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)

Transcriptional activation of the human thymidine kinase (hTK) promoter plays an important role in the cell cycle control of thymidine kinase expression. Using the luciferase reporter cotransfection assay, we found that the activity of the hTK promoter in IMR-90 normal human diploid fibroblasts was increased by the constitutively over-expressed cyclin A or cyclin E but not by cyclin D, suggesting that the former two cyclins may act as positive regulators for the hTK promoter. The sequence responsible for the transcriptional activation by cyclin E was identified to be located between -133 and -92 of the hTK promoter. Regulation of the hTK promoter in HeLa cells appeared to be different from that in IMR-90 fibroblasts. Firstly, the hTK promoter in HeLa was already highly activated and could not be further activated by ectopically expressed cyclin A or E. Secondly, the -133 to -92 region of the hTK promoter was important for the promoter strength in HeLa cells but not in IMR-90 cells. The steady-state levels of cyclins A and E were readily detected in HeLa cells but not in normal IMR-90 fibroblasts. Based on these results, we propose that the cellular environment of the HeLa cell allows the hTK promoter to stay fully activated for transcription regardless of ectopically expressed cyclin A or E and that transcriptional activation of thymidine kinase gene is deregulated in these tumor cells.
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PMID:Different regulation of the human thymidine kinase promoter in normal human diploid IMR-90 fibroblasts and HeLa cells. 759 1

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

Expression of thymidine kinase (TK) gene in normal human diploid, cells is both cell cycle and age dependent and appears to be transcriptionally regulated. Several studies have indicated that the G1/S control sequence may reside within the region of about 130 bp upstream of the transcription initiation site. We have previously shown that a trans-acting factor, CBP/tk (CCAAT binding protein for TK gene), binds to either one of the two inverted CCAAT boxes in a cell cycle- and age-dependent manner (Pang and Chen, 1993, J. Biol. Chem., 268:2909-2916). An upstream 25 bp fragment (-109/-84), containing both Yi-like and E2F-like binding sites, has recently been proposed to be essential for the G1/S regulation of human TK gene. To assess the contribution of various cis-elements in human TK promoter to the G1/S regulation, we have examined the binding activity of these cis-elements in the nuclear extracts derived from human IMR-90 cells at low passage number. Our results indicated that no binding activity could be detected using either the 25 bp fragment (-109/-94) or the authentic Yi sequence. However, Yi binding activity was observed in SV-40 transformed IMR-90 cells. In contrast, the 28 bp fragment (-91/-64) that contains the distal inverted CCAAT box exhibited a strong binding in serum-stimulated young IMR-90 cells. The binding of CBP/tk to the 28 bp fragment was abolished by a single base mutation in the CCAAT box. The CBP/tk binding of the 28 bp fragment could not be displaced by either the 25 bp fragment or the authentic Yi element. A deletion of the 5'-flanking region of the 28 bp fragment up to 5 bases also abolished the binding activity. The CBP/tk binding in IMR-90 cells was supershifted by antiserum against NF-Ya, but not by antiserum made against p107, pRb, cyclin A, p33cdk2, or p34cdc2. Taken together, our results suggest that the G1/S regulatory cis-element in human TK promoter may be confined only to CBP/tk binding sites.
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PMID:Analysis of sequence-specific binding activity of cis-elements in human thymidine kinase gene promoter during G1/S phase transition. 777 6

Glucocorticoids inhibit transcription of the proto-oncogene c-myc in lymphoid cells of thymic origin. To determine if this effect is associated with changes in the properties of the transcription factor E2F, extracts were prepared from control and glucocorticoid-treated P1798 murine T lymphoma cells, and the macromolecular state of E2F was assessed by gel-mobility shift. Control extracts exhibit two predominant gel-mobility shift entities of which one corresponds to "free" E2F. A second entity, complex C, has properties similar to those described for the complex containing E2F, p107, cyclin A, and Cdk2. Complex C disappears after addition of dexamethasone and is replaced by complex D. The mobility of this complex and its sensitivity to SV40 T antigen suggest that complex D corresponds to an E2F-p105Rb-1 complex. Extracts from control and glucocorticoid-treated cells yield identical DNase I protection patterns on the c-myc P2 promoter. Furthermore, such extracts transcribe the c-myc P2 promoter in vitro with equal activity. The relative abundance of the E2F complexes was measured after addition of dexamethasone. Complex C disappears as cells withdraw from S phase, and complex D appears at this time. The genes encoding thymidine kinase (Tk-1) and p34cdc2 (cdc2) are regulated with kinetics similar to those observed for changes in the macromolecular state of E2F. However, regulation of c-myc expression occurs long before any change in E2F. The macromolecular state of E2F may regulate expression of genes at the G1/S boundary. However, the data are not consistent with the hypothesis that association of E2F with tumor suppressor gene products such as p107 or p105Rb-1 is relevant to glucocorticoid regulation of c-myc transcription.
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PMID:The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription. 800 8

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
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PMID:G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. 828 95

