In line with the findings of Jönsson et al.,
5 we did not detect any mutations in the
RASEF gene other than a known SNP.
5 22 Using this SNP, we detected allelic imbalance in some of the tumors that were heterozygous for this marker (UM13 and -21). Because the imbalances were not complete, we suspect tumor heterogeneity in the primary tumors in contrast to the cell lines, all of which, with one exception, displayed a homozygous genotype. Gene expression analysis revealed that 5 of 11 uveal melanoma cell lines had high
RASEF expression, whereas the others hardly showed expression. As almost all the low-expression cell lines displayed the homozygous T-allele, there appears to be an association between expression and genotype. This apparent association, however, could also be based on the small number of cell lines that were tested and the fact that three cell lines were derived from the same patient (Mel 270, OMM 2.3, and OMM 2.5). In the primary tumors, expression varied widely and often exceeded the expression seen in the cell lines. Among the uveal melanomas with low
RASEF expression a homozygous genotype prevailed, but this fact does not favor a specific allele. This finding may indicate that there is no risk factor linked to either allele and that the low expression is more likely due to a somatic alteration. As we had not observed any mutations in the cell lines, we subsequently considered epigenetic modifications as the cause of low
RASEF expression. Indeed, all cell lines that did not express
RASEF contained a methylated promoter, whereas all cell lines with expression lacked this methylation, confirming our hypothesis. Hereafter, we performed demethylation experiments with 5-azacytidine, which revealed a highly induced expression in a cell line with methylated
RASEF. Demethylation of an unmethylated cell line resulted in the opposite effect. The demethylating agent is highly toxic and may explain downregulation of
RASEF expression in the unmethylated cell line. Toxicity of 5-azacytidine and demethylation of all the other genes during treatment are the reasons that we reserve functional analysis using genetically modified cell lines for follow-up research.