Expression of telomerase activity in 4NQO induced carcinoma in rat tongue

用5周龄雄性Sprague-Dowley大鼠研究了端粒酶活性与化学诱导舌癌细胞凋亡的相关性。给动物50 ppm的4-硝基喹啉1-氧化物溶液作为饮用水，4、8、12、16、20和24周后处死。制备舌组织，分别采用TUNEL法和免疫组化法观察凋亡细胞和p53阳性细胞的变化。端粒酶活性也用延伸PCR法检测。即从各组大鼠中提取端粒酶含液，在30℃下进行端粒酶拉伸反应。用rTaq扩增后，将样品涂在丙烯酰胺凝胶上。在100V恒温下进行电泳。用SYBRgreen染色观察。4NQO给药后8周舌上皮组织增生，12周发育异常，16周癌变。P53过表达在4周内升高，并在整个实验期间保持高水平。16周前凋亡细胞增多，16周后凋亡细胞减少。包括4周组在内的所有动物均观察端粒酶活性。因此，端粒酶活性开始与p53过表达同时出现，并开始发生组织学变化。提示在化学诱导癌的早期，组织的凋亡和永生化应该是同等参与的。

Correlation between telomerase activity and apoptosis was examined in chemically induced tongue carcinomas using 5-week-old male Sprague-Dowley rats. Animals were given 50 ppm 4-nitroquinoline 1-oxide solution as drinking water for 4, 8, 12, 16, 20, and 24 weeks and sacrificed. Tongue tissue was prepared to investigate apoptotic cells and p53 positive cells by TUNEL method and immunohistochemical method, respectively. Telomerase activity was also examined by stretch PCR method. Namely, telomerase containing solution was extracted from each group of rats, and stretch reaction by telomerase was performed at 30℃. After amplification by PCR with rTaq, samples were applied to an acrylamide gel. Electrophoresis was carried out at 100V under a constant temperature. The gel was stained with SYBRgreen and observed. Epithelial tissue of the tongue showed hyperplastic change in 8 weeks, dysplastic change in 12 weeks, and carcinogenesis in 16 weeks after the beginning of 4NQO administration. p53 overexpression increased in 4 weeks and was kept high level through experimental period. Apoptotic cells also increased till 16 weeks, but decreased thereafter. Telomerase activity was observed in all animals including 4-week group. As a result, telomerase activity began to appear coincident with p53 overexpression proceeding the initiation of histological changes. So it is suggested that apoptosis and immortalization of the tissue should be equally involved in the early stage of chemically induced carcinoma.