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)

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

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

We have previously isolated two different aFGF cDNA clones from kidney and brain. The two corresponding mRNA, designated aFGF 1.A and 1.B, are the predominant species in kidney and brain, respectively. During the characterization of aFGF mRNA in glioblastoma cells, we demonstrated that aFGF mRNA in U1242MG and D65MG glioblastoma cells contain 5'-untranslated sequences different from those of 1.A and 1.B. Through a strategy combining chromosome walking, identification and sequencing of evolutionarily conserved DNA regions, and a reverse transcription and polymerase chain reaction (RT-PCR)-based assay for RNA expression, we have isolated two novel aFGF cDNA clones. The cDNA clone representing aFGF mRNA 1.C was isolated from U1242MG cells; another aFGF cDNA, designated 1.D, was isolated from D65MG cells. Promoter 1C has extensive sequence homology to the hamster aFGF gene promoter that was shown to respond to testosterone stimulation by chloramphenicol acetyltransferase reporter gene assays. Using RT-PCR, we showed that normal, benign and cancerous prostate tissues do not express aFGF 1.C mRNA. In contrast, a prostate carcinoma cell line (PC-3) expresses 1.C mRNA. RT-PCR using 1.D-specific primers showed that kidney, brain and prostate do not express 1.D mRNA even though kidney and brain are the most abundant source for aFGF protein. RNase protection analysis further showed that 1.D mRNA is the predominant aFGF transcript in D65MG glioblastoma cells and in NFF-6 neonatal foreskin fibroblast cells. The genomic DNA corresponding to these two cDNA clones and the 5'-flanking regions were also isolated and their sequences determined. These DNA clones will provide important reagents for studying the regulatory elements of aFGF gene expression.
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PMID:Cloning of two novel forms of human acidic fibroblast growth factor (aFGF) mRNA. 768 Jan 20

The human prostate-specific kallikreins, human glandular kallikrein-1 (hKLK2) and prostate-specific antigen (hKLK3), have been shown to be regulated by androgens. To determine whether the androgen induction of these genes is transcriptionally regulated via an androgen response element, an hKLK2 promoter DNA fragment was linked to a promoterless chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression vector in an androgen receptor-less human prostate cell line, PC-3. Dose response and steroid specificity experiments showed that the hKLK2 promoter confers androgen receptor-mediated gene induction in a ligand-specific manner. Moreover, 5' deletion constructs of the hKLK2 promoter DNA and internal deletion constructs devoid of the 5' half-site of the putative androgen responsive element (ARE) were used to show that the putative ARE is indeed acting as a functional ARE in prostate cells. In addition, multiple AREs from both hKLK2 and hKLK3 were able to reconstitute androgenic induction, further strengthening the argument that the AREs are functional. Although previous studies have shown that hKLK3 mRNA is expressed at a higher level than that of hKLK2, our results suggest that the hKLK2 ARE may have higher androgenic inducibility than the hKLK3 ARE. These results suggest that other cis-acting elements may be involved in coordinating in vivo androgenic induction of hKLK2 and hKLK3 genes.
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PMID:Androgen induction of a human prostate-specific kallikrein, hKLK2: characterization of an androgen response element in the 5' promoter region of the gene. 768 46

We investigated the role of glucocorticoids in controlling the proliferation of androgen-independent PC-3 human prostate cancer cells via the action of transforming growth factor beta 1 (TGF beta 1). The presence of glucocorticoid receptor (GR) in PC-3 cells was detected by immunoblotting analysis using a rabbit anti-GR polyclonal antibody against the synthetic human GR peptide (hGR383-393). In PC-3 cells, GR bound radiolabeled dexamethasone with an affinity similar to wild-type GR. In addition, GR-ligand complex bound radiolabeled DNA as detected by DNA band-shift analysis on gel electrophoresis and trans-activated the mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase chimeric gene in transiently transfected PC-3 cells. Dexamethasone (0.1 up to 100 nM) and TGF beta 1 (0.5 up to 50 ng/ml) inhibited PC-3 cell proliferation. TGF beta 1 and dexamethasone both increased the distribution of PC-3 cells into the G1/G0 phase of the cell cycle. Platelet-derived growth factor (PDGF) stimulated the proliferation of PC-3 cells and overcame dexamethasone's inhibition of PC-3 cell growth. Dexamethasone's inhibition (10(-7) M) of PC-3 cell growth was completely neutralized by RU 486 (10(-6)M) and partly neutralized by anti-TGF beta 1 polyclonal antibody. Furthermore, dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells. Because dexamethasone's inhibition was neutralized at least in part by an anti-TGF beta 1 polyclonal antibody and dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells, we conclude that GR function in human PC-3 prostate cancer cells is mediated at least in part by TGF beta 1 expression.
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PMID:Mediation of glucocorticoid receptor function by transforming growth factor beta I expression in human PC-3 prostate cancer cells. 775 11

