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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.
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PMID:Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor. 1076 10

Disruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-beta1) has also been shown to trigger the reactivation of EBV in Burkitt lymphoma cell lines; however, the effect of TGF-beta1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-beta1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-beta1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-beta1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-beta1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests that TGF-beta1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-beta1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-beta1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-beta1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-beta1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-beta1 induces BZLF1 expression by an indirect mechanism.
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PMID:Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway. 1084 60

Decorin is a small leucine-rich extracellular matrix proteoglycan, the expression of which is down-regulated in proliferating and malignantly transformed cells. In the present study we show that the expression of decorin in fibroblasts is suppressed by epidermal growth factor (EGF) and PMA, and that the effect of both is potently inhibited by blocking the extracellular signal-regulated protein kinase (ERK)1,2 signalling pathway (Raf/MEK1,2/ERK1,2) with the specific MAPK/ERK kinase (MEK)1,2 inhibitor, PD98059. In addition, specific activation of ERK1,2 by adenovirus-mediated expression of constitutively active MEK1 in dermal fibroblasts results in marked reduction in decorin mRNA abundance and production. Co-transfection of NIH-3T3 fibroblasts with human decorin promoter/chloramphenicol acetyltransferase (CAT) construct (pDEC--879/CAT) in combination with the expression vectors for constitutively active Raf-1 and MEK1 markedly suppressed decorin promoter activity. Co-transfections of human decorin promoter 5'-deletion constructs with constitutively active MEK1 expression vector identified the region -278 to -188 as essential for ERK1,2 mediated down-regulation of decorin promoter activity. These results show that activation of the ERK1,2 signalling pathway by a mitogenic growth factor, a tumour promoter or transformation suppresses decorin gene expression in fibroblasts, which in turn may promote proliferation and migration of normal and malignant cells.
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PMID:Activation of extracellular signal-regulated protein kinase1,2 results in down-regulation of decorin expression in fibroblasts. 1086 Dec 6

Vascular Endothelial Growth Factor (VEGF)/Vascular Permeability Factor plays an important role in angiogenesis and cell proliferation of cancer cells. Glioblastoma cells are most malignant and show resistance to radiation therapy inducing VEGF to cause angiogenesis and brain edema. In the present study, the regulatory mechanism of the expression of VEGF by ionizing radiation was studied in three human glioblastoma cells. Induction of VEGF mRNA by ionizing radiation was dependent on dose and incubation time. Activator protein-1 (AP-1) was activated by 10 Gy of ionizing radiation in 1 h in T98G glioblastoma cells on an electrophoretic mobility shift assay. We constructed chimeric genes containing various regions of the VEGF promoter gene and the coding region for chloramphenicol acetyltransferase (CAT) and transiently transfected them to T98G cells. CAT assay with the VEGF promoter gene containing an AP-1 site demonstrated that the promoter activity of the VEGF gene was enhanced by ionizing radiation. Immunological analysis of the activity of mitogen-activated protein kinase, ERK1/2, showed that this activity is up-regulated by ionizing radiation. These results suggest that ERK1/2 pathway is involved in the up-regulation of VEGF expression ionizing radiation mediated by AP-1, which may lead to further neovascularization and proliferation of glioblastoma cells resistant to radiation therapy.
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PMID:Mitogen-activated protein kinase, ERK1/2, is essential for the induction of vascular endothelial growth factor by ionizing radiation mediated by activator protein-1 in human glioblastoma cells. 1088 23

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.
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PMID:Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities. 1114 17

By use of cDNA array technology we have screened 588 genes to determine the effect of autocrine production of human growth hormone (hGH) on gene expression in human mammary carcinoma cells. We have used a previously described cellular model to study autocrine hGH function in which the hGH gene or a translation-deficient hGH gene was stably transfected into MCF-7 cells. Fifty two of the screened genes were regulated, either positively () or negatively (), by autocrine production of hGH. We have now characterized the role of one of the up-regulated genes, chop (gadd153), in the effect of autocrine production of hGH on mammary carcinoma cell number. The effect of autocrine production of hGH on the level of CHOP mRNA was exerted at the transcriptional level as autocrine hGH increased chloramphenicol acetyltransferase production from a reporter plasmid containing a 1-kilobase pair fragment of the chop promoter. The autocrine hGH-stimulated increase in CHOP mRNA also resulted in an increase in CHOP protein. As a consequence, autocrine hGH stimulation of CHOP-mediated transcriptional activation was increased. Stable transfection of human CHOP cDNA into mammary carcinoma cells demonstrated that CHOP functioned not as a mediator of hGH-stimulated mitogenesis but rather enhanced the protection from apoptosis afforded by hGH in a p38 MAPK-dependent manner. Thus transcriptional up-regulation of chop is one mechanism by which hGH regulates mammary carcinoma cell number.
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PMID:Autocrine human growth hormone (hGH) regulation of human mammary carcinoma cell gene expression. Identification of CHOP as a mediator of hGH-stimulated human mammary carcinoma cell survival. 1129 45

The reduction in levels of the potentially toxic amyloid-beta peptide (Abeta) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Abeta precursor protein (betaAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces betaAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in betaAPP processing. Phenserine treatment resulted in decreased secretion of soluble betaAPP and Abeta into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of betaAPP protein expression by phenserine was posttranscriptional as it suppressed betaAPP protein expression without altering betaAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5'-mRNA leader sequence of betaAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Abeta levels by regulating betaAPP translation via the recently described iron regulatory element in the 5'-untranslated region of betaAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of betaAPP expression that can result in a substantial reduction in the level of Abeta.
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PMID:Phenserine regulates translation of beta -amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development. 1140 70

