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

GM-CSF is a cytokine with pleiotropic biological activities and is increasingly used in clinical trials. The present study demonstrates the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) to induce elevation of interleukin-10 (IL-10) mRNA and protein production in the monocytic cell line U937. As shown by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), IL-10 mRNA increases up to 10 times when stimulated with rGM-CSF (100 U/ml) compared to nonstimulated control cells. Maximal IL-10 mRNA expression occurs at 6 h and remains high for 2 h. Thereafter IL-10 mRNA is downregulated and reaches basal level at approximately 24 h. IL-10 protein was measured by ELISA. The protein yield is dose-dependent on the rGM-CSF concentration. Combined stimulation of U937 cells with both GM-CSF and TNF-alpha results in an additive elevation of the IL-10 protein yield. Application of a neutralising antibody against TNF-alpha revealed that GM-CSF induces IL-10 expression independently from TNF-alpha. By using a luciferase reporter gene it was shown that rGM-CSF enhances IL-10 promoter activity 2-3-fold in a transient transfection assay.
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PMID:Recombinant human granulocyte-macrophage colony-stimulating factor triggers interleukin-10 expression in the monocytic cell line U937. 979 52

Recent studies indicate that reactive oxygen intermediates (ROI) serve as second messengers in cell signaling. ROI have been implicated in the activation of NF-kappaB as well as c-Jun N-terminal kinase (JNK) in response to IL-1 and TNF-alpha stimulation. In this report we examine whether intracellular ROI are involved in CD40 receptor signaling. We show that CD40 engagement on resting splenic B lymphocytes and murine B lymphoma WEHI 231 cells generates ROI. Blocking ROI production by preincubation with the antioxidant N-acetyl-L-cysteine inhibits JNK activation, NF-kappaB-driven luciferase activity, and IL-6 secretion following CD40 ligation, suggesting a role for ROI in CD40-mediated signaling events. Furthermore, transfection of WEHI 231 cells with a plasmid encoding Mn-superoxide dismutase interferes with CD40-induced NF-kappaB activation, providing further support for ROI involvement in this pathway. Collectively, these data demonstrate that ROI may serve as second messengers linking CD40 engagement on B cells to important downstream activation events.
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PMID:Production of reactive oxygen intermediates following CD40 ligation correlates with c-Jun N-terminal kinase activation and IL-6 secretion in murine B lymphocytes. 986 55

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-180 tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed locally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. The significance of this phenomenon for cancer gene therapy was discussed.
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PMID:Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumors. 986 30

ICAM-1 is an inducible cell surface protein that is involved in cell extravasation into inflamed tissues as well as immune responses. ICAM-1 expression is upregulated by proinflammatory cytokines such as TNF-alpha and IL-1beta in numerous cell types including the astrocyte, which functions as an immune effector cell in the central nervous system (CNS). We investigated the mechanism by which the ICAM-1 gene is transcriptionally regulated in astrocytes in response to TNF-alpha and IL-1beta. Human ICAM-1 promoter constructs linked to the reporter gene luciferase were transiently transfected into astrocytes, stimulated with TNF-alpha and IL-1beta, and ICAM-1 promoter activity examined. We determined that binding sites for both NF-kappaB (-186 bp region) and C/EBP (-198 bp region) are involved in TNF-alpha and IL-1beta-mediated ICAM-1 upregulation. Electrophoretic mobility shift assays using antibodies against NF-kappaB and C/EBP isoforms showed that p65 homodimers and p65/p50 heterodimers bind to the NF-kappaB site, and C/EBPdelta homodimers and C/EBPbeta/delta heterodimers bind to the C/EBP site. Transient transfection assays demonstrated that overexpression of p65 could transactivate the promoter activity of ICAM-1 reporter constructs. p50 overexpression had no effect on the basal levels of ICAM-1 transcription, but inhibited, in a dose dependent manner, p65 mediated transcription. Overexpression of C/EBPbeta slightly inhibited basal levels of ICAM-1 promoter activity, however, when C/EBPbeta and p65 were cotransfected, C/EBPbeta completely abolished the transactivating effects of p65. These results demonstrate that cytokine-induced ICAM-1 expression in astrocytes is regulated by interactions between NF-kappaB and C/EBP transcription factors.
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PMID:Transcriptional regulation of intercellular adhesion molecule-1 in astrocytes involves NF-kappaB and C/EBP isoforms. 991 95

