Expression of MUC5AC, an early marker of pancreatobiliary cancer, is regulated by DNA methylation in the distal promoter region in cancer cells

Yamada, Norishige; Nishida, Yukari; Yokoyama, Seiya; Tsutsumida, Hideaki; Houjou, Izumi; Kitamoto, Sho; Goto, Masamichi; Higashi, Michiyo; Yonezawa, Suguru

doi:10.1007/s00534-010-0278-0

Cited by 24 publications

(27 citation statements)

References 36 publications

(65 reference statements)

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“…The expression level of GAPDH mRNA in each cell line was used as a control in the RT-PCR analysis. The methylation pattern of each gene was consistent with the results of massARRAY analysis [2,4,6-10]. …”

Section: Resultssupporting

confidence: 85%

“…Expression of mRNA was detected by semiquantitative RT-PCR analysis, with GAPDH mRNA used as a control. The expression level of mRNA shows a high propensity for regulation by DNA methylation [2,4,6-10] (Figure 3). The results of MSE analysis in each cell line (Figure 3) were consistent with DNA methylation patterns obtained by massARRAY analysis [2,4,7-10].…”

Section: Resultsmentioning

confidence: 99%

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The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

et al. 2012

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BackgroundMethylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity.MethodsHere, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices.ResultThe MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted.ConclusionsThe MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

et al. 2012

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“…Recently, Yamada et al showed that the CpG methylation status of around −3.7 kb in the MUC5AC promoter is associated with MUC5AC expression in some cancer cell lines, suggesting that the expression of MUC5AC is epigenetically regulated in the distal promoter region [45]. In fact, we found increase in MUC5AC expression by 5Aza-dC and/or TSA treatment, indicating some epigenetic mechanism involved in MUC5AC gene regulation (Fig.…”

Section: Discussionsupporting

confidence: 48%

Gli Regulates MUC5AC Transcription in Human Gastrointestinal Cells

et al. 2014

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MUC5AC is a well-known gastric differentiation marker, which has been frequently used for the classification of stomach cancer. Immunohistochemistry revealed that expression of MUC5AC decreases accompanied with increased malignant property of gastric mucosa, which further suggests the importance of MUC5AC gene regulation. Alignment of the 5′-flanking regions of MUC5AC gene of 13 mammal species denoted high homology within 200 bp upstream of the coding region. Luciferase activities of the deletion constructs containing upstream 451 bp or shorter fragments demonstrated that 15 bp region between −111 and −125 bp plays a critical role on MUC5AC promoter activity in gastrointestinal cells. We found a putative Gli-binding site in this 15 bp sequence, and named this region a highly conserved region containing a Gli-binding site (HCR-Gli). Overexpression of Gli homologs (Gli1, Gli2, and Gli3) clearly enhanced MUC5AC promoter activity. Exogenous modulation of Gli1 and Gli2 also affected the endogenous MUC5AC gene expression in gastrointestinal cells. Chromatin immunoprecipitation assays demonstrated that Gli1 directly binds to HCR-Gli: Gli regulates MUC5AC transcription via direct protein-DNA interaction. Conversely, in the 30 human cancer cell lines and various normal tissues, expression patterns of MUC5AC and Gli did not coincide wholly: MUC5AC showed cell line-specific or tissue-specific expression whereas Gli mostly revealed ubiquitous expression. Luciferase promoter assays suggested that the far distal MUC5AC promoter region containing upstream 4010 bp seems to have several enhancer elements for gene transcription. In addition, treatments with DNA demethylation reagent and/or histone deacetylase inhibitor induced MUC5AC expression in several cell lines that were deficient in MUC5AC expression. These results indicated that Gli is necessary but not sufficient for MUC5AC expression: namely, the multiple regulatory mechanisms should work in the distal promoter region of MUC5AC gene.

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“…Recently, methylated CpG mapping of MUC2 and MUC5AC promoters have been determined in pancreatic cancer cell lines [73,74]. It was shown that promoter methylation acts in combination with associated H3 histone deacetylation [74,75].…”

Section: Epigenetic Regulation Of Mucin Genes In Pancreatic Cancermentioning

confidence: 99%

“…It was shown that promoter methylation acts in combination with associated H3 histone deacetylation [74,75]. …”

Section: Epigenetic Regulation Of Mucin Genes In Pancreatic Cancermentioning

confidence: 99%

Mucins and Pancreatic Cancer

Jonckheere

Skrypek

Seuningen

2010

Cancers

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Pancreatic cancer is characterized by an often dramatic outcome (five year survival < 5%) related to a late diagnosis and a lack of efficient therapy. Therefore, clinicians desperately need new biomarkers and new therapeutic tools to develop new efficient therapies. Mucins belong to an ever increasing family of O-glycoproteins. Secreted mucins are the main component of mucus protecting the epithelia whereas membrane-bound mucins are thought to play important biological roles in cell-cell and cell-matrix interactions, in cell signaling and in modulating biological properties of cancer cells. In this review, we will focus on the altered expression pattern of mucins in pancreatic cancer, from the early neoplastic lesion Pancreatic Intraepithelial Neoplasia (PanIN) to invasive pancreatic carcinomas, and the molecular mechanisms (including genetic and epigenetic regulation) and signaling pathways known to control their expression. Moreover, we will discuss the recent advances about the biology of both secreted and membrane-bound mucins and their key roles in pancreatic carcinogenesis and resistance to therapy. Finally, we will discuss exciting opportunities that mucins offer as potential therapeutic targets in pancreatic cancer.

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Expression of MUC5AC, an early marker of pancreatobiliary cancer, is regulated by DNA methylation in the distal promoter region in cancer cells

Cited by 24 publications

References 36 publications

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

Gli Regulates MUC5AC Transcription in Human Gastrointestinal Cells

Mucins and Pancreatic Cancer

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