Damage as a Cause of Erectile Dysfunction Gianmaria Salvio , Alessandro Ciarloni, Melissa Cutini and Giancarlo Balercia * Division of Endocrinology, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, via Conca 71, Umberto I Hospital, 60126 Ancona, Italy; [email protected] (G.S.); [email protected] (A.C.); [email protected] (M.C.) * Correspondence: [email protected] Abstract: Erectile Dysfunction (ED) is defined as the inability to maintain and/or achieve a satis- factory erection. This condition can be influenced by the presence of atherosclerosis, a systemic pathology of the vessels that also affects the cavernous arteries and which can cause an alteration of blood flow at penile level. Among the cardiovascular risk factors affecting the genesis of atherosclero- sis, hyperhomocysteinemia (HHcys) plays a central role, which is associated with oxidative stress and endothelial dysfunction. This review focuses on the biological processes that lead to homocysteine- induced endothelial damage and discusses the consequences of HHcys on male sexual function Keywords: Erectile Dysfunction; hyperhomocysteinemia; endothelial dysfunction 1. Introduction Erectile Dysfunction (ED) is defined as the persistent inability to obtain or maintain penile erection sufficient for a satisfactory sexual performance [1] and represents a common condition in middle-aged men, with a prevalence that increases exponentially with age. Citation: Salvio, G.; Ciarloni, A.; According to data from the Massachusetts Male Aging Study (MMAS), 52% of men between Cutini, M.; Balercia, G. 40 and 70 years old report some form of ED with a percentage that increases proportionally Hyperhomocysteinemia: Focus on with aging and reaches 70% at 70 years of age [2].
    [Show full text]
  • Expanding the Genetic Code Lei Wang and Peter G
    Reviews P. G. Schultz and L. Wang Protein Science Expanding the Genetic Code Lei Wang and Peter G. Schultz* Keywords: amino acids · genetic code · protein chemistry Angewandte Chemie 34 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/anie.200460627 Angew. Chem. Int. Ed. 2005, 44,34–66 Angewandte Protein Science Chemie Although chemists can synthesize virtually any small organic molecule, our From the Contents ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, 1. Introduction 35 modifications are largely restricted to substitutions among the common 20 2. Chemical Approaches 35 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and 3. In Vitro Biosynthetic eukaryotic organisms. Over 30 novel amino acids have been genetically Approaches to Protein encoded in response to unique triplet and quadruplet codons including Mutagenesis 39 fluorescent, photoreactive, and redox-active amino acids, glycosylated 4. In Vivo Protein amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom- Mutagenesis 43 containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps 5. An Expanded Code 46 entire organisms with new or enhanced properties. 6. Outlook 61 1. Introduction The genetic codes of all known organisms specify the same functional roles to amino acid residues in proteins. Selectivity 20 amino acid building blocks. These building blocks contain a depends on the number and reactivity (dependent on both limited number of functional groups including carboxylic steric and electronic factors) of a particular amino acid side acids and amides, a thiol and thiol ether, alcohols, basic chain.
    [Show full text]
  • Questions with Answers- Nucleotides & Nucleic Acids A. the Components
    Questions with Answers- Nucleotides & Nucleic Acids A. The components and structures of common nucleotides are compared. (Questions 1-5) 1._____ Which structural feature is shared by both uracil and thymine? a) Both contain two keto groups. b) Both contain one methyl group. c) Both contain a five-membered ring. d) Both contain three nitrogen atoms. 2._____ Which component is found in both adenosine and deoxycytidine? a) Both contain a pyranose. b) Both contain a 1,1’-N-glycosidic bond. c) Both contain a pyrimidine. d) Both contain a 3’-OH group. 3._____ Which property is shared by both GDP and AMP? a) Both contain the same charge at neutral pH. b) Both contain the same number of phosphate groups. c) Both contain the same purine. d) Both contain the same furanose. 4._____ Which characteristic is shared by purines and pyrimidines? a) Both contain two heterocyclic rings with aromatic character. b) Both can form multiple non-covalent hydrogen bonds. c) Both exist in planar configurations with a hemiacetal linkage. d) Both exist as neutral zwitterions under cellular conditions. 5._____ Which property is found in nucleosides and nucleotides? a) Both contain a nitrogenous base, a pentose, and at least one phosphate group. b) Both contain a covalent phosphodister bond that is broken in strong acid. c) Both contain an anomeric carbon atom that is part of a β-N-glycosidic bond. d) Both contain an aldose with hydroxyl groups that can tautomerize. ___________________________________________________________________________ B. The structures of nucleotides and their components are studied. (Questions 6-10) 6._____ Which characteristic is shared by both adenine and cytosine? a) Both contain one methyl group.
