Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Deoxycytidine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1993

Role of deoxycytidine kinase in an in vitro model for AraC- and DAC-resistance: substrate-enzyme interactions with deoxycytidine, 1-beta-D-arabinofuranosylcytosine and 5-aza-2'-deoxycytidine.

Deoxycytidine kinase activity (dCk) was monitored in cell lines from a rat acute myeloid leukemia model of acquired resistance to cytosine arabinoside (AraC) and decitabine (DAC). In both AraC-resistant cell lines (RCL/A and its subclone RA/7), as well as in a DAC-resistant cell line (RCL/D) which we generated from the drug-sensitive RCL/0 cell line, a total deficiency of dCk activity and a cross-resistance for AraC and DAC was demonstrated. Furthermore, the metabolization of deoxycytidine (dC) was severely impaired in all these cell lines. Km values for dC (9.4 microM in RCL/0 cells) had increased 70- to 100-fold in RCL/D (Km = 673.2 microM), in RCL/A (Km = 947.2 microM) and in RA/7 (Km = 817.5 microM). Vmax values were unaltered in RCL/D and RA/7, and twofold increased in RCL/A. Addition of hydroxyurea (HU) to cell cultures stimulated dCk salvage pathway activity in RCL/0 cells for dC, AraC, and DAC by increasing Vmax values approximately 160% leaving Km constants unchanged. In all resistant cell lines, HU pre-incubation did not influence the level of dCk activity, leaving Km and Vmax values unaltered. These data indicate that deficiency of dCk activity is crucial in the mechanism of drug resistance in this model.

Acute Disease↗
PubMed1995

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene.

The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2'-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q + chromosome in the AraC-resistant cell line and a (Xq:11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the rat dck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of the dck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence of dck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in the dck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functional dck deficiency that may be the consequence of mutations occurring in the dck gene.

Animals↗
PubMed1997

Chemical modification of deoxycytidine at different sites yields adducts of different stabilities: characterization of N3- and O2-deoxycytidine and N3-deoxyuridine adducts of butadiene monoxide.

Eight adducts were characterized from the reaction of deoxycytidine with the chemical carcinogen, butadiene monoxide (BM). They were identified as diastereomeric pairs of N3-(2-hydroxy-3-buten-1-yl)deoxycytidine, N3-(2-hydroxy-3-buten-1-yl)deoxyuridine, N3-(1-hydroxy-3-buten-2-yl)deoxyuridine, and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine based on UV spectra, 1H NMR, FAB/MS, and stability studies. The N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts were unstable at pH 7.4, 37 degrees C, and deaminated to the corresponding N3-deoxyuridine adducts with half-lives of 2.3 and 2.5 h. The N3-(1-hydroxy-3-buten-2-yl)deoxycytidine diastereomers were not detected, apparently because of faster rates of deamination compared to the N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. The corresponding four N3-deoxyuridine adducts were stable for up to 168 h. The O2-deoxycytidine adducts were unstable and decomposed with a half-life of 11 h. The N3-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts were initially the major adducts formed upon reaction of deoxycytidine with BM at 37 degrees C in phosphate buffer (pH 7.4), but the corresponding N3-deoxyuridine adducts showed a lag in formation due to the time needed for deamination. The N3-(1-hydroxy-3-buten-2-yl)deoxyuridine and O2-deoxycytidine adducts had linear formation rates, but were formed to a lesser extent. Heating the reaction mixture at 80 degrees C for 1 h converted all N3-deoxycytidine adducts to the stable N3-deoxyuridine adducts. Incubation of deoxycytidine with an excess of BM at pH 7.4, 37 degrees C, followed by the extraction and heating steps allowed calculation of the pseudo-first-order kinetic rate constants for the four uridine adducts. If the heating step was eliminated, then the pseudo-first-order kinetic rate constants could be calculated for the N3-(2-hydroxy-3-buten-1-yl)deoxycytidine and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. The rate constants for N3-(2-hydroxy-3-buten1-yl)deoxycytidine and the corresponding N3-(2-hydroxy-3-buten-1-yl)deoxyuridine were five- to sixfold the rate constants for the N3-(1-hydroxy-3-buten-2-yl)deoxyuridine and O2-(2-hydroxy-3-buten-1-yl)deoxycytidine adducts. Thus, the results show that the reaction of deoxycytidine with BM yields adducts at different sites with different rates of formation and stabilities. Understanding the chemical interactions of deoxycytidine with BM and the stability of the various adducts may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of BM and the development of useful biomarkers of exposure.

Carcinogens↗
PubMed1982

Capacity of deoxycytidine to selectively antagonize cytotoxicity of 5-halogenated analogs of deoxycytidine without loss of antiherpetic activity.

