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)

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor whose ability to bind hormone is thought to be dependent on association with the 90-kDa heat shock protein (hsp90). In the present study, we have generated a novel form of the GR, in which the receptor remains complexed to hsp90 but has lost its ability to bind hormone, by treatment of intact cells with the calmodulin (CaM) antagonist phenoxybenzamine (POBA). Treatment of these cells, mouse L929 cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter construct, with increasing concentrations of POBA resulted in a concentration-dependent inhibition of dexamethasone (Dex)-induced CAT gene expression, with 100 microM POBA resulting in approximately 80% inhibition. This inhibitory effect of POBA was markedly reduced if POBA was added after a short incubation with Dex, suggesting that the primary effect of POBA was on hormone-induced transformation of the GR. Using a subcellular fractionation technique, POBA inhibition of CAT gene expression was found to correlate with an inhibition of Dex-induced GR nuclear translocation. However, inhibition of translocation was not the primary effect of POBA on the GR signal pathway, as POBA was found to reduce GR hormone-binding capacity after treatment of intact cells. The inhibitory effect of POBA on hormone-binding function correlated closely with the inhibitory effect of this drug on CAT gene expression and was not due to an oxidation of sulfhydryl groups, a condition known to reduce GR hormone-binding capacity. Incubation of cytosols from untreated cells with POBA did not decrease GR steroid-binding capacity, demonstrating that this inhibitory effect was not the result of a competitive antagonism at the ligand-binding site. Quantitation of GR protein in the cytosols of POBA-treated cells revealed that the decrease in steroid-binding function was not due to a loss of GR protein. Surprisingly, the amount of GR-bound hsp90 was also unaltered in response to POBA. Taken together, the above observations provide evidence for a novel state of the GR within intact cells in which hsp90 interaction is but one step in the generation or maintenance of hormone-competent receptors. In addition, these results point to the potential use of POBA, and possibly other CaM inhibitors, as antagonists of steroid receptor actions.
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PMID:In vivo evidence for the generation of a glucocorticoid receptor-heat shock protein-90 complex incapable of binding hormone by the calmodulin antagonist phenoxybenzamine. 883 41

To identify new receptor tyrosine kinases (RTKs), we screened cDNAs from mouse mammary tumor cells and mouse brain. A homology search of the complete cDNA sequences obtained showed that one cDNA was a murine homologue of recently reported human sky [Ohashi, K. et al. (1994) Oncogene 9, 699-705]. Another cDNA obtained was also related to sky but had a 5' upstream sequence similar to brt [Fujimoto, J. and Yamamoto, T. (1994) Oncogene 9, 693-698]. Analysis of the 5' region of the sky genomic DNA revealed that brt-type and sky-type sequences are encoded by the sky gene in different exons. The upstream region of the sky-type coding exon is highly GC-rich and contains potential recognition sites for the Sp1 trans-acting factor, but lacks TATA and CAAT boxes, features commonly found in promoters of other RTKs. To examine whether this upstream region functions as a promoter, we fused it with chloramphenicol acetyltransferase (CAT) gene and transfected the construct into COS-7 cells. The results of the CAT assay showed that the sky upstream region retains a significant promoter activity. Furthermore, primer extension analysis revealed that the transcription starts at -240 nt upstream from the sky translation initiation codon. These observations suggest that the brt- and sky-types of mRNA are transcribed from a single sky gene by an alternative promoter usage.
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PMID:Structure and expression of a murine homologue of sky receptor tyrosine kinase gene. 888 9

