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 lines 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 site in intron 1 of wt1. This site contains potential motifs for cytosine methylation and treatment of mesothelioma cells with 5-azadeoxycytosine restored the normal EcoRI digestion pattern of wt1 in these cells indicating that cleavage was inhibited by methylation at this site. Southern 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 did not express wt1 were also methylated at this EcoRI site. Our identification of a site frequently methylated in malignant cells, independent of gene expression, provides a new model system to study determinants of site-specific methylation in tumors.