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)

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.
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PMID:Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues. 135 86

Although several hypomethylating agents such as 5-azadeoxycytidine and 5-fluorodeoxycytidine have been shown to activate transcription after incorporation into viral or cellular DNA, agents which selectively affect the methylation status of virus-infected cells have not been described. Studies on the antiviral effect of the methyldeoxycytidine (mdCyd) analogue trifluoromethyldeoxycytidine (F3mdCyd) showed significant antiviral activity against herpes simplex virus type 1 (HSV-1). This analogue of both dCyd and dThd is selectively incorporated into the DNA of herpesvirus infected cells due to the unique specificity of the herpesvirus thymidine kinase (TK) because the HSV-1 TK is both a dCyd and dThd kinase. In contrast, the deoxycytidine kinase of uninfected cells preferentially phosphorylates dCyd and has a poor affinity for F3mdCyd. F3mdCyd hemisubstituted M13 DNA displayed the same properties as mdCyd-substituted M13 DNA with respect to cleavage by restriction enzymes, and acted as an efficient template for eukaryotic DNA methyltransferase (S-adenosyl-L-methionine DNA (cytosine-5) methyltransferase: EC 2.1.1.37). Using the persistently infected CEM cell model system, the extent of DNA methylation was shown to increase in a dose-related manner when HSV-1-infected CEM cells were treated with increasing concentrations of F3mdCyd. Higher levels of methylation correlated with significant decreases in HSV-1 titers. Isoschizomer analyses followed by Southern blotting and hybridization with genomic HSV-1 DNA showed that DNA from HSV-1-infected, analogue-treated Vero cells was resistant to cleavage by restriction enzymes at a time when productive virus was not present in culture. We infer from these results that the methylation-like properties of the incorporated F3mdCyd occur concomitantly with, and appear to be involved in, the mechanisms of the analogue's antiviral effect towards HSV-1.
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PMID:Methylation of HSV-1 DNA as a mechanism of viral inhibition: studies of an analogue of methyldeoxycytidine: trifluoromethyldeoxycytidine (F3mdCyd). 138 26

Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200-1000 microM) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 microM ara-C following the exposure of cells to ara-U (1 mM) resulted in 4.5 pmol araC DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 microM ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10-100 microM) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.
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PMID:Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-beta-D-arabinofuranosyluracil. 156 88

Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25, 30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dideoxycytidine (ddCyd) with a Vmax approximately 10-fold lower than that of the two latter enzymes. The Km and Vmax values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.
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PMID:Deoxynucleoside phosphorylating enzymes in monkey and human tissues show great similarities, while mouse deoxycytidine kinase has a different substrate specificity. 165 2

Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for allelic exclusion in Chinese hamster ovary cells. 170 54

Deoxynucleoside kinases are required for the 5'-phosphorylation of deoxynucleoside analogs used in chemotherapy. Cytoplasmic thymidine kinase (TK1), deoxycytidine kinase (dCK) and mitochondrial thymidine kinase (TK2) were completely purified from human leukemic spleen and their capacities to phosphorylate 43 nucleoside analogs were compared. TK1 showed the most restricted substrate specificity but tolerated 3'-modifications of the sugar ring and some 5-substitutions of the pyrimidine ring. TK2 showed a much broader specificity and phosphorylated pyrimidine bases with bulky 5-substitutions, including cytosine analogs, while sugar analogs with substituents other than OH in the 2' and 3' positions were very poor substrates. dCK showed a very broad specificity phosphorylating several cytosine analogs with 2' and 3' modifications as well as acyclic sugar analogs. Purine deoxyribonucleosides were also efficiently phosphorylated by dCK but in this case sugar modifications led to drastically decreased activity.
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PMID:Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs. 202 74

Exposure of V79 cells to hyperoxia (80% O2) for 30 h increased the level of thymidine kinase, a deoxynucleoside salvage enzyme, by approximately 3-fold as compared to cells exposed to room air, but did not cause any significant change in deoxycytidine kinase, the other known deoxynucleoside salvage enzyme. Exposure of cells to anoxia, on the other hand, produced only a slight reduction in thymidine kinase activity. Perturbation in cellular metabolism following exposure to hyperoxia was indicated by marked inhibition of cellular growth and the presence of cellular hypertrophy. Although growth was also inhibited by anoxia, the cell size distribution was minimally altered. The effect of hyperoxia on thymidine kinase suggests that (1) this enzyme may play a role in the modulation of cellular hypertrophy and function following exposure to hyperoxia, and (2) analysis of relative levels of thymidine kinase and deoxycytidine kinase activities may be of value in differentiating between cellular hypertrophy and hyperplasia under some circumstances.
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PMID:Elevation of hyperoxia of thymidine kinase activity in hypertrophic V79 lung fibroblasts. 236 88

