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Seladin-1 expression is regulated by promoter methylation in adrenal cancer.

Simi L, Malentacchi F, Luciani P, Gelmini S, Deledda C, Arvia R, Mannelli M, Peri A, Orlando C - BMC Cancer (2010)

Bottom Line: We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza).The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03).On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Biochemistry, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, Florence, 50139 Italy.

ABSTRACT

Background: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC.

Methods: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands.

Results: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01).

Conclusion: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

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Schematic representation of CpG island in seladin-1 promoter and methylation status in H295R and SW13 cell lines. (Panel A) The large CpG island includes the translation start codon (ATG), spanning from -868 bp to +918 bp. The arrows F1 and R1 indicate primers which did not evidence any methylation sensitive sites. F2 and R2 are primers used for MSP and Q-MSP analysis. (Panel B) Methylation specific PCR performed in H295R and SW13 cell lines. Primers for methylated (upper gel) and unmethylated DNA (lower gel) recognized in both cell lines corresponding methylated and unmethylated seladin-1 promoter sequences. After 6 days of treatment with 5-Aza (+5Aza) we observed a reduction of methylated form in both cell lines in comparison to controls (-5Aza). Conversely, the intensity of bands corresponding to unmethylated DNA increased in intensity, confirming the demethylation of corresponding sequences. (NTC = no template control, MW = molecular weight marker, M = cell line methylated DNA, UM = cell line unmethylated DNA).
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Figure 1: Schematic representation of CpG island in seladin-1 promoter and methylation status in H295R and SW13 cell lines. (Panel A) The large CpG island includes the translation start codon (ATG), spanning from -868 bp to +918 bp. The arrows F1 and R1 indicate primers which did not evidence any methylation sensitive sites. F2 and R2 are primers used for MSP and Q-MSP analysis. (Panel B) Methylation specific PCR performed in H295R and SW13 cell lines. Primers for methylated (upper gel) and unmethylated DNA (lower gel) recognized in both cell lines corresponding methylated and unmethylated seladin-1 promoter sequences. After 6 days of treatment with 5-Aza (+5Aza) we observed a reduction of methylated form in both cell lines in comparison to controls (-5Aza). Conversely, the intensity of bands corresponding to unmethylated DNA increased in intensity, confirming the demethylation of corresponding sequences. (NTC = no template control, MW = molecular weight marker, M = cell line methylated DNA, UM = cell line unmethylated DNA).

Mentions: Methyl Primer Express® Software v1.0 (Applied Biosystems, Foster City, CA, USA) was used to evaluate methylation-sensitive sites in seladin-1 sequence from -4384 bp and +1826 bp. The region from -4384 bp to -1150 did not reveal any CpG island. In the remaining sequence, we identified a large 1786 bp long CpG island from -868 bp o +918 bp. We analyzed two separate sequences in this region by MSP: the first, from -404 bp to -136 bp did not evidence methylation sensitive site (data not shown). In the second region, partially comprising exon1 and the transcription starting site (Figure 1; see also Ref [4] for more sequence details) we selected a primer set for methylated form with the following sequences: F2 meth 5'-CGGGTTGTGGGTTATAGGC-3', localised at -97 and R2 meth 5'-ACGAACACCCAACGCTAATAAAT-3' at +81 nucleotide from the same site (amplicon length 202 bp). The unmethylated form of the same sequence was amplified using the primers: F2unmeth 5'-TTGTGGGTTATAGGTGTAGAGT-3' at -93 nucleotide from translation start site and R2unmeth 5'-CCAAACACACACATAATAATAAA-3' at +141 (amplicon length of 258 bp).


Seladin-1 expression is regulated by promoter methylation in adrenal cancer.

Simi L, Malentacchi F, Luciani P, Gelmini S, Deledda C, Arvia R, Mannelli M, Peri A, Orlando C - BMC Cancer (2010)

Schematic representation of CpG island in seladin-1 promoter and methylation status in H295R and SW13 cell lines. (Panel A) The large CpG island includes the translation start codon (ATG), spanning from -868 bp to +918 bp. The arrows F1 and R1 indicate primers which did not evidence any methylation sensitive sites. F2 and R2 are primers used for MSP and Q-MSP analysis. (Panel B) Methylation specific PCR performed in H295R and SW13 cell lines. Primers for methylated (upper gel) and unmethylated DNA (lower gel) recognized in both cell lines corresponding methylated and unmethylated seladin-1 promoter sequences. After 6 days of treatment with 5-Aza (+5Aza) we observed a reduction of methylated form in both cell lines in comparison to controls (-5Aza). Conversely, the intensity of bands corresponding to unmethylated DNA increased in intensity, confirming the demethylation of corresponding sequences. (NTC = no template control, MW = molecular weight marker, M = cell line methylated DNA, UM = cell line unmethylated DNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875219&req=5

Figure 1: Schematic representation of CpG island in seladin-1 promoter and methylation status in H295R and SW13 cell lines. (Panel A) The large CpG island includes the translation start codon (ATG), spanning from -868 bp to +918 bp. The arrows F1 and R1 indicate primers which did not evidence any methylation sensitive sites. F2 and R2 are primers used for MSP and Q-MSP analysis. (Panel B) Methylation specific PCR performed in H295R and SW13 cell lines. Primers for methylated (upper gel) and unmethylated DNA (lower gel) recognized in both cell lines corresponding methylated and unmethylated seladin-1 promoter sequences. After 6 days of treatment with 5-Aza (+5Aza) we observed a reduction of methylated form in both cell lines in comparison to controls (-5Aza). Conversely, the intensity of bands corresponding to unmethylated DNA increased in intensity, confirming the demethylation of corresponding sequences. (NTC = no template control, MW = molecular weight marker, M = cell line methylated DNA, UM = cell line unmethylated DNA).
Mentions: Methyl Primer Express® Software v1.0 (Applied Biosystems, Foster City, CA, USA) was used to evaluate methylation-sensitive sites in seladin-1 sequence from -4384 bp and +1826 bp. The region from -4384 bp to -1150 did not reveal any CpG island. In the remaining sequence, we identified a large 1786 bp long CpG island from -868 bp o +918 bp. We analyzed two separate sequences in this region by MSP: the first, from -404 bp to -136 bp did not evidence methylation sensitive site (data not shown). In the second region, partially comprising exon1 and the transcription starting site (Figure 1; see also Ref [4] for more sequence details) we selected a primer set for methylated form with the following sequences: F2 meth 5'-CGGGTTGTGGGTTATAGGC-3', localised at -97 and R2 meth 5'-ACGAACACCCAACGCTAATAAAT-3' at +81 nucleotide from the same site (amplicon length 202 bp). The unmethylated form of the same sequence was amplified using the primers: F2unmeth 5'-TTGTGGGTTATAGGTGTAGAGT-3' at -93 nucleotide from translation start site and R2unmeth 5'-CCAAACACACACATAATAATAAA-3' at +141 (amplicon length of 258 bp).

Bottom Line: We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza).The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03).On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Biochemistry, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, Florence, 50139 Italy.

ABSTRACT

Background: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC.

Methods: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands.

Results: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01).

Conclusion: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

Show MeSH
Related in: MedlinePlus