Abstract
The p16/CDKN2 (cycline dependent kinase number 2) gene is known to be one of the negative regulators of the cell cycle. Aberrant 5´CpG island methylation is one of the most important mechanisms of p16/CDKN2 gene promoter region alteration. We studied 8 oral squamous cell carcinoma cell lines and 25 primary tumor tissues for the p16/CDKN2 gene and its expression by PCR-SSCP, MSP, RT-PCR, and immunohistochemical methods to determine the mechanism and the potential biological significance of p16/CDKN2 gene inactivation. In primary tumors, no p16/CDKN2 gene mutations were found by PCR-SSCP. However, hypermethylation of the CpG sites of p16/CDKN2 gene was observed in 48% (12/25) cases of primary tumors and in 50% (4/8) of cell lines. To verify the p16 mRNA expression, we employed RT-PCR and observed decreased or lacked p16 mRNA in 44% (11/25) of primary tumor tissues. In addition, hypermethylation was observed in 6 of the above 11 cases (55%). An immunohistochemistry assay was also performed with the primary tumor tissues, and a semi-quantitative method was used to evaluate the staining intensity of p16 protein. We observed 52% (13/25) negative nucleal staining. When we compared these results with clinicopathological stages, there was no statistical significance. These findings suggest that hypermethylation of p16/CDKN2 promoter region may be associated with p16/CDKN2 gene alteration.
