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)

P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues. In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor. A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.
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PMID:Progesterone regulates the murine multidrug resistance mdr1b gene. 809 15

To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.
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PMID:Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters. 814 66

MDA-MB-231 human breast cancer cells express the aryl hydrocarbon (Ah) receptor; however, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) does not induce CYP1A1 gene expression or chloramphenicol acetyltransferase (CAT) activity in cells transiently transfected with pRNH11c, and Ah-responsive plasmid derived from the 5'-flanking region of the human CYP1A1 gene. However, when MDA-MB-231 cells were treated with 10 nM TCDD and co-transfected with pRHN11c and a human estrogen receptor (hER) expression plasmid (delta hER), there was approximately a 10-fold increase in CAT activity. The restoration of Ah-responsiveness in MDA-MB-231 cells by expression of nuclear hER was highly specific since parallel studies in which plasmids that express the progesterone receptor and Jun nuclear proteins did not restore Ah-responsiveness to this cell line. Moreover, in cells transiently transfected with the pRNH11c and delta hER plasmids and 10 nM TCDD, overexpression of the Jun protein inhibited the effects of the hER on Ah-responsiveness. Plasmids that express truncated forms of the hER were also active in MDA-MB-231 cells but were not as effective as the complete hER. These studies reveal a unique function for the ER in MDA-MB-231 cells in which expression of this protein results in restoration of Ah-responsiveness.
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PMID:Restoration of aryl hydrocarbon (Ah) responsiveness in MDA-MB-231 human breast cancer cells by transient expression of the estrogen receptor. 820 98

A 2.0 kilobase-pair (kb) fragment encompassing the promoter and 5' flanking region of the uteroferrin (UF) gene was previously demonstrated to confer progesterone (P) responsiveness to chimeric UF gene promoter-reporter gene constructs when transfected in endometrial cells. In the present study, transient transfection experiments with the chloramphenicol acetyltransferase reporter gene linked to the sequentially deleted UF gene 5' flanking region and to genomic fragments within this region subcloned into the heterologous SV40 promoter were used to define the progesterone-responsive elements (PRE). The identified PREs are located distal to the promoter in the region between -1754 to -1601 bp and -893 to -678 bp of the UF gene and exhibit only limited similarities to the half-sites of the consensus palindromic PRE. The non-consensus PREs bind the progesterone receptor (PR) and independently exhibit P-dependent enhancer activities within the context of homologous and heterologous promoters in endometrial and placental cell lines. The unique features of these functional PREs suggest that formation of the P-PR complex with its cognate sequences upstream of the UF gene may be less dependent on the sequence per se but may require the binding of nuclear factors proximal to the PRE to stabilize PRE-PRE interactions.
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PMID:Non-consensus progesterone response elements mediate the progesterone-regulated endometrial expression of the uteroferrin gene. 821 75

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.
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PMID:Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. 834 15

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.
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PMID:The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. 838 50

Previous studies have shown that members of the steroid receptor family of transcriptional regulators can function synergistically when bound to multiple arrays of specific DNA binding sites known as hormone response elements, usually located upstream of target genes. We have constructed a mammalian expression vector containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (termed GRE5) placed upstream of the adenovirus 2 major late promoter "TATA" region. In transiently transfected HeLa cells in the presence of dexamethasone, the GRE5 promoter was at least 50-fold more efficient than the mouse mammary tumor virus long terminal repeat in expressing bacterial chloramphenicol acetyltransferase activity. When the GRE5 vector was introduced stably into the HeLa cell genome, chloramphenicol acetyltransferase activity was induced from 10- to >50-fold by dexamethasone in six of eight responsive clones. The levels of both basal and induced expression varied from one clone to the next, probably due to an effect of chromosomal location on promoter activity. When propagated stably in HeLa cells in an Epstein-Barr virus episomal vector, the GRE5 promoter was > 50-fold inducible and its activity was strictly dependent on the presence of dexamethasone. We also show that the GRE5 promoter stably propagated in HeLa cells is inducible by progesterone in the presence of a transiently transfected progesterone receptor expression vector. The GRE5 promoter should be widely applicable for the strictly controlled high-level expression of target genes in eukaryotic cells that contain either the glucocorticoid or progesterone receptors.
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PMID:A steroid-inducible promoter for the controlled overexpression of cloned genes in eukaryotic cells. 839 Jun 72

