Detection of oncogene amplification and its clinical significance in oral squamous cell carcinomas

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Cyclin D1 is an oncogene which involved in the control of G1/S transition in the cell cycle. Gene amplification and overexpression of cyclin Dl have been detected in various malignant rumors, inducing esophageal carcinoma, pancreatic carcinoma etc., and seemed to be associated with poor prognosis of these tumors. In this study, we first examined overexpression of cyclin D1 protein in oral squamous cell carcinomas with the immunohistochemical technique. Cyclin D1 was weakly expressed in the minor compartment of basal and parabasal cells of normal mucosal epithelia of the oral cavity. Up-regulation of cyclin D1 was not observed in hyperkeratotic and dysplastic epithelia, but it was highly expressed in squamous cell carcinomas (SCC) of the oral cavity : approximately 80% of oral SCCs showed a pronounced expression of cyclin D1. Cyclin D1 was demonstrated exclusively in the nuclei of cancer cells. The distribution of positive cells varied between tumors ; they were localized only in the pe … More riphery of rumor nests or throughout the rumor nests. We generated "the expression score" to assess the degree of cyclin D1 overexpression semi-quantitatively, and divided 34 cases of oral SCCs into 2 groups ; the high-expression and low-expression groups. Noteworthly, the cumulative 5-year survival rates of the high-expression and low-expression groups were 49.4% and 85.5%, respectively, by the Kaplan-Meier method.Next, to establish a method for the detection of gene amplification, we rested the polymerase chain reaction (PCR) technique against genomic DNA, extracted from oral SCC cell lines, using both of cyclin D1 primers and gamma-interferon primers (as the internal control for a single copy gene) in the same PCR tube. Following sets of primers were used 5'-TTC TgC CTT TgA TgT TAC-3' and 5'-Agg CTg AAT CAA TgT CTT-3' for 115bp cyclin D1 DNA fragment, and 5'-TCT TTT CTT TCC CgT TAg gT-3' and 5'-CTg ggA TgC TCT TCg ACC TC-3' for 150bp gamma-interferon DNA fragment. PCR products were analysed by 4% agarose gel electrophoresis. Two signals corresponding to cyclin D1 or gamma-interferon were successfully detected under the optimum concentration of primers. This result showed that the amplification of certain genes could be quantitatively decided by the PCR technique, which would be useful for low-integrated DNA samples from the paraffin block and the small amount of DNA such as cytological samples. Less