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

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-ras cDNA, the iNOS expression linked to interleukin-1b (IL1b) 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-ras. Electrophoretic mobility-shift assays demonstrated that NF-jB binding activity induced by IL1b or LPS was also increased in K-ras-transfected cells, along with the binding of CREB-1, CREM-1, ATF1, ATF-2, and Jun D to a cAMP-responsive element (CRE)-like site and the binding of C/EBPb to a C/EBPbinding consensus site. Furthermore, the anchorageindependent growth of K-ras-transfected cells was markedly increased by IL-1b or LPS treatment, and decreased by ONO-1714, an iNOS inhibitor. In addition, tumor growth in nude mice injected with K-rastransfected cells was significantly suppressed by NOS inhibition with 50 p.p.m. ONO-1714 or 100 p.p.m. L-Nnitroarginine methyl ester. These results suggest that an activating mutation of K-ras can markedly enhance the iNOS expression mediated by IL-1b or LPS, through the activation of promoters on NF-jB, C/EBP, and CRElike sites, and that nitric oxide contributes to the colony formation and tumor growth of K-ras-transformed cells. Oncogene (2003) 22, 7667–7676. doi:10.1038/sj.onc.1207051