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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of acidosis on the expression of the vascular endothelial growth factor (VEGF) gene was determined. FG human pancreatic adenocarcinoma cells were incubated for various time periods in media at a physiologically relevant pH level (6.7-7.4). The expression of VEGF mRNA and protein secretion was inversely correlated with pH in a pH- and time-dependent manner. Transient acidosis also activated the VEGF promoter/enhancer luciferase reporter, which was consistent with an increased VEGF gene transcription rate and VEGF mRNA half-life. These data indicated that acidosis transcriptionally and posttranscriptionally regulates VEGF expression, suggesting that an acidic tumor microenvironment contributes to tumor angiogenesis and progression.
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PMID:Regulation of vascular endothelial growth factor expression by acidosis in human cancer cells. 1143 38

An efficient gene delivery system is a prerequisite for myocardial gene therapy. Among the various procedures studied so far, catheter-based percutaneous gene delivery to the myocardium through the coronary vessels seems the most relevant to routine clinical practice; however, the optimal conditions remain to be determined. We selectively infused adenoviral vectors encoding luciferase (1 x 10(9) PFU) or beta-galactosidase (1 x 10(10) PFU) into coronary arteries of adult rabbits in various experimental conditions. Coronary artery occlusion for 30 sec, during and after adenovirus delivery, was required to observe luciferase activity in the target area of the circumflex artery (4.0 +/- 1.0 x 10(5) vs. 1.1 +/- 0.2 x 10(4) RLU/mg with and without coronary occlusion, respectively, p < 0.01, and 1.0 +/- 0.1 x 10(3) RLU/mg using nonselective infusion). When adenoviruses were delivered using high-pressure infusion (82 +/- 12 vs. 415 +/- 25 mmHg before and during infusion, respectively, p < 0.01), luciferase activity increased to 8.5 +/- 2.5 x 10(5) RLU/mg (p < 0.05 vs coronary occlusion alone). Coronary venous sinus occlusion with saline buffer retroinfusion starting before and during anterograde adenovirus delivery resulted in a further 4.7-fold increase in luciferase activity (4.4 +/- 0.8 x 10(6) RLU/mg, p < 0.01) with 5-25% blue-stained myocytes in the target area, compared with 0-5% with the other procedures. Histamine or VEGF-A(165) pretreatment, used to increase vascular permeability, slightly increased gene transfer efficiency (8.5 +/- 2.0 x 10(5) and 9.0 +/- 2.5 x 10(5) RLU/mg respectively, p < 0.05 vs. coronary occlusion alone). We conclude that catheter-mediated adenoviral gene transfer to cardiac myocytes through coronary vessels can be a very efficient procedure for myocardial gene therapy, particularly when the vector residence time and perfusion pressure in the vessels are increased.
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PMID:How to optimize in vivo gene transfer to cardiac myocytes: mechanical or pharmacological procedures? 1153 64

Overexpression of vascular endothelial growth factor (VEGF) is associated with disease progression in human glioblastomas. We recently showed that VEGF promoter activity is inversely correlated with tumor extracellular pH (pH(o)) in vivo in the human glioma (U87 MG) xenografts. Here we show that substitution of the neutral culture medium (pH 7.3) with acidic pH medium (pH 6.6) up-regulates VEGF mRNA and protein production in human glioblastoma cells as reflected by Northern blot analysis and enzyme-linked immunosorbent assay. Functional analysis of the VEGF promoter reveals that the sequence between -961 bp and -683 bp upstream of the transcription start site is responsible for the transcriptional activation of the VEGF gene by acidic pH. This region contains the binding site for AP-1. Consequently, AP-1 luciferase reporter gene was activated by acidic pH. Gel-shift analysis confirmed that AP-1 DNA binding activity is induced under acidic pH. While investigating the upstream signaling pathways, we found that ERK1/2 MAPK is activated and translocates to the nucleus to activate Elk-1, and inhibition of the activation of ERK by specific inhibitors of MEK1 blocks the up-regulation of VEGF by low pH. Dominant negative forms of Ras and Raf abolished the activation of VEGF promoter by acidic pH. These results show that acidic pH activates Ras and the ERK1/2 MAPK pathway to enhance VEGF transcription via AP-1, leading to increased VEGF production.
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PMID:Acidic extracellular pH induces vascular endothelial growth factor (VEGF) in human glioblastoma cells via ERK1/2 MAPK signaling pathway: mechanism of low pH-induced VEGF. 1174 77