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.
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PMID:Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. 1007 45

Salivary glands contain two major epithelial cell types: acinar cells which produce the primary salivary secretion, including amylase, and ductal cells which reabsorb electrolytes but also secrete kallikrein. Here we investigated salivary acinar cell differentiation in vitro using the activity of the salivary amylase and tissue kallikrein promoters as markers of acinar cell and ductal cell differentiation, respectively. Each of the promoter sequences was cloned into a replication-deficient adenoviral vector containing the luciferase reporter gene. Previous studies showed that a human submandibular gland cell line (HSG) differentiated into acinar cells when cultured on a reconstituted basement membrane matrix (Matrigel). The luciferase activity of the amylase promoter vector (AdAMY-luc) was low in HSG cells cultured on plastic, where they grow as an epithelial monolayer. The promoter activity increased approximately tenfold when HSG cells were cultured on Matrigel and developed an acinar phenotype. Under the same conditions, the luciferase activity of the kallikrein promoter (AdKALL-luc) was not induced. Because HSG cells demonstrate acinar cell morphology, but not amylase gene expression, when cultured on laminin-1, certain soluble components of Matrigel were tested for their ability to induce the amylase promoter during in vitro differentiation of acinar cells. We find that epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), which are present in the basement membrane, and hepatocyte growth factor (HGF) increase activity of the amylase promoter. Other basement membrane-derived growth factors such as TGF-beta, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PGDF), as well as tumor necrosis factor (TNF-alpha), keratinocyte growth factor (KGH), nerve growth factor (NGF) and interferon gamma (IFN-gamma) were inactive. This system will be further exploited to study the mechanisms by which extracellular matrix molecules and growth factors regulate salivary acinar cell differentiation.
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PMID:Growth factor regulation of the amylase promoter in a differentiating salivary acinar cell line. 1009 15

Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-kappaB subunit p65 (RelA) and inhibits its transcriptional activity. RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait. We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay. RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain. RAI mRNA was preferentially expressed in human heart, placenta, and prostate. Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay. RAI inhibited the action of NF-kappaB p65 but not that of p53 in transient luciferase gene expression assays. Similarly, RAI inhibited the endogenous NF-kappaB activity induced by tumor necrosis factor-alpha. RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells. RAI protein appeared to be located in the nucleus and colocalized with NF-kappaB p65 that was activated by TNF-alpha. These observations indicate that RAI is another inhibitor of NF-kappaB in addition to IkappaB proteins and may confer an alternative mechanism of regulation.
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PMID:Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor. 1033 63

Gene expression was assessed in three types of mouse solid tumors after direct injection of naked plasmid DNA encoding the luciferase gene (pCMV-Luc) and its DC-chol liposome complexes. Intratumoral injection of 5 or 100 micrograms naked pCMV-Luc into subcutaneously inoculated mouse colon tumor (CT-26), fibrosarcoma (MCA-15) and bladder carcinoma (MBT-2) resulted in significant gene expression. A DC-chol liposome formulation (5 micrograms pCMV-Luc complexed with 25 micrograms DC-chol liposome) showed lower level of gene expression in the tumor models. Based on the results using the reporter gene, we examined the antitumor effect after direct interferon-gamma (IFN-gamma) gene transfer into CT-26 tumors. A significant IFN-gamma production and growth inhibition were obtained following direct intratumoral injection of IFN-gamma gene with naked plasmid DNA (pCMV-Mu gamma). Interestingly, pCMV-Mu gamma/DC-chol liposome complexes exhibited more pronounced growth inhibitory effect despite lower IFN-gamma production. Induction of CT-26 specific antitumor immunity by IFN-gamma gene transfer was confirmed by rejection of a CT-26 tumor challenge in the mice showing complete regression of CT-26 tumors after both treatments. Further analysis demonstrated that a significant cDNA-independent induction of IFN-beta and TNF-alpha occurred following injection with the liposome complexes, suggesting a nonspecific suppressive effect on CT-26 tumor growth by these cytokines. Thus, the present study has demonstrated that tumor tissue might be a promising target for direct IFN-gamma gene transfer with plasmid-based nonviral vectors. It is also suggested that immunomodulatory effects by various cytokines could be involved in antitumor effects after direct intratumoral injection of plasmid DNA formulations.
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PMID:Gene expression and antitumor effects following direct interferon (IFN)-gamma gene transfer with naked plasmid DNA and DC-chol liposome complexes in mice. 1034 84