    [Show full text]
  • Standard Abbreviations
    Journal of CancerJCP Prevention Standard Abbreviations Journal of Cancer Prevention provides a list of standard abbreviations. Standard Abbreviations are defined as those that may be used without explanation (e.g., DNA). Abbreviations not on the Standard Abbreviations list should be spelled out at first mention in both the abstract and the text. Abbreviations should not be used in titles; however, running titles may carry abbreviations for brevity. ▌Abbreviations monophosphate ADP, dADP ‌adenosine diphosphate, deoxyadenosine IR infrared diphosphate ITP, dITP ‌inosine triphosphate, deoxyinosine AMP, dAMP ‌adenosine monophosphate, deoxyadenosine triphosphate monophosphate LOH loss of heterozygosity ANOVA analysis of variance MDR multiple drug resistance AP-1 activator protein-1 MHC major histocompatibility complex ATP, dATP ‌adenosine triphosphate, deoxyadenosine MRI magnetic resonance imaging trip hosphate mRNA messenger RNA bp base pair(s) MTS ‌3-(4,5-dimethylthiazol-2-yl)-5-(3- CDP, dCDP ‌cytidine diphosphate, deoxycytidine diphosphate carboxymethoxyphenyl)-2-(4-sulfophenyl)- CMP, dCMP ‌cytidine monophosphate, deoxycytidine mono- 2H-tetrazolium phosphate mTOR mammalian target of rapamycin CNBr cyanogen bromide MTT ‌3-(4,5-Dimethylthiazol-2-yl)-2,5- cDNA complementary DNA diphenyltetrazolium bromide CoA coenzyme A NAD, NADH ‌nicotinamide adenine dinucleotide, reduced COOH a functional group consisting of a carbonyl and nicotinamide adenine dinucleotide a hydroxyl, which has the formula –C(=O)OH, NADP, NADPH ‌nicotinamide adnine dinucleotide
    [Show full text]
  • Effect of Ischemia on Nucleosides and Bases in Rat Liver and Hepatoma 3924A1
    [CANCER RESEARCH 47, 3092-3096, June 15. 1987] Effect of Ischemia on Nucleosides and Bases in Rat Liver and Hepatoma 3924A1 Konrad Pillwein, Hiremagalur N. Jayaram, and George Weber2 laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, IN 46223 ABSTRACT MATERIALS AND METHODS A high-pressure liquid Chromatographie method was developed which Materials. Nucleobases and nucleosides were obtained from Sigma achieved a separation and quantitation of 20 biologically important Chemical Co., St. Louis, MO. Ammonium acetate, ammonium phos nucleosides and bases. The concentrations of pyrimidine nucleosides and phate, sodium acetate, and acetonitrile (HPLC grade) were purchased bases, namely deoxycytidine, cytosine, cytidine, uracil, and uridine (22.6, from Fisher Scientific Co., Springfield, NJ. Radial Pak, Resolve Ct8 5 10.1, 5.2, 2.9, and 2.4 nmol/ml, respectively) were high in plasma, Mm(8.0 mm x 10 cm), Partisil 10-SAX (8.0 mm x 10 cm) columns, whereas purine nucleosides and bases were present in concentrations less and HPLC apparatus consisting of 720 System Controller, 730 Data than 2.5 nmol/ml. In erythrocytes, the pools of xanthine, hypoxanthine, Module, 71OB-WISP auto injector, 510 pumps, and 440 dual channel and xanthosine were 32-, 27-, and 22-fold larger, respectively, whereas absorbance detector were obtained from Waters Co., Milford, MA. cytidine, uridine, and deoxycytidine were only 21, 12, and 5% of plasma Animals and Tumors. Inbred male rats of the ACI/N strain weighing concentrations. The results suggest a compartmental system for transport 180-200 g (HaríanIndustries, Cumberland, IN) were housed in indi of some of the purine and pyrimidine nucleosides and bases in the whole vidual cages and fed laboratory chow; water was available ad libitum.
    [Show full text]
  • Dynamics of Pyrimidine Deoxynucleoside Triphosphate
    Proc. Nati Acad. Sci. USA Vol. 80, pp. 1347-1351, March 1983 Cell Biology Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts (nucleoside incorporation/compartmentation/amethopterin block) BjORN NICANDER AND PETER REICHARD Medical Nobel Institute, Department of Biochemistry I, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden Contributed by Peter A. Reichard, November 18, 1982 ABSTRACT The 3H-labeled nucleosides cytidine, deoxycyti- tivity into DNA to this value (1, 2). Such a procedure becomes dine, and thymidine are rapidly incorporated into DNA via dCTP imperative when one wishes to distinguish between effects of or dTTP pools. Between 30 and 60 minafter addition of tracer different manipulations-e. g., pharmacological interference- amounts ofa labeled nucleoside to the medium ofrapidly growing on precursor synthesis and DNA replication. Knowing the spe- 3T6 cells, dNTPpools attained a constant specific activity resulting cific activity of a dNTP under steady-state conditions may in from a steady-state equilibrium between incorporation of nucleo- addition permit determination of absolute rather than relative side, de novo synthesis, and linear incorporation of isotope into rates of DNA synthesis. DNA. Removal oflabeled deoxycytidine or thymidine depleted the The of dNTP pools ofisotope within a few minutes and incorporation into possibility compartmentation of dNTP pools poses DNA stopped. When de novo synthesis ofdTTP was blocked with additionalicomplications (3). Fractionation ofcells in nonaque- amethopterin, the intracellular dTTP pool rapidly reached the ous media led to -the suggestion of separate cytoplasmic and. specific activity of thymidine of' the medium and isotope-incor- nuclear dNTP pools in Chinese hamster ovary cells (4).
