Figure 1.

NT5E expression is silenced by methylation in C81–61 cells, but deregulated in highly metastatic isogenic C8161 cells. (A) Expression of NT5E correlates inversely with CpG methylation in C81–61 and C8161. The upper panels are RT–PCR analysis of NT5E and the control gene GAPDH in five melanoma cell lines and normal human melanocytes (NHEM) as indicated. NT5E mRNA is expressed abundantly in all cell lines except C81–61. Lower panels are MSP analysis of the NT5E CpG island in the same cell lines. There is complete methylation in C81–61 but no detectable methylation in C8161. (B) Bisulphite sequencing analysis of the NT5E CpG island in C81–61 and C8161 cell lines. Bisulphite sequencing was performed as described in Materials and Methods and the figure shows a diagrammatic representation of the NT5E CpG island. CpG sites are shown as vertical lines. Methylated CpG dinucleotides are shown as black blocks, unmethylated CpGs as open blocks. Five levels of methylation are indicated: 0-no black blocks; 1–25%-1 black block; 25–50%-2 black blocks; 50–75%-3 black blocks; 75–100%-4 black blocks. Positions of the MSP and bisulphite-sequencing primers are indicated. The CpG island is almost completely methylated in C81–61 cells and entirely unmethylated in C8161, consistent with expression of NT5E mRNA in each cell line. (C) Expression of NT5E mRNA is reactivated by demethylation. Exponentially growing C81–61, C8161 and Mel501 cells were treated with 5′azacytidine (5′ AZA) (black blocks) or untreated (open blocks). cDNA was prepared and expression of NT5E mRNA determined as described in Materials and Methods.