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PubMed1989

Nucleotide and predicted amino acid sequences of the Marek's disease virus and turkey herpesvirus thymidine kinase genes; comparison with thymidine kinase genes of other herpesviruses.

In this paper we present the nucleotide sequences of the thymidine kinase (TK) genes of two avian herpesviruses: a highly oncogenic strain of Marek's disease virus (MDV strain RB1B) and its serologically related vaccine virus, the herpesvirus of turkeys (HVT strain Fc-126). The predicted coding regions of the two genes are 1029 and 1050 nucleotides respectively, corresponding to polypeptides of 343 and 350 amino acids in length. Putative nucleotide- and nucleoside-binding sites have been identified within the two predicted amino acid sequences. The MDV and HVT TK amino acid sequences exhibit 58.2% amino acid identity. Comparison with other available herpesvirus TK sequences reveals a greater homology to those of the alphaherpesviruses than to those of the gammaherpesviruses. No overall homology was found when compared with the chicken cytoplasmic TK sequence.

Amino Acid Sequence↗
PubMed1989

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously.

We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.

Animals↗
PubMed2000

Streptococcus gordonii strains resistant to fluorodeoxyuridine contain mutations in the thymidine kinase gene and are deficient in thymidine kinase activity.

Mutants of Streptococcus gordonii resistant to 5-fluorodeoxyuridine (FUdR(r)) were isolated. Each strain contained a point mutation resulting in the premature termination of the thymidine kinase (TK) open reading frame (tdk). In vitro translation of the mutant tdk coding regions resulted in synthesis of truncated TK polypeptides deficient in TK activity.

Animals↗
PubMed1983

Hepatic infection by thymidine kinase-positive and thymidine kinase-negative herpes simplex virus after partial hepatectomy.

Herpes simplex virus (HSV) infection of mouse liver after partial hepatectomy was studied. Partial hepatectomy resulted in the rapid onset of cellular DNA synthesis and the appearance of many mitotic figures (peak, 3 days after surgery). Similar changes were not seen in control animals. After partial hepatectomy, the mice were infected with thymidine kinase-positive (TK+) and -negative (TK-) HSV to investigate virus titers in liver tissue during liver cell replication. In control unoperated mice, liver titers of TK+ HSV (2 X 10(3) PFU/g) were greater than those of mice inoculated with TK HSV (4 X 10(1) to 5 X 10(2) PFU/g). After partial hepatectomy, TK+ and TK- HSV titers increased, and peak TK+ and TK- HSV titers were similar (6 X 10(5) to 8 X 10(5) PFU/g). Hepatic infection was further investigated by infectious center (IC) assays. The numbers of ICs for TK+ HSV increased 50-fold after partial hepatectomy, whereas the increase was less for TK- HSV. From the results of these studies, we hypothesize that the increase in hepatic TK+ HSV after hepatectomy may have been largely due to the increase in ICs, whereas the increase in hepatic TK- HSV was due, in part, to the increase in ICs, but may also have been due to the enhanced synthesis of TK- HSV in replicating liver cells.

Animals↗
PubMed1986

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.

Adenoviruses, Human↗
PubMed1988

Molecular analysis of the thymidine kinase gene of thymidine kinase-deficient mutants of varicella-zoster virus.

Five thymidine kinase (TK)-deficient mutants of varicella-zoster virus derived from the Oka vaccine strain were examined to determine the state of transcription of the TK gene, and the DNA sequences of two of the mutants and the parent strain were investigated. Restriction endonuclease analysis revealed no major alteration of whole genome organization, but did reveal a minor change of the cleaved fragment in size. Northern blot analysis showed that the transcripts from the TK gene of all five mutants were of the same size as that of the parent strain (1.6 kb). DNA sequences of the TK gene of the parent strain and those of two mutants were determined. One had a base substitution (C to T) in the coding region of TK, resulting in the termination of amino acid elongation in the 68th amino acid of 341. The other had a two-base deletion (AT) in the coding region, resulting in a frame shift from the 126th amino acid and termination at the 162nd amino acid. Both mutants had premature stop codons which might result in the synthesis of nonfunctional TK polypeptides. Thus, the molecular basis of TK deficiency was determined.

