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)

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.
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PMID:Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled. 338 23

Infection of monkey kidney (BSC-40) cells with vaccinia virus strain WR resulted in a marked increase in ribonucleoside diphosphate reductase (EC 1.17.4.1) activity as measured by CDP reduction in cell-free extracts. After a synchronous infection, increased activity was detected at 2 h, peaked at 4 to 5 h, and then declined between 6 and 8 h to the endogenous cellular level. The induction, detectable at 0.5 PFU/cell, correlated strongly with multiplicity of infection to 10 PFU/cell and continued to increase to 50 PFU/cell. It paralleled the previously described induction of viral DNA polymerase and thymidine kinase, suggesting that the reductase may also be a product of early transcription of the viral genome. The inhibition of DNA synthesis throughout infection resulted in prolonged accumulation of reductase activity and delayed and incomplete down-regulation at 8 h, suggesting that repression involves late functions. Rescue of fluorodeoxyuridine-inhibited DNA synthesis with exogenous thymidine restored the normal pattern. Preferential association of the induced reductase with the cytoplasmic sites of vaccinia virus DNA replication (virosomes) was not detected. The induced enzyme is similar in several respects to other eucaryotic ribonucleotide reductases, but is distinct from host cell reductase in response to certain modulators of reductase activity (M. B. Slabaugh and Christopher K. Mathews, J. Virol. 52:501-506, 1984). Full activity required an activator, exogenous reducing equivalents, and iron. Hydroxyurea, EDTA, dATP, and dTTP inhibited CDP reduction, setting this reductase apart from T4 reductase, which is not inhibited by dATP, and from herpesvirus reductase, which requires no activation and is insensitive to deoxyribonucleoside triphosphate inhibition.
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PMID:Vaccinia virus induces ribonucleotide reductase in primate cells. 638 75

A series of forty 5'-ester derivatives of 5-ethyl-2'-deoxyuridine (EDU) have been evaluated for their inhibitory effects on the growth and metabolism of murine leukemia L1210 cells. Several EDU esters proved as potent as EDU in their inhibitory effects on L1210 cell growth (inhibitory dose-50:5-10 micrograms/ml), suggesting that these esters were readily hydrolyzed to release the parent compound EDU. That the EDU esters had to be hydrolyzed first to EDU was further suggested by the dependence of their antiproliferative action on the thymidine kinase activity of the cells. It was further ascertained that EDU and its esters acquired their antiproliferative effects by an interaction with dCTP biosynthesis, possibly at the CDP ribonucleotide reductase step. Under conditions where thymidine was readily incorporated, we were unable to demonstrate any incorporation of EDU into L1210 cell DNA.
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PMID:Antitumor cell and antimetabolic effects of 5-ethyl-2'-deoxyuridine and 5'-substituted 5-ethyl-2'-deoxyuridine derivatives. 646 97

We have selected and characterized a thymidine-sensitive S49 mutant line, MC-3-3. MC-3-3 cells are 35-fold more sensitive to the cytotoxic effects of thymidine and 15-fold more sensitive to the cytotoxic effects of 5-bromodeoxyuridine than wild type S49 cells. In contrast, the MC-3-3 mutant line does not exhibit increased sensitivity to the cytotoxic action of 5-fluorodeoxyuridine. The MC-3-3 mutant line possesses levels of thymidylate synthetase and thymidine kinase activity which are equivalent to the levels in wild type S49 cells, but the ribonucleotide reductase activity in MC-3-3 cells, using CDP as a substrate, is only 10-30% of that in wild type cells. Using ADP as a substrate, the ribonucleotide reductase activity in permeabilized MC-3-3 cells is slightly higher than that in wild type S49 cells. The deoxyribonucleotide pools in exponentially growing MC-3-3 cells are approximately 40-50% of those in wild type S49 cells. By hybrid analysis, we determined that the thymidine sensitivity of the MC-3-3 cells is recessive. The MC-3-3 mutant line displays a rate of spontaneous mutation which is 15-30-fold higher than that of wild type S49 cells. The MC-3-3 mutant cells are also 5-10-fold more sensitive than wild type cells to the cytotoxic effects of tunicamycin and compactin. These results suggest that the MC-3-3 mutant line possesses a mutation in the dTTP binding site in ribonucleotide reductase; abnormal regulation of this enzyme results in an increase in the rate of spontaneous mutation.
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PMID:Mutator phenotype in a mutant of S49 mouse T-lymphoma cells with abnormal sensitivity to thymidine. 670 78

Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine, Zovirax) is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovir-resistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (IC50 = 0.25 microM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 microM). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3H]acyclovir diphosphate dimyristoylglycerol relative to acyclovir. Acyclovir monophosphate added to the culture medium (outside the cell) did not circumvent the acyclovir resistance of the TK-negative DM21 mutant, presumably due to its conversion to acyclovir by phosphatases. Acyclovir diphosphate diacylglycerol prodrugs may be useful in treating TK-deficient mutant and wild-type strains of HSV.
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PMID:Acyclovir diphosphate dimyristoylglycerol: a phospholipid prodrug with activity against acyclovir-resistant herpes simplex virus. 826 34

The proximal CCAAT element located 38 bp upstream of the transcription initiation site contributes to the human thymidine kinase (htk) promoter activity, because site-directed mutagenesis of a 10-bp region containing this CCAAT motif (TKC1) reduced the promoter activity by 55%. Through binding site competitions and antigenic cross-reactivity, the major factor that binds TKC1 from both HeLa and hamster nuclear extracts is identified as NF-Y/CBF. In serum-stimulated cells, the binding of NF-Y/CBF to TKC1 increased gradually, reaching a plateau at the S phase. In cell transfection assays, a dominant-negative mutant of NF-Y/CBF inhibited the htk promoter in a dosage-dependent manner, providing direct evidence that NF-Y/CBF is required for maximal htk promoter activity. Recently, it has been demonstrated that the site occupied by NF-Y/CBF also binds the serum-inducible dbpA and dpbB. We show here that recombinant dbpA interacts with the htk promoter, and overexpression of dbpA can stimulate htk promoter activity mediated through TCK1. In contrast, CDP/cut, the CCAAT displacement protein with known repressor property, binds the htk promoter through both the proximal and distal CCAAT elements. Our discovery that CDP/cut binds the htk promoter primarily in quiescent cells and that overexpression of CDP/cut inhibits htk promoter activity provides an explanation for the reported dramatic increase in htk promoter activity in serum-starved cells when both CCAAT elements were mutated. Thus, a combination of suppression in quiescent cells and activation in serum-stimulated cells mediated through various CCAAT-binding proteins may account in part for the induction of htk promoter activity as quiescent cells reenter the cell cycle.
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PMID:Positive and negative regulation of the human thymidine kinase promoter mediated by CCAAT binding transcription factors NF-Y/CBF, dbpA, and CDP/cut. 941 21

The coding region of the human histone H4 gene FO108 undergoes dynamic changes in chromatin structure that correlate with modifications in gene expression. Such structural alterations generally reflect transcription factor interactions with gene regulatory sequences. To test for regulatory elements within the coding region, we performed transient transfection experiments in HeLa cells using constructs with histone H4 sequences fused upstream of a heterologous thymidine kinase promoter and CAT reporter gene. H4 gene sequences from -10 to +210 repressed transcription 4.8-fold. Further deletion and mutational analysis delineated three repressor elements within this region. Using oligonucleotide competition analysis and specific antibody recognition in electrophoretic mobility shift assays, as well as methylation interference and DNase I footprinting analyses, we have identified the CCAAT displacement protein (CDP/cut) as the factor that interacts with these three repressor elements. CDP/cut binding to these repressor sites is proliferation-specific and cell-cycle-regulated, increasing in mid to late S phase. Our results indicate that the proximal 200 nucleotides of the histone H4-coding region contain transcriptional regulatory elements that may contribute to cell-cycle control of histone gene expression by interacting with repressor complexes containing CDP/cut homeodomain transcription factors.
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PMID:Repressor elements in the coding region of the human histone H4 gene interact with the transcription factor CDP/cut. 987 97

The CCAAT displacement protein/cut homologue (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21(WAF1,CIP1), c-myc, thymidine kinase, and histones. Our previous studies indicate that CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. Here, we report the interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function.
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PMID:Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP. 1085 58