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)

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.
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PMID:Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens. 140 42

Since we have observed effects of growth factors and cAMP as well as estradiol (E2) on regulation of expression of some genes stimulated by the estrogen receptor (ER), we have undertaken studies to examine directly whether activators of protein kinases can modulate transcriptional activity of the ER. We find that activators of protein kinase-A [cholera toxin plus 3-isobutyl-1-methylxanthine (CT+IBMX)] and protein kinase-C [12-O-tetradecanoylphorbol-13-acetate (TPA)] markedly synergize with E2 in ER-mediated transcriptional activation. When a reporter plasmid [with a minimal promoter containing a TATA region and estrogen-responsive elements (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene] was transfected into MCF-7 human breast cancer cells, which contain endogenous ER, E2 evoked a dose-dependent increase in CAT activity. While treatment with protein kinase-A or -C activator alone evoked only very low CAT activity, the maximal (approximately 25-fold) CAT activity stimulated by E2 alone was increased 2- to 3-fold (to approximately 60 times the control value) upon cotreatment with either of the protein kinase activators. Interestingly, antiestrogen abolished all of the CAT activity induced by E2 and protein kinase activators. Immunoblots showed that TPA reduced ER levels to 30% of control values after 24 h, while CT+IBMX increased levels about 1.5-fold. Scatchard binding analysis revealed no change in the binding affinity of E2 to ER by these agents. Gel mobility shift competition assays with extracts prepared from cells that had been treated with E2 and protein kinase activators did not reveal any quantitative or qualitative changes in the binding of ER to the ERE in vitro. In ER-deficient Chinese hamster ovary (CHO) cells transfected with the reporter gene and varying amounts of an ER expression vector, the level of CAT activity obtained by cotreatment with E2 and CT+IMBX was 3-fold higher than that observed with E2 alone over the range of different ER amounts tested. This ER-mediated synergism was still retained in an amino-terminal A/B-domain-deleted ER mutant lacking the hormone-independent transcriptional activation function (TAF-1), but was greatly reduced in two hormone-binding domain (region E) mutants that exhibit significantly diminished ligand-dependent transcriptional activation. TPA did not show any synergistic activation with E2 in CHO cells, indicating differences in responses between cell types.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. 768 75