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1.
Oncogene ; 34(17): 2251-60, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-24909169

ABSTRACT

CYLD is a deubiquitinating (DUB) enzyme that has a pivotal role in modulating nuclear factor kappa B (NF-κB) signaling pathways by removing the lysine 63- and linear-linked ubiquitin chain from substrates such as tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Loss of CYLD activity is associated with tumorigenicity, and levels of CYLD are lost or downregulated in different types of human tumors. In the present study, we found that high CYLD expression was associated with better overall survival and relapse-free neuroblastoma patient outcome, as well as inversely correlated with the stage of neuroblastoma. Retinoic acid-mediated differentiation of neuroblastoma restored CYLD expression and promoted SUMOylation of CYLD. This posttranslational modification inhibited deubiquitinase activity of CYLD against TRAF2 and TRAF6 and facilitated NF-κB signaling. Overexpression of non-SUMOylatable mutant CYLD in neuroblastoma cells reduced retinoic acid-induced NF-κB activation and differentiation of cells, but instead promoted cell death.


Subject(s)
Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Neuroblastoma/enzymology , Sumoylation , Tumor Suppressor Proteins/biosynthesis , Ubiquitination , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Deubiquitinating Enzyme CYLD , Humans , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Neuroblastoma/genetics , Neuroblastoma/pathology , Signal Transduction/drug effects , Signal Transduction/genetics , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Tretinoin/pharmacology , Tumor Suppressor Proteins/genetics
2.
Oncogene ; 32(12): 1601-8, 2013 Mar 21.
Article in English | MEDLINE | ID: mdl-22580608

ABSTRACT

B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. As high levels of Bcl-3 expression and activation have been detected in different types of human cancer, Bcl-3 has been labeled a proto-oncogene. Our study uncovered a markedly upregulated Bcl-3 expression in human prostate cancer (PCa), where inflammatory cell infiltration was observed. Elevated Bcl-3 expression in PCa was dependent on the proinflammatory cytokine interleukin-6-mediated STAT3 activation. Microarray analyses, using Bcl-3 knockdown in PCa cells, identified the inhibitor of DNA-binding (Id) family of helix-loop-helix proteins as potential Bcl-3-regulated genes. Bcl-3 knockdown reduced the abundance of Id-1 and Id-2 proteins and boosted PCa cells to be more receptive to undergoing apoptosis following treatment with anticancer drug. Our data imply that inactivation of Bcl-3 may lead to sensitization of cancer cells to chemotherapeutic drug-induced apoptosis, thus suggesting a potential therapeutic strategy in PCa treatment.


Subject(s)
Gene Expression Regulation, Neoplastic , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 2/genetics , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogenes , Transcription Factors/genetics , B-Cell Lymphoma 3 Protein , Cell Line, Tumor , Humans , Interleukin-6/genetics , Male , NF-kappa B p52 Subunit/physiology , Promoter Regions, Genetic , Proto-Oncogene Mas , Proto-Oncogene Proteins/physiology , STAT3 Transcription Factor/physiology , Transcription Factors/physiology
3.
Oncogene ; 30(44): 4523-30, 2011 Nov 03.
Article in English | MEDLINE | ID: mdl-21577203

ABSTRACT

CYLD is a deubiquitination enzyme that regulates different cellular processes, such as cell proliferation and cell survival. Mutation and loss of heterozygosity of the CYLD gene causes development of cylindromatosis, a benign tumour originating from the skin. Our study shows that CYLD expression is dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans. Reduced CYLD expression in basal cell carcinoma was mediated by GLI1-dependent activation of the transcriptional repressor Snail. Inhibition of GLI1 restored the CYLD expression-mediated Snail signaling pathway, and caused a significant delay in the G1 to S phase transition, as well as proliferation. Our data suggest that GLI1-mediated suppression of CYLD has a significant role in basal cell carcinoma progression.


Subject(s)
Gene Expression Regulation, Neoplastic , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Carcinoma, Basal Cell/genetics , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Deubiquitinating Enzyme CYLD , Humans , Skin Neoplasms/genetics , Snail Family Transcription Factors , Zinc Finger Protein GLI1
4.
Acta Anaesthesiol Scand ; 45(10): 1215-20, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11736672

