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-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.

This work was supported in part by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), grants-in-aid for Cancer Research and for the

Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan, and a grant for the Research on Advanced Medical Technology from the

Ministry of Health, Labour, and Welfare of Japan.

Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo, 104-0045, Japan

Mami Takahashi, Michihiro Mutoh, Yutaka Shoji, Takashi Sugimura & Keiji Wakabayashi

Minase Research Institute, Ono Pharmaceutical Co. Ltd, 1-1, Sakurai 3-chome, Shimamoto-cho, Mishima-gunn, Osaka, 618-8585, Japan

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