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PubMed1998

The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase.

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK- cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK- cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N-, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2'-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.

Acyclovir↗

PubMed1971

Regulation of thymidine metabolism in Escherichia coli K-12: evidence that at least two operons control the degradation of thymidine.

In Escherichia coli K-12, the rise in activity of thymidine phosphorylase, phosphodeoxyribomutase, and deoxyribose-5-phosphate aldolase caused by exogenous thymidine is dependent on the synthesis of new enzyme protein. Phosphodeoxyribomutase is induced by the purine ribonucleosides adenosine and guanosine, whereas the other two enzymes are not. The mutase activity induced by thymidine and by the purine ribonucleosides has been shown to be the same enzyme by four different criteria. This independent induction of phosphodeoxyribomutase suggests that the gene for this enzyme is in an operon different from the one that may contain the genes for thymidine phosphorylase and deoxyribose-5-phosphate aldolase.

Aldehyde-Lyases↗

PubMed1979

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.

Cell Line↗

PubMed1990

Conformational transitions in thymidine bulge-containing deoxytridecanucleotide duplexes. Role of flanking sequence and temperature in modulating the equilibrium between looped out and stacked thymidine bulge states.

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and through-space connectivities in the otherwise self-complementary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 degrees C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 degrees C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.

Base Sequence↗

PubMed1994

Erroneous results of 3H-thymidine incorporation are related to position of thymidine residues in oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) targeted to complementary mRNA sequences have proved to be a powerful approach in assessing the function and the role of unique genes in cell proliferation, differentiation, or transformation. Despite their importance in the development of future therapies, little is known about their fate after uptake by cells. Here, we have examined the contribution of individual nucleotide residues from synthetic nonspecific ODNs on assays commonly used to measure cell proliferation. A dramatic decrease of the 3H-thymidine (3H-T) incorporation was obtained with nonspecific ODNs, while no effect on cell proliferation was observed as assessed by three other techniques. We demonstrate that the presence and position of thymidine in the ODNs directly interfere with the intracellular thymidine pool, leading to faulty data of 3H-T incorporation. As an alternative method, we used 3H-deoxyuridine (3H-dU), which is integrated more efficiently in DNA than thymidine. We observed that 3H-dU incorporation was also decreased. In conclusion, caution should be exercised in the interpretation of the results of 3H-T and 3H-dU incorporation in the presence of oligodeoxynucleotides.

Cell Cycle↗

PubMed2005

Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities.

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.

Electrophoresis, Capillary↗

PubMed1989

Correlations of dihydropyrimidine dehydrogenase, thymidine phosphorylase and thymidine kinase activities in strongly and weakly malignant cultured murine neuroblastoma cells.

The C-1300 neuroblastoma tumor which arises spontaneously in the A/J mouse has been maintained in this mouse strain. Two different cell populations have been recognized in cultured C-1300 mouse neuroblastoma (MNB): (1) round, "neuroblast-like" cells, growing in suspension (poorly attached), that have a highly malignant behavior when injected into the A/J mouse (T1 cells); and (2) flat, "epithelioid" cells that attach well to surfaces and show low malignancy towards the inoculated animals (T2 cells). The specific activities of the pyrimidine metabolizing enzymes thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidine kinase (TK) were examined in both MNB cell lines by a new radiochromatographic method. Enzymatic activities of TP and DPD in the cytosols of T2 (weakly malignant) cells were up to 15 times higher than those of T1 (strongly malignant) cells, whereas the mean TP/DPD activity ratio was 16 in either cell line. TP and DPD activity levels increased with time of growth in culture in T2 cells while no such increase was seen in the T1 cells. Maximal TK activity was similar in both cell lines but dropped more rapidly in the T2 cells as cell densities increased. The enzymatic activity levels of TP and DPD but not of TK correlated inversely with neoplastic expression of MNB cells. The observed patterns of pyrimidine metabolizing enzymes in MNB cells could result in an increased thymidine pool in T1 cells whenever TK activity is suppressed, whereas such conditions would favor the generation of thymine in the T2 cells.

Animals↗

PubMed2006

An evaluation of thymidine phosphorylase as a means of preventing thymidine rescue from the thymidylate synthase inhibitor raltitrexed.

The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.

Animals↗

PubMed1986

Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues.

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk- and LMtk- cells with 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5'-amino-5'-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2'-deoxyuridine (IdUrd) caused a similar increase only in tk- cells (LMtk-, BHKtk-). In vivo and in vitro stabilization studies using a [35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects.

