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)

Herpes simplex virus (HSV) mutants or recombinant vectors might be useful oncolytic agents. Three general types of HSV vectors can be potentially used for this purpose: (1) mutants in viral transcription factors, such as ICP0 and ICP4; (2) mutants in enzymes involved in nucleic acid metabolism, such as thymidine kinase (TK) and ribonucleotide reductase (RR); and (3) mutants in neurovirulence factors, such as gamma 34.5. We tested the destructive ability of each type against rat 9L gliosarcoma cells in culture. We found that the HSV vectors defective in TK or RR were more efficient at tumor cell lysis in culture than the other types of HSV vectors. This increased efficiency provided the rationale for evaluating the TK and RR mutants in vivo following their stereotactic inoculation into 9L gliosarcomas implanted in rat brains. We employed the X-gal enzymatic histochemical assay to show that HSV-mediated lacZ gene expression was present in cells within the tumor mass in a relatively selective fashion. Immunoreactive HSV capsid and core antigens were present both in cells within the tumor, as well as in cells such as neurons and astrocytes, directly adjacent to the tumor mass. Long-term survival studies revealed that rats treated with either the TK or RR mutant lived significantly longer than control rats (p = 0.014, Kruskal-Wallis one-way analysis of variance). These results indicate that HSV vectors, defective in enzymes needed in nucleic acid metabolism, can preferentially mediate lacZ gene expression in cells within the tumor. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase or ribonucleotide reductase. 758 98

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

We determined the cell cycle-dependent fluctuation of mRNAs that encode different enzymes of the deoxynucleotide metabolism in permanent cell lines of human and murine origin. In normal growing cells, dihydrofolate reductase, thymidine kinase, and both subunits of ribonucleotide reductase all show exactly the same variation. The mRNAs rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1. Deoxycytidine kinase mRNA does not follow this pattern, but remains essentially unchanged. Conversely, in DNA tumor virus-transformed cells, the levels of all these mRNAs remain relatively constant throughout all phases. These data provide evidence that DNA tumor viruses suppress a transcriptional down-regulation common to enzymes responsible for the DNA precursor pathway. The usefulness of analysis of mRNA levels of these genes for the detection of DNA tumor virus transformation is indicated.
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PMID:A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation. 769 88

We examined the capability of pseudorabies virus (PRV) to replicate in vitro in porcine peripheral blood mononuclear cells (PBMC) and characterized the phenotype of infected cells. In addition, we investigated whether inactivation of various PRV proteins or the expression of a foreign gene affected this replication. Finally, we studied the replication of PRV strains in concanavalin A (Con A)-stimulated lymphocytes. The replication of PRV mutants with inactivated glycoproteins gE or gG, thymidine kinase (TK), ribonucleotide reductase (RR) or US3-encoded protein kinase (PK), and the replication of PRV vector strains expressing the envelope glycoprotein E1 of hog cholera virus (HCV) were studied. By adherence of PBMC to plastic, monocytes and lymphocytes were largely separated. Infected monocytes were analysed with an immunostaining monolayer assay and infected lymphocytes were analysed with immunofluorescence staining and flow cytometry. We found that the wild-type NIA-3 virus replicated in both lymphocyte and monocyte cultures. NIA-3 infected relatively more monocytes (> 90%) than non-adherent B cells (46-65%) and T cells (17-28%); approximately equal numbers of CD4+ and CD8+ T cells were infected. Although E1 is probably involved in adsorption of HCV to host cells, the expression of E1 by PRV vector strains did not change the level of replication. Inactivation of TK and RR, but not inactivation of gE, gG or PK, severely affected the replication in both monocytes and lymphocytes. Con A stimulation of lymphocytes restored the reduced replication of the TK mutant, but not of the RR mutant. Moreover, Con A stimulation of lymphocytes reduced the replication of the wild-type NIA-3 virus. We concluded that both viral TK and RR activity are important for efficient replication of PRV in resting lymphocytes. Furthermore, Con A-stimulated lymphocytes can restore the viral TK defect and PRV replication can also be influenced by cellular metabolism.
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PMID:Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells. 778 71

