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PubMed1976

Nucleic acids. 16. Orally active derivatives of ara-cytidine.

Water-soluble derivatives of ara-cytidine (cytarabine, Cytosar) were prepared and tested for antitumor, immunosuppressive, and antiarthritic activities in animals after oral administration. The compounds tested included the 5'-palmitate, 5'-benzoate, and 5'-adamantoate esters of ara-cytidine, made water soluble by use of their hydrochloride salts of peptidyl derivatives, and two basic 5' esters (5'-nicotinoate and 5'-quinuclidinate) as their hydrochloride salts. Five of the compounds had antitumor activity superior to that found with ara-cytidine itself after oral administration in the L1210 leukemic mouse assay. One of these, 5'-adamantoyl-ara-cytidine hydrochloride, had antitumor activity after oral administration approaching that achieved with parenterally administered ara-cytidine.

Administration, Oral↗
PubMed2006

Activation-induced cytidine deaminase: structure-function relationship as based on the study of mutants.

Activation-induced cytidine deaminase (AID; gene symbol AICDA) is the key molecule required to induce immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM) of the variable regions of Ig genes. Its deficiency causes a form of hyper-IgM (HIGM) syndrome. The study of natural AID mutants associated with HIGM as well as engineered mutants led to the characterization of the active domains of the protein. AID, through its cytidine deaminase activity, induces a targeted DNA lesion as an early step required for both CSR and SHM. Besides its cytidine deaminase activity, AID plays a further essential role in CSR, likely by recruiting CSR-specific cofactors by its C-terminus. A similar binding of SHM-specific cofactors to the N-terminal part is suggested by the functional characteristics of N(ter) AID artificial mutants. These data require confirmation in vivo. Finally, AID acts as a homo-, di-, or multimeric complex. Together, these data strongly suggest that AID, a master molecule for antibody diversification, exerts its activity on CSR not only as a cytidine deaminase enzyme but also as a docking protein, recruiting specific cofactors to a multimeric complex.

Base Sequence↗
PubMed1995

Evidence for a site-specific cytidine deamination reaction involved in C to U RNA editing of plant mitochondria.

Transcripts of higher plant mitochondria are modified post-transcriptionally by RNA editing. To distinguish between the mechanisms by which the cytidine to uridine transition could occur a combined transcription/RNA editing assay and an in vitro RNA editing system were investigated. Mitochondria isolated from etiolated pea seedlings and potato tubers were supplied with [alpha-32P]CTP to radiolabel the mitochondrial run-on transcripts. High molecular weight run-on transcripts were isolated and hydrolyzed, and nucleotide identities were analyzed by one- and two-dimensional thin layer chromatography. The amount of label comigrating with UMP nucleotides increases with extended incubation times. Analogous products were obtained by incubation of [alpha-32P]CTP or [5-3H]CTP radiolabeled in vitro transcripts with a mitochondrial lysate from pea mitochondria. 5-3H label of the cytosine base was detected in the UMP spot after incubation of in vitro transcripts with mitochondrial lysate. These results are consistent with a deamination reaction involved in this post-transcriptional C to U modification process. To prove that cytidines are deaminated specifically in vitro transcripts were reisolated after incubation and analyzed by reverse transcription-polymerase chain reaction. Sequence analysis clearly shows that only cytidines at editing sites are edited while residual cytidines are not modified and suggests that site-specific factors are involved in RNA editing of plant mitochondria.

Base Sequence↗
PubMed1993

The p27 catalytic subunit of the apolipoprotein B mRNA editing enzyme is a cytidine deaminase.

The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666. This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48. A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C. F., and Davidson, N. O. (1993) Science 260, 1816-1819). Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man. p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts. The homologous E. coli cytidine deaminase does not confer editing activity. The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts. We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase.

APOBEC-1 Deaminase↗
PubMed1987

Determination of plasma cytidine deaminase activity by HPLC.

Cytidine deaminase is an enzyme of nucleic acid metabolism, the measurement of which has been proposed as a useful test for the early detection of pre-eclamptic toxaemia in pregnancy. The enzyme converts the nucleoside cytidine to uridine, with the release of ammonia, and it is the measurement of this latter compound that forms the basis of the conventional methods for the assay of cytidine deaminase. The low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. We have developed a new method for determining cytidine deaminase activity, utilising high performance liquid chromatography to measure the production of uridine. This method uses much shorter incubation times and is unaffected by ammonia contamination. This paper describes the development of the method and its comparison with the established assay. The relative merits of each are discussed. Finally, the adaptation of incubation and chromatographic conditions, in order to measure other enzymes of nucleic acid metabolism which are of clinical interest, is briefly mentioned.

