Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.
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PMID:Cell cycle-dependent metabolism of pyrimidine deoxynucleoside triphosphates in CEM cells. 919 7

The present study was undertaken to assess the predictive value of pretherapeutic determinants of ara-C metabolism and proliferative activity of leukemic blasts for early response to antileukemic therapy in the setting of granulocyte-macrophage colony-stimulating factor (GM-CSF)-based priming before and during TAD-9 induction in 36 consecutive patients with de novo acute myeloid leukemia (AML). Ara-C metabolism was assessed by the activities of deoxycytidine kinase (DCK), deoxycytidine deaminase (DCD), DNA polymerase alpha (Poly alpha), and overall polymerase (overall Poly). The fraction of cells in S phase (%S phase) and thymidine kinase (TK) activity were determined as a measure of proliferative activity. Early response to therapy was defined by the percentage of leukemic blasts in the bone marrow 5 to 7 days after completion of TAD-9 with less than 5% signaling an adequate response and greater than 5% indicating an inadequate early reduction, respectively. While neither %S phase, DCK, nor overall Poly activity were predictive for early response, TK and Poly alpha activities were significantly higher for cases with adequate blast cell clearance. The respective median values were for TK 3.8 versus 1.85 pmol/min/mg protein (P = .012), and for Poly alpha 1.9 versus 0.69 pmol/min/mg protein (P = .014). An inverse relation was detected for DCD activity which was significantly lower in responding patients with a median of 0.33 nmol/min/mg protein (range, 0.0 to 29.5) as compared to a median of 5.1 nmol/min/mg protein (range, 0.11 to 8.45) in early nonresponders, (P = .009). Taking the respective median values as arbitrary cut-points for high or low enzyme activities, responders and nonresponders could be discriminated prospectively. Hence, 14 of 16 cases (88%) with DCD activities below the median of 1.56 nmol/min/mg protein responded as compared to only 3 of 14 (22%) patients with higher DCD activities (P = .0004). From the 15 patients with TK activity above the overall median of 3.2 pmol/min/mg protein, 11 cases (73%) achieved an adequate blast cell clearance while only 6 of 17 cases (35%) with lower values responded (P = .035). Similarly, 12 of 15 patients (80%) with high Poly alpha levels (>1.22 pmol/min/mg protein) responded to induction therapy as compared to only 5 of 14 patients (36%) with lower enzyme activities (P = .02). By logistic regression analysis of enzyme activities, DCD activity was found to be the most sensitive parameter to predict an adequate blast cell clearance (P = .032). Activities of DCD and TK were not only associated with initial response but were also found predictive for remission duration. Hence, from 11 patients with low TK levels 8 (73%) relapsed within 1 year, whereas only 2 of 11 (18%) patients with high TK activity experienced a recurrence of their disease (P = .015). Six of 9 (66%) patients with higher than median DCD levels relapsed within 1 year, whereas 10 of 14 patients (71%) with lower DCD levels had a longer remission duration (P = .085). Analysis of DCD gene expression at the mRNA level by a semi-quantitative reverse transcriptase-polymerase chain reaction method showed that a high transcription rate of the DCD gene was associated with high enzyme activities and vice versa. Hence, the observed intraindividual differences in DCD activity are a reflection of differences in gene activity and transcription rate rather than of variants in translation. Although further analyses are needed to elucidate the molecular mechanisms that determine the variation of enzyme activities in individual patients, the present study strongly suggests that pretherapeutic determination of TK and Poly alpha as well as of DCD allows to predict response to TAD-9 + GM-CSF induction therapy and may provide the means for the development of a risk adapted treatment strategy.
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PMID:Activity of thymidine kinase and of polymerase alpha as well as activity and gene expression of deoxycytidine deaminase in leukemic blasts are correlated with clinical response in the setting of granulocyte-macrophage colony-stimulating factor-based priming before and during TAD-9 induction therapy in acute myeloid leukemia. 929 31

