ABSTRACT
BACKGROUND: The development of non-invasive diagnostic methods for endometriosis requires sensitive and disease specific biomarkers. Here, we describe the use of aspirated endometrial fluid from women with and without endometriosis as a novel biological sample for biomarker discovery. METHODS: Differential protein expression profiling of aspirates from women with early endometriosis (n = 14), advanced endometriosis (n = 32) and without evidence of the disease (n = 32) was assessed by two-dimensional gel electrophoresis (2-DE). A biomarker validation study was performed in an independent cohort (early endometriosis n = 6 and advanced endometriosis n = 14, controls n = 15). RESULTS: The analysis resulted in the identification of 31 proteins showing statistically significant differences in expression. The proteins identified are related to cell signalling, cell death and cell movement, processes that may be involved in the onset and/or progression of endometriosis. The differences in expression observed for 14-3-3 (signal transduction) and moesin (cytoskeletal structure) were confirmed in an independent group of endometriosis patients. CONCLUSIONS: Endometrial fluid represents a novel sample for proteomic analysis offering reliable, disease specific information on protein expression, facilitating the discovery of biomarkers for endometriosis. The results described here complement previous proteomic studies, providing new endometriosis-related proteins to be validated as diagnostic markers.
Subject(s)
Biomarkers/metabolism , Body Fluids/metabolism , Endometriosis/diagnosis , Endometriosis/metabolism , Endometrium/metabolism , Proteins/metabolism , Adolescent , Adult , Blotting, Western , Case-Control Studies , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Middle Aged , Protein Array Analysis , Proteins/isolation & purification , Proteomics , Sensitivity and Specificity , Young AdultABSTRACT
The N-alpha-acetyltransferase NatB, composed in Saccharomyces cerevisiae by the Nat3p and Mdm20p subunits, is an important factor for yeast growth and resistance to several stress agents. However, the expression and functional role of the mammalian counterpart has not yet been analysed. Here, we report the identification of Nat3p human homologue (hNAT5/hNAT3) and the characterization of its biological function. We found that hNAT5/hNAT3 silencing in HeLa cells results in inhibition of cell proliferation and increased sensitivity to the pro-apoptotic agent MG132. Moreover, inhibition of hNAT5/hNAT3 expression induces p53 activation and upregulation of the antiproliferative protein p21(WAF1/CIP1). The changes of the cellular transcriptome after hNAT5/hNAT3 knockdown confirmed the involvement of this protein in cell growth and survival processes. Among the genes differentially expressed, we observed upregulation of several p53-dependent antiproliferative and pro-apoptotic genes. In the c-myc transgenic mice, which is a model of inducible hepatocarcinoma, we found that hNAT5/hNAT3 was upregulated when the tumour was induced. In accordance with this observation, we noticed increased hNAT5/hNAT3 protein level in neoplastic versus non-neoplastic tissue in a high proportion of patients with hepatocellular carcinoma. Consequently, our results suggest that hNAT5/hNAT3 is required for cellular proliferation and can be implicated in tumour growth.
Subject(s)
Acetyltransferases/metabolism , Cell Proliferation , Cell Transformation, Neoplastic , Gene Expression Regulation, Neoplastic , Acetyltransferases/analysis , Acetyltransferases/genetics , Adenoviridae/genetics , Animals , Apoptosis/drug effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Silencing , HeLa Cells , Humans , Kidney/cytology , Leupeptins/pharmacology , Mice , Mice, Transgenic , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , RNA, Small Interfering/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. METHODS: We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. RESULTS: STAT3alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma. CONCLUSIONS: STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.
