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1.
Sci Rep ; 7(1): 8526, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819304

RESUMO

Dynamic remodelling of the extracellular matrix (ECM) is a key feature of cancer progression. Enzymes that modify the ECM, such as matrix metalloproteinases (MMPs), have long been recognised as important targets of anticancer therapy. Inflammatory cytokines are known to play a key role in regulating protease expression in cancer. Here we describe the identification of gamma-activated site (GAS)-like, signal transducer and activator of transcription (STAT) binding elements (SBEs) within the proximal promoters of the MMP-1 and MMP-3 genes, which in association with AP-1 components (c-Fos or Jun), bind STAT-1 in a homodimer like complex (HDLC). We further demonstrate that MMP expression and binding of this complex to SBEs can either be enhanced by interleukin (IL)-6, or reduced by interferon gamma (IFN-γ), and that IL-6 regulation of MMPs is not STAT-3 dependent. Collectively, this data adds to existing understanding of the mechanism underlying cytokine regulation of MMP expression via STAT-1, and increases our understanding of the links between inflammation and malignancy in colon cancer.


Assuntos
Regulação da Expressão Gênica , Interleucina-6/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Fator de Transcrição STAT1/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Ligação Proteica
2.
Neoplasia ; 15(9): 1110-24, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24027435

RESUMO

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL) and various inflammatory disorders including HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I oncoprotein Tax is known to cause permanent activation of many cellular transcription factors including nuclear factor-κB (NF-κB), cyclic adenosine 3',5'-monophosphate response element-binding protein, and activator protein 1 (AP-1). Here, we show that NF-κB-binding cofactor inhibitor of NF-κB-ζ (IκB-ζ) is constitutively expressed in HTLV-I-infected T cell lines and ATL cells, and Tax transactivates the IκB-ζ gene, mainly through NF-κB. Microarray analysis of IκB-ζ-expressing uninfected T cells demonstrated that IκB-ζ induced the expression of NF-κB. and interferon-regulatory genes such as B cell CLL/lymphoma 3 (Bcl3), guanylate-binding protein 1, and signal transducer and activator of transcription 1. The transcriptional activation domain, nuclear localization signal, and NF-κB-binding domain of IκB-ζ were required for Bcl3 induction, and IκB-ζ synergistically enhanced Tax-induced Bcl3 transactivation in an NF-κB-dependent manner. Interestingly, IκB-ζ inhibited Tax-induced NF-κB, AP-1 activation, and HTLV-I transcription. Furthermore, IκB-ζ interacted with Tax in vitro and this interaction was also observed in an HTLV-I-transformed T cell line. These results suggest that IκB-ζ modulates Tax-dependent and Tax-independent gene transcription in T cells. The function of IκB-ζ may be of significance in ATL genesis and pathogenesis of HTLV-I-associated diseases.


Assuntos
Produtos do Gene tax/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Linfócitos T/metabolismo , Proteína 3 do Linfoma de Células B , Linhagem Celular , Ativação Enzimática , Proteínas de Ligação ao GTP/biossíntese , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Proteínas I-kappa B/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Fator de Transcrição STAT1/biossíntese , Transdução de Sinais , Fator de Transcrição AP-1/biossíntese , Fatores de Transcrição/biossíntese , Transcrição Gênica , Ativação Transcricional
3.
Mol Cancer Ther ; 12(7): 1288-98, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23598529

RESUMO

STAT1 plays a pivotal role in signal transduction and transcriptional activation in response to type I and II IFNs. Regulation of STAT1 expression has significant consequences in human cancer cells, where STAT1 deficiencies have been associated with cellular resistance to type I IFN. Distinct promoter, enhancer, and repressor regions have previously been described in the regulatory part of the human STAT1 gene extending as far as the second intron. A putative IFN-stimulated response element sequence in the STAT1 promoter is inducible by type I IFN and binds the IFN-α/ß-induced complex, ISGF3. Together with the previously characterized IRF-E/GAS/IRF-E (IGI) motif, these positive regulatory elements provide a means for intracellular amplification of STAT1 expression, which is necessary for increasing cell responsiveness to the IFNs. In contrast, the transcriptional repressor REST binds to an RE-1 element in the STAT1 repressor region and in doing so represses transcription from the STAT1 gene regulatory region in melanoma cells lines. Repression significantly decreased in a REST-null cell line. Altering REST function from a transcriptional repressor into an activator as REST-VP16 increased expression from RE-1-targeted reporters. RNA expression of 65 melanoma cell lines by microarray and selected lines with known IFN responsiveness showed significant inverse correlations between STAT1/REST that were related to cellular responses to IFN. Thus REST, through the intronic RE-1 element, provides a means for downregulating STAT1 expression, affecting melanoma responsiveness to IFN. Intracellular levels of REST may be a useful marker to test for IFN resistance and as a novel therapeutic target in IFN-resistant melanomas.


Assuntos
Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Melanoma/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT1/genética , Animais , Imunoprecipitação da Cromatina , Expressão Gênica , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Melanoma/metabolismo , Regiões Promotoras Genéticas , Ratos , Proteínas Repressoras/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Ativação Transcricional , Transfecção
4.
J Interferon Cytokine Res ; 25(2): 92-102, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15695930

RESUMO

Luciferase reporter constructs are widely used for analysis of gene regulation when characterizing promoter and enhancer elements. We report that the recently developed codon-modified Renilla luciferase construct included as an internal standard for cotransfection must be used with great caution with respect to the amount of DNA transfected. Also, the dual-luciferase reporter vectors encoding Photinus pyralis firefly or Renilla reniformis luciferase showed a linear increase in dose-response with increasing amounts of transfected DNA, but at higher levels of transfected DNA, a reduction in expressed levels of luciferase activity resulted. In addition, treatment with type I interferon (IFN) was found to significantly reduce levels of P. pyralis firefly and Renilla luciferase activity. In contrast, cells transfected with a green fluorescent protein (GFP) reporter construct showed no significant IFN-associated change. The reduction in luciferase activity resulting from IFN treatment was not due to IFN-mediated cytotoxicity, as no change in cellular propidium iodide (PI) staining was observed by flow cytometry. IFN treatment did not alter the levels of firefly luciferase activity in cell culture supernatants or the luciferase mRNA levels determined by quantitative real-time RT-PCR analysis. Based on these results, it is probable that the IFN-induced reduction in levels of luciferase activity detected in reporter assays occurs via a posttranscriptional mechanism. Thus, it is important to be aware of these complications when using luciferase reporter systems in general or for analyzing cytokine-mediated responsive regulation of target genes, particularly by the type I IFNs.


Assuntos
Genes Reporter , Vetores Genéticos , Luciferases de Vaga-Lume , Luciferases de Renilla , Luciferases/genética , Animais , Linhagem Celular Tumoral , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Citometria de Fluxo , Corantes Fluorescentes , Fluorometria , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Interferon gama/farmacologia , Luciferases/efeitos dos fármacos , Melanoma , Plasmídeos , Propídio/metabolismo , RNA Mensageiro/metabolismo , Renilla/enzimologia , Renilla/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção
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