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)

A hormone-inducible transcriptional system has been established, based on the stable transfection of the rat androgen receptor (rAR) and a reporter plasmid containing the mouse mammary tumour virus promoter linked to the chloramphenicol acetyltransferase gene (pMMTV-CAT) into steroid receptor-negative CV-1 cells. First, the rAR was stably introduced into CV-1 cells. Single clones were tested for stable expression of functionally active AR by analysing the effect of dihydrotestosterone on induction of transiently transfected pMMTV-CAT. Stable transfection and the expression of AR was confirmed by steroid-binding assays. In a second step, a clone expressing physiological amounts of AR protein (30 fmol/mg protein) was stably transfected with pMMTV-CAT to yield a permanent cell line that stably expresses functional AR and MMTV-CAT sequences. This cell line provides a powerful tool for the efficient and accurate determination and quantification of the effects of androgens and anti-androgens on reporter gene transcription. This was demonstrated by investigating the action of the three anti-androgens hydroxyflutamide, casodex and cyproterone acetate. The three compounds were shown to reverse the effects of the androgen R1881 on gene expression but were themselves devoid of agonistic activity.
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PMID:Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: inhibition of cat expression by anti-androgens. 132 16

Although the androgen receptor (AR) has been detected by ligand-binding assays, there is little known about the expression and regulation of the AR gene in human breast-cancer cells. AR mRNA, measured by Northern analysis in 18 cell lines, was found to be expressed predominantly in oestrogen- and progesterone-receptor-positive (ER+, PR+) lines as a single species of approximately 10.5 kb but was also comparatively abundant in I ER- and PR-negative cell line, MDA-MB-453. Dexamethasone (Dex), Organon 2058 (Org 2058), dihydrotestosterone (DHT), and all-trans-retinoic acid (RA) down-regulated AR mRNA levels in T-47D (ER+, PR+) cells 6 hr after treatment, whereas oestradiol (E2) had no effect. In MDA-MB-453 (ER-, PR-) cells, regulation of AR mRNA by RA differed from the other cell lines: RA increased the level of AR mRNA. DHT-binding assays indicated a corresponding increase in AR protein. Transfection of the androgen-responsive mouse mammary tumour virus long-terminal repeat (MMTV LTR) linked to a chloramphenicol acetyltransferase (CAT) reporter gene was used to examine the effect of altered AR levels on androgen action. The increased level of AR following RA pre-treatment in MDA-MB-453 cells resulted in enhanced induction of CAT activity by DHT and, conversely, a decrease in the level of AR following RA pretreatment in T-47D cells resulted in reduced induction of CAT activity by DHT. These data demonstrate that AR is expressed predominantly in ER+ and PR+ cell lines and its expression is regulated by ligands also known to regulate ER or PR, including progestins and retinoids. Androgen responsiveness measured by a transfected reporter gene was altered according to the extent of up- or down-regulation of AR expression, demonstrating that responsiveness is dependent on receptor concentration.
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PMID:Regulation of androgen receptor gene expression by steroids and retinoic acid in human breast-cancer cells. 142 32

Intron and 5'-flanking regions of the androgen-regulated C3 subunit gene contain potential cis-acting transcription control sequences including several 15-base pair (bp) partial palindromes resembling response elements for glucocorticoid (GRE) and progesterone (PRE) receptors. Specific DNA binding of the androgen receptor (AR) and androgen-dependent activation of transcription indicate that some of these GRE/PRE-like sequences are capable of functioning as androgen response elements (ARE). A 0.3-kilobase pair (kbp) 5'-flanking fragment including the promoter region contains one such sequence (element A) and a 0.5-kbp region of the first intron contains two sequences (elements B and C). Androgen-dependent enhancement of transcription was assayed by cotransfection of CV1 cells with a rat AR expression vector, pCMVrAR, and C3 genomic fragments or synthetic elements cloned into the reporter vector ptkCAT. Enhancement of chloramphenicol acetyltransferase activity with the 0.5-kbp first intron fragment was 16 +/- 4-fold, while with the 0.3-kbp 5'-flanking fragment no response was detected and element C alone was greater than B or A. Binding of AR in the mobility shift assay correlated with androgen-dependent enhancement of chloramphenicol acetyltransferase activity. The intensity of transcriptional enhancement with the 0.5-kbp intron fragment suggested that other regulatory sequences within this intron region potentiated the ARE activities of elements B and C. ARE activity of the strongest C3 gene response element (C) was similar to that of a potent GRE (element M) of the mouse mammary tumor virus gene.
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PMID:Response elements of the androgen-regulated C3 gene. 133 54

Inadequate androgen action in genetic and gonadal males causes an intersex phenotype. We have analyzed the androgen receptor (AR) gene in male pseudohermaphrodites with normal specific binding of dihydrotestosterone in their genital skin fibroblasts. In five patients with Reifenstein syndrome we have detected a point mutation in the DNA binding domain. They are from two unrelated families and presented with perineoscrotal hypospadias and undescended testes. After puberty they showed small testes, no palpable prostate, micropenis, azoospermia, and gynecomastia. The mutation was discovered when cDNA fragments from three brothers were sequenced. For rapid detection of the mutation in heterozygous and hemizygous carriers, allele-specific PCRs and restriction-analysis techniques have been developed. Relatives of the patients, a group of normal blood donors, and other patients were screened with these methods. Among 41 intersex patients with incomplete virilization, another two brothers presenting with this mutation were identified. The mutation is a guanine-to-adenine transition at nucleotide 2314, which changes the alanine codon (GCC) immediately after the first cysteine of the second zinc finger motif of the AR into a threonine codon (ACC). The mutation was recreated in an AR expression vector, and wild-type as well as mutant ARs were expressed in COS-7 cells. Cotransfection experiments were made using a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene. The ability of the mutant receptor to stimulate transcription of the reporter gene was reduced by about two-thirds, as compared with the wild-type receptor.
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PMID:Point mutation in the DNA binding domain of the androgen receptor in two families with Reifenstein syndrome. 159 12

