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)

Many transformed mouse lung cells, including LM2 cells, contain activating mutations in the Ki-ras gene and show reduced responsiveness to growth inhibition by glucocorticoids. LM2GR cells, which are LM2 cells stably transfected with a rat glucocorticoid receptor (GR) gene, were used to determine whether increasing glucocorticoid responsiveness can influence aspects of the transformed phenotype. LM2GR cells grew slower and had a lower final saturation density than the parental LM2 cells. Expression of growth-related genes was examined by northern blot analysis. The cells were serum-deprived and treated with fetal bovine serum (FBS), steroid-stripped FBS (ssFBS), dexamethasone, or 12-O-tetradecanoylphorbol-13-acetate. The level and pattern of Ki-ras mRNA expression was similar in both LM2 and LM2GR cells, but histone H4 mRNA was expressed in a more regulated fashion in LM2GR cells. The induction of c-jun and c-fos mRNA expression lasted longer in the LM2GR cells treated with ssFBS; however, the maximal induction was greater in the LM2 cells treated with FBS. LM2GR cells demonstrated similar activator protein-1 (AP-1) activity but higher GR activity than LM2 cells as determined by using AP-1-chloramphenicol acetyltransferase (CAT) and mouse mammary tumor virus-CAT transient transfection assays, consistent with the higher level of GR mRNA in LM2GR cells. Both cell lines exhibited the ability to grow in soft agar and to form tumors in nude mice. These results indicate that introduction of a functional GR transgene into LM2 cells can increase glucocorticoid responsiveness and alter the expression of genes involved in growth regulation but cannot overcome anchorage-independent cell growth or tumorigenicity, apparently because of the presence of an activated Ki-ras gene.
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PMID:Effect of increased glucocorticoid responsiveness in transformed mouse lung cells. 761 16

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and -176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and -132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is beta (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cells, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis. Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.
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PMID:CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter. 762 3

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.
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PMID:Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures. 764 13

It has recently been discovered that the steroid receptor-associated heat shock protein, hsp56, belongs to the FK506 family of immunophilin proteins. The ability of hsp56 to bind the immunosuppressive macrolide FK506 has led to the speculation that the steroid receptor and immunophilin signal transduction pathways are functionally interrelated. We have tested this idea by assessing the effects of FK506 on glucocorticoid receptor (GR)-mediated expression of the murine mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. We report that combined treatment with FK506 and low concentrations of dexamethasone (10(-8) or 10(-7) M) results in a large enhancement of MMTV-CAT gene expression over that seen in response to dexamethasone (Dex) alone. FK506 potentiation of MMTV-CAT expression did not occur at 10(-6) M Dex or in the complete absence of hormone. We also show that potentiation of Dex-mediated MMTV-CAT expression occurs in response to rapamycin, that glucocorticoid-regulated enhancer sequences are sufficient for the FK506-mediated potentiation effect, and that this effect can be blocked by RU486 antagonist. Finally, we provide evidence that FK506 potentiation of GR-mediated gene expression is the result of increased translocation to the nucleus of the GR.
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PMID:Potentiation of glucocorticoid receptor-mediated gene expression by the immunophilin ligands FK506 and rapamycin. 768 Oct 58

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.
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PMID:Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene. 770 74

The effect of heat shock on the transcriptional activity of glucocorticoid receptor was assessed using HeLa cells stably transfected with the chloramphenicol acetyltransferase (CAT) gene the transcription of which is controlled by two glucocorticoid-responsive elements placed directly upstream of a core promoter. Heat shock inactivated the high-affinity glucocorticoid binding capacity of the cells and nullified the rate of accumulation of CAT mRNA in the presence of hormone. Hormonal responsiveness was restored on return to normal temperature concomitantly with recovery of high-affinity glucocorticoid binding capacity. Heat inactivation of the receptor was coincident with loss of its solubility and apparently unrelated to receptor degradation.
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PMID:Temporary loss of glucocorticoid receptor-mediated regulation of gene expression in heat-shocked cells. 772 19