Chromatography, High Pressure Liquid↗
PubMed2003

Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition.

Tetrahydrouridine (THU) is an inhibitor of cytidine deaminase (CDA), the enzyme responsible for the deactivation of ara-C and other cytidine analogues in vivo, and therefore is capable of improving the therapeutic efficacy of these antitumor agents. In aqueous solution formulations, THU exists as a mixture of epimers differing in stereochemistry of the 4-OH substituent. The aims of this study were to investigate the interconversion kinetics of the epimers of THU, the CDA inhibitory effects of these epimers, and the stability and degradation mechanisms of THU epimer mixtures in aqueous solution with the ultimate goal of developing optimal conditions for a parenteral formulation of THU. A stability indicating HPLC assay utilizing a derivatized beta-cyclodextrin column was developed to separate the two epimers of THU and to monitor their reversible isomerization to their beta-ribopyranosyl counterparts and their hydrolysis to form N-glycosidic bond cleavage products. MS and one- and two-dimensional (1)H- and (13)C-NMR measurements were conducted to identify THU epimers and degradation products and to quantitatively model the degradation kinetics. The interconversion reaction between the two THU epimers is acid catalyzed with a first-order rate constant for conversion of epimer 1(1) to epimer 1(2) of (7.4 +/- 0.3) x 10(-3) h(-1) and an equilibrium constant ([1(2)]/[1(1)] of 1.7 +/- 0.1 at pH 7.4 and 25 degrees C. Epimer interconversion was therefore sufficiently slow at pH 7.4 to allow the isolation of each and evaluation of their CDA inhibitory activities utilizing 1% (w/v) mouse kidney homogenates as a source for cytidine deaminase and cytidine as a substrate. Inhibition constants for the two THU epimers (1(1) and 1(2)) were determined to be 8 +/- 1 x 10(-7) M and 6.2 +/- 0.2 x 10(-8) M, respectively. Studies at elevated temperature suggested that THU degradation from epimer mixtures is biphasic with the initial rate of disappearance being acid catalyzed and first order in initial THU concentration, thus ruling out dimerization as a potential reaction mechanism. NMR/MS analyses revealed that the major degradation products included the beta-ribopyranosyl THU isomers (two epimers), the reduced pyrimidinone base (tetrahydrouracil), and various anomers of D-ribose formed through N-glycosidic bond cleavage, and the products of subsequent reactions of the base. Kinetic modeling of the data obtained from both HPLC and NMR measurements indicated that in an acidic solution THU beta-ribofuranosyl --> beta-ribopyranosyl isomerization is a rapid equilibrium reaction, which proceeds through an intermediate observable in 1H-NMR, and is followed by slower N-glycosidic bond hydrolysis. All the reactions between THU, its ribopyranosyl isomers, the intermediate, and the base are acid catalyzed and appear to proceed through the same sugar ring-opened intermediate (carbinolamine), consistent with previous literature.

Animals↗
PubMed1975

An inverse dose response of 5-methyl cytidine on multiple primary spontaneous tumors and their regressions in mice.

Ten series mice of C3H/ST inbreds with spontaneous tumors have received 5-methyl cytidine (total 500 mice). Graded single doses of (1) 3.86, (2) 2.57, (3) 2.06, (4) 1.54, (5) 1.03, (6) 1.54, (7) 1.03, (8) 0.51, (9) 0.26, and (10) 0.13 mg/injection (three times weekly) were used. In series 1-5 the 5-methyl cytidine was dissolved in distilled water, and in series 6-10, the nucleoside was dissolved in physiological salt solution. In doses administered, series 4-5 overlapped with series 6-7. The number of mice that showed a regression of tumor increased in the successive series (48%-63% in series 1-5 and 25%-73% in series 6-10). As the number of mice with regressing tumors increased, the percentage of mice showing multiple primary tumors also increased. The administration of the nucleoside in physiological salt solution altered the inhibitory effect of 5-methyl cytidine on spontaneous tumors. For example, in physiological salt solution the 0.51 mg/injection gave as much inhibitory action as 1.03 mg/injection in distilled water. An inverse dose response is thus indicated between the amount of 5-methyl cytidine injected and the inhibition of spontaneous tumors in mice (percentage of regression and number of multiple primary spontaneous tumors are the criteria of inhibition considered).

