Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transforming growth factor-beta (TGF-beta) superfamily of growth factors is responsible for a variety of physiologic actions, including cell cycle regulation. Activin is a member of the TGF-beta superfamily that inhibits the proliferation of breast cancer cells. Activin functions by interacting with its type I and type II receptors to induce phosphorylation of intracellular signaling molecules known as Smads. Smads regulate transcription of many genes in a cell- and tissue-specific manner. In this study, the role of activin A in growth regulation of breast cancer cells was investigated. Activin stimulated the Smad-responsive promoter, p3TP, 2-fold over control in T47D breast cancer cells. Activin inhibited cellular proliferation of T47D breast cancer cells after 72 hours, an effect that could be abrogated by incubation with the activin type I receptor inhibitor, SB431542. Activin arrested T47D cells in the G0-G1 cell cycle phase. Smad2 and Smad3 were phosphorylated in response to activin and accumulated in the nucleus of treated T47D cells. Infection of T47D cells with adenoviral Smad3 resulted in cell cycle arrest and activation of p3TP-luciferase, whereas a adenoviral dominant-negative Smad3 blocked activin-mediated cell cycle arrest and gene transcription. Activin maintained expression of p21 and p27 cyclin-dependent kinase inhibitors involved in cell cycle control, enhanced expression of p15, reduced cyclin A expression, and reduced phosphorylation of the retinoblastoma (Rb) protein. Smad3 overexpression recapitulated activin-induced p15 expression and repression of cyclin A and Rb phosphorylation. These data indicate that activin A inhibits breast cancer cellular proliferation and activates Smads responsible for initiating cell cycle arrest.
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PMID:Activin A mediates growth inhibition and cell cycle arrest through Smads in human breast cancer cells. 1614 Sep 69

Pasteurella multocida toxin (PMT) is a potent mitogen, which is known to activate phospholipase Cbeta by stimulating the alpha-subunit of the heterotrimeric G protein G(q). PMT also activates RhoA and RhoA-dependent pathways. Using YM-254890, a specific inhibitor of G(q/11), we studied whether activation of RhoA involves G proteins other than G(q/11). YM-254890 inhibited PMT or muscarinic M3-receptor-mediated stimulation of phospholipase Cbeta at similar concentrations in HEK293m3 cells. In these cells, PMT-induced RhoA activation and enhancement of RhoA-dependent luciferase activity were partially inhibited by YM-254890. In Galpha(q/11)-deficient fibroblasts, PMT induced activation of RhoA, increase in RhoA-dependent luciferase activity, and increase in ERK phosphorylation. None of these effects were influenced by YM-254890. However, RhoA activation by PMT was inhibited by RGS2, RGS16, lscRGS, and dominant negative G(13)(GA), indicating involvement of Galpha(12/13) in the PMT effect on RhoA. In Galpha(12/13) gene-deficient cells, PMT-induced stimulation of RhoA, luciferase activity, and ERK phosphorylation were blocked by YM-254890, indicating the involvement of G(q). Infection with a virus harboring the gene of Galpha(13) reconstituted the increase in RhoA-dependent luciferase activity by PMT even in the presence of YM-254890. The data show that YM-254890 is able to block PMT activation of Galpha(q) and indicate that, in addition to Galpha(q), the Galpha(12/13) G proteins are targets of PMT.
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PMID:Pasteurella multocida toxin-induced activation of RhoA is mediated via two families of G{alpha} proteins, G{alpha}q and G{alpha}12/13. 1614 Dec 14