We investigated the role of glucocorticoids in controlling the proliferation of androgen-independent PC-3 human prostate cancer cells via the action of transforming growth factor beta 1 (TGF beta 1). The presence of glucocorticoid receptor (GR) in PC-3 cells was detected by immunoblotting analysis using a rabbit anti-GR polyclonal antibody against the synthetic human GR peptide (hGR383-393). In PC-3 cells, GR bound radiolabeled dexamethasone with an affinity similar to wild-type GR. In addition, GR-ligand complex bound radiolabeled DNA as detected by DNA band-shift analysis on gel electrophoresis and trans-activated the mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase chimeric gene in transiently transfected PC-3 cells. Dexamethasone (0.1 up to 100 nM) and TGF beta 1 (0.5 up to 50 ng/ml) inhibited PC-3 cell proliferation. TGF beta 1 and dexamethasone both increased the distribution of PC-3 cells into the G1/G0 phase of the cell cycle. Platelet-derived growth factor (PDGF) stimulated the proliferation of PC-3 cells and overcame dexamethasone's inhibition of PC-3 cell growth. Dexamethasone's inhibition (10(-7) M) of PC-3 cell growth was completely neutralized by RU 486 (10(-6)M) and partly neutralized by anti-TGF beta 1 polyclonal antibody. Furthermore, dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells. Because dexamethasone's inhibition was neutralized at least in part by an anti-TGF beta 1 polyclonal antibody and dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells, we conclude that GR function in human PC-3 prostate cancer cells is mediated at least in part by TGF beta 1 expression.
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PMID:Mediation of glucocorticoid receptor function by transforming growth factor beta I expression in human PC-3 prostate cancer cells. 775 11

We have developed a simple and highly sensitive tissue culture-based assay for the biological activity of steroids and synthetic steroidal compounds. A DNA cassette, containing a synthetic steroid-inducible promoter controlling the expression of a bacterial chloramphenicol acetyltransferase gene (GRE5-CAT), was inserted into an Epstein-Barr virus (EBV) episomal vector which replicates autonomously in primate and human cells. We then used this promoter/reporter system to generate two stably transfected human cell lines. In the cervical carcinoma cell line HeLa, which expresses high levels of glucocorticoid receptor, the GRE5 promoter is inducible over 100-fold by the synthetic glucocorticoid dexamethasone. In the breast carcinoma cell line T47D, which expresses progesterone and androgen receptors, the GRE5 promoter is inducible over 100-fold by either progesterone or dihydrotestosterone. In both cell lines basal expression of CAT activity is strictly dependent on the presence of steroid, so that very low levels of induction can be detected. Thus, the cell lines can be used to test for low levels of agonist activity in steroid antagonists. These cell lines can be used to screen compounds for steroid agonist or antagonist activity by testing extracts of cells grown in microtiter wells directly using a colorimetric CAT assay. This system should provide a sensitive and efficient method for screening and analysis of the activity of large numbers of natural or synthetic steroid agonists or antagonists.
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PMID:A simple and sensitive high-throughput assay for steroid agonists and antagonists. 776 3

We analyzed glucocorticoid receptor function using ligand binding assays, DNA band-shift analysis and trans-activation of the murine mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase reporter gene in transiently transfected MG-63 human osteosarcoma cells. Dexamethasone increased the distribution of MG-63 cells in the G1/G0 phase of the cell cycle, thus decreasing the rate of DNA synthesis and cell growth. Its effect on MG-63 cell growth was neutralized by RU486 and anti-transforming growth factor beta 1 (TGF beta 1) antibody. In addition, (i) dexamethasone increased the levels of active TGF beta 1 in MG-63-conditioned media without significantly altering the expression of TGF beta 1 mRNA in MG-63 cells and (ii) TGF beta 1 inhibited proliferation of MG-63 cells. Therefore, we conclude that glucocorticoid receptor function is mediated by the activation of latent-TGF beta 1 in MG-63 osteosarcoma cells.
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PMID:Mediation of glucocorticoid receptor function by the activation of latent transforming growth factor beta 1 in MG-63 human osteosarcoma cells. 776 43

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.
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PMID:Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors. 782 16


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