IDENTIFICATION OF A TUMOR-SPECIFIC METHYLATION SITE IN THE WILMS-TUMOR SUPPRESSOR GENE

Malignant mesothelioma is one of the very few extrarenal neoplasms in which the Wilms tumor suppressor gene (wt1) is expressed. We examined wt1 for alterations in rat mesotheliomas, a well characterized animal model for the human disease. Southern analysis revealed a 3.5 kb EcoRI wt1 fragment readily detectable in majority of mesothelioma cell line s and primary mesotheliomas but not in normal rat tissues. Cloning and sequencing of this fragment revealed that the presence of this EcoRI fragment resulted from an inability of this enzyme to cut at a EcoRI s ite in intron 1 of wt1. This site contains potential motifs for cytosi ne methylation and treatment of mesothelioma cells with 5-azadeoxycyto sine restored the normal EcoRI digestion pattern of wt1 in these cells indicating that cleavage was inhibited by methylation at this site. S outhern analysis using HpaII/MspI digestion revealed no differences in methylation between mesothelioma cell lines and normal mesothelium at other CpG sites in wt1 5' region. Renal cell carcinoma lines which di d not express wt1 were also methylated at this EcoRI site. Our identif ication of a site frequently methylated in malignant cells, independen t of gene expression, provides a new model system to study determinant s of site-specific methylation in tumors.