Aberrant cell growth signaling in oral cancer

本研究旨在阐明口腔鳞状细胞癌（OSCC）中异常生长信号的详细机制。获得了以下结果。(1)我们首先在人类癌症中发现了ERK 2在CD区322位密码子由Glu突变为Lys，并伴随着电荷改变和PAGE迁移速度加快。类似的突变已在果蝇中被鉴定为sevenmaker，并且据报道突变蛋白与MKP（一种针对ERK的磷酸酶）具有弱结合。除HSC 6外，我们还检测了3个EGFR扩增的细胞系，发现EGF处理后EGFR在846和1068位高水平磷酸化，下调大部分被抑制。此外，我们发现了一种细胞系，其中MKP 1/2没有被可检测地诱导。上述生长信号的变化可能至少部分地促成了ERK的组成性激活。(2)通过酵母双杂交系统，我们分离到两个蛋白，尚未知道与ERK 2相互作用。EGF/ERK介导的磷酸化作用。EGF/ERK信号转导途径。进一步的实验表明，该蛋白在M期被超磷酸化（SP），并且用G2/M抑制剂处理诱导SP物种。此外，我们还发现NCL 4的C端1/3增强了诱导细胞凋亡的作用。(3)检测15例口腔鳞癌组织中ERK磷酸化水平，并与临床病理资料进行相关性分析。与组织病理分化程度相关，与TNM分期无关。(4)35个耐药基因的RNA表达水平在不同的依托泊苷敏感性的OSCC细胞系中没有改变，但依托泊苷调节的2个基因的表达根据对依托泊苷的敏感性而差异表达。(5)在印度的OSCC中发现了5个肿瘤抑制相关基因的高甲基化，而在正常组织中没有发现，这可以作为诊断标志物。

This study was undertook to elucidate detailed mechanisms of aberrant growth signaling in oral squamous cell carcinoma (OSCC). The following results were obtained. (1) We, first in human cancer, found an ERK2 mutation within CD domain of codon 322 from Glu to Lys, which is accompanied with charge alteration and faster migration in PAGE. The similar mutation has been identifed in Drosphila as sevenmaker and the mutant protein reported to have weak binding to MKP, a phosphatase to inactivate ERK. In addition to HSC6, we examined 3 cell lines with EGFR amplification, and found that EGFR was phosphorylated at high levels for 846 and 1068 positions upon EGF treatment and the down regulation was mostly suppressed. Moreover, we found a cell line in which MKP1/2 was not detectably induced. The above changes in growth signaling of OSCC might have contributed at least in part to the constitutive activation of ERK. (2) By yeast 2 hybrid system we isolated two proteins that had not been known to interact with ERK2. Nafi was phosphorylated through EGF/ERK. signaling, while attenuated EGF/ERK signaling. Further experiments showed the protein to be super-phsphoaated (SP) in M phase and the treatment with G2/M inhibitors induced SP species. Besides, we found the C-terminal 1/3 of Nafi enhanced to induce apoptosis. (3) 15 tumor specimen of oral squamous cell carcinoma were examined for levels of phosphorylation of ERK which was related to clinicopathological data. There was good correlation to histopathological differentiation grade but not to TNM classification. (4) RNA expression levels of 35 drug-resitnat genes were not changed in OSCC cell linss irrespective of sensitivity to etoposide, but the expression of etoposide-regulated 2 genes was differentially expressed according to the sensitivity to etoposide. (5) Hypermethylation of 5 tumor-suppressor related genes was found in OSCCs of India but not in normal tissues, which could be used as diagnostic markers.