Fujita M, Goto K, Yoshida K, Okamura H, Morimoto H, Kito S, Fukuda J, Haneji T
Department of Histology and Oral Histology, The University of Tokushima 3-18-15, Tokushima 770-8504, Japan.
Oral Oncol. 2004 Feb;40(2):199-206. doi: 10.1016/s1368-8375(03)00152-0.
In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.
在本研究中，我们使用蛋白质免疫印迹法和逆转录-聚合酶链反应分析，检测了经冈田酸处理的人口腔鳞状细胞癌SCC-25细胞中Fas受体和Fas配体的蛋白质及mRNA表达。用冈田酸处理可增强SCC-25细胞中Fas受体和Fas配体的蛋白质及mRNA表达。在经冈田酸处理的细胞中，全细胞裂解物中IkappaB-α的量减少，而细胞核中NF-κB的水平增加。冈田酸处理还改变了NF-κB的细胞定位，使其从细胞质转移至细胞核。为研究经冈田酸处理的SCC-25细胞中NF-κB的激活情况，我们使用核提取物和NF-κB DNA结合位点的共有寡核苷酸进行了电泳迁移率凝胶阻滞试验。当细胞用20 nM冈田酸处理4小时后，核蛋白与NF-κB寡核苷酸的结合增加。我们用含有NF-κB结合位点的Fas受体基因启动子区域的pFLF1转染细胞。荧光素酶报告基因检测表明，用pFLF1转染并用20 nM冈田酸处理的细胞中的活性呈时间依赖性增加，且该活性比对照细胞中的活性高出三倍以上。我们的结果表明，在经冈田酸处理的SCC-25细胞早期激活的NF-κB刺激了细胞中Fas受体的启动子活性，导致这些细胞发生凋亡性死亡。
