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)

Nuclear transport, phosphorylation, ligand binding, and degradation rate of the recombinant androgen receptor (AR) were analyzed in transfected COS cells in the presence of various steroids and antiandrogens. Transcriptional activation was assessed in CV1 cells by cotransfection with an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter vector. Hormone binding specificity of recombinant AR was essentially identical to endogenous AR. AR localized in the nucleus in the presence of methyltrienolone (R1881, a synthetic androgen), dihydrotestosterone, testosterone, hydroxyflutamide, cyproterone acetate, estradiol, progesterone, and RU486. In the absence of hormone or with the antiandrogen, flutamide, AR remained largely in the cytoplasm with a perinuclear distribution. AR was degraded rapidly (t1/2 = 1 h) except in the presence of androgen (t1/2 = 6 h) which accounted for an apparent 2-4-fold androgen-induced increase in AR phosphorylation, indicating that AR phosphorylation was not enhanced by androgen. CAT activity was stimulated by R1881, dihydrotestosterone, testosterone, cyproterone acetate, estradiol, progesterone, and RU486 in a dose-dependent manner. The antiandrogens, flutamide and hydroxyflutamide, lacked agonist activity and inhibited R1881-induced activation of CAT and androgen stabilization of AR. Steroids and antiandrogens with moderate to low affinity for AR promoted both nuclear transport and transcriptional activation but only at high hormone concentrations. Hydroxyflutamide acted as a true antiandrogen since it lacked agonist activity and was an inhibitor of androgen-induced transcriptional activation.
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PMID:Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. 173 Jun 84

The androgen receptor (AR) is a signal-transducing protein required for sexual differentiation, development, and expression of the male phenotype. A series of human AR deletion mutants were created either by site-directed mutagenesis using restriction enzyme digestion, the polymerase chain reaction, or, for a series of unidirectional NH2-terminal deletions, exonuclease III digestion. Receptor mutants were expressed in monkey kidney COS cells as truncated AR proteins between 20 and 107 kDa as revealed on immunoblots, where wild type AR was a doublet of 114 and 108 kDa. Subcellular localization by immunocytochemical staining demonstrated androgen-dependent nuclear uptake of AR from a perinuclear region of the cytoplasm. A nuclear targeting signal similar in sequence and position to the glucocorticoid receptor and homologous to the SV40 large T antigen was required for androgen-induced nuclear uptake of wild type AR. AR mutants lacking the NH2-terminal and/or steroid binding domains were constitutively nuclear with reduced transcriptional activity. Transcriptional activation by wild type AR was androgen-dependent in cotransfection studies of CV1 cells using the chloramphenicol acetyltransferase reporter gene linked to the mouse mammary tumor virus promoter. Deletion mutagenesis revealed within the NH2-terminal region a domain required for full transcriptional activity and within the steroid binding domain, an inhibitory function, deletion of which yielded a constitutively active receptor. Inhibition of wild type AR by coexpression with an inactive NH2-terminal fragment suggested competition for nuclear factors required for transcriptional regulation. These studies demonstrate a concerted interplay among the domains of the AR protein in regulating gene transcription.
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PMID:Transcriptional activation and nuclear targeting signals of the human androgen receptor. 198 13

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in the human prostate cancer cell line LNCaP examine the ability of dihydrotestosterone (DHT), hydroxyflutamide (HO-FLU), cyproterone acetate (Cypro.A), and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). DHT stimulated transcription activation of MMTV-CAT gene in LNCaP cells in a dose-dependent manner. HO-FLU, Cypro.A, and RU 23908-10, though only partially, also stimulated the transcription activation of MMTV-CAT. Despite this, 100- to 1,000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A, and RU 23908-10 competed with DHT for AR binding in LNCaP cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in LNCaP cells, following treatment with antiandrogens. Increasing doses of HO-FLU stimulated the expression of the 114-kDa AR by 2.5-fold, but did not affect the 108-kDa AR. Increasing doses of Cypro.A and RU 23908-10 decreased the levels of both the 114-kDa and the 108-kDa AR. Although the exact nature of 108-kDa and 114-kDa AR in LNCaP cells is still unknown, these data suggest that the regulatory actions of each individual antiandrogen on AR expression in LNCaP cells may be different.
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PMID:Antiandrogens inhibit human androgen receptor-dependent gene transcription activation in the human prostate cancer cells LNCaP. 814 66

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in receptor deficient monkey kidney cells CV-1 in culture examine the ability of dihydrotestosterone (DHT) and of the antiandrogens hydroxylflutamide (HO-FLU), cyproterone acetate (Cypro.A) and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). CV-1 cells cotransfected with wild type hAR (hAR1-910) and MMTV-CAT, were treated with varying concentrations of DHT. DHT stimulated transcription activation of MMTV-CAT gene in a dose-dependent fashion. Cypro.A though only partially, also stimulated the transcription activation of MMTV-CAT. In the absence of steroids, HO-FLU induced the MMTV-CAT transcription in transfectants only 4% above the basal level. RU 23908-10 revealed the least agonistic activity at concentrations between 10 nM and 1 microM. Despite this, 100- to 1000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A and RU 23908-10 competed with [3H]DHT for AR binding with hAR expressed in CV-1 cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in extracts prepared from hAR1-910 transfected CV-1 cells. Incubation of labeled synthetic palindromic androgen responsive element (ARE) with the hAR containing CV-1 cell extracts followed by u.v. cross-linking demonstrated the specificity of AR-DNA interaction. Analysis by gel mobility shift assays showed that the interaction of AR-antiandrogen complexes with labeled ARE was specific.
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PMID:Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation. 827 4

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

Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARDelta641-902) decreased pCMV-ARE2-CAT activity by 50%. The rARDelta46-408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells.
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PMID:Interaction of androgen receptors with androgen response element in intact cells. Roles of amino- and carboxyl-terminal regions and the ligand. 918 99

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.
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PMID:Selective activation of the probasin androgen-responsive region by steroid hormones. 1034 90

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