title: Cyclopentenyl cytosine increases the phosphorylation and incorporation into DNA of 1-beta-D-arabinofuranosyl cytosine in a human T-lymphoblastic cell line.
abstract: The cytotoxic effect of 1-beta-D-arabinofuranosyl cytosine (araC) depends on the intracellular phosphorylation into its active compound araCTP, on the degree of degradation of araCTP and on the incorporation of araCTP into DNA. Deoxycytidine triphosphate (dCTP) inhibits the phosphorylation of araC (by feedback inhibition of the enzyme deoxycytidine kinase) and the incorporation of araCTP into DNA (by competition for DNA polymerase). In a T-lymphoblastic cell line, we studied whether the cytotoxicity of araC (2 nM-50 microM) could be enhanced by decreasing the concentration of dCTP, using the nucleoside-analogue cyclopentenyl cytosine (CPEC), an inhibitor of the enzyme CTP synthetase. Preincubation of the cells with CPEC (100-1,600 nM) for 2 hr increased the concentrations of araCMP 1.6-9.5-fold, which was significant for each concentration of CPEC used. The concentration of araCDP remained low, whereas the concentration of araCTP changed depending on the concentration of araC used. With 2-15 microm of araC and a preincubation with 400 nM of CPEC, the araCTP concentration increased by 4-15% (not significant), and the total amount of araC nucleotides increased significantly by 21-45%. When using a concentration of araC of 2 nM after a preincubation with CPEC of 100 nM, the concentration of araCMP increased by 60% (p = 0.015), whereas that of araCTP decreased by 10% (p = 0.008). This was compensated by an increase of 41% (p = 0.005) of araCTP incorporation into DNA, which represented 43% of all araC metabolites. Moreover, by performing pulse/chase experiments with 400 nM of CPEC and 2 microM of araC, the retention of cytosolic araCTP and the incorporated amount of araCTP into DNA were increased by CPEC. The modulation by CPEC of araC metabolism was accompanied by a synergistic increase of araC-induced apoptosis and by an additive effect on the araC-induced growth inhibition.
authors.affiliation: Laboratory of Genetic Metabolic Diseases, Division of Clinical Chemistry, Academic Medical Centre, University of Amsterdam, Emma Kinderziekenhuis AMC, Amsterdam, The Netherlands. a.c.verschuur@amc.uva.nl
authors.forename: Arnauld C
authors.initials: AC
authors.lastname: Verschuur
authors.forename: Albert H
authors.initials: AH
authors.lastname: Van Gennip
authors.forename: René
authors.initials: R
authors.lastname: Leen
authors.forename: P A
authors.initials: PA
authors.lastname: Voûte
authors.forename: Josanne
authors.initials: J
authors.lastname: Brinkman
authors.forename: André B P
authors.initials: AB
authors.lastname: Van Kuilenburg
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003561:Cytarabine / Q000378:metabolism* / Q000494:pharmacology; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D003845:Deoxycytosine Nucleotides; D003561:Cytarabine; D003562:Cytidine; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine; D004247:DNA