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)

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

Most oral contraceptives (OC) contain a progestin in combination with an estrogen, and the progestin component in OC includes one of the following 19-nortestosterone derivatives: norethynodrel; norethindrone; or norgestrel (levonorgestrel). It is well known that estrogens promote the growth of breast cancer. However, progestins have recently also been implicated in the development of breast cancer. We have compared and contrasted the ability of synthetic progestins to stimulate the proliferation of cultured human breast cancer cells and examined their possible mechanism of action. We found that some progestins used in OC were able to stimulate the growth of estrogen receptor-positive (ER+) MCF-7 and T47DA18 human breast cancer cells but not ER- MDA-MB-231, BT-20, and T47DC4 human breast cancer cells. However, two other progestins, MPA and R5020, which are not used in OC, were either not able to stimulate or only slightly stimulated growth. The potency of norethynodrel [median effective dose (EC50) = 4 x 10(-8) M] and norethindrone (EC50 = 3 x 10(-8) M) was greater than norgestrel (EC50 = 2 x 10(-7) M) in MCF-7 cells. E2 (EC50 = 8 x 10(-13) M) was an even more potent stimulator of growth. More importantly, the progestin-induced growth stimulation was blocked by the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-estra-4, 9-dien-3-one (RU486). To determine whether the proliferative action of progestins was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene containing an estrogen response element derived from vitellogenin 2A gene. The progestins which stimulated the growth of breast cancer cells also increased chloramphenicol acetyltransferase activity. The induction of chloramphenicol acetyltransferase activity was blocked by the addition of the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin RU486. This study provides direct evidence that the 19-nortestosterone derivatives in OC have estrogenic properties and suggests that activation of ER, but not progesterone receptor, is the growth-stimulatory mechanism for these synthetic progestins. Our results may help to explain the conflicting evidence linking OC and breast cancer risk. A rigorous evaluation of the "total" estrogenic potential of OC might produce a better correlation with breast cancer risk.
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PMID:Estrogenic potential of progestins in oral contraceptives to stimulate human breast cancer cell proliferation. 142

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).
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PMID:Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor. 157 85

The genomic DNA of a vaccine strain of canine adenovirus type 2 (Vaxitas; ICI Tasman) has been shown to contain two copies of the E1a region, the second being at the far right end of the genome. DNA sequence analysis of the right terminal 2.8 kbp of this vaccine strain showed that numerous point mutations have occurred in the second copy, which would preclude the synthesis of any functional products. However, expression vectors in which the E1a promoter from the right terminus were linked to the chloramphenicol acetyltransferase gene showed that the promoter was fully functional. Furthermore, the activity of the reiterated E1a promoter was considerably greater than that of the normal E4 promoter. This dramatic change in the regulation of E4 expression may be an important factor in determining the altered host cell specificity displayed by this vaccine strain virus.
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PMID:A mutant of canine adenovirus type 2 with a duplication of the E1a region exhibits altered expression of early region 4. 183 8

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.
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PMID:Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells. 246 Jul 49

We have previously reported that addition of 8-bromocyclic AMP enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells in vitro. Isoproterenol is known to stimulate the synthesis of hepatic intracellular cyclic AMP via beta-adrenergic receptors. To study the possible effect of beta-adrenergic receptors on the expression of the angiotensinogen gene in mouse hepatoma cells, we transiently transfected them with a fusion gene with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase coding sequence as a reporter, pOCAT (ANG N-1498/+18). The addition of isoproterenol (10(-9) to 10(-5) mol/L) alone had no stimulatory effect on the expression of pOCAT (ANG N-1498/+18). In the presence of dexamethasone (10(-6) mol/L), however, isoproterenol enhanced the stimulatory effect on the dexamethasone on the expression of pOCAT (ANG N-1498/+18). The enhancing effect of isoproterenol was inhibited by the presence of propranolol (beta 1- and beta 2-adrenergic receptor antagonist) and ICI 118,551 (beta 2-adrenergic receptor antagonist) but not by the presence of atenolol (beta 1-adrenergic receptor antagonist). Furthermore, the addition of Rp-cAMP (an inhibitor of protein kinase A I and II) blocked the enhancing effect of isoproterenol. These studies demonstrated that isoproterenol enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells via beta 2-adrenergic receptor and cyclic AMP-dependent protein kinase pathways. Our data may be important in understanding the molecular mechanism(s) of the stimulatory effect of catecholamines/glucocorticoid-induced expression of the angiotensinogen gene in the liver.
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PMID:Beta-adrenergic receptors and angiotensinogen gene expression in mouse hepatoma cells in vitro. 784 40