Animals↗
PubMed1976

Inhibition of uridine-cytidine kinase by 5-azacytidine 5'-triphosphate.

5-Azacytidine 5'-triphosphate (5-aza-CTP) inhibited the phosphorylation of uridine, cytidine, and 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. The inhibition appeared to be competitive with respect to the adenosine 5'-triphosphate and noncompetitive with respect to nucleoside substrates. 5-aza-CTP was a potent inhibitor of 5-aza-C phosphorylation but a weak inhibitor of uridine and cytidine phosphorylation. These results suggest that the feedback inhibition of uridine-cytidine kinase by 5-aza-CTP may limit the amount of intracellular nucleotide analog formed in drug-treated cells.

Azacitidine↗
PubMed1987

Effect of cytidine on the modification of phospholipid metabolism induced by ischemia.

[1-14C-]Arachidonic acid was injected into the lateral ventricle of the gerbils (meriones unguiculatus) two hours before producing brain ischemia by the bilateral ligation of the carotid arteries. Ten minutes before the carotid ligation a group of animals received an additional intraventricular injection of cold cytidine (2.5 mumol/brain). Control animals with and without cytidine, together with the ischemic group, were decapitated directly into liquid nitrogen ten minutes after carotid ligation or sham surgery. Cytidine is able to both stimulate arachidonic acid incorporation into lipids and noticeably correct the release of this acid from polar lipids induced by ischemia. Based on these findings, it is possible to assume that cytidine exerts an effect on the biosynthesis of phosphoglycerides as well as on their catabolic activities.

Animals↗
PubMedDate not supplied

Models for enzyme-copper-nucleic acid interaction. 1H NMR line broadening study of the interaction of some copper complexes with cytidine.

Sodium (salicylideneglycylglycinato) copper(II), bis(2,9-dimethyl-1, 10-phenanthroline)copper (II), and (1,5-bis (benzimidazol-2-yl)-3-thiapentane) copper(II) were interacted with cytidine in D2O. For all the complexes, the broadening of the 1H signals of cytidine is found to be in the order H(5) greater than H(6) greater than H(1'); this suggests the binding of N (3) and O2 sites of cytidine to copper. The possibility that the ribose moiety is also involved in coordination cannot be ruled out. The complete broadening of the H(5) signals for the last complex above is because of the coordinative unsaturation of the complex. It has been concluded that steric factors and coordinative unsaturation in the complexes affect the extent of interaction with cytidine.

Binding Sites↗
PubMed1999

Cytidine deaminase - the methodological relevance of AraC deamination for ex vivo experiments using cultured cell lines, fresh leukemic blasts, and normal bone marrow cells.

The clinical effects of cytosine arabinoside (AraC) are highly dependent on schedule and dose. Many regimens administered to patients are derived from artificial model systems involving permanent leukemic cell lines. The differences in pharmacokinetics between the in vivo situation and such cell lines are largely neglected. However, cytidine deaminase activity in particular has a major impact on AraC pharmacokinetics by degrading AraC to its inactive metabolite AraU, and it has been shown to be of prognostic relevance in the treatment of acute myeloid leukemia. This study therefore investigated cytidine deaminase activities and AraC deamination in a variety of the most commonly used leukemic cell lines and fresh blasts and their impact on the results of an in vitro model system. It was found that cells from different cell lines (BLIN, CEM, HL60, K562, RAJI, REH, U937) vary greatly in cytidine deaminase activity (e.g., 1.89 nmol per min/mg in K562 versus 0.01 in BLIN cells) and degrade between 18.5 (BLIN) and 96.5% (REH) of AraC to AraU in the incubation medium. This degradation results in highly different AraC exposures for different cells (e.g., AUC of 960 ng per h/ml in REH versus 4048 ng per h/ml in BLIN cells) in spite of identical starting concentrations of the drug. Formation of AraCTP as the main cytotoxic metabolite of AraC is significantly influenced by the differences in cell type-dependent cytidine deaminase activity (e.g., 35.6 ng/10(7) cells in REH versus 180.2 ng/10(7) cells in BLIN cells). In contrast to permanent cell lines, fresh leukemic blasts and normal bone marrow mononuclear cells featured low AraC degradation in the model system.

