-
Magyar Onkologia 2004Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the...
Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the major source of dTTP comes also from dC via dCMP deaminase. Moreover, based on its broad substrate specificity, this enzyme is responsible for the activation of several nucleoside analogues of therapeutical importance, influencing the sensitivity of malignant tissues towards chemotherapy. The expression of dCK is highest in different lymphoid cells/tissues, in embryonic cells and in most malignant cells (2, 7, 13-15, 18). The activity of dCK is not cell cycle-regulated. In contrast to this, dCK activity was found to be elevated several fold upon short-term treatments of normal human lymphocytes with therapeutic nucleoside analogs, and other genotoxic agents as well as by DNA damaging agents including the DNA polymerase inhibitor aphidicolin, the topoisomerase II inhibitor etoposide and gamma-irradiation, which might be a potentially important phenomenon with respect to the clinical practice, too. These findings indicated that the main trigger of activation could be the damaged DNA itself, and the biological relevance might be to supply the dNTPs for the enhanced DNA repair. Activation of dCK was paralleled by elevated levels of intracellular dATP, raising the possibility that dCK activation is linked to the induction of apoptosis. With regard to the mechanism of enzyme activation, no changes were found in the protein and mRNA levels of dCK upon stimulation, while the activation process was calcium dependent and comprised a protein phosphorylation step. A positive correlation was found between the enzymatic activity and the native immunoreactivity of dCK, strongly arguing that dCK undergoes a conformational change during activation, which results in the formation of a catalytically more active steric structure (8-11, 22, 26, 32-34, 35, 36).
Topics: Antineoplastic Agents; Aphidicolin; Cell Proliferation; Deoxycytidine Kinase; Etoposide; Gamma Rays; Humans; Lymphocytes; Nucleosides; Nucleotides; Phosphorylation
PubMed: 15520873
DOI: No ID Found -
The Journal of Biological Chemistry Aug 2004Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue...
Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.
Topics: Amino Acid Sequence; Bacillus subtilis; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cytidine Deaminase; Dimerization; Guanine Deaminase; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Conformation; Protein Folding; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 15180998
DOI: 10.1074/jbc.M405304200 -
Journal of Clinical Laboratory Analysis 2003The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on... (Clinical Trial)
Clinical Trial Comparative Study
Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: comparison of real-time reverse transcription-PCR vs. relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products.
The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.
Topics: Algorithms; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Breast; Breast Neoplasms; Cisplatin; DCMP Deaminase; DNA, Complementary; DNA-Binding Proteins; Deoxycytidine; Deoxycytidine Kinase; Endonucleases; Female; Gene Expression Regulation, Neoplastic; Humans; Proteins; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoside Diphosphate Reductase; Sensitivity and Specificity; Sequence Analysis, DNA; Tumor Suppressor Proteins; Gemcitabine
PubMed: 12938148
DOI: 10.1002/jcla.10091 -
The American Journal of Pathology Jul 2003Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage...
Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage diagnosis, lack of serum markers, and limited information regarding biliary tumor pathogenesis. RNA-based global analyses of gene expression have led to the identification of a large number of up-regulated genes in several cancer types. We have used the recently developed Affymetrix U133A gene expression microarrays containing nearly 22,000 unique transcripts to obtain global gene expression profiles from normal biliary epithelial scrapings (n = 5), surgically resected biliary carcinomas (n = 11), and biliary cancer cell lines (n = 9). Microarray hybridization data were normalized using dCHIP (http://www.dCHIP.org) to identify differentially up-regulated genes in primary biliary cancers and biliary cancer cell lines and their expression profiles was compared to that of normal epithelial scrapings using the dCHIP software as well as Significance Analysis of Microarrays or SAM (http://www-stat.stanford.edu/ approximately tibs/SAM/). Comparison of the dCHIP and SAM datasets revealed an overlapping list of 282 genes expressed at greater than threefold levels in the cancers compared to normal epithelium (t-test P <0.1 in dCHIP, and median false discovery rate <10 in SAM). Several pathways integral to tumorigenesis were up-regulated in the biliary cancers, including proliferation and cell cycle antigens (eg, cyclins D2 and E2, cdc2/p34, and geminin), transcription factors (eg, homeobox B7 and islet-1), growth factors and growth factor receptors (eg, hepatocyte growth factor, amphiregulin, and insulin-like growth factor 1 receptor), and enzymes modulating sensitivity to chemotherapeutic agents (eg, cystathionine beta synthase, dCMP deaminase, and CTP synthase). In addition, we identified several "pathway" genes that are rapidly emerging as novel therapeutic targets in cancer (eg, cytosolic phospholipase A2, an upstream target of the cyclooxygenase pathway, and ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E, two important downstream mediators of the mitogenic Akt/mTOR signaling pathway). Overexpression of selected up-regulated genes was confirmed in tissue microarrays of biliary cancers by immunohistochemical analysis (n = 4) or in situ hybridization (n = 1), and in biliary cancer cell lines by reverse transcriptase PCR (n = 2). The majority of genes identified in the present study has not been previously reported in biliary cancers, and represent novel potential screening and therapeutic targets of this cancer type.
