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 thymidine kinase (HSV-TK) gene was ligated with four repeats of the Myc-Max response elements (a core nucleotide sequence CACGTG), and its utility for gene therapy was examined by the treatment of either c-, L- or N-myc-overexpressing the small cell lung cancer (SCLC) cell line with ganciclovir (GCV). The chloramphenicol acetyltransferase assay demonstrated that the overexpression of any myc genes activated transcription from the CAT gene depending on the Myc-Max binding sites. The transduction of the HSV-TK gene ligated with the CACGTG core rendered all three SCLC lines to be more sensitive to GCV than parental ones in vitro. In addition, the growth of c- or L-myc-overexpressing SCLC cells containing the hybrid HSV-TK gene were significantly suppressed by GCV in vivo. When parental SCLC cells were mixed with HSV-TK-expressing tumor cells at a ratio of 1:3, GCV treatment inhibited tumor growth by 90% compared with parental cells only, indicating the existence of the "bystander effect." These data suggest that the CACGTG-driven HSV-TK gene may be useful for the treatment of SCLC overexpressing any type of myc family oncogenes.
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PMID:Eradication of Myc-overexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements. 854 91

Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C-reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N)5AA motifs. We report an IL-6 responsive element in the proximal region of the human CRP 5'-flanking region that bears a TT(N)4AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-gamma was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6.
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PMID:STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6. 862 22

Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.
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PMID:Activation of smooth muscle alpha-actin promoter in ras-transformed cells by treatments with antimitotic agents: correlation with stimulation of SRF:SRE mediated gene transcription. 872 Jan 48

The urokinase-type plasminogen activator contributes to tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellular signal-regulated kinases (ERKs) in the regulation of urokinase-type plasminogen activator expression in a squamous cell carcinoma cell line (UM-SCC-1) that contains a transcriptionally activated urokinase-type plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promoter, which had progressive 5' deletions or which had been point-mutated, indicated the requirement of binding sites for AP-1 (-1967) and PEA3 (-1973) for its maximal activation. Expression of a mutant jun protein, which lacks the transactivation domain, caused a dose-dependent repression of a CAT reporter driven by either the urokinase-type plasminogen activator promoter or three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter indicating the importance of AP-1-binding transcription factor(s) in the regulation of urokinase-type plasminogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotide spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via phosphorylation of p62TCF, but not ERK2, in UM-SCC-1 cells. Moreover, the expression of a dominant-negative ERK1, but not ERK2, repressed urokinase-type plasminogen activator promoter activity. Similarly, interfering with the function of the c-raf serine-threonine kinase, which lies upstream of ERK1, by the expression of a kinase-inactive c-raf repressed the activity of a CAT reporter driven by either the urokinase-type plasminogen activator promotor or tandem AP-1 repeats. These data suggest that urokinase-type plasminogen activator expression in UM-SCC-1 cells is regulated partly by an ERK1, but not ERK2, -dependent signaling pathway.
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PMID:Regulation of urokinase-type plasminogen activator expression by an ERK1-dependent signaling pathway in a squamous cell carcinoma cell line. 876 47

The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.
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PMID:Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions. 883 39

Retinoic acid (RA) is known to have potent effects on development and differentiation. RA exerts its effects on transcription through two distinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), that bind to specific RA-responsive elements (RARE) in target genes. alpha-Fetoprotein (AFP), a hepatocyte differentiation, maturation, and carcinogenesis marker, is transcriptionally upregulated by RA in McA-RH8994 hepatoma cells. Using deletion mapping analysis, we have identified a RARE-like sequence that is located between -2406 and -2378 of the transcription initiation site of the rat AFP gene. Sequence analysis demonstrated that this cis-acting element consists of three direct repeats and one inverted repeat of a GGGTCA-like half-site. The putative RARE can specifically bind to both RXR homodimers and RAR/RXR heterodimers as determined by gel mobility shift assays. A DR1 direct repeat was more efficient than a DR5 direct repeat oligonucleotide in competition for binding of the putative RARE to RXR and RAR/RXR. A mutagenesis study indicated that to have a full-strength induction, all the repeats were required. To further analyze the function of this element in vivo, a reporter gene construct of the putative RARE combined with the thymidine kinase promoter was cotransfected with RAR and RXR expression plasmids in CV1 cells. CAT assays demonstrated that overexpression of RXRalpha conferred the best RA response, consistent with our previous observation that 9-cis-RA is more potent than all-trans-RA for inducing the expression of the AFP gene. In addition, the RXR selective ligand LG100153 alone can stimulate the expression of the AFP gene. Our data suggest that an RXR-mediated pathway exists for modulation of AFP gene expression through a specific element.
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PMID:RXR-mediated regulation of the alpha-fetoprotein gene through an upstream element. 894 36