Acute Disease↗
PubMed2001

Comparative study of a novel nucleoside analogue (Troxatyl, troxacitabine, BCH-4556) and AraC against leukemic human tumor xenografts expressing high or low cytidine deaminase activity.

PURPOSE: Troxacitabine (beta-L-dioxolane cytidine, BCH-4556; Troxatyl, BioChem Pharma Inc.) is a novel nucleoside analogue, which in experiments demonstrated potent antitumor activity against both leukemias and solid tumors. Since troxacitabine is a cytidine nucleoside analogue like AraC (1-beta-D-arabinofuranosylcytosine), which is currently used in the treatment of acute myelogenous leukemia, we compared the in vivo antileukemic activity of troxacitabine with that of AraC in human leukemia xenograft models. METHODS: The antiproliferative activity of troxacitabine and AraC was analyzed on hemapoietic cell lines by use of a thymidine incorporation assay. For in vivo studies, we compared troxacitabine with AraC by using equitotoxic schedules of the two nucleosides optimized for therapeutic activity. The antileukemic activity of both drugs was evaluated by measurement of their effect on the percent increased lifespan. RESULTS: AraC had good in vitro antiproliferative activity (IC50 = 14 nM) but was ineffective in vivo against the HL60 promyelocyte leukemia cell line (treated vs control, T/C = 105%). Troxacitabine, which in contrast to AraC is not a substrate for cytidine deaminase, showed potent in vitro and in vivo activity in the same model (IC50 = 53 nM and T/C = 272% to 422%). The poor in vivo activity of AraC against HL60 leukemia cells could be due to the high cytidine deaminase (CDA; EC 3.5.4.5) activity in this cell line. This hypothesis was tested with CCRF-CEM T-lymphoblastoid leukemia cells which have undetectable levels of CDA activity. Short-term exposure of these leukemia cell lines to both drugs indicated that AraC was indeed significantly more effective in the CCRF-CEM cell line than in HL60. In contrast, the antiproliferative activity of troxacitabine was similar for both cell lines. These observations were extended to in vivo studies. Mice bearing CCRF-CEM tumor xenografts were treated with AraC and troxacitabine. In this model, T/C values were comparable for both drugs and ranged from 138% to 157%. CONCLUSIONS: Our findings indicate that troxacitabine is likely to be effective not only against solid tumors with high CDA activity but also in leukemias which have developed resistance to AraC due to increased CDA levels; this suggests that troxacitabine is a promising agent for the treatment of cancer. Indeed, significant antileukemic activity has been observed with troxacitabine in a phase I clinical trial in patients with primary refractory or relapsed acute myeloid leukemias (AML).

Animals↗
PubMed1991

Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs.

Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.

Amino Acids↗
PubMed1992

Pyrimidine nucleotide synthesis is preferentially supplied by exogenous cytidine in adult rat cultured cardiomyocytes.

The metabolism of pyrimidine nucleotides was studied in non-contracting myocytes isolated from adult rat hearts and compared to that observed in freshly prepared myocardium. The myocytes were cultured for up to 96 hrs in a commercial medium containing 50 microM cytidine, uridine, adenosine and adenine; 20 microM guanosine, thymidine and D-ribose; and 5 microM hypoxanthine, xanthine, guanine, thymine and uracil. Nucleotide pool sizes were measured by HPLC. Nucleotide and RNA labelling were followed by incorporation of [U-14C]-cytidine or [U-14C]-uridine added in trace amounts to the medium. The adenine nucleotide pool was 2.4-fold larger than in situ after 7 hrs of incubation and then returned to values 30% higher than that found in the myocardium after 25 hrs. Cytosine and uracil nucleotide pools after 25 hrs of culture were respectively 2 and 4-fold larger than in situ and remained at these levels thereafter. Intracellular cytidylate and uridylate equilibrated very rapidly with exogenous [U-14C]-cytidine but not with [U-14C]-uridine. We conclude that, under the experimental conditions used here, the synthesis of pyrimidine nucleotides in isolated myocytes is mainly supplied by exogenous nucleosides. Furthermore, extracellular cytidine is rapidly converted to both uracil and cytosine nucleotides while uridine serves only as the precursor for uracil nucleotide synthesis.

