Transfection of K-rasasp12 cDNA markedly elevates IL-1β- and lipopolysaccharide-mediated inducible nitric oxide synthase expression in rat intestinal epithelial cells

• Published in: Oncogene Vol. 22; no. 48; pp. 7667 - 7676
• Main Authors: TAKAHASHI, Mami, MUTOH, Michihiro, SHOJI, Yutaka, KAMANAKA, Yoshihisa, NAKA, Masao, MARUYAMA, Takayuki, SUGIMURA, Takashi, WAKABAYASHI, Keiji
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 23.10.2003; Nature Publishing Group
• Subjects: Activating transcription factor 2; Biological and medical sciences; Carcinogenesis; CCAAT/enhancer-binding protein; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Colon; Cyclic AMP response element-binding protein; Electrophoretic mobility; Epithelial cells; Fundamental and applied biological sciences. Psychology; IL-1β; Intestine; Lipopolysaccharides; Molecular and cellular biology; Mutation; NF-κB protein; NG-Nitroarginine methyl ester; Nitric oxide; Nitric-oxide synthase; Regulatory sequences; Transfection; Transformed cells; Rat; Enzyme; Rodentia; Gene expression; IL-1β; Carcinogenesis; Nitric-oxide synthase; inducible nitric oxide synthase; Vertebrata; Mammalia; Transfection; Complementary DNA; K-ras; Lipopolysaccharide; Epithelial cell; Oxidoreductases; Onc gene
• ISSN: 0950-9232; 1476-5594
• 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-1β (IL-1β) 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-κB binding activity induced by IL-1β 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/EBPβ to a C/EBP-binding consensus site. Furthermore, the anchorage-independent growth of K-rasAsp12-transfected cells was markedly increased by IL-1β 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-1β or LPS, through the activation of promoters on NF-κB, C/EBP, and CRE-like sites, and that nitric oxide contributes to the colony formation and tumor growth of K-ras-transformed cells.