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Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays.

Aleman A, Adrien L, Lopez-Serra L, Cordon-Cardo C, Esteller M, Belbin TJ, Sanchez-Carbayo M - Br. J. Cancer (2007)

Bottom Line: In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases.Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival.In vitro analyses supported the role of methylation in silencing SOX9 gene.

View Article: PubMed Central - PubMed

Affiliation: Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain.

ABSTRACT
CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n=12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n=11) and primary bladder tumours (n=101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.

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Related in: MedlinePlus

The treatment with the demethylating agent AZA reactivates gene expression of SOX9. The upper part displays the reverse transcription polymerase chain reaction analysis of SOX9 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a transcript loading control. The hypermethylated J82 cell line did not express SOX9, and restored SOX9 transcript expression after AZA exposure. The mid-section shows western blot analysis of protein expression. Tubulin expression was used as a protein loading control. The hypermethylated cell line did not express the coded protein. The treatment with the demethylating agent reactivated SOX9 protein expression. The unmethylated RT4 cell line did not show changes in transcript or protein expression. The bottom part displays immunofluorescence analysis of SOX9 expression after AZA exposure. The methylated cell line did not show any staining for the protein, while the unmethylated ones showed its characteristic staining pattern.
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fig4: The treatment with the demethylating agent AZA reactivates gene expression of SOX9. The upper part displays the reverse transcription polymerase chain reaction analysis of SOX9 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a transcript loading control. The hypermethylated J82 cell line did not express SOX9, and restored SOX9 transcript expression after AZA exposure. The mid-section shows western blot analysis of protein expression. Tubulin expression was used as a protein loading control. The hypermethylated cell line did not express the coded protein. The treatment with the demethylating agent reactivated SOX9 protein expression. The unmethylated RT4 cell line did not show changes in transcript or protein expression. The bottom part displays immunofluorescence analysis of SOX9 expression after AZA exposure. The methylated cell line did not show any staining for the protein, while the unmethylated ones showed its characteristic staining pattern.

Mentions: Treatment of methylated and unmethylated bladder cancer cell lines with a DNA-demethylating agent served to further link SOX9 hypermethylation and gene silencing. Exposure of methylated bladder cancer cell lines to the demethylating drug, AZA, restored expression of SOX9 at the transcript level in the J82 cell line. RT4 was used as the control cell line to assess the specificity of AZA exposure, but not to modify gene expression of this candidate gene in unmethylated bladder cancer cells (Figure 4). Western blot and immunofluorescence analyses were performed to confirm that protein expression was also restored after AZA exposure. Overall, the results indicated a high correlation of methylation data with gene expression, observations especially supported by AZA reactivation analyses.


Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays.

Aleman A, Adrien L, Lopez-Serra L, Cordon-Cardo C, Esteller M, Belbin TJ, Sanchez-Carbayo M - Br. J. Cancer (2007)

The treatment with the demethylating agent AZA reactivates gene expression of SOX9. The upper part displays the reverse transcription polymerase chain reaction analysis of SOX9 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a transcript loading control. The hypermethylated J82 cell line did not express SOX9, and restored SOX9 transcript expression after AZA exposure. The mid-section shows western blot analysis of protein expression. Tubulin expression was used as a protein loading control. The hypermethylated cell line did not express the coded protein. The treatment with the demethylating agent reactivated SOX9 protein expression. The unmethylated RT4 cell line did not show changes in transcript or protein expression. The bottom part displays immunofluorescence analysis of SOX9 expression after AZA exposure. The methylated cell line did not show any staining for the protein, while the unmethylated ones showed its characteristic staining pattern.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361432&req=5

fig4: The treatment with the demethylating agent AZA reactivates gene expression of SOX9. The upper part displays the reverse transcription polymerase chain reaction analysis of SOX9 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a transcript loading control. The hypermethylated J82 cell line did not express SOX9, and restored SOX9 transcript expression after AZA exposure. The mid-section shows western blot analysis of protein expression. Tubulin expression was used as a protein loading control. The hypermethylated cell line did not express the coded protein. The treatment with the demethylating agent reactivated SOX9 protein expression. The unmethylated RT4 cell line did not show changes in transcript or protein expression. The bottom part displays immunofluorescence analysis of SOX9 expression after AZA exposure. The methylated cell line did not show any staining for the protein, while the unmethylated ones showed its characteristic staining pattern.
Mentions: Treatment of methylated and unmethylated bladder cancer cell lines with a DNA-demethylating agent served to further link SOX9 hypermethylation and gene silencing. Exposure of methylated bladder cancer cell lines to the demethylating drug, AZA, restored expression of SOX9 at the transcript level in the J82 cell line. RT4 was used as the control cell line to assess the specificity of AZA exposure, but not to modify gene expression of this candidate gene in unmethylated bladder cancer cells (Figure 4). Western blot and immunofluorescence analyses were performed to confirm that protein expression was also restored after AZA exposure. Overall, the results indicated a high correlation of methylation data with gene expression, observations especially supported by AZA reactivation analyses.

Bottom Line: In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases.Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival.In vitro analyses supported the role of methylation in silencing SOX9 gene.

View Article: PubMed Central - PubMed

Affiliation: Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain.

ABSTRACT
CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n=12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n=11) and primary bladder tumours (n=101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.

Show MeSH
Related in: MedlinePlus