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British Journal of Pharmacology Oct 2009The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and... (Review)
Review
The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; DNA Mismatch Repair; Fluorouracil; Humans; Models, Biological; Neoplasms; Signal Transduction
PubMed: 19775280
DOI: 10.1111/j.1476-5381.2009.00423.x -
Bulletin Du Cancer Aug 2002The drugs concerned by this review are cytarabine (ara-C), gemcitabine and fludarabine. Seventy-eighty per cent of a dose of ara-C are excreted under the form of ara-U... (Review)
Review
The drugs concerned by this review are cytarabine (ara-C), gemcitabine and fludarabine. Seventy-eighty per cent of a dose of ara-C are excreted under the form of ara-U (main metabolite). Plasma concentrations of ara-C are not related to drug pharmacodynamics (response to treatment) in contrast to intracellular levels of ara-CTP (active metabolite) which are associated with cytotoxic activity. Gemcitabine is able to autoactivate its own mechanism of action. Gemcitabine is characterized by a short half-life of elimination (15-20 min) and plasma pharmacokinetics of the drug are not linked to pharmacodynamics. Prolonged administration of gemcitabine is pharmacokinetically and pharmacologically justified and should deserve more intense clinical investigations. Total body clearance of F-ara-A (main circulating metabolite of fludarabine) is linked to creatinine clearance and drug-related neutropenia are more frequent in patients with creatinine clearance below 50 mL/min. So far there are no relationships between intracellular levels of F-ara-CTP and response to treatment.
Topics: Antimetabolites; Antimetabolites, Antineoplastic; Biotransformation; Cytarabine; Cytidine Deaminase; DCMP Deaminase; Deoxycytidine; Deoxycytidine Kinase; Female; Half-Life; Humans; Male; Metabolic Clearance Rate; Neoplasm Proteins; Phosphorylation; Prodrugs; Vidarabine; Gemcitabine
PubMed: 12449033
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1982A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at...
A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
Topics: Centrifugation, Density Gradient; Cytosol; DNA Replication; Deoxyribonucleotides; Escherichia coli; Kinetics; Multienzyme Complexes; T-Phages; Virus Replication
PubMed: 6757252
DOI: No ID Found -
Journal of Experimental & Clinical... Jun 2009The aim of this study was to determine a predictive indicator of gemcitabine (GEM) efficacy in unresectable pancreatic cancer using tissue obtained by endoscopic...
BACKGROUND
The aim of this study was to determine a predictive indicator of gemcitabine (GEM) efficacy in unresectable pancreatic cancer using tissue obtained by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA).
METHODS
mRNAs extracted from 35 pancreatic tubular adenocarcinoma tissues obtained by EUS-FNA before GEM-treatment were studied. mRNAs were amplified and applied to a Focused DNA Array, which was restricted to well-known genes, including GEM sensitivity-related genes, deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1 (hENT1), hENT2, dCMP deaminase, cytidine deaminase, 5'-nucleotidase, ribonucleotide reductase 1 (RRM1) and RRM2. mRNA levels were classified into high and low expression based on a cut-off value defined as the average expression of 35 samples. These 35 patients were divided into the following two groups. Patients with partial response and those with stable disease whose tumor markers decreased by 50% or more were classified as the effective group. The rest of patients were classified as the non-effective group. The relationship between GEM efficacy and mRNA expression was then examined by chi-squared test.
RESULTS
Among these GEM sensitivity-related genes, dCK alone showed a significant correlation with GEM efficacy. Eight of 12 patients in the effective group had high dCK expression, whereas 16 of 23 patients in non-effective group had low dCK expressions (P = 0.0398).
CONCLUSION
dCK mRNA expression is a candidate indicator for GEM efficacy in unresectable pancreatic cancer. Quantitative mRNA measurements of dCK using EUS-FNA samples are necessary for definitive conclusions.
Topics: Adenocarcinoma; Aged; Antimetabolites, Antineoplastic; Biopsy, Fine-Needle; Deoxycytidine; Female; Gene Expression; Humans; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Prognosis; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate; Tumor Suppressor Proteins; Ultrasonography; Gemcitabine
PubMed: 19531250
DOI: 10.1186/1756-9966-28-83 -
Applied and Environmental Microbiology May 2015Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were...
Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.
Topics: Amino Acid Sequence; Bacillus; Bacterial Proteins; Biosynthetic Pathways; Crystallography, X-Ray; Cytosine; DCMP Deaminase; Deoxyribonucleotides; Deoxyuracil Nucleotides; Kinetics; Molecular Sequence Data; Nucleotide Deaminases; Substrate Specificity
PubMed: 25746996
DOI: 10.1128/AEM.00268-15 -
Open Biology Jan 20165-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a...
