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)

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to inflammation, its expression is increased by 1000-fold, primarily because of a 200-fold increase in the rates of SAA gene transcription. We have shown that when 304 bp of 5' flanking region of the rat SAA1 gene is fused to a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, CAT activity is induced in a cell-specific manner in response to conditioned media prepared from activated mixed lymphocyte cultures and recombinant interleukin-1. In this study, deletion of the SAA1 promoter to -120 bp with respect to the transcriptional start site did not diminish promoter activity; however, deletion to -94 bp renders the promoter completely inactive. Functional analysis have demonstrated that a 66-bp DNA fragment spanning -138 bp to -73 bp could confer cytokine responsiveness to a heterologous thymidine kinase promoter. Within this 66-bp responsive element resided an NF kappa B-like-binding site and a C/EBP-like-binding site. Although each binding site alone could confer responsiveness when stimulated with conditioned media and TPA, the response was much weaker than that observed when both sites were present. Moreover, site-specific mutations of either binding site completely abolished SAA1 promoter activity. Taken together, these results suggest a functional importance for and cooperative interaction of these two nuclear-factor binding sites in the cytokine-induced expression of the rat SAA1 gene.
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PMID:Cooperative effects of C/EBP-like and NF kappa B-like binding sites on rat serum amyloid A1 gene expression in liver cells. 140 89

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5'-TGAGTAA-3' at -59/-53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (-1020 to -780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at -803/ -797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(-59/ -53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(-59/ -53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(-59/ -53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(-59/ -53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.
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PMID:Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription. 142 Mar 63

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. During inflammation, its expression is increased by 1000-fold as the result of greatly increased gene transcription. In this study, we analyzed the cis-acting regulatory elements and trans-acting factors important for the expression of the rat SAA1 gene. A DNA fragment containing 304 base pairs (bp) of 5'-flanking sequences of the SAA1 gene was fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and the resulting construct, pSAA1/CAT (-304), was used to assess the function of the 5'-flanking sequences by transient transfection assay. pSAA1/CAT (-304) was not expressed or expressed at very low levels in both the liver- and nonliver-derived cells. However, when stimulated with conditioned medium prepared from mixed lymphocyte cultures, recombinant interleukin 1, or 12-O-tetradecanoylphorbol-13-acetate, expression of the pSAA1/CAT (-304) hybrid gene was induced 15-20-fold, but only in liver-derived cells. Further functional analysis demonstrated that a 66-bp DNA fragment conferred cytokine responsiveness onto a heterologous thymidine kinase promoter both in liver and nonliver cells. Footprint analysis with the Hep3B nuclear proteins revealed four protected regions in the 5'-flanking region of the SAA1 gene. The pattern of protection was identical with nuclear extracts prepared from either unstimulated or conditioned medium-treated Hep3B cells. Two of these footprint regions were identified as binding sites for C/EBP or C/EBP-related proteins, with the distal region having about 10-fold higher binding affinity than the proximal region. One additional cis-element formed a specific protein-DNA complex only with the nuclear proteins from TPA- or conditioned medium-treated Hep3B cells. This cis-element shares sequence identity with nuclear factor NF kappa B binding sites. The finding of a NF kappa B binding site within the 66-bp cytokine-responsive fragment further suggests its functional importance in the regulation of SAA1 gene expression. Our results suggest that C/EBP- and NF kappa B-related proteins may be important regulatory factors that contribute both to tissue specificity and to the high rate of SAA transcription in response to inflammatory mediators.
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PMID:Expression of rat serum amyloid A1 gene involves both C/EBP-like and NF kappa B-like transcription factors. 186 49

We have previously isolated 3T3 cell variants unable to respond to specific mitogens. In this report we analyze the dominant and/or recessive nature of these variants. Two independently isolated EGF nonproliferative variants are unable to bind EGF. Hybrids between 3T3R5 cells (thymidine kinase deficient, ouabain-resistant) and these variants express EGF receptors; the "EGF receptorless" phenotype of these variants is recessive. Hybrids between these two variants do not bind EGF; they are defective in a common, non-complementing function. A TPA nonproliferative 3T3 variant is also recessive; hybrids with 3T3R5 mount a mitogenic response to TPA. In contrast a fourth variant, which can neither bind labeled EGF nor respond to TPA, is dominant for both characteristics. Hybrids between this latter variant and 3T3R5 can neither bind EGF nor mount a mitogenic response to TPA.
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PMID:Dominant and recessive mitogen-nonproliferative variants of 3T3 cells. 387 70

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes.
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PMID:A cell-based reporter assay for the identification of protein kinase C activators and inhibitors. 789 70

HC-11 mouse mammary epithelial cells stably transfected with a glucocorticoid-inducible Ha-ras construct encoding a transforming (val12) p21Ha-ras were cotransfected with a c-fos-CAT construct containing the human c-fos promoter up to position -711 and the CAT reporter gene. Expression of Ha-ras by dexamethasone leads to a transcriptional activation of the fos-CAT construct which was found to be sensitive to the PKC inhibitor ilmofosine (BM41440) and abrogated by PKC depletion following long-term exposure to TPA. The responsiveness to Ha-ras is retained if only the portion of the fos promoter covering the serum response element (SRE) and the adjacent fos AP-1 (FAP) site are put in front of a CAT gene linked to a thymidine kinase (TK) promoter. Further depletion of the FAP-site does not affect the inducibility by Ha-ras. Transcriptional activation of the SRE-FAP-TK-CAT as well as the SRE-TK-CAT constructs by Ha-ras is sensitive to the PKC-inhibitor ilmofosine (BM41440) and blocked by long-term exposure to TPA. Long-term exposure to TPA depletes cells of PKC alpha and significantly reduces the PKC epsilon levels. Long-term exposure in bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT-construct by Ha-ras. It is concluded that (i) transforming Ha-ras induces c-fos in HC-11 cells via PKC (presumably epsilon), (ii) the signal is mediated to the serum response element (SRE) of the fos promoter and (iii) the fos AP-1 (FAP) site is not required for this mechanism.
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PMID:Role of protein kinase C in ras-mediated fos-expression. 794 78