The mouse cytochrome oxidase (COX) Vb promoter contains three sequence motifs with partial or full consensus for YY-1 and GTG factor binding and a CArG box, located between positions -480 and -390. Individually, all three motifs stimulated transcription of the TKCAT promoter, and bound distinctly different proteins from the liver and differentiated C2C12 nuclear extracts. Collectively, these motifs, together with the downstream flanking sequence, -378 to -320, suppressed the transcription activity of heterologous promoters, thymidine kinase-chloramphenicol acetyltransferase (TKCAT) and COXIV/CAT. The transcription activities of both TKCAT and COXIV/CAT constructs were induced 3-4-fold during induced myogenesis of C2C12 cells. The downstream CArG-like motif binds transcription factor YY-1, while the upstream YY-1-like motif binds to a yet unidentified factor. Co-expression with intact YY-1, but not that lacking the DNA binding domain suppressed the transcriptional activity. Mutations targeted to the CArG-like motif abolished the suppressive effect of the negative enhancer and the inducibility of the promoter during myogenic differentiation. Our results suggest that the activity of the negative enhancer may determine the level of expression of the COX Vb gene in different tissues.
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PMID:Regulation of murine cytochrome oxidase Vb gene expression in different tissues and during myogenesis. Role of a YY-1 factor-binding negative enhancer. 903 8

Previous analysis of the rat PAP I promoter indicated that the region between nt -180 and -81 possessed silencer activity in cells that did not express PAP I. Based on this finding, we performed a series of experiments to characterize functionally that region and analyze the nuclear proteins interacting with it. Transient transfection assays were conducted in the fibroblast Rat2 cell line, in which PAP I is not expressed, and in the pancreatic cell line AR-42J, expressing PAP I, using the CAT gene as reporter. Experiments in Rat2 cells revealed that the sequence with silencer activity was located within the rep27 region (position -180/-153). Suppressor activity was observed when rep27 was inserted upstream from the core PAP I promoter, in both orientations. By contrast, inserting the rep27 region in front of the promoters of SV40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-dependent. In pancreatic AR-42J cells, rep27 act as a positive element but did not alter CAT expression when inserted in front of the core PAP I promoter or heterologous promoters. Electrophoretic mobility shift assays allowed identification of specific DNA-protein complexes. The shifted complex migrated at the same position with both Rat2 and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with rat nuclear extracts from healthy pancreas, pancreas with acute pancreatitis, liver, kidney, spleen, and small intestine. Results suggest that the rep27 cis-acting element contributes to the tissue specific expression of the PAP I gene. That activity could be mediated by the synergistic action of several transcription factors, one of which being present in all cells.
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PMID:Characterization of a silencer regulatory element in the rat PAP I gene which confers tissue-specific expression and is promoter-dependent. 912 83

The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)- and extracellular signal-regulated kinase- (ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we first determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning -144 to -73 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides -91 to -68 and containing a consensus AP-1 motif at -79. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinase-deficient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-deficient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinase-deficient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK- and ERK-dependent signaling pathways.
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PMID:Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. 913 92

The hypothesis of a primary action of steroid hormones on mitochondrial gene expression has been supported by the detection of the glucocorticoid receptor in liver mitochondria and the demonstration of the interaction of the receptor with putative mitochondrial HREs. We now show that two putative mitochondrial glucocorticoid response elements present within the cytochrome oxidase subunit I gene (GREI and GREII), linked to a thymidine kinase promoter and to the CAT gene, transfected to LATK- cells, confer dexamethasone inducibility to the CAT gene. As the plasmids were stably transfected, hormone induction was analysed in the nuclear background. This effect is dose dependent and is abolished by the glucocorticoid antagonist RU38486.
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PMID:The mitochondrion as a primary site of action of glucocorticoids: mitochondrial nucleotide sequences, showing similarity to hormone response elements, confer dexamethasone inducibility to chimaeric genes transfected in LATK- cells. 919 95


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