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.
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PMID:Human deoxycytidine kinase is located in the cell nucleus. 934 41

Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating beta-D-2'-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of d-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for beta-L-2'-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of L-nucleoside analogues as antivirals.
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PMID:Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L-nucleoside analogues. 935 70

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.
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PMID:Life on the salvage path: the deoxynucleoside kinase of Lactobacillus acidophilus R-26. 942 44

In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5'-fluorodeoxyuridine, were phosphorylated. There was negligible 3'-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides.
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PMID:Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. 946 77

2'-Fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) is the first L-nucleoside analog with low cytotoxicity discovered to have potent antiviral activities against both hepatitis B virus and Epstein-Barr virus but not human immunodeficiency virus. This spectrum of activity is different from those of the other L-nucleoside analogs examined. L-FMAU enters cells through equilibrative-sensitive and -insensitive nucleoside transport as well as through nonfacilitated passive diffusion. L-FMAU is phosphorylated stepwise in cells to its mono-, di-, and triphosphate forms. In the present study the enzymes responsible for the first step of L-FMAU phosphorylation were identified. This is the first thymidine analog shown to be a substrate not only for cytosolic thymidine kinase and mitochondrial deoxypyrimidine kinase but also for deoxycytidine kinase. This finding suggests that the antiviral activity of L-FMAU will not be limited by the loss or alteration of any of these deoxynucleoside kinases.
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PMID:Unique metabolism of a novel antiviral L-nucleoside analog, 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil: a substrate for both thymidine kinase and deoxycytidine kinase. 955 92

Recently, it has been shown, that 2-Chloro-deoxyadenosine (1), a series of analogues, and other DNA synthesis inhibitors, increased the deoxycytidine kinase (dCK) enzyme activity in different cells, without influencing thymidine kinase isoenzymes (TK1, TK2), dCMP-deaminase and thymidylate synthase (TS) activities (2,3). The dCK activity was 2-4 times higher in analogue treated cells, than in controls, which can not be explained by metabolic pool imbalance induced by the drugs. New mRNA and protein synthesis of dCK could not be detected, thus post-translational modification has been suggested for potentiation the activity of the dCK (1). Because secondary modifications of enzymes usually involve the signalling processes in cells, the universal G-protein activator fluorine ions were tested. dCK activity of human lymph node lymphocytes were increased 2-times, if cells were incubated in the presence of NaF for 1-2 hrs in cultures, while TK activity was not changed. The formation of dUTP from dCyd, was also enhanced by NaF, in parallel of dCK potentiation.
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PMID:Deoxycytidine kinase can be also potentiated by the G-protein activator NaF in cells. 959 3

The effect of different nucleoside analogues on deoxycytidine kinase (dCK) and thymidine kinase (TK) was compared in normal human lymphocytes and various leukemic cell lines. G-phase enriched tonsilar lymphocyte subpopulation treated by CdA showed more profound stimulation of dCK activity than S-phase cells. No substantial changes in TK activity were detected. CdA treatment increased the activity of dCK 4-fold in peripheral blood mononuclear cells (PBMC) and 2-fold in promyelocytic cell line HL60, too. However, no significant stimulation was detected either in CCRF-CEM or in K562 cell lines. 2-Cl-2'deoxy-2'F-adenine arabinoside (CAFdA), 2F-adenine arabinoside (F-araA) and cytosine arabinoside (AraC) had the same effect as CdA, although higher concentrations were needed for maximal activation. In contrast, treatment by dCyd caused slight inhibition of dCK. The possibility of interference of nucleoside analogues with the mechanisms of posttranslational modification of dCK was proposed.
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PMID:Activation of deoxycytidine kinase by various nucleoside analogues. 959 44

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to approximately 5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.
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PMID:Human thymidine kinase can functionally replace herpes simplex virus type 1 thymidine kinase for viral replication in mouse sensory ganglia and reactivation from latency upon explant. 965 18


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