Induction of p16^<INK4a> gene expression by oncogenic stress is involved in the tumor suppression mechanism of skin cancer

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In order to examine the role of p16^<INK4a> gene in the skin carcinogenesis, we tried to generate a mouse model to visualize p 16^<INK4a> gene expression in living mice by bioluminescence. We utilized the BAC clone which contains approximately 200kbp genomic DNA including p16^<INK4a> gene locus, and constructed recombinant BAC clone by inserting luciferase gene in frame at the downstream of the last coding exon of the p16^<INK4a> gene. Using this recombinant BAC clone, we have generated a transgenic mouse line that produces p16-luciferase fusion protein to visualize p16^<INK4a> expression in living mice. To visualize the expression of p16^<INK4a> -Luciferase fusion, mice were subjected with luciferin, the subatrate of luciferase, and exposed for 15 minutes to CCD camera under anesthesia. We confirmed that light emission from each organ is well correlated with the endogenous expression of p16^<INK4a> genep16^<INK4a> gene is one of the most important tumor suppressor genes, and is inacti … More vated in more than 50% of human cancers. p16^<INK4a> gene is known to be induced by oncogenic signal such as activated Ras signal as a fail safe mechanism against oncogenesis. In this study, we investigated how p16^<INK4a> gene is involved and induced in skin cancer development in vivo. By utilizing the transgenic mouse described above, the real-time visualization of p16^<INK4a> gene was performed. We have used the chemical-induced skin carcinogenesis model using DMBA-TPA protocol which develops skin papillomas in the mouse skin. DMBA is known to induce a mutation in H-Ras gene and activate Ras signal. The bioluminescence from the mice was increased in the late papilllomas (later than 12 weeks after DMBA-TPA treatment was started). We confirmed that the intensity of bioluminescence was well correlated with the endogenous p16^<INK4a> gene expression in the papillomas These results suggests that p16^<INK4a> gene is induced in late papillomas, and that it inhibits the tumor progression from benign to malignant skin tumors Less