IGF-I is known to stimulate the expression of oxygen- and nutrient-sensitive genes in several cell types. In this study we investigated the signaling pathways and transcriptional mechanisms that mediate IGF-I induction of vascular endothelial growth factor (VEGF) expression in human osteoblast-like cells. IGF-I (50 ng/ml) induced a rapid increase (3-fold) in VEGF mRNA in osteoblasts that was accompanied by an increase in the level of hypoxia-inducible factor-2alpha (HIF-2alpha) protein without changes in HIF-2alpha mRNA expression. These effects were mimicked by chemical inhibition of proteosomal degradation of HIF-2alpha. Transcriptional activation of a proximal VEGF promoter-luciferase construct was greatly enhanced by cotransfection with an HIF-2alpha, but not an HIF-1alpha, construct. IGF-I acutely stimulated Akt phosphorylation, which was abolished by pretreatment of cells with the PI3K inhibitor LY294002. Pretreatment of the cells with LY294002 also greatly attenuated IGF-I induction of HIF-2alpha and blunted IGF-I-induced VEGF promoter activity. Finally, forced expression of a constitutively active PI3K expression construct induced VEGF promoter to levels similar to those observed with IGF-I alone. These data indicate that IGF-I, by activation of the PI3K pathway, induces VEGF expression in osteoblasts through a transcriptional control mechanism common to those that activate VEGF and other hypoxia response genes.
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PMID:Induction of vascular endothelial growth factor by IGF-I in osteoblast-like cells is mediated by the PI3K signaling pathway through the hypoxia-inducible factor-2alpha. 1179 94

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.
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PMID:Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. 1180

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.
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PMID:Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide. 1180 28

It was previously demonstrated that p53 status in human multiple myeloma (MM) cells regulates distinct cell cycle responses to CD40 activation. In this study, the production of vascular endothelial growth factor (VEGF) and migration in MM cells triggered by CD40 activation was examined, and the influence of p53 status in regulating this process was determined. Two human MM cell lines that express wild-type p53 at permissive (28 degrees C) and mutant p53 at restrictive (37 degrees C) temperatures were used as a model system. CD40 activation induces a 4-fold (RPMI 8226) and a 6-fold (SV) increase in VEGF transcripts, respectively, under restrictive, but not permissive, temperatures. VEGF expression is significantly induced after CD40 activation in patient MM cells expressing mutant p53. Increased VEGF transcripts result in increased protein and secretion levels, as evidenced by immunoblotting and enzyme-linked immunosorbent assay. In a double-chamber transmigration assay, CD40 activation of MM cells induced a 3-fold (RPMI 8226) and a 5-fold (SV) increase in migration under restrictive, but not permissive, conditions. A 2- to 8-fold induction in migration of patient MM cells expressing mutant p53 was similarly observed. Transduction of MM cells with a luciferase reporter under the control of a human VEGF promoter further indicated that CD40-induced VEGF expression was mediated through a transcriptional control mechanism. Finally, adenovirus-mediated wild-type p53 overexpression down-regulated CD40-induced VEGF expression and transmigration in MM cells expressing mutant p53. These studies demonstrate that CD40 induces VEGF secretion and MM cell migration, suggesting a role for CD40 in regulating MM homing and angiogenesis.
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PMID:CD40 activation induces p53-dependent vascular endothelial growth factor secretion in human multiple myeloma cells. 1183 Apr 95

Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A2A adenosine receptors and HMEC-1 preferentially express A2B receptors. Neither cells expressed A1 or A3 receptors. The nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Cotransfection of each type of adenosine receptors with a luciferase reporter in HMEC-1 showed that A2B receptors, but not A1, A2A, or A3, activated IL-8 and VEGF promoters. These effects were mimicked by constitutively active alphaG(q), alphaG12, and alphaG13, but not alphaG(s) or alphaG(i1-3). Furthermore, stimulation of phospholipase C indicated coupling of A2B receptors to G(q) proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes to functional heterogeneity of human endothelial cells. A2B receptors, predominantly expressed in human microvascular cells, modulate expression of angiogenic factors via coupling to G(q), and possibly via G12/13.
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PMID:Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation. 1190 16

Hypoxia induces a group of physiologically important genes that include erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Hypoxia-inducible factor 1 (HIF-1) was identified as a hypoxia-activated transcription factor; however, the molecular mechanisms that underlie hypoxia signal transduction in mammalian cells remain undefined. In this study, we found that a flavoprotein, NADPH-P450 reductase (NPR), could regulate the induction of EPO mRNA under hypoxic conditions. Hypoxic EPO mRNA induction in Hep3B cells was inhibited by diphenyleneiodonium chloride, which is an inhibitor of NADPH-dependent enzymes. NPR antisense cDNA was transfected into Hep3B cells, and NPR-deficient hepatocyte cells (NPR(-) cells) were established. NPR(-) cells lacked EPO induction under hypoxia, and HIF-1alpha in NPR(-) cells did not respond to either transcriptional activation or translocation to the nucleus based on electrophoretic mobility shift assays and reporter gene assay including hypoxia response element. In contrast, NPR overexpression in Hep3B cells enhanced the DNA binding activity of HIF-1alpha by luciferase reporter gene assay. A study with HeLa S3 cells produced the same results. Furthermore, anti-NPR IgG inhibited EPO induction. EPO induction inhibited by diphenyleneiodonium chloride was recovered by bovine serum albumin-NADPH (a covalent binding complex of bovine serum albumin and NADPH) as well as NADPH. These results suggested that NPR located at the plasma membrane regulates EPO expression in hypoxia, including HIF-1 activation and translocation. We further studied the expression of NPR and VEGF mRNAs in human tumor tissues and found that the NPR mRNA levels were correlated with the VEGF mRNA levels, suggesting that NPR might be an important factor in the hypoxic induction of genes such as VEGF in vivo.
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PMID:NADPH-cytochrome P-450 reductase in the plasma membrane modulates the activation of hypoxia-inducible factor 1. 1197 99

We investigated the effects of vascular endothelial growth factor (VEGF) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in human microvascular endothelial cells (HMEC-1). Treatment of HMEC-1 with VEGF resulted in a dose- and time-dependent up-regulation of COX-2 mRNA and protein levels. This up-regulation was accompanied by a 1.6-fold increase in PGE(2) synthesis. Pretreatment of HMEC-1 with a selective COX-2 inhibitor, NS-398, abolished VEGF-induced PGE(2) synthesis, suggesting specific up-regulation of COX-2 activity by VEGF in HMEC-1. Transient transfection assays using deletion mutants of the human COX-2 promoter fused to the luciferase reporter gene indicated critical requirement of a regulatory region spanning -828/-123 bp for VEGF induction of COX-2 promoter activity in HMEC-1. Site-directed mutation analysis demonstrated that a GATA cis-acting element at -685/-680 bp was essential for VEGF- induced COX-2 promoter activity in HMEC-1. These observations are of particular importance given the recent demonstrations of critical requirement of COX-2 isoenzyme for tumor growth and angiogenesis.
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PMID:Vascular endothelial growth factor up-regulates cyclooxygenase-2 expression in human endothelial cells. 1210 71


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