The ability of glucocorticoids (GCs) to induce death in lymphoid-origin cells is the basis for their frequent use in the therapy of various human hematological malignancies. However, the occurrence of primary or secondary GC resistance limits their clinical usefulness. Prior investigations into the mechanism of GC resistance in established human leukemic cell lines revealed loss-of-function mutations in the GC receptor (GR) gene. In this study, we analyzed the GC-resistant human acute T-cell leukemia line CEM-C1, which has been reported to express biochemically functional GR and, thus, was thought to owe its GC resistance to signal transduction changes distal from the GR. Radioligand binding assays revealed a 2-3-fold lower expression of GR in CEM-C1 than in the GC-sensitive sister cell line CEM-C7H2. Analysis of transcriptional activity using mouse mammary tumor virus-long terminal repeat-controlled chloramphenicol acetyltransferase expression in transient transfection assays confirmed the expression of functional GR in CEM-C1 but at levels lower than those in CEM-C7H2 cells. Upon molecular analyses of the GR gene and its transcripts, we found that CEM-C1 cells were heterozygous for the ligand binding domain L753F point mutation in exon 9, which is also present in GC-sensitive CEM-C7H2. No mutations, however, were found on the second GR allele of CEM-C1. To test the possibility that resistance in CEM-C1 cells might be caused by insufficient expression of GR, we established several cell lines stably transfected with rat GR expression vectors. These cell lines differed in exogenous GR expression as determined by Northern blotting and radioligand binding assays. The GR expression level in individual lines correlated well with their sensitivity to GC-induced apoptosis. Thus, GC resistance of CEM-C1 cells might be due to subthreshold expression of functional GR rather than defects in signal transduction pathways distal from the GR. Since several clinical investigations showed a correlation between reduced GR expression and poor response to GC-containing treatment, the CEM-C1 line may represent a valid model for GC resistance in human acute T-cell leukemia.
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PMID:Resistance to glucocorticoid-induced apoptosis in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression. 889 60

We analysed the glucocorticoid receptor (GR) function and its ability to modulate cell-cell interactions between the PA-III rat prostate cancer and UMR 106 osteoblast-like rat osteosarcoma cells as an in vitro model for studying GR function in PA-III cell-induced tumor and blastic reaction in rat bone. Intact GR was detected by ligand binding assays, DNA band-shift, and GR trans-activation analysis of PA-III and UMR 106 cells transiently transfected with the mouse mammary tumor virus thymidine kinase-chloramphenicol acetyltransferase reporter gene. Dexamethasone and transforming growth factor beta 1 (TGFbeta1) inhibited the growth of PA-III and UMR 106 cells. Dexamethasone's inhibition of PA-III and UMR 106 cells was reversed by anti-TGFbeta1 antibody and exogenous insulin-like growth factor I (IGF-I). Exogenous IGF-I, urokinase-type plasminogen activator (uPA), UMR 106 conditioned media (CM) and PA-III CM stimulated the proliferation of PA-III and UMR 106 cells. The mitogenic activity exerted by uPA and PA-III CM in UMR 106 cells was completely neutralized by anti-IGF-I specific antibody. In addition, dexamethasone up-regulated TGFbeta1 mRNA and down-regulated uPA mRNA expression in PA-III cells without affecting TGFbeta1 and uPA mRNA expression in UMR 106 cells. These data suggested that TGFbeta1, uPA, and IGF-I mediate at least in part cell-cell interactions and GR function in PA-III prostate cancer and UMR 106 osteosarcoma cells.
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PMID:Glucocorticoid receptor function possibly modulates cell-cell interactions in osteoblastic metastases on rat skeleton. 917 22

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.
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PMID:The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression. 927 5

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that in its unactivated form resides primarily in the cytoplasm. After being bound by steroid, the GR undergoes a conformational change and translocates to the nucleus, where it influences gene transcription. Because the GR mediates negative feedback exerted by circulating glucocorticoid hormones on the hypothalamic-pituitary-adrenal (HPA) axis, it has been hypothesized that abnormalities in GR expression and/or function may underlie the HPA axis hyperactivity described in patients with major depression. In further support of this hypothesis, animal studies have shown that long term in vivo treatment with antidepressants enhances glucocorticoid feedback inhibition, possibly through a direct effect on the GR. To examine this latter possibility, we evaluated translocation of the GR from the cytoplasm to the nucleus after 24-hr in vitro treatment of L929 cells (mouse fibroblasts) with the tricyclic antidepressant desipramine (0.1-10 microM) in the presence or absence of the synthetic steroid dexamethasone. In addition, GR-mediated gene transcription was measured with the use of L929 cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene. Desipramine was found to (i) induce GR translocation from the cytoplasm to the nucleus in the absence of steroids (with no effect alone on GR-mediated gene transcription) and (ii) potentiate dexamethasone-induced GR translocation and dexamethasone-induced GR-mediated gene transcription. Treatment with desipramine for 24-96 hr had no effect on the expression of GR protein as measured by cytosolic radioligand receptor binding. We suggest that one important aspect of the effects of antidepressants in vivo may be to facilitate GR-mediated feedback inhibition on the HPA axis, by facilitating GR translocation and function, and thereby reverse glucocorticoid hypersecretion in depression.
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PMID:Steroid-independent translocation of the glucocorticoid receptor by the antidepressant desipramine. 938 19