Topics: Biliary Tract Neoplasms; Carcinoma; Gene Expression Profiling; Humans; Immunohistochemistry; In Situ Hybridization; Oligonucleotide Array Sequence Analysis; Phylogeny; Tumor Cells, Cultured; Up-Regulation
PubMed: 12819026
DOI: 10.1016/S0002-9440(10)63645-0 -
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 -
Blood Feb 2003Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to...
Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P =.007) and accumulated 2.3-fold more Ara-CTP (P =.011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P =.026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P =.001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r(s) = -0.58, P =.006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n = 14) and germ line MLL cases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P =.001) and a 2.7-fold higher hENT1 (P =.046) mRNA expression in patients with MLL gene-rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL gene-rearranged infant ALL.
Topics: Arabinofuranosylcytosine Triphosphate; Cell Survival; Child; Child, Preschool; Cytarabine; DCMP Deaminase; DNA-Binding Proteins; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Female; Gene Expression; Gene Rearrangement; Histone-Lysine N-Methyltransferase; Humans; Infant; Male; Myeloid-Lymphoid Leukemia Protein; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogenes; Pyrimidine Nucleotides; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured
PubMed: 12406912
DOI: 10.1182/blood-2002-05-1600 -
FEBS Letters May 2002Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation...
Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation product 5-mdCMP into dTMP which is further cleaved into thymine and the abasic sugar-phosphate. Both 5-methylcytidine 5' and 3' monophosphates are good substrates for the deaminase. 5-methylcytidine is not a good deaminase substrate and 5-methylcytosine (5mC) is not a substrate. A purified fraction of the deaminase free of nucleases deaminates 5mC present in intact methylated double-stranded DNA. 5-mdCMP deaminase co-purifies on SDS-PAGE with dCMP deaminase and has an apparent molecular weight of 25 kDa. The enzyme requires no divalent cations and has a Km of 1.4 x 10(-7) M for 5-mdCMP and a Vmax of 7 x 10(-11) mol/h/microg protein. The possible biological implications of the deaminase's activities in the present system are discussed.
Topics: Aminohydrolases; Cytidine; DNA; Deoxycytidine Monophosphate; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Male; Molecular Weight; Oligonucleotides; Spermatozoa; Substrate Specificity; Thymine; Uracil
PubMed: 12023031
DOI: 10.1016/s0014-5793(02)02737-0 -
Antimicrobial Agents and Chemotherapy Jun 2002beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro...
Pharmacology of beta-L-thymidine and beta-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus.
beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.
Topics: Antiviral Agents; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Deoxycytidine; Half-Life; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Liver Neoplasms; Phosphorylation; Thymidine; Tumor Cells, Cultured
PubMed: 12019082
DOI: 10.1128/AAC.46.6.1728-1733.2002 -
The Journal of Biological Chemistry Apr 2000A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G. F., Duceman, B....
A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G. F., Duceman, B. W., Wang, A. M., Martinez, J. M., and Maley, F. (1990) J. Biol. Chem. 265, 47-51) to be similar in size, properties, and amino acid composition to the T2-phage-induced deaminase. Neither enzyme is active in the absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP. However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to either a glutamate (R115E) or a glutamine (R115Q), the resulting mutant enzymes were active in the absence of dCTP, with each mutant possessing a turnover number or k(cat) that is about 15% that of the wild-type deaminase. When compared on the basis of specific activity, however, the mutants are about 40-50% of the wild-type (WT)-enzyme's specific activity. Molecular weight analysis on the wild-type and mutant deaminases using HPLC size exclusion chromatography revealed that the wild-type deaminase was basically a hexamer, particularly in the presence of dCTP, regardless of the extent of dilution. Under similar conditions, R115E remained a dimer, whereas R115Q and F112A varied from hexamers to dimers particularly at concentrations normally present in the assay solution. Activity measurements appear to support the conclusion that the hexameric form of the enzyme is activated by dCTP, while the dimer is not. Another feature emphasizing the difference between the WT and mutant deaminases was observed on their denaturation-renaturation in EDTA, which revealed the mutants to be restored to 50% of their original activities with the WT deaminase only marginally restored.
Topics: Arginine; Bacteriophage T4; Binding Sites; Chromatography, Gel; DCMP Deaminase; Deoxycytosine Nucleotides; Edetic Acid; Glutamic Acid; Glutamine; Humans; Kinetics; Mutagenesis, Site-Directed; Mutation; Protein Denaturation; Protein Renaturation; Protein Structure, Secondary; Time Factors; Zinc
PubMed: 10777550
DOI: 10.1074/jbc.275.17.12598 -
The Journal of Biological Chemistry Jun 2000Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to...
Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.
Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Bacteriophage T4; Binding, Competitive; Cytidine Triphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; Guanosine Triphosphate; Kinetics; Nucleotides; Ribonucleotide Reductases; Substrate Specificity; Time Factors; Uridine Triphosphate
PubMed: 10748029
DOI: 10.1074/jbc.M001490200