Abstract:The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of… Show more
“…Interestingly, treatment of our EpCAM-negative ovarian cancer cells with a DNA methylation inhibitor induced EpCAM expression, both on mRNA and protein level. The role of DNA methylation in silencing epcam has been previously published by us for the intermediate epcam expressing SKOV3 ovarian cancer cells, and is in line with observations in other tumour types (Spizzo et al , 2007; Tai et al , 2007; van der Gun et al , 2008). In normal cells (HOSE), we did not find DNA hypermethylation, even though in several ovarian cancer cell lines, including SKOV3, CaOV3 and OVCAR3, EpCAM mRNA was reported to be 3 log higher compared with HOSE cells (Kim et al , 2003).…”
Section: Discussionsupporting
confidence: 88%
“…In our ovarian cancer cell line panel, EpCAM expression was inversely correlated with the DNA methylation status of the promoter and part of exon 1, as reported for several other tumour types (Spizzo et al , 2007; Tai et al , 2007; Yu et al , 2008; van der Gun et al , 2008). Interestingly, treatment of our EpCAM-negative ovarian cancer cells with a DNA methylation inhibitor induced EpCAM expression, both on mRNA and protein level.…”
Section: Discussionsupporting
confidence: 82%
“…Alternatively, we also demonstrated that endogenous EpCAM expression can be actively downregulated by nuclear delivery of a DNA methyltransferase (van der Gun et al , 2008). Here, we investigate epigenetic mechanisms and transcription factors underlying the overexpression of EpCAM in ovarian cancer.…”
mentioning
confidence: 90%
“…For various tumour types, EpCAM overexpression has been associated with DNA hypomethylation of the promoter and treatment of EpCAM-negative cells with a DNA methylation inhibitor induced EpCAM expression (Spizzo et al , 2007; Tai et al , 2007; van der Gun et al , 2008). Alternatively, we also demonstrated that endogenous EpCAM expression can be actively downregulated by nuclear delivery of a DNA methyltransferase (van der Gun et al , 2008).…”
Background:The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma.Methods:Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2′-deoxycytidine (5-AZAC) was used to induce EpCAM expression.Results:Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe.Conclusion:This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.
“…Interestingly, treatment of our EpCAM-negative ovarian cancer cells with a DNA methylation inhibitor induced EpCAM expression, both on mRNA and protein level. The role of DNA methylation in silencing epcam has been previously published by us for the intermediate epcam expressing SKOV3 ovarian cancer cells, and is in line with observations in other tumour types (Spizzo et al , 2007; Tai et al , 2007; van der Gun et al , 2008). In normal cells (HOSE), we did not find DNA hypermethylation, even though in several ovarian cancer cell lines, including SKOV3, CaOV3 and OVCAR3, EpCAM mRNA was reported to be 3 log higher compared with HOSE cells (Kim et al , 2003).…”
Section: Discussionsupporting
confidence: 88%
“…In our ovarian cancer cell line panel, EpCAM expression was inversely correlated with the DNA methylation status of the promoter and part of exon 1, as reported for several other tumour types (Spizzo et al , 2007; Tai et al , 2007; Yu et al , 2008; van der Gun et al , 2008). Interestingly, treatment of our EpCAM-negative ovarian cancer cells with a DNA methylation inhibitor induced EpCAM expression, both on mRNA and protein level.…”
Section: Discussionsupporting
confidence: 82%
“…Alternatively, we also demonstrated that endogenous EpCAM expression can be actively downregulated by nuclear delivery of a DNA methyltransferase (van der Gun et al , 2008). Here, we investigate epigenetic mechanisms and transcription factors underlying the overexpression of EpCAM in ovarian cancer.…”
mentioning
confidence: 90%
“…For various tumour types, EpCAM overexpression has been associated with DNA hypomethylation of the promoter and treatment of EpCAM-negative cells with a DNA methylation inhibitor induced EpCAM expression (Spizzo et al , 2007; Tai et al , 2007; van der Gun et al , 2008). Alternatively, we also demonstrated that endogenous EpCAM expression can be actively downregulated by nuclear delivery of a DNA methyltransferase (van der Gun et al , 2008).…”
Background:The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma.Methods:Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2′-deoxycytidine (5-AZAC) was used to induce EpCAM expression.Results:Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe.Conclusion:This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.
“…Important to mention is that the mitochondrial expression of M.SssI or M.CviPI was not associated with any toxicity, which is in contrast to the nuclear expression of e.g. M.SssI 33 in mammalian cells.Figure 4Mitochondrial localization of mitochondria-targeted DNA methyltransferases. ( a ) Confocal microscopy of HCT116 cells expressing MLS-mCherry-M.SssI.…”
Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the detection of mtDNA methylation as well as hydroxymethylation. Importantly, differential mtDNA methylation has been linked to aging and diseases, including cancer and diabetes. However, functionality of mtDNA methylation has not been demonstrated. Therefore, we targeted DNA methylating enzymes (modifying cytosine in the CpG or GpC context) to the mtDNA. Unexpectedly, mtDNA gene expression remained unchanged upon induction of CpG mtDNA methylation, whereas induction of C-methylation in the GpC context decreased mtDNA gene expression. Intriguingly, in the latter case, the three mtDNA promoters were differentially affected in each cell line, while cellular function seemed undisturbed. In conclusion, this is the first study which directly addresses the potential functionality of mtDNA methylation. Giving the important role of mitochondria in health and disease, unravelling the impact of mtDNA methylation adds to our understanding of the role of mitochondria in physiological and pathophysiological processes.
The presence and number of circulating tumor cells (CTCs) in the blood of patients with solid tumors are predictive of their clinical outcomes. To date, the CellSearch system is the only US Food and Drug Administration-approved CTC enumeration system for advanced breast, prostate, and colon cancers. However, sensitivity issues due to epithelial cellular adhesion molecule (EpCAM)-based enrichment and limited capability for subsequent molecular analysis must be addressed before CTCs can be used as predictive markers in the clinical setting. We have developed a multicolor CTC detection system using cross-contamination-free flow cytometry, which permits the enumeration and characterization of CTCs for multiple molecular analyses. Tumor cell lines with different expression levels of EpCAM were spiked into peripheral blood obtained from healthy donors. Spike-in samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, and this was followed by fixation and labeling with CD45-Alexa Fluor 700, EpCAM-phycoerythrin, cytokeratin (CK)-fluorescein isothiocyanate antibodies, and/or 7-aminoactinomycin D for nuclei staining. Excellent detection (slope 5 0.760-0.888) and a linear performance (R 2 5 0.994-0.998) were noted between the observed and expected numbers of tumor cells, independent of EpCAM expression. The detection rate was markedly higher than that obtained using the CellSearch system, suggesting the superior sensitivity of our system in detecting EpCAM2 tumor cells. Additionally, the incorporation of an epithelial-mesenchymal transition (EMT) marker allowed us to detect EpCAM2/CK2 cells and EMT-induced tumor cells. Taken together, our multicolor CTC detection system may be highly efficient in detecting previously unrecognized populations of CTCs. V C 2013 International Society for Advancement of Cytometry
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