Amino Acid Sequence↗
PubMed2000

Reactivation of acyclovir-resistant thymidine kinase-deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene.

HSV infections are treated efficiently and prevented by acyclovir, although resistant strains have been reported. Resistance to acyclovir involves mainly mutations in the viral gene encoding thymidine kinase; mutations may lead to an altered or, more frequently, deficient TK. These acyclovir-resistant TK deficient strains are not able to reactivate from a latent infection in an experimental model, compared to TK positive strains. A case is reported of a bone marrow transplant child who developed HSV infection at 11 days post-transplantation. Acyclovir-resistant HSV 1 was isolated on day 19 post-transplantation. The patient was cured of his infection. A resistant virus was detected 20 months later that harboured the same TK gene mutation as the first resistant virus. This mutation is an insertion of one guanine in a homopolymer repeat of seven guanines located at codon 146 of TK. It has previously been reported and associated with the expression of a deficient TK activity and the ability to reactivate in mice. These results corroborate the clinical relevance of this mutation, which is associated with acyclovir-resistant recurrent infections in humans.

Acyclovir↗
PubMed1979

Thymidine and thymine in biologic fluids during high-dose infusions of thymidine in mice, monkeys, and man.

Measurements were made to determine if millimolar thymidine (TdR) concentrations, shown to have antitumor activity in vitro, could be attained in vivo. An adaptation of an ion-exclusion liquid chromatography method was used to quantitate TdR and thymine in various biologic fluids. Millimolar concentrations of TdR were attained in plasma of mice, monkeys, and man by constant infusions of large quantities of TdR (approximately 80 g/m2/day). Cerebrospinal fluid concentrations of TdR at equilibrium were one tenth of the plasma concentration in the monkey. The primary route of removal of TdR at these high plasma concentrations is kidney clearance of intact TdR. Metabolism, which plays a major role in the clearance of TdR at micromolar concentrations in plasma, appears to be saturated at millimolar concentrations of TdR.

Animals↗
PubMed2003

Thymidine phosphorylase-mediated angiogenesis regulated by thymidine phosphorylase inhibitor in human ovarian cancer cells in vivo.

Metastasis or progression of ovarian cancer cells is known to be due to the action of various angiogenic factors. We determined the expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) and vascular endothelial growth factor (VEGF) in cell lines established from 3 serous adenocarcinomas, 3 clear cell carcinomas and 2 mucinous carcinomas of the human ovary. TP activity and the TP mRNA level were much higher in the serous adenocarcinoma cells than in the clear cells and mucinous carcinoma cells, and TP expression was extremely low in the clear cell carcinoma cells. Expression of VEGF mRNA was variable, but not significantly different between the 3 histological types of ovarian cancer. In vivo angiogenesis in the ovarian cancer cells was evaluated by the dorsal air sac assay and revealed that SHIN-3 and HRA serous adenocarcinoma cells, which have high levels of TP expression, induced angiogenesis, while KK clear cell carcinoma cells with low TP expression, did not. The degree of ovarian-cancer-induced angiogenesis seemed to be independent of expression of VEGF in the cells. To confirm that the serous adenocarcinoma-induced angiogenesis is dependent on TP levels, a potent and specific inhibitor of TP was administered orally to mice implanted with a chamber containing SHIN-3 or HRA cells. The TP inhibitor significantly inhibited the angiogenesis induced by the serous adenocarcinoma cells. These results suggest that the angiogenic potency of ovarian cancer cells differs with the histological type and is controlled by expression of TP/PD-ECGF, not by VEGF, and that TP-mediated angiogenesis may be the main factor responsible for progression or metastasis of ovarian serous adenocarcinomas.

