Takahashi Mami, Mutoh Michihiro, Shoji Yutaka, Kamanaka Yoshihisa, Naka Masao, Maruyama Takayuki, Sugimura Takashi, Wakabayashi Keiji
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan.
Oncogene. 2003 Oct 23;22(48):7667-76. doi: 10.1038/sj.onc.1207051.
Activating mutations of K-ras are frequent in colon tumors and aberrant crypt foci, and may play important roles in colon carcinogenesis. Here, we investigated the effects of a K-ras codon 12 mutation on inducible nitric oxide synthase (iNOS) expression. When rat intestinal epithelial cells (IEC-6) were transfected with K-rasAsp12 cDNA, the iNOS expression linked to interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS) treatment was markedly increased and prolonged. In contrast, it was only very faint and transient in cells transfected with the control vector or K-rasWT. Electrophoretic mobility-shift assays demonstrated that NF-kappaB binding activity induced by IL-1beta or LPS was also increased in K-rasAsp12-transfected cells, along with the binding of CREB-1, CREM-1, ATF-1, ATF-2, and Jun D to a cAMP-responsive element (CRE)-like site and the binding of C/EBPbeta to a C/EBP-binding consensus site. Furthermore, the anchorage-independent growth of K-rasAsp12-transfected cells was markedly increased by IL-1beta or LPS treatment, and decreased by ONO-1714, an iNOS inhibitor. In addition, tumor growth in nude mice injected with K-rasAsp12-transfected cells was significantly suppressed by NOS inhibition with 50 p.p.m. ONO-1714 or 100 p.p.m. L-NG-nitroarginine methyl ester. These results suggest that an activating mutation of K-ras can markedly enhance the iNOS expression mediated by IL-1beta or LPS, through the activation of promoters on NF-kappaB, C/EBP, and CRE-like sites, and that nitric oxide contributes to the colony formation and tumor growth of K-ras-transformed cells.
K-ras的激活突变在结肠肿瘤和异常隐窝灶中很常见，可能在结肠癌发生过程中起重要作用。在此，我们研究了K-ras密码子12突变对诱导型一氧化氮合酶（iNOS）表达的影响。当用K-rasAsp12 cDNA转染大鼠肠上皮细胞（IEC-6）时，与白细胞介素-1β（IL-1β）或脂多糖（LPS）处理相关的iNOS表达显著增加且持续时间延长。相比之下，在转染对照载体或K-rasWT的细胞中，iNOS表达非常微弱且短暂。电泳迁移率变动分析表明，在K-rasAsp12转染的细胞中，IL-1β或LPS诱导的NF-κB结合活性也增加，同时CREB-1、CREM-1、ATF-1、ATF-2和Jun D与cAMP反应元件（CRE）样位点结合，C/EBPβ与C/EBP结合共有位点结合。此外，IL-1β或LPS处理显著增加了K-rasAsp12转染细胞的非锚定依赖性生长，而iNOS抑制剂ONO-1714则降低了这种生长。此外，用50 ppm ONO-1714或100 ppm L-NG-硝基精氨酸甲酯抑制一氧化氮合酶可显著抑制注射了K-rasAsp12转染细胞的裸鼠体内肿瘤生长。这些结果表明，K-ras的激活突变可通过激活NF-κB、C/EBP和CRE样位点上的启动子，显著增强IL-1β或LPS介导的iNOS表达，并且一氧化氮有助于K-ras转化细胞的集落形成和肿瘤生长。
