Abstract
The 14-3-3 proteins constitute a family of abundant, highly conserved and broadly expressed acidic polypeptides that are involved in the regulation of various cellular processes such as cell-cycle progression, cell growth, differentiation, and apoptosis. One member of this family, the 14-3-3 isoform sigma, is expressed only in epithelial cells and is frequently down-regulated in a variety of human cancers. To determine the prevalence of 14-3-3 sigma silencing in bladder cancer progression, we have studied the expression of this protein in normal urothelium and bladder transitional cell carcinomas (TCCs) of various grades and stages using two-dimensional gel electrophoresis in combination with Western blotting and immunohistochemistry. We show that the expression of 14-3-3 sigma is down-regulated in invasive TCCs, particularly in lesions that are undergoing epithelial-to-mesenchymal conversion. Altered expression of 14-3-3 sigma in invasive TCCs is not due to increased externalization of the protein nor to an aberrant proliferative potential of neoplastic cells. Furthermore, we found that impaired 14-3-3 sigma expression is not associated with increased levels of the dominant-negative transcriptional regulator Delta Np63. Down-regulation of 14-3-3 sigma was confirmed by indirect immunofluorescence using a peptide-based rabbit polyclonal antibody specific for this protein. We also show that the expression of 14-3-3 sigma is highly up-regulated in pure squamous cell carcinomas. Taken together, these results provide evidence that deregulation of 14-3-3 sigma may play a key role in bladder cancer progression, in particular in differentiation events leading to epithelial-to-mesenchymal transition and stratified squamous metaplasia.
Highlights
The 14-3-3 proteins constitute a family of abundant, highly conserved and broadly expressed acidic polypeptides that are involved in the regulation of various cellular processes such as cell-cycle progression, cell growth, differentiation, and apoptosis
We present evidence indicating that deregulation of the tumor suppressor protein 14-3-3 may play a key role in bladder cancer progression, in particular in events leading to epithelial-to-mesenchymal transition (EMT) and stratified squamous metaplasia
Immunohistochemical analysis was performed on 6-m-thick sections of frozen tissue according to standard methods using either a rabbit polyclonal antibody raised against a C-terminal peptide (H2N-CNAGEEGGEAPQEPQS-CONH2) specific for 14-3-3 (Eurogentec, Brussels, Belgium) and/or a mouse monoclonal antibody against cytokeratin 5 (CK5; Neomarkers, Freemont, CA)
Summary
Bladder Tumor Biopsies—Bladder specimens collected over a period of 5 years at Skejby Hospital, Aarhus, Denmark were analyzed. Proteomic Analysis and Quantitation of the Levels of 14-3-3— Two-dimensional gel electrophoresis (2D PAGE) was performed as previously described [24]. Immunohistochemical analysis was performed on 6-m-thick sections of frozen tissue according to standard methods using either a rabbit polyclonal antibody raised against a C-terminal peptide (H2N-CNAGEEGGEAPQEPQS-CONH2) specific for 14-3-3 (Eurogentec, Brussels, Belgium) and/or a mouse monoclonal antibody against cytokeratin 5 (CK5; Neomarkers, Freemont, CA). Double staining, using appropriate fluorescein isothiocyante (FITC)- and TRITC-labeled secondary antibodies (Dako, Glostrup, Denmark) were performed to investigate the relative localization of 14-3-3 and CK5. Proteins were resolved by 2D PAGE, blotted onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA), and detected with a 14-3-3-specific rabbit peptide polyclonal antibody prepared by Eurogentec using Supersignal WestPico detection reagents according to manufacturer’s instructions (Pierce, Rockford, IL)
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