We have analyzed the nucleotide sequence of complementary and genomic DNAs of the human androgen receptor (AR) gene in two siblings (patients 9006 and 9030) with receptor-positive complete androgen insensitivity (Rec(+)-CAI). Northern analysis indicated that mRNA of the AR was normal in size. However, its expression was relatively reduced in both patients. Consistent with the normal androgen-binding capacity (496 and 552 fmol/mg DNA for patients 9006 and 9030, respectively) but decreased DNA-binding ability (168 fmol/mg DNA) measured in genital skin fibroblasts, no mutation was found in both N-terminal and ligand-binding domains of the AR. However, a single base substitution (G-->A) was found in the second zinc finger of the DNA-binding domain at nucleotide 2372 of the AR cDNA in both cases. This resulted in the replacement of a highly conserved arginine residue (amino acid 614) by a histidine. When the mutated receptor plasmid was cotransfected into PC-3 cells together with the reporter chloramphenicol acetyltransferase gene, chloramphenicol acetyltransferase activity was not induced by 5 alpha-dihydrotestosterone treatment, confirming that the mutation renders the AR nonfunctional and can, therefore, be held responsible for the clinical features in these patients. These results highlight the importance of Arginine-614 in the second zinc finger of the DNA-binding domain of the AR in the protein-DNA interaction.
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PMID:A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance. 841 10

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.
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PMID:Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells. 956 51

Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25-dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3-1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10(-9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer.
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PMID:The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer. 1074 14

Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. To understand the regulation of expression of the PAcP gene, we studied the cis-regulatory elements of its promoter. A DNA fragment from -2899 to +87 base pairs (bp) of PAcP gene was fused to the chloramphenicol acetyltransferase (CAT) reporter gene and introduced into PC-3 and LNCaP human prostate cancer cells. The expression of the CAT gene driven by the PAcP promoter was assessed in transient expression assays. Sequential 5' deletions of the promoter were constructed and analyzed to reveal the positive and the negative regulatory elements that are involved in regulating the transcription of the PAcP gene. Our data showed that the proximal sequence -1305/+87 bp directs a high level of the CAT activity in both cell lines. Deletion of the region from -1305 to -779 resulted in approximately a 10- and three-fold decrease of the PAcP promoter activity in PC-3 and LNCaP cells, respectively. Interestingly, an inverse correlation of the CAT activity with the cell growth was observed when the reporter gene was driven by the -1305/+87 fragment, but not by the -779/+87 fragment. Two regions of transcriptional suppression were identified and located in positions from -2899 to -2583, and from -2583 to -1305 bp. Furthermore, the activity of the core promoter region from -779 to +87 bp can be activated by a SV-40 enhancer. The results, thus, clearly demonstrate the presence of positive and negative cis-elements in the promoter region of the PAcP gene.
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PMID:Expression of human prostatic acid phosphatase gene is regulated by upstream negative and positive elements. 1076 May 75

Prolactin stimulates citrate accumulation in prostate cells by increasing the expression of mitochondrial aspartate aminotransferase (mAAT). In this study, we further investigated the mechanism of prolactin regulation of mAAT expression in rat lateral prostate and LNCaP and PC-3 prostate cancer cells. Prolactin and 12-O-tetra-decanoylphorbol 13-acetate (TPA) increased the mAAT mRNA level twofold to fourfold. In addition, prolactin and TPA increased protein kinase C (PKC) activity in prostate cells 20% to 60% and 40% to 210%, respectively. The effects of both prolactin and TPA on mAAT mRNA were eliminated by downregulation of PKC. The effect of prolactin and TPA on gene transcription was determined using mAAT-chloramphenicol acetyltransferase (CAT) reporter-gene constructs, transiently transfected into PC-3 cells. The 59 untranslated region of the precursor form (pmAAT) of the mAAT gene contains five sequences that are homologous to the consensus TPA response elements (TRE). Reporter constructs with various combinations of these sequences were used to assay prolactin stimulation of CAT transcription in PC-3 cells. Prolactin increased CAT expression in PC-3 cells transfected with a reporter gene containing four of the TRE consensus sequences. Another CAT reporter gene, which contained two of the putative TREs, was also stimulated by prolactin, but a third reporter, containing the two other TRE sequences, was not induced by prolactin. These results suggest that prolactin regulates mAAT at the transcriptional level. Moreover, because both prolactin and TPA induced PKC activity, and because the effects of prolactin and TPA were eliminated when PKC was downregulated, we postulate that the prolactin effect on mAAT expression is mediated via the diacylglycerol PKC signal transduction pathway in rat lateral prostate and human prostate cancer cells.
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PMID:Protein Kinase C Mediates Prolactin Regulation of Mitochondrial Aspartate Aminotransferase Gene Expression in Prostate Cells. 1085 Dec 92


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