The interaction between epithelial cells and micro-organisms is often a crucial initiating event in infectious diseases. Infection with Porphyromonas gingivalis, a Gram-negative anaerobe, is strongly associated with severe periodontal disease. This bacterium possesses an array of virulence factors, some of which can induce apoptosis. The tumour necrosis factor (TNF) receptor family is involved in the regulation of cellular homeostasis, cell surface molecules involved in phagocytosis, Fas ligand (L) expression and activation of the caspase cascade resulting in DNA fragmentation and cell blebbing. The current study examined the role of nuclear factor-kappaB (NFkappaB) in FasL-mediated apoptotic cell death in primary human gingival epithelial cells (HGEC) induced by heat-killed P. gingivalis, probably through TLR signalling pathways. A marked up-regulation of TLR2 and Fas-FasL was detected in HGEC stimulated with P. gingivalis. Activation of NFkappaB by P. gingivalis in HGEC was demonstrated by an NFkappaB promoter luciferase assay as well as by phosphorylation of p65 as detected by Western blotting. Activation of cleaved caspase-3 and caspase-8 resulted in apoptotic cell death of HGEC. The survival proteins c-IAP-1/c-IAP-2 were decreased in HGEC exposed to P. gingivalis. HGEC apoptosis induced by P. gingivalis was inhibited by an anti-human FasL monoclonal antibody. Blockade of NFkappaB by helenalin resulted in down-regulation of FasL whereas a caspase-8 inhibitor did not decrease FasL. Taken together, these studies show that P. gingivalis can induce epithelial cell apoptosis through Fas-FasL up-regulation and activation of caspase-3 and caspase-8.
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PMID:Porphyromonas gingivalis enhances FasL expression via up-regulation of NFkappaB-mediated gene transcription and induces apoptotic cell death in human gingival epithelial cells. 1651 59

Squamous cell carcinoma antigens SCCA1 and SCCA2 are highly homologous serine proteinase inhibitors which have been widely utilized as serological markers for squamous cell cancers, but it has recently been demonstrated that only SCCA2 is truly specific for certain forms of lung cancer. Using a construct containing the 5'-flanking region of the SCCA2 gene between -460 and +0 bp and the luciferase reporter gene, SCCA2 promoter activity was detected in SCCA2-producing SCC cell lines (LK-2, LC-1), but not in SCCA2-nonproducing lung adenocarcinoma cell lines (A549, ABC-1, and RERF-LC-MS) or normal cells (WI-38, SAEC, and NHEK-Adult). Infection with a recombinant adenovirus vector, Ad-SCCA2-DsRed, resulted in cell-specific expression of the SCCA2 promoter-driven DsRed marker gene only in LK-2 and LC-1 cells. The same strategy was used for SCCA2-driven expression of a proapoptotic gene, (KLAKLAK)2, which can cause mitochondrial disruption by triggering mitochondrial permeabilization and swelling, resulting in the release of cytochrome c and induction of apoptosis. Infection with Ad-SCCA2-KLAKLAK2 specifically reduced the growth of the two human lung SCC cell lines compared to the SCCA2 nonproducing cell lines both in vitro and in vivo, suggesting that the SCCA2 promoter had a tumor-specific effect. These results suggest that transduction of SCCA2 promoter-controlled suicide genes by adenoviral vectors can confer transcriptionally targeted cytotoxicity in SCCA2-producing lung SCC cells, and represents a novel strategy for gene transfer specifically targeted to SCC in the lung.
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PMID:Transcriptional targeting of adenovirus vectors with the squamous cell carcinoma-specific antigen-2 promoter for selective apoptosis induction in lung cancer. 1671 Mar 47

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine 2000 and FuGENE 6; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61-86%) than the SeVV-GFP (4-24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7-43%) compared to Ad-GFP (4-16%). The Lipofectamine 2000 revealed a higher transfection efficiency than the FuGENE 6 for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.
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PMID:Optimizing vector application for gene transfer into human hepatoblastoma cells. 1689 19

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREB(S133A)) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREB(WT)) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREB(S133A) markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.
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PMID:cAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival. 1706 10

