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Transfection of K-rasAsp12 cDNA markedly elevates IL-1β- and lipopolysaccharide-mediated inducible nitric oxide synthase expression in rat intestinal epithelial cells

Oncogene volume 22, pages 7667–7676 (2003)Cite this article

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-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.

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Acknowledgements

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.

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Authors and Affiliations

  1. 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

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

    Yoshihisa Kamanaka, Masao Naka & Takayuki Maruyama

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  1. Mami Takahashi
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Takahashi, M., Mutoh, M., Shoji, Y. et al. Transfection of K-rasAsp12 cDNA markedly elevates IL-1β- and lipopolysaccharide-mediated inducible nitric oxide synthase expression in rat intestinal epithelial cells. Oncogene 22, 7667–7676 (2003). https://doi.org/10.1038/sj.onc.1207051

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