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)

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

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.
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PMID:Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115). 163 20

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

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

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.
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PMID:Expression of recombinant androgen receptor in cultured mammalian cells. 217 2

Proliferation of LNCaP 104-S cells, a clonal subline of the human prostate cancer cell line, was very slow in androgen-depleted medium but increased 10-13-fold in the presence of 0.1 nM of a synthetic androgen, R1881. This induction of proliferation was diminished at higher concentrations of R1881, indicating the biphasic nature of the androgen effect. After 20-30 passages in androgen-depleted medium, these cells progressed to 104-I cells, which exhibited much lower proliferative sensitivity to 0.1 nM R1881. After another 20-30 passages, LNCaP 104-I cells gave rise to 104-R cells, which proliferated rapidly without additional androgen. Proliferation of 104-R cells was induced 2-fold by 0.01 nM R1881 but was repressed by 0.1 nM R1881 and above. Thus, androgen induction and repression of proliferation could be seen at lower concentrations of androgen as the cells progressed. During the transition of 104-S cells to 104-R cells, the androgen receptor mRNA level increased 2.5-fold whereas the androgen receptor protein level increased 15-fold in the absence of androgen. Androgen receptor transcriptional activity, measured by androgen induction of prostate-specific antigen mRNA and chloramphenicol acetyltransferase activity in transfected cells, increased up to 20-fold during the progression. LNCaP cells, therefore, appear to be able to adapt to reduced androgen availability by increasing their sensitivity to androgen, raising questions concerning the therapeutic strategies used against prostate cancer. Androgen induction of c-myc expression in 104-R cells occurred at a 10-fold lower concentration (0.01 nM) than in 104-S cells (0.1 nM). In all stages, cell proliferation and c-myc expression were repressed by androgen at a high concentration (20 nM), but the repression of cell proliferation was blocked by retroviral overexpression of c-myc.
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PMID:Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. 751 Oct 45

The androgen receptor (AR) is a developmental and tissue-specific transcription factor which is activated by binding testosterone or dihydrotestosterone. Several different methods of transcriptional regulation of the AR have been shown, including regulation by androgens, follicle-stimulating hormone, epidermal growth factor, and the cAMP pathway. In order to further characterize the transcriptional regulation of the AR, portions of the mouse androgen receptor (mAR) promoter were cloned into the promoterless pBLCAT3 vector and assayed for chloramphenicol acetyltransferase activity. The results indicate that in addition to the previously characterized promoter (+1) there is a second distinct promoter located 3' to the first promoter. Amplification of the 5'-end of the AR gene indicates that RNA originating from the second promoter is initiated from 162 and 170 bases downstream from the 5'-most previously characterized site. Northern blot analysis indicated that RNA initiated from the two promoters is differentially expressed in several cell lines and multiple tissues. Androgen ablation by castration showed that both promoters are controlled by androgens in the kidney. Sequence analysis revealed that the second promoter does not contain a TATA or CAAT box. Further characterization of this promoter may provide important insights into the transcriptional regulation of the androgen receptor since previous studies have often included only the first promoter.
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PMID:The mouse androgen receptor gene contains a second functional promoter which is regulated by dihydrotestosterone. 798 Dec 21

Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the epithelial cells of the deferent duct under androgenic regulation. To better understand androgenregulated MVDP gene expression, the location and sequences of androgen response elements (AREs) in the 5'-flanking DNA were determined. Sequence analysis revealed two putative AREs as follows: one between positions -1186 and -1171 (distal ARE) and the other between -111 and -97 (proximal ARE). To study hormonal regulation, fragments of the MVDP promoter region, extending from residue -1804 to +41, were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a human androgen receptor expression vector into T47D cells in a transient expression assay. A minimal region (-121 to +41) was identified as being sufficient for androgen-regulated gene expression. A mutation in proximal ARE almost completely abolished androgen induction of CAT. One copy of the sequence TGAAGT tcc TGTTCT, cloned in the opposite orientation in front of the thymidine kinase promoter, confers androgen responsiveness to the CAT reporter gene. Androgen transcriptional activity was not detected with the distal ARE. The data provide strong evidence that transcriptional regulation of the MVDP gene occurs via the sequence TGAAGT tcc TGTTCT.
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PMID:Identification of a functional androgen response element in the promoter of the gene for the androgen-regulated aldose reductase-like protein specific to the mouse vas deferens. 811 28

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


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