Cultures of endogenous GnRH neurons and the GT1 GnRH neuronal cell line release GnRH in pulses (intrinsic pulsatile release) with an interpulse frequency similar to that seen in castrated animals. In both GT1 cells and transgenic rats, lowering cAMP levels by expression of a phosphodiesterase decreased the frequency of intrinsic GnRH pulsatility. We asked whether the cyclic nucleotide-gated cation (CNG) channels expressed in GT1 cells participated in cAMP modulation of intrinsic GnRH pulsatility. Because expression of the CNGA2 subunit is essential for formation of functional CNG channels, we developed an adenovirus (Ad) vector expressing a short interference RNA (siRNA) to the CNGA2 subunit (Ad-CNG-siRNA) or as an infection control, to the coding region of luciferase (Ad-Luc-siRNA). Infection with the Ad-CNG-siRNA of COS cells transfected with a CNGA2 expression vector significantly inhibited CNGA2 protein levels by 74% by Western blot. Infection of GT1-1 cells with Ad-CNG-siRNA resulted in a 68% decrease in the levels of CNGA2 mRNA, a 44% decrease in protein levels, and a clear decrease in immunostaining with an antibody to CNGA2. Infection of GT1-1 cells with Ad-CNG-siRNA decreased spontaneous Ca2+ oscillations compared with Ad-Luc-siRNA-infected or uninfected cells by 71%. Furthermore infection with Ad-CNG-siRNA resulted in a 2-fold increase in the interpulse interval in GnRH secretion (49.4+/-9.1 min) compared with uninfected cells (25.9+/-2.5 min) or Ad-Luc-siRNA (29.3+/-2.8 min)-infected cells. These data provide the first direct evidence that the CNG channel is a downstream signaling molecule in the regulation of the frequency of intrinsic GnRH pulsatility by cAMP.
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PMID:Frequency of intrinsic pulsatile gonadotropin-releasing hormone secretion is regulated by the expression of cyclic nucleotide-gated channels in GT1 cells. 1739 96

Challenged by the lack of success of experimental gene therapy of cystic fibrosis, we set out to investigate one of the potential causes of this failure, the barrier function of the airway epithelium and the way this is affected by bacterial infection. In an in vitro model of the airway epithelium we determined the effect of Pseudomonas aeruginosa or Escherichia coli on the transfection efficiency of polyethylenimine (PEI)-plasmid DNA complexes, carrying a luciferase gene, as well as on the barrier function of the epithelial cell layer, using transepithelial resistance (TER), cytotoxicity, bacterial transmigration, and morphological appearance as parameters. The level of luciferase expression was more than one order of magnitude higher in the cells which, before transfection, were incubated with P. aeruginosa. TER was strongly reduced by P. aeruginosa, whereas E. coli had no effect. Pseudomonas aeruginosa also effectively destroyed the structure of the tight junctions, as visualized by immunostaining of the zonula occludens. By the same token, small but significant numbers of P. aeruginosa cells were found to migrate through the epithelial layer, whereas no E. coli cells were observed at the transcompartment of the wells. Release of lactate dehydrogenase from the epithelial cells, a parameter of cell damage, occurred in a dose-dependent manner on incubation with P. aeruginosa, but not with E. coli. To evaluate the relevance of these results for the in vivo situation, we infected C57BL/6 mice with P. aeruginosa or E. coli 48 hr before transfecting them intratracheally with PEI-DNA polyplexes. Infection with P. aeruginosa caused a 5-fold increase in luciferase expression whereas infection with E. coli had no effect. Fluorescence microscopy of lung sections, after administration of fluorescein isothiocyanate-labeled polyplexes, showed that prior treatment with P. aeruginosa effectuated penetration of the complexes deeper into the epithelium than in untreated animals. In P. aeruginosa-treated animals fluorescence was detected not only in the airway epithelium itself but also in the parenchyma. We conclude that infection with P. aeruginosa causes disruption of the tight junctions between the cells and thus of the barrier function of the epithelium. As a consequence, PEI-DNA complexes injected intratracheally into infected animals gain access to the basolateral side of the cells and to spaces across the epithelial lining, giving rise to substantially increased transfection efficiency.
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PMID:Pseudomonas aeruginosa infection destroys the barrier function of lung epithelium and enhances polyplex-mediated transfection. 1763 71

Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5'-regulatory region of the sheep NOS3 gene.
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PMID:Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1. 1793 57

Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L(6)E(9) myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.
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PMID:Regulation of host cell cyclin D1 by Trypanosoma cruzi in myoblasts. 1823 52


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