We have used a negative glucocorticoid response element (nGRE) from the bovine prolactin promoter linked to the gene for chloramphenicol acetyltransferase (PRL3CAT) to study the inhibition of gene expression by steroid hormone receptors. This nGRE increased basal expression from a heterologous promoter in COS-7 cells. In the presence of cotransfected glucocorticoid (GR), androgen, or progesterone receptor (PR) expression vectors and their cognate ligands, the expression of PRL3CAT could be repressed, indicating that these steroid receptor subfamily members could function through the same negative response element. No repression was observed with the estrogen receptor, showing that the repressive effect was specific for members of the GR-subfamily. Mutation of three amino acids within the GR-DNA binding domain that determine the specificity of GR-GRE interaction abolished the ability of the GR to inhibit the expression of PRL3CAT, demonstrating the requirement for DNA binding of the GR in the mechanism of repression. The antiglucocorticoid/antiprogestin RU486 when bound to PR or GR also repressed the expression of the PRL3CAT, but higher concentrations of RU486 were required to obtain an effect with the GR when compared to the PR. RU486 was unable to antagonize the effect of progestins on PRL3CAT and only partially antagonized the glucocorticoid repression. Thus, regarding the repression of PRL3CAT, RU486 acted as an agonist when bound to the PR and as a partial agonist when bound to the GR.
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PMID:Inhibition of gene expression by steroid hormone receptors via a negative glucocorticoid response element: evidence for the involvement of DNA-binding and agonistic effects of the antiglucocorticoid/antiprogestin RU486. 839 27

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.
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PMID:Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line. 846 59

Previously, we demonstrated that the progestin components (19-nortestosterone derivatives) in oral contraceptives are able to stimulate human breast cancer cell proliferation via an estrogen receptor (ER)-mediated mechanism. We now examine RU486, an antiprogestin, to determine whether it has estrogenic properties because it is also a 19-nortestosterone derivative. We found that RU486 stimulated the growth of MCF-7 human breast cancer cells at a concentration of 10(-6) M, which is similar to the pharmacological concentration (micromolar range) found in women taking RU486. The antiestrogens 4-hydroxytamoxifen and ICI 164,384 blocked RU486-induced cell proliferation. The estrogenic activity of RU486 is not due to impurities or aromatization to estrogenic metabolites. To determine whether the proliferative action of RU486 was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene under the control of an estrogen response element derived from the Xenopus laevis vitellogenin 2A gene. We found that RU486 was able to induce chloramphenicol acetyltransferase activity at the concentrations that stimulated cell proliferation, and this induction was blocked by the addition of 4-hydroxytamoxifen and ICI 164,384. The estrogenic potential of RU486 to regulate ER target gene expression was also investigated. We found that, like 17 beta-estradiol (E2), RU486 was able to alter the expression and synthesis of progesterone receptor. The level of progesterone receptor (145 and 186 fmol/mg cytosol protein, respectively) was increased significantly compared to the control value (3 fmol/mg cytosol protein) with the addition of 10(-6) M RU486 or 10(-10) M E2, as determined by an enzyme immunoassay. The levels of transforming growth factor-beta 2 (TGF beta 2) and TGF beta 3 mRNA, but not TGF beta 1 mRNA, were decreased dramatically with the addition of 10(-6) M RU486. This is consistent with the effects of E2 on TGF beta expression. Therefore, RU486 has estrogen-like activities in its regulation of ER target gene expression. These results demonstrate that RU486 is a weak estrogen in human breast cancer cells and suggest that the RU486-induced cell proliferation is mediated via ER. The novel finding that RU486 exhibits some estrogen-like activity may be important for the interpretation of its action at high dosages as an abortifacient and also if RU486 is going to be evaluated clinically, again at high doses, for the treatment of breast cancer.
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PMID:Estrogenic actions of RU486 in hormone-responsive MCF-7 human breast cancer cells. 850 63


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