Human papillomavirus (HPV) 8 belongs to the HPV types frequently associated with skin cancers of Epidermodysplasia verruciformis (EV)-patients. There are 33 nucleotides (M33 motif) in the 5'-part of the non-coding regulatory region of HPV8, which appear highly conserved among EV-specific HPVs and are consistently followed by an AP1 binding site. These sequences were shown to constitute an essential activator of transcription driven by the HPV8 late promoter P7535. The M33/AP1 element displayed properties of a constitutive enhancer, being also able to stimulate the activity of the heterologous thymidine kinase promoter in a position-independent manner. No protein binding could be detected within the 5'-part of the M33/AP1 region, which contributed significantly to the overall activity of the HPV8 enhancer. As shown by DNasel-footprinting, the central and the 3'-part of the enhancer region were involved in interactions with nuclear proteins. Three specific complexes could be observed in gel retardation tests with nuclear extracts from epithelial cells. One of these interactions involved the AP1 protein. Analysis of deletion and point mutations revealed binding of the AP1 protein to be essential for transcriptional activation, but DNA-protein interactions within M33 were important for maximal stimulation. The response to the phorbol ester TPA also required a cooperation of M33 and AP1.
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PMID:Constitutive transcriptional activator of Epidermodysplasia verruciformis-associated human papillomavirus 8. 839 5

As the clinical manifestations of adult T-cell leukemia (ATL) can be quite diverse, useful indicators for the therapy and prognosis are required for the disease. In this review, the clinical and biological significance of serum tumor markers at diagnosis in ATL patients is described. Serum lactic dehydrogenase (S-LDH), serum thymidine kinase (S-TK) and serum parathyroid hormone-related protein (S-PTHrP) at diagnosis of ATL showed a correlation with among leukocyte count, absolute number of abnormal lymphocytes with polymorphic nuclei, platelet count, serum calcium and the length of survival after the initial diagnosis. Serum beta 2-microglobulin (S-beta 2M) correlated with age, platelet count and survival. A statistical correlation existed between these four serum tumor markers. Other serum tumor markers such as immunosuppressive acidic protein (S-IAP), ferritin (S-Ft) and tissue polypeptide antigen (S-TPA) showed no correlation with clinical and histological data in ATL patients.
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PMID:Clinical and biological significance of serum tumor markers in adult T-cell leukemia. 888 54

GnRH stimulates both transcription and secretion of the alpha-subunit in pituitary cells, but the precise role of the calcium- signaling mechanisms mediating these actions are unclear. We have examined the role of calcium using alpha T3-1 gonadotropes transfected with alpha-promoter constructs linked to a luciferase reporter gene and concomitant measurement of alpha-subunit secretion. The calcium channel agonist, BayK8644 (1 microM) significantly stimulated alpha-subunit transcription (4.9-fold, P < 0.05) but to a lesser extent than either GnRH (100 nM, 20.7-fold, P < 0.001) or phorbol-12-myristate-13-acetate (TPA, 100 nM, 8.7-fold, P < 0.05). The transcriptional response to a combination of BayK8644 and TPA was approximately additive. Despite stimulating alpha-subunit gene expression, BayK8644 had no effect on alpha-subunit secretion at 24 h, and co-addition of BayK8644 and TPA did not produce any further stimulation of alpha-subunit secretion (3.0-fold, P < 0.001) compared with TPA alone (3.2-fold, P < 0.001). Pretreatment of alpha T3-1 cells with the calcium channel blocker, nifedipine (1 microM for 5 min), essentially blocked GnRH-stimulated alpha-promoter activity without affecting GnRH-stimulated alpha-subunit release. In contrast, thapsigargin pretreatment (1 microM for 5 min), which depletes intracellular calcium stores, significantly reduced basal and GnRH-stimulated secretion without affecting the ability of GnRH to increase alpha-promoter activity. Incubation of alpha T3-1 cells in calcium-depleted media showed that the transcriptional response was dependent on extracellular calcium concentration, with maximum stimulation by GnRH seen at a calcium concentration of 1.7 mM. Deletion analysis indicated that sequences between -420 and -244 bp were involved in mediating the response to BayK8644. Constructs containing only upstream alpha-promoter sequences from -517 to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited loss of responsiveness to BayK8644 below -298 bp. These upstream elements were also found to be important for basal expression of the alpha-promoter and for mediating the response to TPA but were distinct from GnRH responsiveness of the human promoter in alpha T3-1 cells. These studies suggest differential regulation of GnRH-stimulated alpha-subunit gene transcription and secretion by extracellular calcium influx and intracellular calcium mobilization. The transcriptional response to extracellular calcium influx is mediated through two or more elements between -420 and -244 bp, which are also involved in basal and TPA-stimulated expression of the alpha-subunit promoter.
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PMID:Calcium and glycoprotein hormone alpha-subunit gene expression and secretion in alpha T3-1 gonadotropes. 892 57

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.
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PMID:Vitamin E mediated response of smooth muscle cell to oxidant stress. 1058 72


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