Transfection of K-rasAsp12 cDNA markedly elevates IL-1β- 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(Asp12) 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-ras(WT). Electrophoretic mobility-shift assays demonstrated that NF-kappaB binding activity induced by IL-1beta or LPS was also increased in K-ras(Asp12)-transfected cells, along with the bindingof CREB-1, CREM-1, ATF-1, ATF-2, and Jun D to a cAMP-responsive element (CRE)-like site and the bindingof C/EBPbeta to a C/EBP-binding consensus site. Furthermore, the anchorage-independent growth of K-ras(Asp12)-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-ras(Asp12)-transfected cells was significantly suppressed by NOS inhibition with 50 p. p. m. ONO-1714 or 100 p. p. m. L-N-G-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.