The 14-3-3sigma gene promoter is methylated in both human melanocytes and melanoma.
BACKGROUND
Recent evidence demonstrates that 14-3-3sigma acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3sigma has been largely overlooked in melanoma.


METHODS
The methylation status of 14-3-3sigma CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3sigma mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3sigma gene expression.


RESULTS
14-3-3sigma is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3sigma gene expression and the active induction of 14-3-3sigma mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3sigma was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3sigma gene.


CONCLUSIONS
14-3-3sigma is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3sigma is a rare event in melanoma, indicating 14-3-3sigma might have a tentative role in the pathogenesis of melanoma.
Liu
Suhu
S
Ochsner Cancer Institute, Department of Surgery 1514 Jefferson Highway, BH334 New Orleans, LA 70121, USA. suhu_liu@dfci.harvard.edu
Howell
Paul
P
Ren
Suping
S
Fodstad
Oystein
O
Riker
Adam I
AI
D048948:14-3-3 Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D006801:Humans; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic
D048948:14-3-3 Proteins