ABSTRACT
BACKGROUND: Deregulation of the canonical Wnt/beta-catenin-pathway is known to play an important role in the progression of various tumour cell types including prostate cancer (PCa). Recently, the tumour-suppressor p53 was shown to down-regulate beta-catenin-signalling in colon cancer. As p53 is frequently mutated in late stage PCa we investigated the effect of wild-type p53 (p53wt) as well as p53-mutants on beta-catenin-signalling in PCa-cell lines. METHODS: The effects of p53wt and p53-mutants on Wnt/beta-catenin-signalling were studied using reporter gene assays. Expression of beta-catenin levels was monitored by Western blotting. RESULTS: Overexpression of p53wt as well as p53(249Ser) (a structural mutant) and p53(273His) (a DNA-contact-mutant) almost completely inhibited beta-catenin-mediated transcriptional activity of the T-cell factor (TCF) whereas p53(175His), a structural mutant, and a p53-mutant with a C-terminal deletion in the tetramerization domain (Deltap53) were unable to do so. Co-transfection experiments with p53wt and a dominant negative p53-mutant reversed the down-regulation of TCF-signalling, while Deltap53 was unable to interfere with p53wt-function. Down-regulation of TCF-signalling by p53wt and p53(273His) was accompanied by a reduction in beta-catenin protein level. CONCLUSIONS: p53wt, p53(273His)- and p53(249Ser)-mutants are able to down-regulate beta-catenin-signalling in PCa-cells probably via degradation of beta-catenin. The degradation of beta-catenin in PCa by p53 is not linked to transcriptional activity of p53. So far the mechanism how p53 interferes with beta-catenin-signalling is unknown. For the first time we provide experimental evidence that the C-terminus of p53 plays an important role in the down-regulation of beta-catenin-mediated TCF-signalling in PCa-cell lines possibly via p53 transrepressional function.
Subject(s)
Mutation , Prostatic Neoplasms/metabolism , TCF Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , beta Catenin/metabolism , Blotting, Western , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic , Genes, p53 , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Male , Promoter Regions, Genetic , Prostatic Neoplasms/genetics , Signal Transduction , TCF Transcription Factors/genetics , Transcription Factor 7-Like 2 Protein , Transcriptional Activation , Transfection , beta Catenin/agonistsABSTRACT
BACKGROUND: Epigenetic silencing of the RAS association domain family 1A (RASSF1A) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation. Contradictory results have been reported for RASSF1A methylation in normal kidney, thus it is not clear whether a significant difference between RASSF1A methylation in normal and tumor cells of the kidney exists. Moreover, RASSF1A expression has not been characterized in tumors or normal tissue as yet. RESULTS: Using combined bisulfite restriction analysis (COBRA) we compared RASSF1A methylation in 90 paired tissue samples obtained from primary kidney tumors and corresponding normal tissue. Bisulfite sequence analysis was carried out using both pooled amplicons from the tumor and normal tissue groups and subclones obtained from a single tissue pair. Expression of RASSF1A was analyzed by the use of tissue arrays and immunohistochemistry. We found significantly increased methylation in tumor samples (mean methylation, 20%) compared to corresponding normal tissues (mean methylation, 11%; P < 0.001). Densely methylated sequences were found both in pooled and individual sequences of normal tissue. Immunohistochemical analysis revealed a significant reduced expression of RASSF1A in most of the tumor samples. Heterogeneous expression patterns of RASSF1A were detected in a subgroup of histologically normal tubular epithelia. CONCLUSION: Our methylation and expression data support the hypothesis that RASSF1A is involved in early tumorigenesis of renal cell carcinoma.
Subject(s)
DNA Methylation , Kidney Neoplasms/pathology , Promoter Regions, Genetic , Tumor Suppressor Proteins/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Line , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Kidney/metabolism , Kidney/pathology , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Male , Middle Aged , Sequence Analysis, DNA/methods , Sulfites/chemistry , Tissue Array Analysis , Tumor Suppressor Proteins/metabolismABSTRACT
BACKGROUND: The prospective diagnostic efficiency of quantitative glutathione S transferase (GSTP1) promoter hypermethylation analysis in biopsy washing samples has not been determined so far. METHODS: Biopsies were obtained prospectively from 86 patients suspicious for prostate cancer (CaP). After isolation of DNA from biopsy washings and bisulfite conversion methylated and unmethylated GSTP1 sequences were specifically quantitated by real-time fluorescence PCR. Relative degrees of methylation were compared to results of histopathological examination. RESULTS: Increased relative methylation was found for the CaP group (mean 28.1%) compared to biopsies without histological evidence for malignancy (5.2%; P < 0.001). A sensitivity and specificity of 92% and 86% and positive and negative predictive values of 82% and 94% were obtained. Receiver operating characteristic (ROC) analysis demonstrated a value of 0.90 (95% CI 0.82-0.98) for the area under curve (AUC). CONCLUSIONS: Biopsy washing DNA GSTP1 hypermethylation analysis demonstrates a high diagnostic efficacy which is comparable to the retrospective analysis of biopsy tissue specimens. Moreover it is compatible with routine biopsy examination thus permitting further prospective evaluation in CaP diagnosis.
