Chamulitrat Walee, Schmidt Rainer, Tomakidi Pascal, Stremmel Wolfgang, Chunglok Warangkana, Kawahara Tsukasa, Rokutan Kazuhito
Deutsches Krebsforschungszentrum, Department of Applied Tumorvirology, 69120 Heidelberg, Germany.
Oncogene. 2003 Sep 4;22(38):6045-53. doi: 10.1038/sj.onc.1206654.
Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.
在NADPH氧化酶（Nox）家族的五个成员中，Nox1具有促有丝分裂特性，并被认为参与细胞转化过程。我们通过用16型人乳头瘤病毒的E6/E7癌基因永生化的人牙龈角质形成细胞进行乙醇处理，建立了两种致癌模型表型：具有上皮样形态的永生化（EPI）未转化细胞和具有纺锤形成纤维细胞样形态的更高级转化（FIB）细胞。与EPI细胞膜相比，FIB细胞膜含有更高水平的63 kDa Nox1蛋白，并且超氧化物和羟基自由基生成能力高2.8倍。EPI和FIB细胞在增殖阶段比静止汇合期表达更丰富的Nox1蛋白。用抗Nox1抗体进行免疫荧光染色显示，两种细胞类型的整个内部都分布有免疫反应性物质，而它们优先定位于FIB细胞的细胞核中。从EPI和FIB细胞分离的细胞核含有63 kDa的Nox1蛋白。与EPI细胞相比，FIB细胞中Jun N末端激酶（JNK）和细胞外信号调节激酶蛋白的表达水平升高。此外，JNK2在FIB细胞中持续磷酸化。总之，我们的数据强烈表明Noxl在人类角质形成细胞更高级转化阶段的细胞激活相关的氧化还原介导信号传导中起作用。