Bolus doses of 5-chlorodeoxycytidine (CldC) administered with modulators of pyrimidine metabolism, followed by X-irradiation, resulted in a 2-fold dose increase effect against RIF-1 tumors in C3H mice. Pool size studies of the fate of [14C]-CldC in BDF1 mice bearing Sarcoma-180 tumors, which demonstrated the rapid formation of 5-chlorodeoxycytidylate (CldCMP), and incorporation of CldC as such in RIF-1 tumor DNA, indicate that CldC is a substrate for deoxycytidine kinase, as our past Km studies have shown. Our data indicate that 5-chlorodeoxyuridine triphosphate (CldUTP) accumulates from both the cytidine deaminase-thymidine kinase pathway, as well as from the deoxycytidine kinase-dCMP deaminase pathway, in tumor tissue. As shown in a previous study, tetrahydrouridine (H4U), a potent inhibitor of cytidine deaminase, can effectively inhibit the enzyme in the normal tissues of BDF1 mice. When H4U was administered with the modulators N-(phosphonacetyl)-L-aspartic acid (PALA) and 5-fluorodeoxycytidine (FdC), the levels of CldC-derived RNA and DNA directed metabolites increased in tumor and decreased in normal tissues compared to when CldC was administered alone. These modulators inhibit the de novo pathway of thymidine biosynthesis, lowering thymidine triphosphate (TTP) levels, which compete with CldUTP for incorporation into DNA. 5-Benzylacyclouridine (BAU), an inhibitor of uridine phosphorylase, was also utilized. DNA incorporation studies using C3H mice bearing RIF-1 tumors showed that the extent of incorporation of 5-chlorodeoxyuridine (CldU) into DNA correlates with the levels of cytidine and dCMP deaminases; this is encouraging in view of their high activity in many human malignancies and the low activities in normal tissues, including those undergoing active replication. Up to 3.9% replacement of thymidine by CldU took place in RIF-1 tumors, whereas incorporation into bone marrow was below our limit of detection. CldC did not result in photosensitization under conditions in cell culture in which radiosensitization to X rays was obtained. Thus, the combination of CldC with modulators of its metabolism has potential as a modality of selective radiosensitization for ultimate clinical use in a wider range of tumors than those of the brain.
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PMID:Radiation, pool size and incorporation studies in mice with 5-chloro-2'-deoxycytidine. 239 14

Deoxycytidine kinase specific activity was high in human peripheral lymphocytes and increased less than 2-fold when the lymphocytes were stimulated by phytohemagglutinin A. Ion-exchange chromatography showed the same profile of deoxycytidine kinase activity in resting and proliferating cells. This enzyme could also efficiently phosphorylate deoxyadenosine and deoxyguanosine. In contrast, the thymidine kinase activity was very low in resting peripheral lymphocytes and increased more than 40-fold upon stimulation. Similar relative changes in the activities of the two enzymes were observed in human T-lymphoblast cells (CCRF-CEM) separated by centrifugal elutriation into cells of different cell cycle phases. The ratio of deoxycytidine to thymidine kinase activities is 20:1 in extracts from resting human lymphocytes and 1:2 in PHA-stimulated cells. This drastic change in deoxyribonucleoside phosphorylating activities during the cell cycle in human lymphocytes is of importance for studies on unscheduled DNA synthesis, for the design of therapies to interfere with viral DNA metabolism, and for a correct interpretation of the compartmentation effects observed in DNA precursor metabolism.
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PMID:Deoxycytidine kinase is constitutively expressed in human lymphocytes: consequences for compartmentation effects, unscheduled DNA synthesis, and viral replication in resting cells. 2861 64

Rat cytomegalovirus (RCMV) induces a cytosol thymidine kinase (TK) in G0-phase rat embryo fibroblasts (REF), but not in a TK deficient rat cell line (R-2), though virus titers in both cell types reached comparable levels. The results indicate that TK is neither virus-coded nor is required for a productive infection in R-2 cells. A deoxycytidine kinase (dCK) is induced in either growing or RCMV-infected REF and R-2 cells, suggesting that dCK is essential for both host-cell and viral DNA synthesis. A deoxyguanosine kinase (dGK) is detectable in low concentrations in either growing or G0-phase REF and R-2 cells suggesting that this enzyme is cell-cycle independent. In contrast, RCMV induces high persisting levels of dGK, particularly in R-2 cells, indicating that this enzyme is of crucial importance for viral DNA synthesis. By comparison of thermostabilities and electrophoretic mobilities (Rf for TK, dCK and dGK were 0.12; 0.97; and 0.54, respectively) the enzymes were found to be substrate specific but of cellular origin. In contrast to TK and dCK, only dGK is inhibited by Acyclovir (Ki = 320 microM). It is suggested that RCMV inducable dGK is an important enzyme determining the in vitro anti-CMV activity of Acyclovir.
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PMID:Rat cytomegalovirus induces cellular purine and pyrimidine nucleoside kinases in rat embryo fibroblasts and TK- rat-2 cells. Correlations with the antiviral activity of Acyclovir. 298 53


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