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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The early androgen-dependent (AD) phase of prostate cancer is dependent on the androgen receptor (AR). However, it is unclear whether AR is fully functional in recurrent prostate cancer after androgen withdrawal. To address this issue we interrogated AR signaling in AD and recurrent prostate cancer xenografts using molecular imaging, chromatin immunoprecipitation, and immunohistochemistry. In the imaging experiments, an adenovirus bearing a two-step transcriptional activation cassette, which amplifies AR-dependent firefly luciferase reporter gene activity, was injected into tumors implanted into severe combined immunodeficiency mice. A charge-coupled device optical imaging system detected the initial loss and then resumption of AR transcriptional activity in D-luciferin-injected mice as tumors transitioned from AD to recurrent growth. The results of chromatin immunoprecipitation and immunohistochemical localization experiments correlated with the Ad two-step transcriptional activation imaging signal. AR localized to the nucleus and bound to the endogenous prostate-specific antigen enhancer in AD tumors but exited the nucleus and dissociated from the enhancer upon castration. However, AR reentered the nucleus and rebound the prostate-specific antigen enhancer as the cancer transitioned into the recurrent phase. Surprisingly, RNA polymerase II and the general factor TFIIB remained bound to the gene throughout the transition. Our data support the concept that AR is fully functional in recurrent cancer and suggest a model by which a poised but largely inactive transcription complex facilitates reactivation by AR at castrate levels of ligand.
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PMID:Interrogating androgen receptor function in recurrent prostate cancer. 1290 31

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.
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PMID:Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation. 1456 46

RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.
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PMID:Inducible shRNA expression for application in a prostate cancer mouse model. 1457 27

Our previous studies demonstrate that introduction of a approximately 70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30-fold (>/=90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis-suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis-suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence-tagged site (STS)-based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis-suppressor activity to a approximately 4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1-12 hybrids that retain the metastasis-suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis-suppressor genes characterized to date, the metastasis-suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.
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PMID:Inhibition of prostate cancer metastatic colonization by approximately 4.2 Mb of human chromosome 12. 1461 10

We have used chromatin immunoprecipitation (ChIP) assay to follow transcription factor loading and monitor changes in covalent histone modifications associated with the prostate-specific antigen and kallikrein (KLK2) genes in response to androgen and antiandrogen in LNCaP cells. The dynamics of testosterone (T)-induced loading of androgen receptor (AR) onto the proximal promoters of the genes differed significantly from that onto the distal enhancers. Significantly more holo-AR was loaded onto the enhancers than the promoters, but the receptor's residence time was more transient on the enhancers. Even though holo-AR recruited some RNA polymerase II (Pol II) onto the enhancers, the principal Pol II transcription complex was assembled on the promoters. The pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the prostate-specific antigen promoter, but not that of the enhancer, whereas the partial antagonists cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading also onto the enhancer. In contrast to the CDX-occupied receptor, both CPA- and RU486-bound AR recruited Pol II and coactivators p300 and glucocorticoid receptor-interacting protein 1 (GRIP1) onto the promoter and enhancer. However, CPA and RU486 also brought about a simultaneous recruitment of the nuclear receptor corepressor (NCOR) onto the promoter as efficiently as CDX. There were dynamic changes in covalent modifications of histone H3: acetylation of lysine 9 and 14, methylation of arginine 17, phosphorylation of serine 10 as well as di- and tri-methylation at lysine 4 of the H3 N-terminal tail were enhanced in response to T, but not after CDX treatment. Collectively, these results indicate that transcriptional activation by AR is accompanied by a cascade of distinct covalent histone modifications and that the pure antiandrogen CDX and the partial antagonists CPA and RU486 exhibit clear differences in their ability to promote recruitment of histone-acetylating and histone-deacetylating complexes in human prostate cancer cells.
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PMID:Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor. 1530 89

PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.
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PMID:Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids. 1534 9

The success of gene therapy using a RNA interference approach relies on small interfering RNA (siRNA) expression from a highly tissue-specific RNA polymerase II promoter rather than from ubiquitous RNA polymerase III. Accordingly, we have developed a prostate-specific vector that expresses siRNAs from the human prostate-specific antigen promoter, a RNA polymerase II promoter. Our data demonstrate androgen-dependent and tissue-specific siRNA-mediated gene silencing in the androgen-responsive prostate cancer cell line, LNCaP. The biological significance was evidenced by altered apoptotic activity through the inhibition of the apoptosis-related regulatory gene. These results demonstrate that siRNA-mediated gene silencing from a tissue-specific RNA polymerase II promoter could be a potential tool for tissue-specific gene therapy.
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PMID:Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter. 1552 Jan 64

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.
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PMID:The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells. 1553 83

Peroxisome proliferator-activated receptor (PPAR) delta is the most widely expressed member of the PPAR family of nuclear receptor fatty acid sensors. Real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARdelta expression was increased 1.5 to 3.2-fold after three hours stimulation with the natural vitamin D receptor (VDR) agonist, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). In silico analysis of the 20 kb of the human PPARdelta promoter revealed a DR3-type 1alpha,25(OH)2D3 response element approximately 350 bp upstream of the transcription start site, which was able to bind VDR-retinoid X receptor (RXR) heterodimers and mediate a 1alpha,25(OH)2D3-dependent upregulation of reporter gene activity. Chromatin immuno-precipitation assays demonstrated that a number of proteins representative for 1alpha,25(OH)2D3-mediated gene activation, such as VDR, RXR and RNA polymerase II, displayed a 1alpha,25(OH)2D3-dependent association with a region of the proximal PPARdelta promoter that contained the putative DR3-type VDRE. This was also true for other proteins that are involved in or are the subject of chromatin modification, such as the histone acetyltransferase CBP and histone 4, which displayed ligand-dependent association and acetylation, respectively. Finally, real-time PCR analysis demonstrated that 1alpha,25(OH)2D3 and the synthetic PPARdelta ligand L783483 show a cell and time-dependent interference in each other's effects on VDR mRNA expression, so that their combined application shows complex effects on the induction of VDR target genes, such as CYP24. Taken together, we conclude that PPARdelta is a primary 1alpha,25(OH)2D3-responding gene and that VDR and PPARdelta signaling pathways are interconnected at the level of cross-regulation of their respective transcription factor mRNA levels.
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PMID:The human peroxisome proliferator-activated receptor delta gene is a primary target of 1alpha,25-dihydroxyvitamin D3 and its nuclear receptor. 1589 Jan 93

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer, where it is a key therapeutic target. Here we report that, in contrast to estrogen receptor transcription complexes which form within minutes and recycle hourly, the levels of regulatory regions bound by AR complexes rise over a 16 hr period and then slowly decline. AR regulation of the prostate specific antigen (PSA) gene involves both a promoter-proximal sequence as well as an enhancer approximately 4 kb upstream. Recruitment of AR and its essential coactivators at both sites creates a chromosomal loop that allows RNA polymerase II (pol II) to track from the enhancer to the promoter. Phosphorylation of the pol II C-terminal domain is required for pol II tracking but not chromosomal looping. Development of improved hormonal therapies for prostate cancer must take in account the specific spatial and temporal modes of AR-mediated gene regulation.
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PMID:Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking. 1613 20


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