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

为了探讨p16^&lt；INK4a&gt；基因在皮肤癌发生中的作用，我们试图建立一种小鼠模型，用生物发光法观察p16^&lt；INK4a&gt；基因在活体小鼠体内的表达。我们利用含有p16^&lt；INK4a&gt；基因座的基因组DNA的BAC克隆，在p16^&lt；INK4a&gt；基因最后一个编码外显子的下游插入荧光素酶基因，构建了重组BAC克隆。利用这个重组的BAC克隆，我们已经建立了一个产生p16-荧光素酶融合蛋白的转基因小鼠系，以显示p16^&lt；INK4a&gt；在活小鼠中的表达。为了观察p16^&lt；INK4a和gt；-荧光素酶融合蛋白的表达，小鼠在麻醉下给予荧光素酶的底物荧光素，并暴露在CCD摄像机下15分钟。我们证实了各器官的光发射与p16^-lt；ink4a&gt；genep16^-lt；ink4a&gt；基因是最重要的抑癌基因之一的内源表达密切相关，并且是不活跃的…在超过50%的人类癌症中表现出更强的敏感性。已知p16^&lt；INK4a&gt；基因是由致癌信号如激活的RAS信号诱导的，是一种抗肿瘤发生的失败安全机制。在这项研究中，我们研究了p16^&lt；INK4a&gt；基因是如何参与和诱导体内皮肤癌的发生发展的。利用上述转基因小鼠，实现了p16^&lt；INK4a&gt；基因的实时可视化。我们使用了使用DMBA-TPA方案的化学诱导的皮肤癌模型，该模型在小鼠皮肤上发展为皮肤乳头状瘤。已知DMBA可诱导H-RAS基因突变并激活RAS信号。在乳头状瘤晚期(在DMBA-TPA治疗开始后12周以后)，小鼠的生物发光增强。我们证实生物发光强度与内源性p16；lt；INK4a和gt；基因在乳头状瘤中的表达有很好的相关性。这些结果表明p16；lt；INK4a和gt；基因在晚期乳头状瘤中被诱导，并且它对皮肤肿瘤从良性向恶性进展的抑制作用较小。

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