The hormone regulation of viruses has been of great interest since the discovery of glucocorticoid stimulation of mouse mammary tumor virus via a hormone response element in the viral long terminal repeat (LTR) promoter region. This report describes the investigation of the hormone responsiveness of bovine leukemia virus (BLV), an oncogenic retrovirus that infects dairy and beef cattle worldwide. It is a member of the human T cell leukemia (HTLV)/BLV group of retroviruses, which encode a protein, Tax, that is essential for regulating transcription of their own proviruses and for transforming host cells. We investigated the responsiveness of BLV to the hormones 17 beta-estradiol, progesterone, prolactin, insulin, and dexamethasone, a potent glucocorticoid. Only dexamethasone, in combination with insulin or insulin/prolactin, consistently stimulated BLV expression, as measured by reverse transcriptase activity, RNA blot hybridization (Northern blots), and CAT (chloramphenicol acetyltransferase) reporter assays of cell lines transiently or stably transfected with the BLV LTR. This effect required the presence of glucocorticoid receptors and Tax. This is the first report of hormone responsiveness in a virus of the HTLV/BLV group.
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PMID:Hormone regulation of bovine leukemia virus via the long terminal repeat. 943 16

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.
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PMID:Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells. 956 51

Tannic acid, which comprises polyphenolic compounds from tea leaves, suppresses the glucocorticoid-induced gene expression of mouse mammary tumor virus (MMTV) integrated into 34I cells. To investigate whether this suppression is due to promoter responsiveness to tannic acid, we performed chloramphenicol acetyltransferase analysis transfecting a MMTV promoter containing a chloramphenicol acetyltransferase expression vector into mouse fibroblast L929 cells. Deletion analysis of the promoter region revealed that a 50-base pair (bp) region located downstream of the TATA element is responsible for the suppressive effect of tannic acid. The tannic acid-sensitive suppressibility was introduced into a thymidine kinase promoter by inserting the 50-bp region into the region on the 5'-upstream side of the promoter. Detailed point mutation analyses revealed that two elements, a 13-bp element and an ACTG motif in the 50-bp region, contribute to tannic acid sensitivity and promoter repressibility, respectively. Interestingly, this repressive ACTG motif is found in the human immunodeficiency virus promoter, the activity of which is also suppressed by tannic acid (Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T., and Tanuma, S. (1996) Biochem. Biophys. Res. Commun. 220, 411-417). Furthermore, electrophoretic mobility shift analysis revealed that a protein factor(s) in nuclear extracts from L929 cells binds to the 50-bp region in a sequence-specific manner and that the amount of DNA-protein complex is increased by tannic acid treatment. Moreover, the negative regulatory sequence ACTG and the tannic acid-sensitive 13-bp element in this region were shown to be responsible for the formation of the DNA-protein complex by electrophoretic mobility shift analysis and footprint analyses. These findings suggest that the suppressive effect of tannic acid on MMTV gene expression is mediated by a protein factor(s) that binds to the negative regulatory element containing the common ACTG motif in a cooperative manner with the tannic acid-sensitive 13-bp element.
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PMID:Identification and characterization of a tannic acid-responsive negative regulatory element in the mouse mammary tumor virus promoter. 957 8

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liver in vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between -1.6 and -1.1 kb of the 5'-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20-30%; no effect of GC was observed with CAT constructs containing -1.15 kb of the 5'-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5'-flanking region. We conclude that pGRE and half-site GREs of gstA2 participate in regulation of this gene; however, a second unidentified responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.
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PMID:Negative regulation of the rat glutathione S-transferase A2 gene by glucocorticoids involves a canonical glucocorticoid consensus sequence. 961 3


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