Animals↗

PubMed2006

Intracellular uptake of thymidine and antiherpetic drugs for thymidine kinase-deficient mutants of herpes simplex virus type 1.

The influence of the thymidine (Thd) kinase (TK) of herpes simplex virus type 1 (HSV-1) on the intracellular uptake and anabolism of nucleosides has been investigated. To compare the differences between the TK-positive (TK(+)) and TK-deficient strains, acyclovir (ACV)-resistant strains were cloned from a cell culture and classified into 2 groups, viz. the TK-partial (TK(p)) and TK-negative (TK(-)). The cellular uptake of thymidine was highly dependent on the viral TK (vTK) activity. The TK(+) strain showed the highest level of intracellular thymidine uptake, the TK(p) strain a moderate level, which varied from strain to strain, and the TK(-) and mock strains showed little uptake. The inhibition of viral replication by ACV, ganciclovir (GCV) and penciclovir (PCV) did not decrease the Thd uptake at all. On the contrary, a notable increase found to be induced by ACV. The influence of the vTK on the uptake of GCV or PCV was much greater than that of ACV. The metabolism was generally less dependent on the vTK activity than the influx. The influx and phosphorylation rates of GCV and PCV were dependent on the substrate specificity of the vTK.

Acyclovir↗

PubMed2004

Binding causes the remote [5'-3H]thymidine kinetic isotope effect in human thymidine phosphorylase.

The remote 5'-3H V/K kinetic isotope effect (KIE) observed in human thymidine phosphorylase (6.1%) is significantly larger than can be explained by the reaction chemistry. One hypothesis connects the 5'-3H KIE in purine nucleoside phosphorylase to that enzyme's SN1 transition state. The transition state of thymidine phosphorylase, however, is an SN2 nucleophilic displacement. Here we report equilibrium binding isotope effects sufficiently large to explain the presence of this substantial KIE in thymidine phosphorylase.

Binding, Competitive↗

PubMed1992

L-thymidine is phosphorylated by herpes simplex virus type 1 thymidine kinase and inhibits viral growth.

We have demonstrated that herpes simplex 1 (HSV1) thymidine kinase (TK) shows no stereospecificity for D- and L-beta-nucleosides. In vitro, L enantiomers are not recognized by human TK, but function as specific substrates for the viral enzyme in the order: L-thymidine (L-T) >> 2'-deoxy-L-guanosine (L-dG) > 2'-deoxy-L-uridine (L-dU) > 2'-deoxy-L-cytidine (L-dC) > 2'-deoxy- L-adenosine (L-dA). HSV1 TK phosphorylates both thymidine enantiomers to their corresponding monophosphates with identical efficiency and the Ki of L-T (2 microM) is almost identical to the Km for the natural substrate D-T (2.8 microM). The L enantiomer reduces the incorporation of exogenous [3H]T into cellular DNA in HeLa TK-/HSV1 TK+ but not in wild-type HeLa cells, without affecting RNA, protein synthesis, cell growth, and viability. L-T markedly reduces HSV1 multiplication in HeLa cells. Our observations could lead to the development of a novel class of antiviral drugs characterized by low toxicity.

Cell Division↗

PubMed1973

Herpes simplex virus as a source of thymidine kinase for thymidine kinase-deficient mouse cells: suppression and reactivation of the viral enzyme.

Thymidine kinase (EC 2.7.1.21)-deficient mouse cells were infected with inactivated herpes simplex virus, after which "transformed" cells that produce viral thymidine kinase were isolated. Shortly after transformation, the expression of the viral enzyme could be suppressed and reactivated with high efficiency. On continued multiplication in nonselective medium, the proportion of cells producing the viral enzyme decayed exponentially. This decay seemed to represent a change in the expression of the viral gene for thymidine kinase rather than the loss of the gene from the cells, since the viral enzyme could be apparently reactivated in every cell, albeit at a very low frequency.

Aminopterin↗

PubMed2003

Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression.

