ABSTRACT
BACKGROUND & AIMS: This study explored the eyes absent 4 (EYA4) gene promoter methylation in noncolitic colorectal tissues and assessed its discrimination for neoplasia in chronic ulcerative colitis (CUC). METHODS: The methylation status of noncolitic specimens was confirmed by direct bisulfite sequencing. Methylation-specific polymerase chain reaction (MSP) primers were designed to evaluate colorectal tissues, including 50 noncolitic patients comprising 24 normal epithelia, 14 polyps, and 12 cancers. The assay was tested on tissues from 67 CUC patients including 31 surveillance neoplasia-positive patients and nonneoplastic controls including 22 CUC surveillance-negative and 14 CUC short-disease duration. Remote colonic tissue was included from each of 27 of the 31 CUC neoplasia cases. The expression of EYA4 was quantified in cell lines by use of reverse-transcription polymerase chain reaction. RESULTS: Within noncolitic tissues, bisulfite sequencing showed EYA4 promoter hypermethylation in 80% (8 of 10) of colorectal cancers but in none (0 of 9) of the normal tissues. MSP was positive in 81% (21 of 26) of cancers and polyps and in only 4% (1 of 14) of normal mucosa. In CUC, MSP was positive in 81% (25 of 31) of neoplastic cases but in none (0 of 36) of the nonneoplastic controls. RNA expression was decreased in methylated compared with unmethylated cell lines (P < .001). Treatment with 5-Aza-2'-deoxycytidine (DAC)/Trichostatin (TSA) increased the overall messenger RNA expression (P = .005). CONCLUSIONS: The EYA4 gene promoter is hypermethylated commonly in sporadic and colitic neoplasia and may be associated with gene silencing. EYA4 methylation represents a candidate marker for CUC surveillance.
Subject(s)
Colitis, Ulcerative/pathology , Trans-Activators/genetics , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biomarkers/analysis , Cell Line , Chronic Disease , Colitis, Ulcerative/complications , Colitis, Ulcerative/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Colonic Polyps/genetics , Decitabine , Humans , Hydroxamic Acids/pharmacology , Methylation , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
APC (adenomatous polyposis coli) promoter methylation has been linked to the early development of colorectal cancers. However, the role of APC methylation and its effect on protein expression in colon cancer metastasis is largely unknown. In this study, we investigated APC promoter methylation by Methylight analysis and analysed the APC protein levels by immunohistochemistry and western blot analysis in 24 liver metastasis and 39 primary colorectal cancers. Promoter methylation of the APC gene was found to be a frequent event in liver metastasis (10/24) and significantly more frequent compared with primary colorectal cancer (7/39, P = 0.047). APC methylation was not found in 14 matched normal colon tissues. APC protein was detected in the cytoplasm of primary and metastatic cancer cells and non-tumorous colon epithelium. By western blot analysis, APC protein levels were found to be decreased in primary tumour tissues compared with the normal colon mucosa. In contrast, APC protein levels were not decreased in the cancer cells that had metastasized to the liver. APC protein levels were independent of the presence of APC promoter methylation or gene mutations. In summary, APC promoter methylation is a frequent epigenetic alteration in colorectal cancer metastasis. However, we observed no significant association between APC promoter methylation or gene mutation and APC protein expression in colorectal metastasis. Therefore, metastatic cancer cells seem to harbour a heterogenous genetic and epigenetic background, in which cancer cells may exhibit APC promoter methylation that is independent of APC expression.
Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , DNA Methylation , Genes, APC/physiology , Neoplasm Metastasis/genetics , Adult , Blotting, Western , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Male , Middle Aged , Promoter Regions, GeneticABSTRACT
A new isoform of the full-length murine thrombopoietin (Tpo) receptor was isolated from a murine spleen cDNA library. This isoform, c-mpl-II, differs from full-length c-mpl (c-mpl-I) by virtue of deletion of 180 nucleotides that encode 60 amino acids located in the extracellular domain of Mpl. Normal murine megakaryocytes were found to express both c-mpl-I and c-mpl-II transcripts. BaF3 cells transfected with c-mpl-I expressed a 95 kDa protein that was displayed on the cell surface and bound 125I-Tpo. BaF3 cells transfected with c-mpl-II expressed a 70 kDa protein. However, these cells were not able to bind 125I-Tpo and surface display of Mpl-II could not be detected. In summary, c-mpl-II is an isoform of murine Mpl expressed by megakaryocytes that lacks a 60 amino acid region required for surface expression of the protein.