The estrogen receptor (ER) typically activates gene transcription by binding to estrogen-responsive elements (EREs). The brain creatine kinase (BCK) promoter is responsive to estrogen but contains no ERE-related sequence. To investigate the mechanism of estrogen induction, we have introduced the estrogen receptor into HeLa cells and primary rat cardiomyocytes and fibroblasts along with 195 bp of BCK promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene. A 10-fold stimulation of CAT activity was observed in the presence of beta-estradiol in both HeLa and rat primary fibroblasts, but no induction was observed in primary rat cardiomyocytes. In contrast, a control vitellogenin gene construct which contains a typical ERE was induced in an ER-dependent manner in all cell types studied. Estrogen induction in HeLa was not sensitive to cycloheximide and was blocked by the ER antagonists tamoxifen and ICI 164,384. Analysis of 5' deletion and linker-scanning mutations indicates sequences between bp -45 and -75 including a TA-rich sequence and a CCAAT sequence to be crucial for stimulation of the BCK promoter by the ER. BCK estrogen induction is dependent on the DNA-binding domain and transactivation domain TAF2 of the ER. However, direct DNA binding is probably not required. Taken together, these results suggest a novel mechanism for ER-mediated gene activation. This mechanism is consensus ERE independent and cell type specific and requires interactions between the ER and molecules capable of interacting with the BCK promoter TA-rich region.
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PMID:A novel, cell-type-specific mechanism for estrogen receptor-mediated gene activation in the absence of an estrogen-responsive element. 793 28

The brain isozyme of creatine kinase (CKB) is a major component of the estrogen-induced proteins in the rat uterus. Hormonal specificity of this response was studied in cotransfection assays using the rat CKB promoter linked to the bacterial chloramphenicol acetyltransferase gene. Response was specific for estrogen as 17 beta-estradiol in the presence of estrogen receptor dramatically stimulated the CKB promoter. This induction was completely blocked by the estrogen antagonist ICI 164,384. Nuclear receptors for progesterone, androgen, glucocorticoid and vitamin D did not significantly activate the CKB promoter in the presence of their respective ligands. Creatine kinase (CK) activity was analyzed in decidualized mouse uterus to assess estrogenic activity in vivo. Upon oil stimulation, uterine horns of day 4 pseudopregnant mice underwent a dramatic outgrowth in response to endogenous progesterone. This response was accompanied by a significant decrease in CK activity from a control value of 1.44 +/- 0.25 to 0.38 +/- 0.08 IU/mg protein (P < 0.001), indicating that the action of estrogen was suppressed. Treatment of females one day prior to oil-stimulation with progesterone receptor antagonists, RU486 (Mifepristone) or ZK299 (Onapristone), or with a monoclonal antibody to progesterone (DB3), abolished decidualization, and also restored the CK activity to the control value. These results suggest that CK can be used as a specific cellular marker to detect unopposed estrogen action in the mouse uterus associated with progesterone withdrawal or receptor blockade.
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PMID:Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment. 803 8

Mechanisms for regulation of heat shock protein (hsp) 83 expression were examined in Leishmania amazonesis. Transcripts of hsp83 accumulated upon temperature elevation; however, in contrast to non-protozoan eukaryotes (i.e. Drosophila, yeast, avian or human cells), no transcriptional activation was observed. The increase in the hsp83 mRNA level evolved from temperature induced variations in mRNA turn-over: the hsp83 transcript was rapidly degraded at normal temperatures, whereas heat shock led to its stabilization. The quick decay of the mRNA at lower temperatures was dependent on active protein synthesis. A similar pattern of regulation was observed for the transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked by sequences from the hsp83 intergenic region (IR), and cloned into the pX transfection vector (pX-ICI). CAT mRNA was abundant at normal temperatures and further accumulated upon temperature elevation. The altered turn-over rates of CAT mRNA at the different temperatures were observed only in the presence of flanking hsp83 IR sequences. The increase in temperature also affected translational regulation of hsps, and synthesis of hsp83 was more efficient at 35 degrees C than at 26 degrees C. However, the effect of translation was transient, and the steady state level of the protein was hardly altered.
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PMID:Expression of heat shock protein 83 in Leishmania is regulated post-transcriptionally. 807 27

Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) has been identified in the non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.
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PMID:Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. 819 46


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