Enzyme kinetic studies from this laboratory (M. Dobersen and S. Greer, Biochemistry 17:920-928, 1978) suggested that deoxycytidine could antagonize the toxicity of 5-halogenated analogs of deoxycytidine without interfering with their antiviral activity. Antagonism by deoxycytidine of the toxicity of 5-chlorodeoxycytidine without impairing its anti-herpes simplex virus type 2 activity is demonstrated in the present studies. Tetrahydrouridine, an inhibitor of cytidine deaminase, was utilized. The high Km for deoxycytidine (0.6 mM) with respect to the herpes pyrimidine nucleoside kinase as compared with the low Km for 5-chlorodeoxycytidine (1.1 microM) accounts for the absence of antagonism of the antiviral activity. The high Km for 5-chlorodeoxycytidine (56 microM) as compared with the low Km of deoxycytidine (2 microM) with respect to mammalian deoxycytidine kinase accounts, in great part, for the antagonism of toxicity. In addition, antagonism of toxicity by deoxycytidine is the result of factors other than the kinetic parameters of nucleoside kinases, as indicated by its antagonism of the cytotoxicity of 5-chlorodeoxyuridine. This may be attributed to replenishment of low dCTP pools, diminished because of effector inhibition of ribonucleoside diphosphate reductase by Cl-dUTP. Resistance of the herpes-encoded enzymes to effector control may also play a role in the selective antagonism. Cell culture studies with high concentrations of tetrahydrouridine and 2'-deoxytetrahydrouridine suggest that competition by deoxycytidine for deaminases may not play a major role. The fact that deoxycytidine antagonizes the toxicity of chlorodeoxyuridine also argues against competition for the deaminases as a major reason for its effect. Limited studies with a topical herpes simplex virus type 2 infection system indicate heightened efficacy of 5-chlorodeoxycytidine (and tetrahydrouridine) when deoxycytidine is coadministered. The concepts of selective antagonism of a chemotherapeutic agent derived from these studies may be applied to other approaches that extent beyond viral chemotherapy.

Animals↗
PubMed1986

Interaction of deoxycytidine and deoxycytidine analogs in normal and leukemic human myeloid progenitor cells.

The inhibitory effects of three deoxycytidine analogs, 1-B-D-arabinofuranosylcytosine (Ara-C), 5-aza-2'-deoxycytidine (DAZ) and Ara-5-azacytosine (AAC) were compared with respect to the clonogenic behavior of human promyelocytic leukemic cells (HL-60), a deoxycytidine kinase deficient subvariant (HL-60/Ara-C), and normal human myeloid progenitor cells (CFU-GM). When cells were continuously exposed to each agent for 7 days, Ara-C was the most inhibitory, DAZ slightly less effective and AAC the least inhibitory on a molar basis. HL-60/Ara-C were also highly cross-resistant to both DAZ and AAC. In the absence of deoxycytidine, all three agents were either equally inhibitory or slightly more inhibitory to the growth of CFU-GM than to HL-60, whereas administration of deoxycytidine in ten to one-hundred fold excess protected CFU-GM to a greater extent than HL-60. In contrast, administration of high concentrations of drugs, e.g. 10(-5)-10(-4) M, in conjunction with excess deoxycytidine exhibited greater toxicity toward CFU-GM than toward HL-60/Ara-C. Coadministration of deoxycytidine in ten-fold excess reduced the total intracellular accumulation and DNA incorporation of each analog in HL-60 cells by approx. 50%, whereas a hundred fold excess was associated with greater than a 90% reduction in these values. These studies demonstrate that deoxycytidine may antagonize the effects of Ara-C, DAZ and AAC in both normal and leukemic human myeloid cells, and that at low drug concentrations the degree of protection may be greater for normal elements. However, regimens employing high drug concentrations in conjunction with deoxycytidine do not appear to exert a selective inhibitory effect toward a highly resistant leukemic subvariant. These data suggest that alternative deoxycytidine/deoxycytidine analog dose relationships and schedules must be sought which are capable of selectively eradicating resistant cells.

Azacitidine↗
PubMed2002

Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene.

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.

3T3 Cells↗
PubMed1997

Lack of enantiospecificity of human 2'-deoxycytidine kinase: relevance for the activation of beta-L-deoxycytidine analogs as antineoplastic and antiviral agents.

We demonstrate that human 2'-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates beta-D-2'-deoxycytidine (D-dCyd), the natural substrate, and beta-L-2'-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a Kl value of 0.12 microM, which is lower than the K(m) value for D-dCyd (1,2 microM). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd, L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [3H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.

Antineoplastic Agents↗
PubMed1993

Deoxycytidine kinase and deoxycytidine deaminase activities in human tumour xenografts.

