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Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene
E V Kleymenova1, X Yuan, M E LaBate
1Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville 78957, USA.
Abstract:
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.
Insights
A specific site in the Wilms tumor suppressor gene (wt1) is methylated in malignant mesothelioma, preventing normal gene digestion. This methylation occurs independently of gene expression, offering a new model for studying cancer-specific DNA methylation.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Malignant mesothelioma is a rare extrarenal neoplasm.
- The Wilms tumor suppressor gene (wt1) is expressed in malignant mesothelioma.
- A well-characterized rat model for human mesothelioma was used.
Purpose of the Study:
- To investigate alterations in the wt1 gene in rat mesotheliomas.
- To identify specific molecular changes associated with malignant mesothelioma development.
- To explore the role of DNA methylation in wt1 regulation in cancer.
Main Methods:
- Southern blot analysis to detect wt1 gene fragments.
- Cloning and sequencing to characterize wt1 alterations.
- DNA methylation analysis using restriction enzymes (EcoRI, HpaII/MspI).
- Treatment with 5-azadeoxycytosine to assess methylation reversibility.
Main Results:
- A 3.5 kb EcoRI fragment of wt1 was detected in most mesothelioma samples but not in normal tissues.
- This fragment resulted from methylation-induced inhibition of EcoRI digestion at an intron 1 site.
- 5-azadeoxycytosine treatment restored normal EcoRI digestion, confirming methylation.
- Other CpG sites in the wt1 5' region showed no differential methylation.
- The EcoRI site was also methylated in renal cell carcinoma lines lacking wt1 expression.
Conclusions:
- A specific EcoRI site in the wt1 gene is frequently methylated in malignant mesothelioma.
- This methylation is independent of wt1 gene expression.
- The findings provide a novel model system for studying site-specific DNA methylation in tumors.
- This methylation pattern may serve as a potential biomarker or therapeutic target.