title: Cyclopentenyl cytosine-induced activation of deoxycytidine kinase increases gemcitabine anabolism and cytotoxicity in neuroblastoma.
abstract: The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [3H]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED50) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED50 with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [3H]dFdC-anabolism increased 6-44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro.
authors.affiliation: Academic Medical Center, Department of Clinical Chemistry and Emma Children's Hospital, University of Amsterdam, 1100 Amsterdam, DE, The Netherlands.
authors.forename: Jörgen
authors.initials: J
authors.lastname: Bierau
authors.forename: Albert H
authors.initials: AH
authors.lastname: van Gennip
authors.forename: René
authors.initials: R
authors.lastname: Leen
authors.forename: Rutger
authors.initials: R
authors.lastname: Meinsma
authors.forename: Huib N
authors.initials: HN
authors.lastname: Caron
authors.forename: André B P
authors.initials: AB
authors.lastname: van Kuilenburg
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D003841:Deoxycytidine; D003562:Cytidine; C045911:cyclopentenyl cytosine; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine