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Molecular and Biochemical Parasitology Jan 2013Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and...
Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and therefore strongly depend on dUDP/dUTP hydrolysis for dUMP production. Here we have addressed by gene replacement the consequences of elimination of dUTPase activity in bloodstream forms of Trypanosoma brucei. We first generated conditional DUT-knockout strains that allowed an effective decrease of dUTPase resulting in proliferation arrest, although gene repression could not be sustained long enough to cause lethality. Alternatively, DUT null mutants could be isolated in the presence of high levels of thymidine while exogenous supplementation with uracil, uridine or deoxyuridine could not complement metabolically the dUTPase deficiency. Upon thymidine removal, trypanosomes exhibited impaired proliferation and eventually died. These data establish a strict requirement for dUTPase in T. brucei viability and support a major role of the enzyme in the provision of pyrimidine nucleotides in kinetoplastids.
Topics: Cell Survival; Gene Knockout Techniques; Pyrimidines; Pyrophosphatases; Trypanosoma brucei brucei
PubMed: 23201394
DOI: 10.1016/j.molbiopara.2012.11.003 -
Molecular and Cellular Biology Nov 2012Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside...
Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.
Topics: Cell Cycle; Checkpoint Kinase 1; DCMP Deaminase; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; Deoxycytosine Nucleotides; Genomic Instability; Nucleotidyltransferases; Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Thymine Nucleotides
PubMed: 22927644
DOI: 10.1128/MCB.01062-12 -
Expert Opinion on Investigational Drugs Apr 2012Nucleoside analogs are widely used for treatment of various malignancies. Benchmark drugs are cytarabine for acute myeloid leukemia and gemcitabine for pancreatic and...
Nucleoside analogs are widely used for treatment of various malignancies. Benchmark drugs are cytarabine for acute myeloid leukemia and gemcitabine for pancreatic and lung cancer. Sapacitabine is a novel cytidine analog currently in development. This editorial focuses on the potential of new nucleoside analogs and on novel possibilities of gemcitabine. Gemcitabine is a nucleoside analog with many faces, which shows a remarkable activity in a variety of cancers, most likely because it has a unique metabolism, a so-called self-potentiation. Gemcitabine is taken up by nucleoside transporters, is activated by deoxycytidine kinase and incorporated into both RNA and DNA. Inhibition of ribonucleotide reductase and dCMP deaminase enhances its activation, while cytidine deaminase converts gemcitabine to its presumably inactive metabolite 2',2'-difluorodeoxyuridine, which in its nucleotide form may inhibit thymidylate synthase. Gemcitabine is widely used in combination, predominantly with a platinum analog, with other combinations less frequently used or being explored. Standard administration of gemcitabine is with a 30-min weekly infusion at 1000 mg/m(2), but alternatives are being explored such as prodrugs (e.g., CO-1.01, which can bypass transport deficiency), the fixed-dose rate infusion (10 mg/m(2)/min), and local routes of administration by a 24-h hepatic artery infusion, by instillation in the bladder or by intraperitoneal administration to treat advanced ovarian cancer. Other alternatives for combinations of gemcitabine in ovarian cancer consist of increasing the inhibition of ribonucleotide reductase with triapine or hydroxyurea. Gemcitabine's action on signaling also provides a rational concept for combination with signal transduction pathways.
Topics: Animals; Antimetabolites, Antineoplastic; Arabinonucleosides; Cytosine; Deoxycytidine; Humans; Neoplasms; Nucleosides; Gemcitabine
PubMed: 22404148
DOI: 10.1517/13543784.2012.666236 -
Nucleosides, Nucleotides & Nucleic Acids 2012A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was...
A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.
Topics: Animals; Antimetabolites; Antineoplastic Agents; Arabinonucleotides; Cell Line, Tumor; DCMP Deaminase; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Female; Leukemia P388; Mice; Neoplasm Transplantation; Transplantation, Heterologous
PubMed: 22257207
DOI: 10.1080/15257770.2011.637099 -
Virology May 2011The anticodon nuclease (ACNase) PrrC is silenced in Escherichia coli by an associated DNA restriction-modification protein, activated by the phage T4-encoded anti-DNA...
