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Similarity of aberrant DNA methylation in Barrett's esophagus and esophageal adenocarcinoma.

Smith E, De Young NJ, Pavey SJ, Hayward NK, Nancarrow DJ, Whiteman DC, Smithers BM, Ruszkiewicz AR, Clouston AD, Gotley DC, Devitt PG, Jamieson GG, Drew PA - Mol. Cancer (2008)

Bottom Line: The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC.This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3.We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Nursing and Midwifery, Flinders University, Bedford Park, South Australia 5042, Australia.

ABSTRACT

Background: Barrett's esophagus (BE) is the metaplastic replacement of squamous with columnar epithelium in the esophagus, as a result of reflux. It is the major risk factor for the development of esophageal adenocarcinoma (EAC). Methylation of CpG dinucleotides of normally unmethylated genes is associated with silencing of their expression, and is common in EAC. This study was designed to determine at what stage, in the progression from BE to EAC, methylation of key genes occurs.

Results: We examined nine genes (APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3, and TMEFF2), frequently methylated in multiple cancer types, in a panel of squamous (19 biopsies from patients without BE or EAC, 16 from patients with BE, 21 from patients with EAC), BE (40 metaplastic, seven high grade dysplastic) and 37 EAC tissues. The methylation frequency, the percentage of samples that had any extent of methylation, for each of the nine genes in the EAC (95%, 59%, 76%, 57%, 70%, 73%, 95%, 74% and 83% respectively) was significantly higher than in any of the squamous groups. The methylation frequency for each of the nine genes in the metaplastic BE (95%, 28%, 78%, 48%, 58%, 48%, 93%, 88% and 75% respectively) was significantly higher than in the squamous samples except for CDKN2A and RBP1. The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC. The methylation extent was an estimate of both the number of methylated alleles and the density of methylation on these alleles. This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3. There was no significant difference in methylation extent for any gene between high grade dysplastic BE and EAC.

Conclusion: We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC. This is also the first report of gene silencing by methylation of ID4 in BE or EAC. This study suggests that metaplastic BE is a highly abnormal tissue, more similar to cancer tissue than to normal epithelium.

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Methylation and expression of APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3 and TMEFF2 in esophageal cancer cell lines OE33 and TE7. The esophageal cancer cell lines OE33 and TE7 were treated with either 1 μmol/L aza-dC or vehicle for 72 hours. The medium was replaced with fresh medium only, and the cells incubated for a further 24 hours before harvesting. Bisulphite modified DNA was amplified using primers and PCR conditions (Table 2) which were specific for bisulphite modified DNA, did not discriminate between methylated and unmethylated sequences, and did not amplify unmodified DNA. The PCR products were melted by increasing the temperature from 60 to 95°C, rising 0.5 or 1°C at each step, waiting 30 seconds on the first step then 5 seconds for each step thereafter. Data was collected and analysed using the Melt Curve Analysis function of the RG-3000 Application Software. The left hand column shows the melt curves for each of the nine genes for OE33, the central column for TE7. Each plot shows the melt curves for the unmethylated (black lines) and methylated (open circles) controls and the cell lines treated with vehicle (red circles) or aza-dC (green circles). The horizontal axis represents temperature and the vertical axis -dF/dT. CDKN2A was not amplified in TE7. Interpretation of the melt curves is described in the Materials and Methods. The right hand column shows the gene expression in cell lines treated with vehicle (red columns) or aza-dC (green columns), as determined by qRT-PCR and normalised to HMBS. Data shown are the means ± SD from three independent experiments. TMEFF2 expression was below detectable limits in either cell line treated with vehicle or aza-dC.
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Figure 1: Methylation and expression of APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3 and TMEFF2 in esophageal cancer cell lines OE33 and TE7. The esophageal cancer cell lines OE33 and TE7 were treated with either 1 μmol/L aza-dC or vehicle for 72 hours. The medium was replaced with fresh medium only, and the cells incubated for a further 24 hours before harvesting. Bisulphite modified DNA was amplified using primers and PCR conditions (Table 2) which were specific for bisulphite modified DNA, did not discriminate between methylated and unmethylated sequences, and did not amplify unmodified DNA. The PCR products were melted by increasing the temperature from 60 to 95°C, rising 0.5 or 1°C at each step, waiting 30 seconds on the first step then 5 seconds for each step thereafter. Data was collected and analysed using the Melt Curve Analysis function of the RG-3000 Application Software. The left hand column shows the melt curves for each of the nine genes for OE33, the central column for TE7. Each plot shows the melt curves for the unmethylated (black lines) and methylated (open circles) controls and the cell lines treated with vehicle (red circles) or aza-dC (green circles). The horizontal axis represents temperature and the vertical axis -dF/dT. CDKN2A was not amplified in TE7. Interpretation of the melt curves is described in the Materials and Methods. The right hand column shows the gene expression in cell lines treated with vehicle (red columns) or aza-dC (green columns), as determined by qRT-PCR and normalised to HMBS. Data shown are the means ± SD from three independent experiments. TMEFF2 expression was below detectable limits in either cell line treated with vehicle or aza-dC.

Mentions: Methylation and expression of the nine genes listed in Table 1 were measured in triplicate cultures of the esophageal cancer cell lines OE33 and TE7, following treatment with either 5-aza-2'-deoxycytidine (aza-dC) or vehicle (Figure 1). Methylation was observed in all genes in OE33, except APC and CDKN2A, while in TE7 it was only observed in ID4, RBP1, SFRP1, and TMEFF2. Treatment with aza-dC reduced methylation and significantly increased the expression of all methylated genes except TMEFF2, which was undetected in either cell line. CDKN2A was not detected in TE7 by methylation PCR or quantitative real-time reverse-transcription PCR (qRT-PCR), suggesting that the gene was deleted in this cell line. Following treatment with aza-dC, transcript levels were not significantly increased for any unmethylated genes, except for APC in OE33.


Similarity of aberrant DNA methylation in Barrett's esophagus and esophageal adenocarcinoma.

Smith E, De Young NJ, Pavey SJ, Hayward NK, Nancarrow DJ, Whiteman DC, Smithers BM, Ruszkiewicz AR, Clouston AD, Gotley DC, Devitt PG, Jamieson GG, Drew PA - Mol. Cancer (2008)

Methylation and expression of APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3 and TMEFF2 in esophageal cancer cell lines OE33 and TE7. The esophageal cancer cell lines OE33 and TE7 were treated with either 1 μmol/L aza-dC or vehicle for 72 hours. The medium was replaced with fresh medium only, and the cells incubated for a further 24 hours before harvesting. Bisulphite modified DNA was amplified using primers and PCR conditions (Table 2) which were specific for bisulphite modified DNA, did not discriminate between methylated and unmethylated sequences, and did not amplify unmodified DNA. The PCR products were melted by increasing the temperature from 60 to 95°C, rising 0.5 or 1°C at each step, waiting 30 seconds on the first step then 5 seconds for each step thereafter. Data was collected and analysed using the Melt Curve Analysis function of the RG-3000 Application Software. The left hand column shows the melt curves for each of the nine genes for OE33, the central column for TE7. Each plot shows the melt curves for the unmethylated (black lines) and methylated (open circles) controls and the cell lines treated with vehicle (red circles) or aza-dC (green circles). The horizontal axis represents temperature and the vertical axis -dF/dT. CDKN2A was not amplified in TE7. Interpretation of the melt curves is described in the Materials and Methods. The right hand column shows the gene expression in cell lines treated with vehicle (red columns) or aza-dC (green columns), as determined by qRT-PCR and normalised to HMBS. Data shown are the means ± SD from three independent experiments. TMEFF2 expression was below detectable limits in either cell line treated with vehicle or aza-dC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Methylation and expression of APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3 and TMEFF2 in esophageal cancer cell lines OE33 and TE7. The esophageal cancer cell lines OE33 and TE7 were treated with either 1 μmol/L aza-dC or vehicle for 72 hours. The medium was replaced with fresh medium only, and the cells incubated for a further 24 hours before harvesting. Bisulphite modified DNA was amplified using primers and PCR conditions (Table 2) which were specific for bisulphite modified DNA, did not discriminate between methylated and unmethylated sequences, and did not amplify unmodified DNA. The PCR products were melted by increasing the temperature from 60 to 95°C, rising 0.5 or 1°C at each step, waiting 30 seconds on the first step then 5 seconds for each step thereafter. Data was collected and analysed using the Melt Curve Analysis function of the RG-3000 Application Software. The left hand column shows the melt curves for each of the nine genes for OE33, the central column for TE7. Each plot shows the melt curves for the unmethylated (black lines) and methylated (open circles) controls and the cell lines treated with vehicle (red circles) or aza-dC (green circles). The horizontal axis represents temperature and the vertical axis -dF/dT. CDKN2A was not amplified in TE7. Interpretation of the melt curves is described in the Materials and Methods. The right hand column shows the gene expression in cell lines treated with vehicle (red columns) or aza-dC (green columns), as determined by qRT-PCR and normalised to HMBS. Data shown are the means ± SD from three independent experiments. TMEFF2 expression was below detectable limits in either cell line treated with vehicle or aza-dC.
Mentions: Methylation and expression of the nine genes listed in Table 1 were measured in triplicate cultures of the esophageal cancer cell lines OE33 and TE7, following treatment with either 5-aza-2'-deoxycytidine (aza-dC) or vehicle (Figure 1). Methylation was observed in all genes in OE33, except APC and CDKN2A, while in TE7 it was only observed in ID4, RBP1, SFRP1, and TMEFF2. Treatment with aza-dC reduced methylation and significantly increased the expression of all methylated genes except TMEFF2, which was undetected in either cell line. CDKN2A was not detected in TE7 by methylation PCR or quantitative real-time reverse-transcription PCR (qRT-PCR), suggesting that the gene was deleted in this cell line. Following treatment with aza-dC, transcript levels were not significantly increased for any unmethylated genes, except for APC in OE33.

Bottom Line: The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC.This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3.We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Nursing and Midwifery, Flinders University, Bedford Park, South Australia 5042, Australia.

ABSTRACT

Background: Barrett's esophagus (BE) is the metaplastic replacement of squamous with columnar epithelium in the esophagus, as a result of reflux. It is the major risk factor for the development of esophageal adenocarcinoma (EAC). Methylation of CpG dinucleotides of normally unmethylated genes is associated with silencing of their expression, and is common in EAC. This study was designed to determine at what stage, in the progression from BE to EAC, methylation of key genes occurs.

Results: We examined nine genes (APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3, and TMEFF2), frequently methylated in multiple cancer types, in a panel of squamous (19 biopsies from patients without BE or EAC, 16 from patients with BE, 21 from patients with EAC), BE (40 metaplastic, seven high grade dysplastic) and 37 EAC tissues. The methylation frequency, the percentage of samples that had any extent of methylation, for each of the nine genes in the EAC (95%, 59%, 76%, 57%, 70%, 73%, 95%, 74% and 83% respectively) was significantly higher than in any of the squamous groups. The methylation frequency for each of the nine genes in the metaplastic BE (95%, 28%, 78%, 48%, 58%, 48%, 93%, 88% and 75% respectively) was significantly higher than in the squamous samples except for CDKN2A and RBP1. The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC. The methylation extent was an estimate of both the number of methylated alleles and the density of methylation on these alleles. This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3. There was no significant difference in methylation extent for any gene between high grade dysplastic BE and EAC.

Conclusion: We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC. This is also the first report of gene silencing by methylation of ID4 in BE or EAC. This study suggests that metaplastic BE is a highly abnormal tissue, more similar to cancer tissue than to normal epithelium.

Show MeSH
Related in: MedlinePlus