1. ¹Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
2. ²Minase Research Institute, Ono Pharmaceutical Co. Ltd, 1-1, Sakurai 3-chome, Shimamoto-cho, Mishima-gunn, Osaka 618-8585, Japan

Correspondence: M Takahashi, E-mail: mtakahas@gan2.ncc.go.jp

Received 31 October 2002; Revised 2 June 2003; Accepted 31 July 2003.

Abstract

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-1 (IL-1) 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-B binding activity induced by IL-1 or LPS was also increased in K-ras^Asp12-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/EBP to a C/EBP-binding consensus site. Furthermore, the anchorage-independent growth of K-ras^Asp12-transfected cells was markedly increased by IL-1 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-1 or LPS, through the activation of promoters on NF-B, C/EBP, and CRE-like sites, and that nitric oxide contributes to the colony formation and tumor growth of K-ras-transformed cells.