    [Show full text]
  • Deoxyadenosine Triphosphate As a Mediator of Deoxyguanosine Toxicity in Cultured T Lymphoblasts
    Deoxyadenosine triphosphate as a mediator of deoxyguanosine toxicity in cultured T lymphoblasts. G J Mann, R M Fox J Clin Invest. 1986;78(5):1261-1269. https://doi.org/10.1172/JCI112710. Research Article The mechanism by which 2'-deoxyguanosine is toxic for lymphoid cells is relevant both to the severe cellular immune defect of inherited purine nucleoside phosphorylase (PNP) deficiency and to attempts to exploit PNP inhibitors therapeutically. We have studied the cell cycle and biochemical effects of 2'-deoxyguanosine in human lymphoblasts using the PNP inhibitor 8-aminoguanosine. We show that cytostatic 2'-deoxyguanosine concentrations cause G1-phase arrest in PNP-inhibited T lymphoblasts, regardless of their hypoxanthine guanine phosphoribosyltransferase status. This effect is identical to that produced by 2'-deoxyadenosine in adenosine deaminase-inhibited T cells. 2'-Deoxyguanosine elevates both the 2'-deoxyguanosine-5'-triphosphate (dGTP) and 2'-deoxyadenosine-5'-triphosphate (dATP) pools; subsequently pyrimidine deoxyribonucleotide pools are depleted. The time course of these biochemical changes indicates that the onset of G1-phase arrest is related to increase of the dATP rather than the dGTP pool. When dGTP elevation is dissociated from dATP elevation by coincubation with 2'-deoxycytidine, dGTP does not by itself interrupt transit from the G1 to the S phase. It is proposed that dATP can mediate both 2'-deoxyguanosine and 2'-deoxyadenosine toxicity in T lymphoblasts. Find the latest version: https://jci.me/112710/pdf Deoxyadenosine Triphosphate as a Mediator of Deoxyguanosine Toxicity in Cultured T Lymphoblasts G. J. Mann and R. M. Fox Ludwig Institute for Cancer Research (Sydney Branch), University ofSydney, Sydney, New South Wales 2006, Australia Abstract urine of PNP-deficient individuals, with elevation of plasma inosine and guanosine and mild hypouricemia (3).
    [Show full text]
  • NIH Public Access Author Manuscript Leuk Res
    NIH Public Access Author Manuscript Leuk Res. Author manuscript; available in PMC 2009 August 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Leuk Res. 2008 August ; 32(8): 1268±1278. doi:10.1016/j.leukres.2007.12.015. Determinants of Sensitivity of Human T Cell Leukemia CCRF-CEM cells to Immucillin-H Min Huang∫, Yanhong Wang*, Jingjin Gu§, Jing Yang*, Karen Noel§, Beverly S. Mitchell∫, Vern L. Schrammf, and Lee M. Graves* ∫Stanford University, Albert Einstein School of Medicine, Bronx, NY 10461. *Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 §Department of Medicine, Division of Hematology/Oncology and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 fDepartment of Biochemistry, Albert Einstein School of Medicine, Bronx, NY 10461. Abstract Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. 3H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas 3H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from an ImmH- resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells.
    [Show full text]
  • Enzymic Ribonucleoside Reduction at the Non-Phosphorylated Level in Acetabularia
    J. Cell Sci. 73, 7-18 (1985) Printed in Great Britain © The Company of Biologists Limited 1985 ENZYMIC RIBONUCLEOSIDE REDUCTION AT THE NON-PHOSPHORYLATED LEVEL IN ACETABULARIA EGON J. DE GROOT AND HANS-GEORG SCHWEIGER Max-Planck-Iiislitut fur Zellbiologie, 6802 Ladenburg bei Heidelberg, West Germany SUMMARY The occurrence of an enzyme that catalyses the conversion of cytidine into deoxycytidine was demonstrated in homogenates of Acetabularia. Cytidine was identified as the substrate by comparing cytidine, cytidine 5'-monophosphate, cytidine S'-diphosphate and cytidine S'-triphos- phate as potential substrates. Experiments with ATP analogues whose inhibitory effect on kinase reactions is well established, supplied evidence that the nucleoside is reduced without a phos- phorylation step before the reduction. Further evidence in this line came from incubations with cytidine in the presence of phosphatase and from trap-type experiments in which the effects of excess non-labelled cytidine 5'-phosphate and deoxycytidine, respectively, on the formation of deoxycytidine phosphates from cytidine were studied. INTRODUCTION Enzymes that convert the ribose moiety of nucleotides into deoxyribose are very important because all deoxyribonucleotides, particularly those of DNA, have to pass through this enzymic reaction (Larsson & Reichard, 1967; Reichard, 1967; Blakley & Vitols, 1968; Follmann, 1974; Hogenkamp & Sando, 1974; Thelander & Reichard, 1979). It is generally accepted that reduction occurs at the nucleoside diphosphate level (Larsson & Reichard,
    [Show full text]