Low frequency of p16 ^INK4a promoter methylation in mammary carcinomas as revealed by positive display of methylated sites

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Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. In order to study methylation of the promoter of the tumour suppressor p16 ^INK4a, we developed a fast and simple method that in contrast to previous studies relies on the positive display of methylated sites (PDM). The method is based on bisulphite treatment of DNA, PCR-amplification of the modified DNA, and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided.

Using this approach, we analysed 33 ductal invasive mammary carcinomas, 4 normal mammary tissues and 4 cell lines for methylation. p16^INK4a methylation was detected in 1/33 carcinomas (3%) and in 0/4 normal tissue samples. We conclude that PDM provides a useful tool for determining the degree and pattern of promoter methylation and is suitable for screening large series of tissue samples.