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Mutational alteration of the p16CDKN2a tumor suppressor gene is infrequent in Ewing's sarcoma
1Department of Pathology, College of Medicine, Kyung Hee University, Dongdaemoon-ku, Seoul 130-702, Korea.
Abstract:
Mutational alteration of the p16CDKN2a tumor suppressor gene has been frequently observed in a variety of human cell lines and tumor tissues. To assess whether alterations of the p16CDKN2a gene play an important role in the pathogenesis of Ewing's sarcoma, we examined the allelic deletion and point mutation of the gene in 27 primary tumors. In quantitative DNA/PCR analysis for three individual exons of the gene, none of the 27 specimens showed detectable reduction in the amplification levels compared to those of normal lymphocytes. To explore the presence of any subtle sequence changes within the protein coding region, we performed a comprehensive screening of sequence alteration in the three exons using DNA/PCR-SSCP analysis. However, no abnormal shift of SSCP bands indicative of sequence change was identified. In addition, no elevation of p16CDKN2a mRNA expression was observed in the RD-ES cell line following 5'-Aza-cytidine treatment, indicating that the promoter of the gene is not abnormally methylated in this cell line. Immunohistochemical study of the same tissue specimens for p16CDKN2a and pRB also revealed grade 2+ or 3+ nuclear staining of both proteins in most of the specimens we examined. Taken together, our results strongly suggest that the mutational inactivation of the p16CDKN2a gene might be a rare event, and thus not play a critical role in the pathogenesis of Ewing's sarcoma.
Insights
Mutational inactivation of the p16CDKN2a tumor suppressor gene is rare in Ewing
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p16CDKN2a gene is a tumor suppressor frequently altered in various cancers.
- Its role in Ewing's sarcoma pathogenesis requires investigation.
Purpose of the Study:
- To determine if p16CDKN2a gene alterations are significant in Ewing's sarcoma development.
- To analyze allelic deletion and point mutations in primary Ewing's sarcoma tumors.
Main Methods:
- Quantitative DNA/PCR analysis of p16CDKN2a exons.
- DNA/PCR-Single-Strand Conformation Polymorphism (SSCP) for sequence alterations.
- 5'-Aza-cytidine treatment to assess promoter methylation.
- Immunohistochemical analysis of p16CDKN2a and pRB protein expression.
Main Results:
- No detectable allelic deletion or sequence alteration of p16CDKN2a was found in 27 primary tumors.
- No abnormal promoter methylation was observed in the RD-ES cell line.
- High levels of p16CDKN2a and pRB nuclear staining were present in most tumor specimens.
Conclusions:
- Mutational inactivation of the p16CDKN2a gene appears to be infrequent in Ewing's sarcoma.
- The p16CDKN2a gene is unlikely to play a critical role in the pathogenesis of this cancer.