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase (TK) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isolated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that complementation may contribute to pathogenicity of PRV.
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PMID:In vivo recombination of pseudorabies virus strains in mice. 785 6

Survival of rats harboring cerebral 9L gliosarcomas can be significantly extended by an intratumoral inoculation with a herpes simplex virus vector, designated as hrR3. This vector, which bears the lacZ reporter gene, is defective in the gene encoding ribonucleotide reductase, allowing for replication in dividing tumor cells but not in postmitotic neural cells. It also possesses an intact viral thymidine kinase (TK) gene, which confers chemosensitivity to ganciclovir. In this study, the ability of ganciclovir to potentiate the antitumor effect of hrR3 was evaluated. In culture, there was a 23% decrease in the growth of 9L cells treated with hrR3 plus ganciclovir compared to hrR3 alone (P < 0.01). The combination of hrR3 plus ganciclovir led to the long-term survival of 48% of rats harboring intracerebral 9L gliosarcomas compared to 20% survival in the hrR3 group (P < 0.05). Ganciclovir treatment had no effect on the growth of tumor cells in vitro or in vivo when a herpes simplex virus vector with a defective TK gene was used. Immunocytochemistry confirmed selective expression of the TK gene in cells within the tumor. These findings indicate that the TK gene can potentiate the antitumor effect of the hrR3 herpes simplex virus vector and provide the basis for placing additional therapeutic genes in the genome of hrR3.
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PMID:Long-term survival of rats harboring brain neoplasms treated with ganciclovir and a herpes simplex virus vector that retains an intact thymidine kinase gene. 795 93

Herpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (< 1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacy in vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p = 0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.
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PMID:Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats. 796 89

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.
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PMID:Global change of gene expression at late G1/S boundary may occur in human IMR-90 diploid fibroblasts during senescence. 807 91

Phylogenetic trees were derived for the Alphaherpesvirinae subfamily of the Herpesviridae using molecular sequences. Sequences from the families of genes encoding glycoprotein B, thymidine kinase, S region protein kinase, immediate-early transcriptional regulator IE175 and ribonucleotide reductase large subunit were examined by means of both maximum parsimony and distance methods, and for both protein and DNA alignments. Trees obtained were evaluated by bootstrap analysis. A clear consensus tree was obtained, with most detail coming from 14 sequences in the glycoprotein B gene set. The tree showed two avian viruses branching first from the lineage leading to the mammalian alphaherpesviruses. The mammalian viruses were split into two groups, which corresponded to the Simplexvirus and Varicellovirus genera. A timescale for events in alphaherpesvirus evolution was tested, based on the proposition that most of the lineages arose by ancient cospeciation with hosts. The virus phylogenetic tree was unambiguously compatible with cospeciation for ten of the 12 mammalian viruses. The tree was also supported by demonstration of an approximate proportionality between magnitudes of pairwise divergences of viral sequences and times since lineages of corresponding pairs of hosts split. On the basis of this timescale it was estimated that the two mammalian alphaherpesvirus groups diverged around the period of the mammalian radiation, and that alphaherpesviral genome sequences have evolved faster than those of mammals by a factor of one to two orders of magnitude.
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PMID:Molecular phylogeny of the alphaherpesvirinae subfamily and a proposed evolutionary timescale. 814 60

We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragments. More than half of these transfections yielded viable virus mutants. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated from cells infected with wild-type PRV. In this way we were able to construct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes simplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants were assayed for virulence in mice. Mutagenesis of the identified homologs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DNA polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsid-associated protein), and UL12 (alkaline nuclease) completely abrogated or strongly reduced virulence.
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PMID:Mutagenesis and characterization of a 41-kilobase-pair region of the pseudorabies virus genome: transcription map, search for virulence genes, and comparison with homologs of herpes simplex virus type 1. 817 60


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