Deoxycytidine kinase (dCK) and deaminase (dCDA) are both key enzymes in the activation and inactivation, respectively, of several deoxycytidine antimetabolites. We determined the total dCK and dCDA activities using standard assays, in 28 human solid tumours grown as xenografts in nude mice, and four corresponding cell lines. dCK activities in colon tumours varied from 11 to 12 nmol/h/mg protein, in ovarian tumours from 3 to 10 nmol/h/mg protein, in soft tissue sarcomas from 2 to 7 nmol/h/mg protein and in squamous cell carcinomas of the head and neck about 45-fold, between 0.4 and 18 nmol/h/mg protein. The dCDA activities showed a larger variation, from 243 to 483, 14 to 1231, 3 to 7 and 1 to 222 nmol/h/mg protein, respectively. The ratios of dCK vs. dCDA activities in these tumours varied from 0.025 to 0.046, 0.004 to 0.240, 0.581 to 1.123 and from 0.012 to 4.227, respectively. In four cell lines (A2780, OVCAR-3, WiDr and UM-SCC-14C), sources for some of the above mentioned tumours, a different pattern in dCK and dCDA was observed than in the corresponding tumours. The variation in dCDA activities was in a smaller range (20-fold) than in the tumours (40-fold). In all cell lines dCK activity was higher than dCDA activity, in contrast to the corresponding tumours, in which the reverse pattern was observed. Previously, some of the tumours were tested for sensitivity to the deoxycytidine analogues 5-aza-deoxycytidine and 2',2'-difluorodeoxycytidine. In the sensitive tumours, both the highest and lowest dCK activity was observed, indicating that dCK activity in solid tumours is high enough to activate deoxycytidine analogues.

Animals↗
PubMed1987

High degree of resistance to 5-aza-2'-deoxycytidine in L1210 cells in vitro associated with almost complete loss of deoxycytidine kinase activity.

In L1210 mouse leukemic cells the resistance towards 5-aza-2'-deoxycytidine was developed in vitro by stepwise selection in increasing concentrations of this drug. According to the respective IC50 values the variant cell line was at least 7000-fold more resistant to 5-aza-2'-deoxycytidine, 3300-fold to arabinosyl 5-azacytosine and 20,000-fold to arabinosyl cytosine in comparison to the parental cell line. The uptake of radioactivity derived from 5-aza-2'-deoxycytidine in variant cells was depressed by 95% as compared to the parental strain and no uptake occurred in case of 2'-deoxycytidine. The phosphorylation of this natural precursor and of its analog in the presence of the cell-free system derived from the variant cell line was nearly completely abolished. In resistant cells the amount of DNA 5-methyl-cytosine relative to cytosine was decreased by 24-44%.

5-Methylcytosine↗
PubMed1999

Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2'-deoxycytidine.

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2'-deoxycytidine (HO5dCyt) in its anionic form. In DNA, this 2'-deoxycytidine analog arises from the oxidation of 2'-deoxycytidine and induces C --> T transitions with 10(2) greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (approximately 1650 cm-1) is enhanced at approximately 65 degrees C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N3-methylcytidine at high pH and N4-methoxy-2'-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO5dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.

DNA↗
PubMed1987

Effect of deoxycytidine on the metabolism and cytotoxicity of 5-aza-2'-deoxycytidine and arabinosyl 5-azacytosine in normal and leukemic human myeloid progenitor cells.

The effect of deoxycytidine (dCyd) on the inhibitory effects of two antileukemic nucleoside analogs, 5-aza-2'-deoxycytidine and ara-5-aza-Cyd, toward the clonogenic growth of normal human bone marrow progenitors (CFU-GM) and leukemic blast progenitors (L-CFU) was examined. Continuous exposure of cells to 10(-6)-10(-5) M 5-aza-deoxycytidine or 10(-5)-5 x 10(-5) M ara-5-aza-Cyd in conjunction with a 10- 100-fold excess of dCyd resulted in significantly greater restoration of CFU-GM growth than L-CFU colony formation at each dose relationship. Normal bone marrow mononuclear cells exposed to 10(-3) M dCyd for 4 hr (along with 5-aza-dCyd or ara-5-aza-Cyd) exhibited intracellular deoxycytidine triphosphate (dCTP) pools 20-fold higher than their leukemic counterparts. However, this finding was not associated with enhanced analog incorporation into leukemic cell DNA. These results suggest that high concentrations of dCyd preferentially protect normal versus leukemic progenitor cells from the inhibitory actions of 5-aza-dCyd and ara-5-aza-Cyd. They also raise the possibility that this in vitro selectivity may be related to enhanced expansion of dCTP pools in normal bone marrow elements and involves factors other than differential short-term analog incorporation into DNA.