Adenocarcinoma, Clear Cell↗
PubMed1995

5-Ethoxy-2'-deoxyuridine, a novel substrate for thymidine phosphorylase, potentiates the antitumor activity of 5-fluorouracil when used in combination with interferon, an inducer of thymidine phosphorylase expression.

Clinical studies have demonstrated that the combination of 5-fluorouracil (FUra) and IFN-alpha has activity in the treatment of advanced colorectal cancer. Treatment of human colon carcinoma cells with IFN caused a 5-fold increase in the level of thymidine phosphorylase (TP) mRNA and an 8-fold increase in TP enzyme activity. Since TP catalyzes the first step in the direct conversion of FUra to deoxyribonucleotides, its induction by IFN is a potential biochemical mechanism for the modulation of the antitumor activity of FUra. In contrast to the activity measured in cell extracts, however, thymine utilization by intact cells was increased less than 2-fold by IFN, suggesting that the metabolic activation of FUra by TP in the IFN-treated cells was similarly suboptimal. This was likely due to a rate-limiting amount of cosubstrate for TP, and in this study, a series of 5-substituted 2'-deoxyuridine analogues were synthesized and tested as potential deoxyribose donors for TP. One of the compounds, the novel pyrimidine analogue 5-ethoxy-2'-deoxyuridine (EOdU), was found to be a substrate for the transferase reaction of TP, to have little or no direct cytotoxicity, to selectively increase the cellular levels of 5-fluoro-dUMP, to enhance the inhibitory effect of FUra on thymidylate synthase activity, and to potentiate the cytotoxicity of FUra and IFN in human colon carcinoma cells. EOdU was tested in vivo against HT-29 cells grown as xenografts in nude mice. The combination of EOdU+FUra+IFN-alpha 2a produced tumor regressions and a significantly greater delay in tumor growth when compared to FUra+IFN-alpha 2a, FUra+EOdU, or FUra or IFN used alone; tumors were 72% smaller in the EOdU+FUra+IFN-alpha 2a-treated animals compared to the saline control group. A comparable antitumor effect was also found when a related nucleoside analogue, 5-propynyloxy-2'-deoxyuridine, was used with FUra+IFN, and it also showed modulating activity when used with only FUra. The antitumor activity of the three agent combination (nucleoside+IFN+FUra) was comparable to that of a higher dose of FUra used alone, but it was substantially less toxic to the animals than the higher dose of FUra, indicating that the modulating agents improved the therapeutic index of FUra.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Cloning of the genes encoding thymidine diphosphoglucose 4,6-dehydratase and thymidine diphospho-4-keto-6-deoxyglucose 3,5-epimerase from the erythromycin-producing Saccharopolyspora erythraea.

Genes involved in deoxysugar metabolism, encoding thymidine diphospho (TDP)-glucose 4,6-dehydratase (gdh) and a putative TDP-4-keto-6-deoxyglucose 3,5-epimerase (kde), were cloned from the erythromycin (Er)-producing Saccharopolyspora erythraea by means of an oligodeoxynucleotide corresponding to a segment of the purified Gdh protein. Determination of the nucleotide sequence established that kde lies 3' to gdh. The function of gdh was confirmed by an enzymatic assay following expression of the gene in Escherichia coli. Southern analysis indicated that Sa. erythraea contains only one copy of gdh and kde. It was not possible to establish whether these genes are required for Er biosynthesis, but they appear to be essential for cellular metabolism, since resolution of a partial diploid containing a wt and a disrupted copy of gdh always maintained the wt gene. These loci do not lie within or near the known boundaries of the cluster of Er-production and -resistance genes, nor do they appear to be flanked by other deoxysugar biosynthesis genes.

Bacterial Proteins↗
PubMed1980

Properties of cells carrying the herpes simplex virus type 2 thymidine kinase gene: mechanisms of reversion to a thymidine kinase-negative phenotype.

We have isolated cells with a thymidine kinase-negative (tk(-)) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10(-3) to 10(-4), and resistance to either compound conferred simultaneous resistance to the other. tk(-) revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences.

Journal Article↗