ABSTRACT

BACKGROUND: It has previously been shown that propofol in clinically relevant concentrations induces a calcium-dependent conformational change in the cytoskeleton. The aim of this study was to further clarify the effect of propofol on the actin cytoskeleton and to determine if this conformational change is mediated by the interaction between the GABA(A)-receptor and propofol. METHODS: Primary cultured cortical neurons from newborn rats were treated with propofol 3 microg x ml(-1) in a time-response titration, with and without preincubation with the GABA(A)-receptor antagonist, bicuculline. Actin-protein content was detected by Western blot analysis and the cellular content of F-actin measured by a spectrophotometric technique. RESULTS: Propofol triggers a relatively slow statistically significant increase in the intracellular F-actin content, maximum after 20-min incubation (160%+/-16.3) (mean+/-SEM) P<0.05. The propofol-induced increase in F-actin was effectively blocked by bicuculline. The increase in intracellular actin content after exposure to propofol as well as the effect of bicuculline were verified by Western blot analysis. CONCLUSION: The present study shows that propofol triggers a time-dependent change of actin. Since this reorganization can be blocked effectively by a GABA(A)-receptor antagonist, this suggests that the GABA(A)-receptor is involved in the pathway leading to cytoskeletal reorganization after propofol treatment. The actin polymerization reached its maximum after 20 min. Therefore, we believe that the propofol-induced changes might be connected with slower cellular responses such as cell-to-cell interaction and/or channel regulation.


Subject(s)
Actins/metabolism , Anesthetics, Intravenous/pharmacology , Neurons/metabolism , Propofol/pharmacology , Actins/drug effects , Animals , Animals, Newborn , Bicuculline/pharmacology , Cells, Cultured , Cerebral Cortex/anatomy & histology , Dose-Response Relationship, Drug , GABA Antagonists/pharmacology , Immunoblotting , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Spectrometry, Fluorescence
5.
J Cell Sci ; 114(Pt 10): 1925-34, 2001 May.
Article in English | MEDLINE | ID: mdl-11329379

ABSTRACT

Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D(4 )(LTD(4)), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD(4 )induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD(4) caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD(4) also induced dissociation of vinculin from alpha-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD(4)- and the TPA-induced dissociation of vinculin from alpha-catenin. Furthermore, LTD(4) caused a colocalisation of vinculin with PKC-alpha in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD(4)-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD(4)-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active beta1 integrins on the cell surface and an enhanced beta1 integrin-dependent adhesion of the cells to collagen IV.


Subject(s)
Intestinal Mucosa/enzymology , Leukotriene D4/pharmacology , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Vinculin/metabolism , Actins/metabolism , Cadherins/metabolism , Carcinogens/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Line , Cytoskeletal Proteins/metabolism , Enzyme Inhibitors/pharmacology , Focal Adhesions/enzymology , Humans , Indoles/pharmacology , Integrin beta1/biosynthesis , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Maleimides/pharmacology , Phosphorylation , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Tyrosine/metabolism , alpha Catenin
6.
Eur J Cell Biol ; 76(3): 185-91, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9716265

ABSTRACT

Epithelial cells play an important role in maintaining the intestinal mucosa barrier, a barrier that is impaired in several inflammatory conditions. The mechanisms behind this impairment are not known, but it can be presumed that structural alterations of the epithelial cells are involved. In support of this notion, we here show the inflammatory mediator leukotriene D4 (LTD4) triggered first a rapid (10 s) increase and immediately thereafter (30 s) a sustained decrease in the cellular filamentous actin (F-actin) level in intestinal epithelial cells. The initial LTD4-induced increase in F-actin content was effectively blocked by preincubating the cells with either pertussis toxin or the tyrosine kinase inhibitor genistein. A possible involvement of the tyrosine kinase-dependent phosphatidylinositol-3-kinase (PI-3-kinase) in the polymerisation of actin was supported by the observations that LTD4 induced a translocation to a membrane fraction of PI-3-kinase and by the findings that wortmannin, a PI-3-kinase inhibitor, totally abolished both this translocation of PI-3-kinase as well as the initial LTD4-induced polymerisation of actin. In addition, pertussis toxin and genistein, both known to interfere with the LTD4-induced calcium signal, completely or partially reversed the actin-depolymerising effect of LTD4. The calcium ionophore ionomycin (30s) induced actin depolymerisation to the same extent as LTD4 (30 s) did, but lacked the initial effect on actin polymerisation. In cells loaded with the calcium chelator MAPT, LTD4 induced a normal actin polymerisation response but the subsequent depolymerisation was completely inhibited. Similar results were obtained when the cells were preincubated with the protein kinase A inhibitor Rp-cAMPS, which has been shown to impair the LTD4-induced calcium signal in these epithelial cells. The present results show that the inflammatory mediator LTD4 triggers a reorganisation of the actin network in intestinal epithelial cells that is likely to contribute to the impairment of the intestinal barrier function.


Subject(s)
Actins/metabolism , Intestinal Mucosa/drug effects , Leukotriene D4/pharmacology , Calcium/physiology , Cell Line , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Enzyme Inhibitors/pharmacology , Epithelial Cells , Genistein/pharmacology , Humans , Intestinal Mucosa/cytology , Intracellular Fluid , Pertussis Toxin , Phosphatidylinositol 3-Kinases/physiology , Virulence Factors, Bordetella/pharmacology
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