The gene for the androgen receptor (AR) in the androgen-sensitive human prostate cancer cell line LNCaP has a single-base mutation that produces a threonine to alanine change in the androgen-binding domain. Androgen-insensitive prostatic cancer (PC-3) cells were cotransfected with an expression vector encoding normal, LNCaP, or chimeric normal/LNCaP AR and a vector carrying a chloramphenicol acetyltransferase (CAT) reporter gene linked to the mouse mammary tumor virus promoter. CAT activity was specifically induced by androgens in PC-3 cells expressing normal AR. In PC-3 cells expressing LNCaP AR, however, CAT activity was also induced by progestins and the antiandrogen hydroxyflutamide, which had little activity in cells expressing normal AR. Steroid-binding competition assays using in vitro synthesized ARs showed that LNCaP AR had a higher affinity than normal AR for progestins, 17 beta-estradiol, and hydroxyflutamide. The antiprogestin and antiglucocorticoid RU 38486 induced CAT activity in PC-3 cells expressing normal AR but not LNCaP AR. These studies indicate that AR mutations may be very important in determining the appropriate method of treatment with steroid hormones or their antagonists.
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PMID:Expression and function of normal and LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells. Altered hormone and antihormone specificity in gene transactivation. 166 32

A full length human androgen receptor (hAR) cDNA was constructed from cDNA and genomic clones. Structurally the 10.6-kilobase (kb) hAR cDNA consists of a long 5'-untranslated region (5'-UTR, 1.1 kb), a previously described open reading frame (ORF, 2.7 kb) (Trapman, J., Klaassen, P., Kuiper, G. G. J. M., van der Korput, J. A. G. M., Faber, P. W., van Rooij, H. C. J., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E., and Brinkmann, A. O. (1988) Biochem. Biophys. Res. Commun. 153, 241-248; Faber, P. W., Kuiper, G. G. J. M., van Rooij, H. C. J., van der Korput, J. A. G. M., Brinkmann, A. O., and Trapman, J. (1989) Mol. Cell. Endocrinol. 61, 257-262), and a very long 3'-untranslated region (3'-UTR, 6.8 kb). The complete 5'- and 3'-UTRs were found to be encoded by the previously reported first and eight protein coding exons of the hAR gene, respectively (Kuiper, G. G. J. M., Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Ris-Stalpers, C., Klaassen, P., Trapman, J., and Brinkmann, A. O. (1989) J. Mol. Endocrinol. 2, R1-R4). Two major sites of transcription initiation were identified in a 13-base pair region. DNA fragments spanning these transcription initiation sites conferred promoter activity upon a promoterless chloramphenicol acetyltransferase reporter gene construct. Two equally effective, functional polyadenylation signals (ATTAAA and CATAAA) at a mutual distance of 221 base pairs were detected. The ATTAAA hexamer sequence gave rise to multiple sites of poly(A) addition, whereas only one position was used following the CATAAA hexamer. In LNCaP prostatic carcinoma cells an alternatively spliced hAR mRNA species was identified which lacks 3 kb of the 3'-UTR.
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PMID:Characterization of the human androgen receptor transcription unit. 171 Feb 13

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.
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PMID:The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. 172 87

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

Sex-limited protein (Slp) is expressed in adult male mice. A 160-basepair fragment 2 kilobases upstream of the gene serves as an androgen-dependent enhancer of chloramphenicol acetyltransferase expression in transient transfection assays in cells with endogenous or cotransfected androgen receptor. One element that is necessary, but not sufficient, for induction is a consensus glucocorticoid (or hormone) response element (HRE). This element binds to the mouse androgen receptor in vitro, but with apparent weak affinity. Induction by the HRE is greatly augmented by an accessory sequence within the 160 basepairs, suggesting that cooperative interactions confer strong response to androgen. Additional elements within the enhancer modulate induction, positively or negatively, and exhibit cell-specific behavior. Of particular interest are two degenerate HREs that are adjacent to the consensus sequence; they show no independent activity, but are functionally significant in conjunction with other elements. The complexity of this enhancer may reflect biological mechanisms that ensure specificity of hormonal response and allow gene expression to respond to changes in hormone concentration.
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PMID:Multiple components of a complex androgen-dependent enhancer. 177 65

TR3 receptor is a human homolog of mouse Nurr77 and N10 protein and the rat NGFI-B protein. A cDNA encoding a chimeric nuclear receptor composed of the N-terminal domain and C-terminal putative ligand-binding domain of the orphan receptor TR3 receptor and the DNA-binding domain of the androgen receptor was constructed. The chimeric receptor, called TR3/AR/TR3 receptor, when expressed in COS-1 monkey kidney cells or PC-3 human prostate tumor cells, cotransfected with an ARE-containing mouse mammary tumor virus long terminal repeat-linked reporter gene encoding chloramphenicol acetyltransferase (CAT), activated CAT expression in the absence of any added factor. The activation was dependent on the amount of expression vector transfected and appeared to be independent of the concentration of serum supplement. Intact TR3 receptor was not active in this system. A TR3/AR/TR3 receptor protein truncated in the putative ligand-binding domain also induced CAT activity. TR3 receptor appears to be a transcriptional factor that activates transcription independently of ligand or binds an endogenous ligand present constitutively in cultured cells.
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PMID:Transcriptional activation by TR3 receptor, a member of the steroid receptor superfamily. 184 11


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