Transfection of K-rasAsp12 cDNA markedly elevates IL-1beta- 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-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-rasWT. Electrophoretic mobility-shift assays demonstrated that NF-kappaB binding activity induced by IL-1beta 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/EBPbeta to a C/EBP-binding consensus site. Furthermore, the anchorage-independent growth of K-rasAsp12-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-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-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.
Takahashi
Mami
M
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. mtakahas@gan2.ncc.go.jp
Mutoh
Michihiro
M
Shoji
Yutaka
Y
Kamanaka
Yoshihisa
Y
Naka
Masao
M
Maruyama
Takayuki
T
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D011905:Genes, ras / Q000235:genetics*; D007375:Interleukin-1 / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D020218:Response Elements / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D001224:Aspartic Acid; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat