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Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas.

Peng D, Hu TL, Jiang A, Washington MK, Moskaluk CA, Schneider-Stock R, El-Rifai W - PLoS ONE (2011)

Bottom Line: Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively).The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells.The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).

Methods and results: Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (r = -0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.

Conclusion: In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

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Treatment with 5-Aza and TSA reverses DNA methylation and gene expression patterns of MT3.An esophageal adenocarcinoma cell line (OE33) in which the MT3 gene is silenced and the MT3 promoter CpG island was highly methylated, were treated with 5-Aza and TSA as described in the Methods section. DNA methylation level was determined by Pyrosequencing in Region 2 (from −127 to −8). MT3 gene mRNA expression level was determined by real-time RT-PCR and normalized to HPRT to generate a relative fold induction. The 5-Aza treatment alone led to the induction of relative expression from 0.01 fold to 1.1 whereas the combination of 5-Aza and TSA led to an increase to 17.8.
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pone-0022009-g004: Treatment with 5-Aza and TSA reverses DNA methylation and gene expression patterns of MT3.An esophageal adenocarcinoma cell line (OE33) in which the MT3 gene is silenced and the MT3 promoter CpG island was highly methylated, were treated with 5-Aza and TSA as described in the Methods section. DNA methylation level was determined by Pyrosequencing in Region 2 (from −127 to −8). MT3 gene mRNA expression level was determined by real-time RT-PCR and normalized to HPRT to generate a relative fold induction. The 5-Aza treatment alone led to the induction of relative expression from 0.01 fold to 1.1 whereas the combination of 5-Aza and TSA led to an increase to 17.8.

Mentions: To validate if DNA methylation is responsible for the gene silencing, an esophageal adenocarcinoma cell line (OE33) was treated with 5-Aza and TSA, as described in the Methods section. As shown in Figure 4, both 5-Aza alone and 5-Aza followed by TSA treatment restored MT3 gene expression in a previously silenced OE33 cell line accompanied by significant demethylation of a previously methylated promoter. TSA treatment alone did not have an effect on MT3 promoter methylation.


Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas.

Peng D, Hu TL, Jiang A, Washington MK, Moskaluk CA, Schneider-Stock R, El-Rifai W - PLoS ONE (2011)

Treatment with 5-Aza and TSA reverses DNA methylation and gene expression patterns of MT3.An esophageal adenocarcinoma cell line (OE33) in which the MT3 gene is silenced and the MT3 promoter CpG island was highly methylated, were treated with 5-Aza and TSA as described in the Methods section. DNA methylation level was determined by Pyrosequencing in Region 2 (from −127 to −8). MT3 gene mRNA expression level was determined by real-time RT-PCR and normalized to HPRT to generate a relative fold induction. The 5-Aza treatment alone led to the induction of relative expression from 0.01 fold to 1.1 whereas the combination of 5-Aza and TSA led to an increase to 17.8.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139601&req=5

pone-0022009-g004: Treatment with 5-Aza and TSA reverses DNA methylation and gene expression patterns of MT3.An esophageal adenocarcinoma cell line (OE33) in which the MT3 gene is silenced and the MT3 promoter CpG island was highly methylated, were treated with 5-Aza and TSA as described in the Methods section. DNA methylation level was determined by Pyrosequencing in Region 2 (from −127 to −8). MT3 gene mRNA expression level was determined by real-time RT-PCR and normalized to HPRT to generate a relative fold induction. The 5-Aza treatment alone led to the induction of relative expression from 0.01 fold to 1.1 whereas the combination of 5-Aza and TSA led to an increase to 17.8.
Mentions: To validate if DNA methylation is responsible for the gene silencing, an esophageal adenocarcinoma cell line (OE33) was treated with 5-Aza and TSA, as described in the Methods section. As shown in Figure 4, both 5-Aza alone and 5-Aza followed by TSA treatment restored MT3 gene expression in a previously silenced OE33 cell line accompanied by significant demethylation of a previously methylated promoter. TSA treatment alone did not have an effect on MT3 promoter methylation.

Bottom Line: Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively).The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells.The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).

Methods and results: Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (r = -0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.

Conclusion: In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

Show MeSH
Related in: MedlinePlus