Adenine Nucleotides↗
PubMed1985

Evidence for a different metabolic behaviour of cytidine diphosphate choline after oral and intravenous administration to rats.

Radioactivity plasma decay was studied in rats after intravenous and oral administration of cytidine diphosphate [methyl-14C]choline at doses of 25 and 300 mg/kg. The kinetics fitted well with a two compartment open model and showed a long lasting elimination phase with a half-life ranging from 2.0 to 2.6 days for the two doses and the two administration routes. Absorption of cytidine diphosphate choline radioactivity was complete after oral treatment with the low dose and accounted for 94.5% of the dose when 300 mg/kg of cytidine diphosphate [methyl-14C]choline were administered. However the distribution of radioactivity in tissues, urine and expired air suggest metabolic differences, at least from a quantitative point of view, between the oral and intravenous treatments. In particular, the higher excretion of radioactivity associated with trimethylamine in urine found when cytidine diphosphate [methyl-14C]choline was given orally, suggest that the compound may be metabolized, at least in part, previous to its gastrointestinal absorption.

Administration, Oral↗
PubMed1979

Alterations in metabolism of cytidine components in rat liver after oral administration of butylated hydroxytoluene (in vivo study).

The administration of the antioxidant, butylated hydroxytoluene (BHT) to rats decreased the utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides in the acid-soluble extract and RNA of the liver. The specific activity of the uridine components was slightly decreased. The depression of the specific activity of the cytidine components depended on the dose of the drug. Simultaneously preformed [U-14C]cytidine in experimental rats was to a higher degree transported to the liver and incorporated into RNA cytosine; its deamination was markedly suppressed. Both phenomena depend on the BHT dose. The concentration of both the uridine and the cytidine components of the acid-soluble extract remained unaffected by the administration of BHT. The utilization of [2-14C]orotic acid for the synthesis of DNA cytosine was depressed after the administration of BHT; by contrast, the specific activity of DNA thymine was higher. The incorporation of [1-14C]palmitic acid into microsomal phospholipids was not substantially influenced over the dose range 25--500 mg BHT/kg. The specific activity of neutral lipids in microsomes increased.

Animals↗
PubMed1985

Utilization of labelled uridine, cytidine and orotic acid for determination of ribonucleic acid synthesis in mouse liver.

[3H]uridine and [3H]orotic acid were equally utilized for labelling of RNA in mouse liver. Incorporation of [3H]cytidine was 2-3 times as high as that of [3H]-labelled uridine or orotic acid. These results differ from findings in rat liver, where both cytidine and orotic acid are better utilized for RNA labelling than is uridine. The ratio between liver RNA [3H]-activity and volatile [3H]-activity was 2, 3 and 13, respectively, at 300 min after injection of labelled uridine, orotic acid and cytidine, indicating an efficient chanelling of cytidine into liver anabolic pathways.

Animals↗
PubMed1997

A cytidine deaminase expressed in the post-infective L3 stage of the filarial nematode, Brugia pahangi, has a novel RNA-binding activity.

A number of genes have been identified that are highly expressed in the post-infective L3 stage of the filarial parasite, Brugia pahangi. Amongst these was a cDNA with homology to the cytidine deaminase (CDD) gene family. Phylogenetic analysis of the various cytosine nucleoside deaminases suggest that Brugia pahangi CDD evolved with significant divergence from the RNA editing family. In order to characterize its function, we have expressed Brugia pahangi CDD in bacteria as a chimera with maltose-binding protein (MBP). Biochemical analysis demonstrates the MBP-CDD fusion protein functions as an authentic cytidine deaminase with an obligate requirement for zinc. In addition to cytidine deaminase activity, however, the fusion protein demonstrates RNA binding activity with specificity for AU-rich sequences and was found to bind an RNA template spanning the edited site of mammalian apolipoprotein B (apoB) mRNA. This RNA binding activity was not found in two different recombinant bacterial CDD proteins. In vitro RNA editing assays revealed that MBP-CDD failed to mediate cytidine deamination of a mammalian apoB RNA template. Furthermore, binding of MBP-CDD to the apoB RNA did not inhibit in vitro editing of this template by apobec-1. The data suggest that the cytosine nucleoside deaminases and RNA editing deaminases have acquired different mechanisms of binding to an AU-rich RNA template, presumably with different functional implications.

Amino Acid Sequence↗