5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.
Topics: Antibodies; Bromodeoxyuridine; Cell Death; Cell Line, Tumor; Cell Nucleus; Cytidine Deaminase; DNA; DNA Replication; Deoxycytidine; Deoxyuridine; Humans; Metabolome; RNA, Small Interfering
PubMed: 26740587
DOI: 10.1098/rsob.150172 -
MSphere Aug 2019Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we...
Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2'-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for thymidylate biosynthesis. This observation contrasts with the existence in of a dimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in , CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5'-triphosphate nucleotidohydrolase does not have a prominent role in dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.
Topics: Cytidine Deaminase; DCMP Deaminase; Gene Knockdown Techniques; Humans; Kinetics; Protozoan Proteins; Pyrimidines; RNA Interference; Recombinant Proteins; Sequence Alignment; Thymidine Monophosphate; Thymine Nucleotides; Trypanosoma brucei brucei
PubMed: 31391279
DOI: 10.1128/mSphere.00374-19 -
The Journal of Biological Chemistry Jan 2015Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here...
Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.
Topics: Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Bacteriophages; Crystallography, X-Ray; DCMP Deaminase; Deoxycytosine Nucleotides; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Gene Expression; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Substrate Specificity; Thymine Nucleotides; Tyrosine; Viral Proteins
PubMed: 25404739
DOI: 10.1074/jbc.M114.617720 -
Molecular Microbiology Jul 2021We show that the ComEB protein is not required for transformation in Bacillus subtilis, despite its expression from within the comE operon under competence control, nor...
We show that the ComEB protein is not required for transformation in Bacillus subtilis, despite its expression from within the comE operon under competence control, nor is it required for the correct polar localization of ComGA. We show further that the synthesis of the putative channel protein ComEC is translationally coupled to the upstream comEB open reading frame, so that the translation of comEB and a suboptimal ribosomal-binding site embedded in its sequence are needed for proper comEC expression. Translational coupling appears to be a common mechanism in three major competence operons for the adjustment of protein amounts independent of transcriptional control, probably ensuring the correct stoichiometries for assembly of the transformation machinery. comEB and comFC, respectively, encode cytidine deaminase and a protein resembling type 1 phosphoribosyl transferases and we speculate that nucleotide scavenging proteins are produced under competence control for efficient reutilization of the products of degradation of the non-transforming strand during DNA uptake.
Topics: Bacillus subtilis; Bacterial Proteins; Cell Membrane; DCMP Deaminase; DNA Transformation Competence; DNA, Bacterial; DNA-Binding Proteins; Membrane Proteins; Multienzyme Complexes; Transformation, Bacterial
PubMed: 33527432
DOI: 10.1111/mmi.14690 -
Genetics Apr 2014RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about...
RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about genome-scale RNA editing in fungi. In this study, we screened for fungal RNA editing sites at the genomic level in Ganoderma lucidum, a valuable medicinal fungus. On the basis of our pipeline that predicted the editing sites from genomic and transcriptomic data, a total of 8906 possible RNA-editing sites were identified within the G. lucidum genome, including the exon and intron sequences and the 5'-/3'-untranslated regions of 2991 genes and the intergenic regions. The major editing types included C-to-U, A-to-G, G-to-A, and U-to-C conversions. Four putative RNA-editing enzymes were identified, including three adenosine deaminases acting on transfer RNA and a deoxycytidylate deaminase. The genes containing RNA-editing sites were functionally classified by the Kyoto Encyclopedia of Genes and Genomes enrichment and gene ontology analysis. The key functional groupings enriched for RNA-editing sites included laccase genes involved in lignin degradation, key enzymes involved in triterpenoid biosynthesis, and transcription factors. A total of 97 putative editing sites were randomly selected and validated by using PCR and Sanger sequencing. We presented an accurate and large-scale identification of RNA-editing events in G. lucidum, providing global and quantitative cataloging of RNA editing in the fungal genome. This study will shed light on the role of transcriptional plasticity in the growth and development of G. lucidum, as well as its adaptation to the environment and the regulation of valuable secondary metabolite pathways.
Topics: Adenosine Deaminase; DCMP Deaminase; Fungal Proteins; Genome, Fungal; High-Throughput Nucleotide Sequencing; Molecular Structure; Phylogeny; RNA Editing; RNA, Fungal; Reishi; Reproducibility of Results
PubMed: 24496007
DOI: 10.1534/genetics.114.161414