We reported previously that tumor cells isolated from metastases of the in vitro transformed squamous cell carcinoma line Pam 212 exhibit an elevation in constitutive production of proinflmmatory cytokines interleukin (IL)-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and KC (the murine homologue of chemokine Gro-alpha). The basis for constitutive expression of these cytokines after tumor progression in vivo is unknown. Regulation of the expression of these proinflammatory cytokines involves transcription factor nuclear factor kappaB (NF-kappaB), which can be activated by cytokines such as tumor necrosis factor (TNF)-alpha. In this study, we compared the constitutive and TNF-alpha-induced expression of proinflammatory cytokines in parental Pam 212 and metastatic LY-2 and LY-8 cell lines and determined the relationship of cytokine expression to activation of NF-kappaB. We found that the metastatic cell lines exhibited an increase in constitutive and TNF-alpha-inducible expression of proinflammatory cytokines when compared with parental Pam 212 cells. The increased cytokine expression was associated with an increase in constitutive and TNF-alpha-inducible activation of NF-kappaB as demonstrated by electrophoretic mobility shift assay and luciferase-reporter gene assay. Constitutive nuclear localization of NF-kappaB p65 was observed in LY-2 and LY-8 cells in culture and in tumor specimens but rarely in Pam 212 cells, consistent with the constitutive activation of NF-kappaB in tumor cels after selection in vivo. Induction of NF-kappaB by TNF-alpha was inhibited by the addition of protease inhibitors calpain inhibitor I and N-tosyl-phechloromethyl ketone and antioxidant 1-pyrrolidinecarbodithioic acid, whereas constitutive activation of NF-kappaB and cytokine KC mRNA expression was inhibited by N-tosyl-phechloromethyl ketone alone. Overexpression of a human Ikappa(B)alpha dominant suppresser in Pam 212 cells inhibited TNF-alpha-induced NF-kappaB binding activity and KC expression. These data indicate that activation of NF-kappaB contributes to increased expression of proinflammatory cytokines during metastatic tumor progression of squamous cell carcinoma, and that distinct mechanisms may be involved in the regulation of constitutive and TNF-alpha-induced activation of NF-kappaB in squamous cell carcinoma.
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PMID:The host environment promotes the constitutive activation of nuclear factor-kappaB and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. 1041 16

Although the precise mechanisms by which steroids mediate their therapeutic effects remain unknown, steroids have been reported to abrogate cytokine-induced activation of the transcription factor NF-kappa B. In some cell types, NF-kappa B activation is necessary to regulate cytokine-mediated cellular functions. However, compelling evidence suggests that the steroid inhibition of NF-kappa B is complex and cell specific. Using EMSA, we show that stimulation with TNF-alpha or IL-1 beta induces NF-kappa B DNA-binding activity in human airway smooth muscle cells. TNF-alpha and IL-1 beta also increased luciferase activity in airway smooth muscle cells transfected with a reporter plasmid containing kappa B enhancer elements. Cytokines activated NF-kappa B by rapidly degrading its cytosolic inhibitor I kappa B alpha, which was then regenerated after 60 min. Cytokine-mediated I kappa B alpha reappearance was completely blocked by the protein synthesis inhibitor cycloheximide. Inhibition of cytokine-mediated I kappa B alpha proteolysis using the protease inhibitors N-tosyl-L -phenylalanine chloromethyl ketone and N-acetyl-L -leucinyl-L -leucinyl-norleucinal also inhibited cytokine-mediated early expression of ICAM-1. Although dexamethasone partially inhibited IL-1 beta- and TNF-alpha-induced up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on cytokine-induced ICAM-1 expression at 18-24 h. In addition, neither cytokine-induced degradation or resynthesis of I kappa B alpha nor NF-kappa B DNA-binding activity were affected by dexamethasone. In cells transfected with the luciferase reporter, dexamethasone did not affect TNF-alpha-induced NF-kappa B-dependent transcription. Interestingly, cytokine-mediated expression of cyclooxygenase-2 was completely abrogated by dexamethasone at 6 h. Together, these data demonstrate that cytokine-mediated NF-kappa B activation and ICAM-1 expression involve activation of a steroid-insensitive pathway.
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PMID:Up-regulation of ICAM-1 by cytokines in human tracheal smooth muscle cells involves an NF-kappa B-dependent signaling pathway that is only partially sensitive to dexamethasone. 1043 53


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