Epigenetic Gene Silencing in Human Melanoma Figure 2. Distribution of the melanoma cell lines according to their methylation status for a. COL1A2, b. TNFRSF10D, c. THBS1, d. UCHL1. The melanoma cell lines were grouped following their mRNA expression level, no or high expression. Methylation levels were determined by Epityper assay. doi:10.1371/journal.pone.0026121.g002 contamination, which will likely have the effect of decreasing the observed overall percentage of methylation of the DNA assessed. TNFRSF10D appeared to be the only gene methylated in the glioma lines (33%) (Table 1). Methylation of candidate gene promoters in other cancer types In order to assess the possible specificity of methylation of the four candidate genes to melanoma, we repeated the same Epityper assays on a limited number of cell lines from cancers of the colon, esophagus and brain (glioma). These different cancer types were assessed to determine whether the candidate genes might play a more general role in tumor suppression. Two cell lines from each tumor type were assessed for their methylation status for COL1A2, THBS1, TNFRSF10D and UCHL1. Using the same cut off as for the melanoma cell lines and tumors, the percentage methylation for the COL1A2 and UCHL1 CpGs islands was 65% and 82% in the esophageal cancer cell lines respectively and 92% and 90% in the colon cancer cell lines respectively. While THBS1 was only methylated in the colon cancer cell lines (43% methylation), Discussion The objective of this study was to combine different array platforms to strengthen the identification of novel TSGs inactivated by promoter methylation in melanoma. Selection of candidate genes was based on reduced expression in a panel of melanoma cell lines which correlated with a high methylation profile, and lack of the same observation in melanocyte cultures. Using these criteria we identified and subsequently confirmed four genes silenced by DNA methylation in melanoma. We found 24%, 31%, 66% and 42% of cell lines and 13%, 15%, 30% and 21% of tumors were methylated for COL1A2, THBS1, TNFRSF10D and UCHL1 respectively. Each of these four genes had previously been linked to melanoma. Muthusamy et al. and Koga et al. [12,21] identified COL1A2 as methylated in 35% (7/20) to 89% (16/20) of melanoma Figure 3. Western blot analysis of THBS1 and UCHL1 protein expression. doi:10.1371/journal.pone.0026121.g003 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26121