Actinomycin D (ActD) is a transcription inhibitor and has been used in the treatment of certain forms of cancer. ActD has been reported to be a potential inhibitor of human immunodeficiency virus type 1 (HIV-1) replication due to its ability to inhibit reverse transcription. In contrast to what was expected, low concentrations of ActD (1 to 10 nM) upregulated HIV-1 replication 8- to 10-fold in MT-2 cells and had no effect on HIV-2 replication or on HIV-1 replication in MT-4, Jurkat, or peripheral blood mononuclear cells. The upregulation of HIV-1 replication was associated with an increase in HIV-1 transcription and a decrease in CD4 and CXCR4 expression. To further evaluate the effects of ActD on emergence of drug resistance in HIV-1 replication, a series of drug resistance assays were performed. Of interest, treatment of MT-2 cells with ActD also led to a high level of resistance to thymidine analogs (>1,000-fold increase in resistance to zidovudine and >250-fold to stavudine) but not to other nucleoside reverse transcriptases (RT), nonnucleoside RT, or protease inhibitors. This resistance appeared to be due to a suppression of host cell thymidine kinase-1 (TK-1) expression. These results indicate that ActD leads to a novel form of thymidine analog resistance by suppressing host cell TK-1 expression. These results suggest that administration of combination drugs to HIV-1-infected patients may induce resistance to antiretroviral compounds via a modification of cellular factors.

Anti-HIV Agents↗

PubMed2003

Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.

UNLABELLED: The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. METHODS: TK(1) activity; S-phase fraction; and uptake of TdR, 3'-deoxy-3'-fluorothymidine (FLT), and 2'-fluoro-5-methyl-1-(beta-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. RESULTS: TK(1) activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK(1) activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK(1) activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK(1) activity. The slope of the relationship between TdR uptake and TK(1) activity was nearly twice that for FLT and more than 40-fold that for FMAU. CONCLUSION: Although not all tumors show a strong TK(1) dependence of proliferation, in all cell lines for which proliferation is highly TK(1) dependent, phosphorylation of TdR or FLT accurately reflects changes in TK(1) enzyme activity.

Animals↗

PubMedDate not supplied

[Thymidine phosphorylation with the participation of rat thymic and hepatic thymidine kinase in the presence of a factor from Cl. perfringens (welchii)].

Factor CPW, isolated from Cl. perfringens/welchii was shown to activate latent thymidine kinase (factor N) from liver tissue of rats beginning from 42 days age. CPW factor activated 3.5-fold also thymidine kinase from rat strumous gland; the enzyme was observed during involution of the tissue. The data suggest that increase in thymidine kinase activity in proliferating tissues is related to activation of latent enzymes.

Age Factors↗

PubMed1982

Radiation-induced reactions of thymine, thymidine and thymidine-5'-monophosphate in aqueous solutions.

Radiochemical reactivities of thymine (T), thymidine (Td), thymidine-3' and 5'-monophosphates (3'-, 5'-TMP) and thymidylyl (3' leads to 5') thymidine (TpT) varied in the following order: [A] T (G-value for decomposition, 1.81) less than Td (2.14) less than 3'-TMP = TpT (2.51) less than 5'-TMP (2.76) in deaerated aqueous solution; [B] TpT (2.82) less than T (3.17) congruent to Td (3.18) less than 3'-TMP (3.49) less than 5'-TMP (3.65) in N2O-saturated aqueous solution; [C] T (2.70) congruent to TpT (2.73) less than Td (2.81) less than 3'-TMP = 5'-TMP (3.47) in deaerated aqueous solution containing sodium formate.

Aerobiosis↗

PubMed1988

Penetration of substances into tumour tissue. Model studies using saccharides, thymidine and thymidine-5'-triphosphate in cellular spheroids.

In order to achieve a better understanding of factors involved in drug penetration into poorly vascularized tumour tissue, the penetration of some model substances was studied in vitro. Multicellular human tumour spheroids were used as model system. The test substances were [3H]thymidine and [14C]glucose, both of which are capable of passing easily through cell membranes, and [3H]thymidine-5'-triphosphate, [3H]sucrose and [3H]inulin, all of which are unable to pass directly through cell membranes. The penetration of these substances was studied using a dry histological and autoradiographical method preserving the distribution of water-soluble substances. The two thymidine compounds penetrated very efficiently into the spheroids, and their penetration patterns were rather similar. The saccharides differed somewhat in their penetration properties. Glucose had the fastest penetration and inulin the slowest. After 15 min, however, inulin was also found isotropically distributed within the spheroids. Thus, extracellular penetration seemed to be a possible way for a substance to reach the central parts of a spheroid. The differences between the saccharides could be due to some extent to differences in molecular weight and solubility.

Carbohydrate Metabolism↗