The anticodon nuclease (ACNase) PrrC is silenced in Escherichia coli by an associated DNA restriction-modification protein, activated by the phage T4-encoded anti-DNA restriction factor Stp and counteracted by T4's tRNA repair enzymes polynucleotide kinase and RNA ligase 1. Hence, only tRNA repair-deficient phages succumb to PrrC's restriction. PrrC's ABC-ATPase motor domains are implicated in driving its activation by hydrolyzing GTP and in stabilizing the activated ACNase by avidly binding dTTP. The latter effect has been associated with dTTP's accumulation early in T4 infection when PrrC is activated. In agreement, delayed dTTP accumulation caused by dCMP deaminase deficiency coincided with impaired manifestation of PrrC's ACNase activity. This impairment did not suffice to suppress the PrrC-mediated restriction of tRNA repair deficient phage but was synthetically suppressive with a leaky stp mutation that only partly impairs PrrC's activation. Presumably, ability to gauge dTTP's changing level helps confine PrrC's toxicity to its viral target.
Topics: Bacteriophage T4; Escherichia coli; Escherichia coli Proteins; Ribonucleases; Thymine Nucleotides
PubMed: 21481433
DOI: 10.1016/j.virol.2011.03.022 -
Cancer Science Mar 2011Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to...
Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.
Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Membrane; Deoxycytidine; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Gemcitabine
PubMed: 21205085
DOI: 10.1111/j.1349-7006.2010.01837.x -
Fundamental & Clinical Pharmacology Apr 2011Deoxycytidine analogs (dCa's) are nucleosides widely used in anticancer and anti (retro) viral therapies. Intracellularly phosphorylated dCa anabolites are considered to... (Review)
Review
Deoxycytidine analogs (dCa's) are nucleosides widely used in anticancer and anti (retro) viral therapies. Intracellularly phosphorylated dCa anabolites are considered to be their main active metabolites. This article reviews the literature on the formation and pharmacological activity of deaminated dCa nucleotides. Most dCa's are rapidly deaminated into deoxyuridine analogs (dUa's) which are only slowly phosphorylated and therefore relatively inactive. dUa nucleotides are, however, also formed via deamination of dCa monophosphates by deoxycytidine monophosphate deaminase (dCMPD). dUa-monophosphates can interact with thymidylate synthase (TS), whereas dUa-triphosphates are incorporated into nucleic acids and interfere with polymerases. Administration of dCa's as monophosphate prodrugs or co-administration of the cytidine deaminase inhibitor tetrahydrouridine (THU) does not prevent dUa nucleotide formation which is, on the other hand, influenced by the dose and dCMPD activity. Taken together, these observations show that the formation of dUa nucleotides is a common phenomenon in treatment with dCa's and these compounds may play a role in treatment outcome. We conclude that more attention should be given to these relatively unknown, but potentially important metabolites.
Topics: Animals; Antineoplastic Agents; Antiviral Agents; DCMP Deaminase; Deamination; Deoxycytidine; Deoxyuracil Nucleotides; Dose-Response Relationship, Drug; Humans; Phosphorylation
PubMed: 20199587
DOI: 10.1111/j.1472-8206.2010.00823.x -
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 -
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 -
Journal of Medicinal Chemistry Dec 2008We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is...
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
Topics: Adenocarcinoma; Amides; Cytidine; DCMP Deaminase; DNA Methylation; Dose-Response Relationship, Drug; Gene Silencing; Genes, p16; Humans; Molecular Structure; Pancreatic Neoplasms; Phosphoric Acids; Reverse Transcriptase Polymerase Chain Reaction; Stereoisomerism; Thymidylate Synthase; Tumor Cells, Cultured
PubMed: 19006382
DOI: 10.1021/jm8005965