Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Skin

Epigenetic silencing of genes by hypermethylation of promoter sequences constitutes an important step in tumourigenesis and has equally been described as first or second hit. The hypermethylation of the Adenomatous Polyposis Coli (APC) promoter is a common event in a variety of tumours, for example in colon cancer or lung cancer. The APC protein is not only a key player in the wnt signalling pathway which when turned on leads to cell proliferation but exhibits diverse functions in the maintenance of chromosomal stability, cytoskeleton regulation and DNA binding. In the majority of tumours, methylated as well as non-methylated alleles can be detected by standard methods implying either monoallelic or inconsistent methylation of cancer cells or the presence of non-tumour cells in the sample. Therefore a quantitative analysis of methylation should be helpful in evaluating the role of promoter hypermethylation in cancer formation. Standard methylation-sensitive (ms) PCR assays are not quantitative. Methods for quantitative analysis of methylation which include ms-real-time PCR or ms-SNuPE (Single Nucleotide Primer Extension) are laborious and costly. A novel ms solid phase PCR to test APC promoter 1A methylation was developed which combines the amplification on a solid phase with subsequent colorimetric detection in an ELISA based format. This method promises to be low-cost and also suitable for medium-throughput. In a first PCR reaction a fragment of the APC promoter is amplified in a non-methylation specific manner thereby preventing PCR bias. The second ms solid phase PCR is carried out with an immobilized non-methylation specific primer and a biotinylated ms primer. After extensive washing the PCR product can be detected via a streptavidin-biotin horseradish peroxidase complex. The applicability for quantitative analysis was tested by applying mixtures of different percentages of methylated and non-methylated DNA. Thereby a calibration curve was set up. The novel ms solid phase PCR promises a fast, accurate and low-cost quantitative analysis of methylation levels in promoters of tumour suppressor genes.