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 have previously shown that a human mammotropic polypeptide hormone, prolactin (PRL) can act synergistically with steroid hormones to regulate gene expression directed by the long terminal repeat of mouse mammary tumor virus (MMTV LTR) in a human ductal carcinoma cell line T47D cells using a chloramphenicol acetyltransferase reporter gene system and gene transfection methods. In the present study, using various recombinant plasmids we analyzed functional elements in the MMTV LTR that is essential for the PRL responses. We show that the PRL-responsive elements are located in the extreme 5' end of the MMTV LTR, a region previously described by others to be a mammary cell-specific enhancer.
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PMID:Prolactin acts on the extreme 5' portion of MMTV LTR involving a mammary cell-specific enhancer. 827 23

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

TR3 orphan receptor is a human homologue of the mouse nur77, N10 and rat NGFI-B, TIS1 genes which may represent an early response gene involved in the control of cell proliferation. We have studied potential target genes for TR3 orphan receptor using the DNA-binding domain replacement method. We found that mouse mammary tumor virus long terminal repeat-linked chloramphenicol acetyltransferase expression can be activated in transfected cells by a chimeric androgen receptor/TR3 orphan receptor/androgen receptor construct (AR/TR3/AR) in the presence of androgen. By deletion analysis, a region with 20 nucleotides in length between positions -1178 and -1159 of the mouse mammary tumor virus long terminal repeat was confirmed as a potential TR3 orphan receptor response element. These results suggest that feasibility of using the DNA-binding domain replacement method to detect target sequences of orphan receptors.
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PMID:The use of a DNA-binding domain replacement method for the detection of a potential TR3 orphan receptor response element in the mouse mammary tumor virus long terminal repeat. 839 47

The functional significance of receptor phosphorylation in mediating the actions of glucocorticoids remains undefined. The identification of seven phosphorylation sites in the mouse glucocorticoid receptor (Bodwell, J. E., Orti, E., Coull, J. M., Pappin, D. J. C., Smith, L. I., and Swift, F. (1991) J. Biol. Chem. 266, 7549-7555) permits a direct examination of the potential regulatory role of glucocorticoid receptor phosphorylation in transactivation. Using oligonucleotide-directed mutagenesis of the mouse glucocorticoid receptor cDNA, we have substituted alanine or aspartate for the residues phosphorylated in this ligand-dependent transcription factor. COS-1 cells were cotransfected with mutant receptor cDNA expression vectors and a reporter plasmid containing the glucocorticoid-inducible mouse mammary tumor virus promoter linked to chloramphenicol acetyltransferase in order to characterize the effect of these substitutions on receptor-mediated gene expression. Substitution of alanine or aspartate at single phosphorylation sites does not prevent receptor transactivation. Receptors containing multiple substitutions of alanine or aspartate at the major phosphorylation sites in the acidic domain elicit levels of hormone-induced reporter gene expression that are comparable to wild-type receptors. Mutant receptors substituted with alanine at the five phosphorylation sites conserved among the rat, human, and mouse receptors exhibit a 22% decrease in transcriptional activity. Receptors mutated at all seven sites display a similar modest reduction. These results demonstrate that receptor phosphorylation at these seven identified residues is not a major determinant in glucocorticoid receptor transcriptional activity at the mouse mammary tumor virus promoter.
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PMID:Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor. 840 99

A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.
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PMID:Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. 845 96

Androgen insensitivity is an X-linked disorder of sexual differentiation resulting from mutations in the androgen receptor (AR) gene. In this paper, we report the clinical phenotype and molecular analysis of two siblings with severe partial androgen insensitivity due to a novel mutation in the ligand-binding domain of the AR gene. Binding studies using cultured genital skin fibroblasts demonstrated reduced AR affinity and binding capacity. Nucleotide sequence analysis of the AR gene of both siblings revealed a point mutation causing a glycine to arginine amino acid substitution at position 907 within a conserved region of the ligand-binding domain. A silent guanine to adenine substitution was also identified in the protein-coding region of exon 1. Using an expression vector in which the identified mutation was recreated by site-directed mutagenesis, the mutant receptor was found to have a reduced binding affinity (Kd = 3.06 nmol/L) for mibolerone compared with that of normal AR (Kd = 1.71 nmol/L) when expressed in COS-7 cells. In cotransfection experiments using CV-1 cells and a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter system, the concentration of dihydrotestosterone required to induce half-maximal chloramphenicol acetyltransferase gene expression was 50-fold higher in cells transfected with the mutant AR complementary DNA than in cells transfected with normal AR complementary DNA. AR messenger ribonucleic acid levels in genital skin fibroblasts determined by both competitive PCR amplification and ribonuclease protection assay were decreased compared with normal values. Our studies demonstrate the importance of this region of the AR gene in normal AR function and AR gene expression.
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PMID:Partial androgen insensitivity caused by an androgen receptor mutation at amino acid 907 (Gly-->Arg) that results in decreased ligand binding affinity and reduced androgen receptor messenger ribonucleic acid levels. 855 Jul 58

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
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PMID:GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. 864 9

Transformed A5 mouse lung cells were examined for mechanisms that may explain their loss of glucocorticoid-induced growth inhibition. These cells were compared to nontransformed C10 mouse lung cells, which retain this response. Southern blot analysis revealed no major differences in the amount or pattern of restriction fragments for the glucocorticoid receptor (GR) gene between the responsive and nonresponsive cells. Northern blot analysis demonstrated that both cell lines expressed GR mRNA at similar levels and that these mRNAs had similar relative stabilities. The mRNA from both cell lines was used for reverse transcription-polymerase chain reaction amplification and direct sequencing with primers for different regions of the GR cDNA. A conservative mutation previously shown not to affect receptor function was detected within the DNA-binding domain region of the GR from both cell lines. Because of the ability of the transcription factors for activator protein-1 to antagonize GR function, c-jun and c-fos mRNA levels were examined. A5 cells were found to have higher levels of c-jun mRNA than C10 cells both during active cell growth and after serum starvation. Stable transfection of the nonresponsive A5 cells with a rat GR expression vector (A5GR7) resulted in strong glucocorticoid-induced growth inhibition, demonstrating that these cells retain the ability to be growth inhibited by these steroids. The A5GR7 transfectants also had higher mouse mammary tumor virus (MMTV)-chloramphenicol acetyltransferase (CAT) activity than the parental A5 cells and lower levels of c-jun during active cell growth. Transient transfection of the C10 cells with c-jun expression vector strongly reduced glucocorticoid-inducible MMTV-CAT activity. These results suggest that the transformed A5 cells apparently contain functional GR but that the high level of c-jun mRNA expression (probably resulting from the activated Ki-ras allele in these cells) may antagonize their ability to respond to the growth-inhibitory signaling of glucocorticoids.
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PMID:Loss of glucocorticoid-dependent growth inhibition in transformed mouse lung cells. 878 64

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.
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PMID:Effects of mitogens on androgen receptor-mediated transactivation. 882 95


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