Acute Disease↗
PubMed2003

Selective activation of deoxycytidine kinase by thymidine-5'-thiosulphate and release by deoxycytidine in human lymphocytes.

Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5'-thiosulphate (5'-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2'-deoxythymidine-5'-thiosulphate (dThd-5'-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5'-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5'-TS nor Urd-5'-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3H-dThd into DNA was suppressed by 90% in cells by dThd-5'-TS, while Urd-5'-TS only slightly influenced the same process. The 3H-dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3H-dCyd labelling of the nucleotide fraction was enlarged in dThd-5'-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different "inhibitors" of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.

Cell-Free System↗
PubMed1983

Labelling of the thymidine and deoxycytidine bases of DNA by [2-14C]deoxycytidine in cultured L1210 cells.

Exposure of cultured L1210 cells to [2-14C]deoxycytidine and analysis or radioactivity incorporated into DNA-pyrimidines revealed that 2.7--5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favored over successive phosphorylation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with [2-14C]deoxycytidine. 3-Deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (greater than or equal to 10 microM) increased the specific activity of DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (greater than or equal to 10 microM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect.

3-Deazauridine↗
PubMed1986

Analysis of 5-fluoro-2'-deoxycytidine and 5-trifluoromethyl-2'-deoxycytidine and their related antimetabolites by high-performance liquid chromatography.

An isocratic, ion-paired, reversed-phase high-performance liquid chromatography technique for the quantitative determination of 5-fluoro-2'-deoxycytidine (FdCyd) and 5-trifluoromethyl-2'-deoxycytidine (F3methyldCyd) and their related antimetabolites is described. Extraction and purification of these compounds from DNA, RNA, and free pools is reviewed diagrammatically. Total analysis time including quantitation of DNA and RNA primary constituents is 45 min. Average combined recoveries for prodrugs and antimetabolites is above 90% with standard deviations of 0.07 and 0.58 and average precisions of 5.51 and 8.30% for FdCyd and F3methyldCyd, respectively. Average coefficients of variation were 3.8 +/- 0.5% for FdCyd and 7.7 +/- 1.0% for F3methyldCyd. Limits of detection were approximately 1 pmol for unlabelled prodrugs and antimetabolites. FdCyd, when generally tritiated with a specific activity of 18 Ci/mmol, was detected in the 5 X 10(-15)-20 X 10(-15) mol (fmol) range depending on sample condition.

Buffers↗
PubMed2001

Activation of the maternally preset program of apoptosis by microinjection of 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine-5'-triphosphate in Xenopus laevis embryos.

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5-aza-2'-deoxycytidine (5-Aza-CdR), which induces hypomethylation of DNA, and 5-methyl-2'- deoxycytidine-5'-triphosphate (5-methyl-dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid-blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end-labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a "ladder" on electrophoresis. Furthermore, the induction of cell dissociation by 5-Aza-CdR and 5-methyl-dCTP was postponed by 2-3 h by co-injection of Bcl-2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5-methyl-cytosine, we confirmed that 5-Aza-CdR induces hypomethylation, whereas 5-methyl-dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5-Aza-CdR-injected and 5-methyl-dCTP-injected embryos. These results show that 5-Aza-CdR and 5-methyl-dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.

Animals↗
PubMed1999

Formation of 5-formyl-2'-deoxycytidine from 5-methyl-2'-deoxycytidine in duplex DNA by Fenton-type reactions and gamma-irradiation.

5-methyl-2'-deoxycytidine (5-Me-dC) is formed by the enzymatic methylation of dC, primarily in CpG sequences in DNA, and is involved in the regulation of gene expression. In the present study, 5-Me-dC and double-stranded DNA fragments containing 5-Me-dC were either gamma-irradiated or aerobically treated with Fenton-type reagents, Fe(II)-EDTA, Fe(II)-nitrilotriacetic acid, Fe(III)-EDTA-H(2)O(2)-catechol or ascorbic acid-H(2)O(2) under neutral conditions. The formation of 5-formyl-2'-deoxycytidine (5-CHO-dC) was observed upon treatment of both 5-Me-dC and DNA fragments containing 5-Me-dC. The yields of 5-CHO-dC from 5-Me-dC and those of 5-formyl-2'-deoxyuridine from dT were comparable. These results suggest that 5-Me-dC in DNA is as susceptible to oxidation as dT in cells, and raise the possibility that 5-CHO-dC may contribute to the high mutagenic rate observed in CpG sequences in genomic DNA.

Ascorbic Acid↗
PubMed2001

Synthesis and properties of oligonucleotides containing 5-formyl-2'-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2'-deoxycytidine.

Oligodeoxynucleotides (ODNs) containing 5-formyl-2'-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2'-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C.G-->T.A and the transversion mutation C.G-->A.T during DNA synthesis.

Base Pairing↗