The cyclins are an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle. The synthesis of A-type cyclins begins at the start of S phase. In mammalian cells, association with the cdc-type kinases suggests that cyclin A complexes are important for DNA replication and regulating other DNA-bound substrates required for S phase. We report here that a 25-bp promoter element previously shown to be important for the G1-S activation of the human thymidine kinase (htk) promoter in growth-stimulated cells is a cellular target of cyclin A and the p33cdk2 complexes. Though the p33cdk2 and other nuclear factor complexes exhibit constitutive binding to the htk G1-S regulatory domain, the binding activity of a cyclin A/p107 protein complex is greatly enhanced when the cells enter S phase, correlating with the increase in the tk mRNA levels and the replication of DNA. The binding activity of the cyclin A complex is maintained throughout S phase. Mutation of the DNA sequences on either half of the 25-bp protein binding site results in the loss of its ability to compete efficiently in vitro for the htk complexes, including that of cyclin A-containing complex. The loss of high-affinity binding for the htk complexes also substantially reduces the S-phase regulation of the htk promoter in vivo. Our results support the hypothesis that a cyclin A complex, in association with the p33cdk2 kinase, mediates the S-phase-regulated transcription of the htk promoter in growth-stimulated cells.
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PMID:Temporal regulation of cyclin A-p107 and p33cdk2 complexes binding to a human thymidine kinase promoter element important for G1-S phase transcriptional regulation. 847 4

Promoter elements that are important for the G1-S induction of the human thymidine kinase (htk) promoter reside within the core of the cell cycle regulatory unit, positioned between -110 and -84 upstream of the TATA element. Within this 27-bp region are three GC-rich motifs, which resemble the E2F binding site. By site-directed mutagenesis, we identified a 14-bp region, between -97 and -84, critical for the htk promoter transcriptional activity. Methylation interference studies indicate that the sequences between -97 and -84 are major protein contact points, correlating with the functional significance of this sequence in vivo. Although the core of the cell cycle regulatory unit contains three E2F-like sites and can form minor S-phase-specific complexes containing p107, cyclin A, and cdk2, the major complex that binds to this region is not competed by E2F binding sites. Through DNA affinity chromatography, we identified a set of protein species of approximately 40 kDa that copurified with the htk DNA binding activity. From gel shift assays and Western blot analysis, this protein species is antigenically distinct from E2F-1, E2F-2, E2F-3, and E2F-4. Our studies raise the possibility that other members of the E2F protein family or a novel protein(s) with preferred binding affinity for the htk promoter exert(s) control on the G1 to S regulation of the htk promoter through their interactions with cyclins and kinases.
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PMID:Identification of a set of protein species approximately 40 kDa as high-affinity DNA binding factor(s) to the cell cycle regulatory region of the human thymidine kinase promoter. 895 43

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.
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PMID:Mouse thymidine kinase stability in vivo and after in vitro translation. 912 45

Small DNA viruses (adenoviruses, simian virus 40, or human papillomaviruses) induce S-phase progression but prevent cell division to provide precursors for viral DNA replication. Herpes simplex viruses types 1 or 2 (HSV-1 or HSV-2) contain genes which encode DNA-metabolizing enzymes, for example, ribonucleotide reductase, thymidine kinase and dUTPase, suggesting that S-phase factors are not required for an efficient infection. However, several studies indicated that HSV induces some events that occur during cell-cycle progression. To determine if HSV-2 induces S-phase entry, we examined serum-arrested African green monkey kidney cells (CV-1) after infection. Two hours after infection steady-state levels of the S-phase-specific cyclin, cyclin A, increased. S-phase cyclin-dependent kinase activity (CDK2) was stimulated 10-fold 8 h after infection but decreased at 16 or 24 h after infection. Mitotic CDK activity (CDC2) was not activated after infection, in part due to decreases in CDC2 protein levels and inactivation of enzymatic activity resulting from tyrosine phosphorylation of CDC2. Furthermore, CDK4 activity was not dramatically affected by infection. These studies indicate that HSV-2 infection selectively activates CDK2 after infection but cell-cycle progression does not occur. We hypothesize that infection activates certain components of the cell cycle which enhance viral gene expression and DNA replication.
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PMID:Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection. 940 Sep 86

Induced cell cycle delays were among the first described cellular responses to ionizing radiation (IR). To understand the sensitivity and the molecular events involved in the response to low doses of IR and to examine the role of p53 and its downstream effector p21Waf1, we measured changes in expression of genes postulated to be involved in the cellular response to IR. Expression levels were examined in normal human diploid fibroblasts irradiated and maintained in quiescent density-inhibited growth up to 24-48 h after exposure to X-ray doses as low as 0.1-0.3 Gy, which have negligible effects on cell survival. Among 31 genes analyzed, we observed down-regulation in response to IR of the mRNA levels of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51. A similar reduction in the expression levels of these genes occurred when irradiated cells were released from confluence and allowed to proliferate. This was not observed in cells in which p53 function was defective and up-regulation of p21Waf1 levels either did not occur (E6 transfected normal human fibroblasts and Li-Fraumeni fibroblasts) or was delayed (ataxia telangiectasia fibroblasts) after irradiation. Down-regulation was also absent in p21Waf1-null mouse embryo fibroblasts (MEFs) but occurred at a lower level in p53-null MEFs, due to slight increases in p21Waf1 levels by a p53-independent pathway. These findings indicate that the down-regulation of these cell cycle regulated genes in irradiated cells is p53-dependent and involves its effector p21Waf1. Although no down-regulation in the expression of genes involved in G2-M was observed in p53 or in p21Waf1-null MEFs, these cells showed a G2-M delay after irradiation, indicating that the expression levels of these genes does not regulate the G2-M delay.
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PMID:Regulation by ionizing radiation of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51 expression in normal human diploid fibroblasts is dependent on p53/p21Waf1. 983 Dec 41


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