The GR is a hormone-activated transcription factor that acts to regulate specific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat shock proteins. As heat shock proteins are produced by cells as an adaptive response to stress, speculation has existed that communication between the heat shock and glucocorticoid hormone signal pathways must exist. Only recently has evidence to support this hypothesis been reported. In almost all cases, the evidence has been of an ability of heat shock to cause a potentiation of the glucocorticoid hormone response. In this proposal, evidence is now presented that heat shock signaling can, in turn, be regulated by glucocorticoids. In mouse L929 cells stably expressing a chloramphenicol acetyltransferase reporter controlled by the human heat shock protein70 promoter and containing known binding sites for heat shock transcription factor 1 treatment with glucocorticoid agonist (dexamethasone) results in a dose-dependent decrease of stress-induced chloramphenicol acetyltransferase gene expression. In these cells, inhibition of heat shock protein70 promoter activity by dexamethasone was completely blocked by GR antagonist (RU486). Similar treatment of L929 cells stably expressing a chloramphenicol acetyltransferase reporter under the control of the constitutively active SV40 promoter showed no such inhibition by dexamethasone. More importantly, dexamethasone was also found to inhibit heat shock-induced expression of the major heat shock proteins-heat shock proteins70, 90, and 110. Thus, the inhibitory effect of dexamethasone appears to apply to most, if not all, heat shock transcription factor 1-regulated genes. Although dexamethasone did not prevent the DNA-binding function of heat shock-activated heat shock transcription factor 1, it did inhibit a constitutively active mutant of human heat shock transcription factor 1 under nonstress conditions, suggesting that dexamethasone repression of heat shock transcription factor 1 was primarily through an inhibition of heat shock transcription factor 1 transcription enhancement activity. To more accurately characterize the stage of GR signaling responsible for inhibition of heat shock transcription factor 1, a series of Chinese hamster ovary cells containing either no GR, wild-type mouse GR, or single-point mutations of GR were employed. Dexamethasone inhibition of heat shock-induced heat shock transcription factor 1 activity was observed in the presence of wild-type GR, but not in Chinese hamster ovary cells lacking GR, suggesting that signaling cascades other than GR were not involved in this effect of dexamethasone. Consistent with this conclusion was the observation that dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or c-jun N-terminal kinase), which are known to negatively regulate heat shock transcription factor 1. Dexamethasone inhibition of heat shock transcription factor 1 was not seen in Chinese hamster ovary cells expressing GR defective for DNA-binding function. Moreover, dissociation of GR/Hsp90/Hsp70 complexes was observed in response to hormone for both the wild-type and DNA binding-defective forms of GR, demonstrating that release of Hsp90 or Hsp70 (both of which are known to keep heat shock transcription factor 1 in its inactive state) could be ruled out as a potential mechanism. Thus, it appears that GR-mediated transactivation or transrepression is required for the inhibitory effect of dexamethasone on heat shock transcription factor 1 activity. Taken as a whole, these results provide evidence for a novel mechanism of cross-talk in which signaling by the GR can attenuate the heat shock response in cells through an inhibition of the transcription enhancement activity of HSF1.
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PMID:Inhibition of heat shock transcription factor by GR. 1146 62

Endothelin-1 (Et-1) is a peptide synthesized by endothelial cells (ECs) both in culture and in vivo. Cyclic strain induces gene expression of Et-1, however, the molecular mechanisms remain unclear. Since cyclic strain induces a sustained increase in intracellular reactive oxygen species (ROS), we hypothesized that the ROS could be a modulator in strain-induced Et-1 gene expression. Human umbilical vein ECs (HUVECs) subjected to cyclic strain had increased Et-1 secretion. Pretreatment of HUVECs with antioxidants, catalase (300 U/ml) or 1,3-dimethyl-2-thiourea (DMTU, 0.1 mm), abolished the strain-induced Et-1 release. ECs strained for 6 h had elevated Et-1 mRNA levels. In contrast, ECs treated with catalase or DMTU did not have increase Et-1 mRNA levels stimulated by cyclic strain. Bovine aortic ECs (BAECs) transfected with fusion plasmid containing Et-1 5'-flanking sequence (4.4 kb) and chloramphenicol acetyltransferase reporter gene produced a maximal Et-1 promoter activity after undergoing strain for 6 h, whereas pretreatment with catalase decreased this activity. BAECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or catalytically inactive mutant of extracellular signal-regulated kinase (mERK2) had inhibited strain-induced Et-1 promoter activity, indicating the Ras/Raf/ERK pathway was involved; moreover, ERK phosphorylation was induced in ECs which were strained. This strain-activated ERK phosphorylation was attenuated in the presence of catalase. Functional analysis of the Et-1 promoter with site-directed mutagenesis indicates that the activator protein-1 (AP-1) binding site had to be within 143 base-pairs upstream of transcription initiation site for strain-induced promoter activity. Pretreatment of ECs with catalase also decreased the strain-induced promoter activity in the minimal construct (-143 bp). Our data demonstrate that strain-induced Et-1 gene expression is modulated by ROS via Ras/Raf/ERK signaling pathway, and indicate the responsiveness of the AP-1 binding site for strain-induced Et-1 expression.
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PMID:Reactive oxygen species mediate cyclic strain-induced endothelin-1 gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in endothelial cells. 1160 23

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.
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PMID:Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1. 1199 34


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