Introduction: The K-ras gene product is mutated in 30-50% of human lung adenocarcinomas. It exists in two isoforms, K-ras 4A and 4B. Their amino acid sequences are very similar, except for marked differences in the hypervariable domain near the carboxyl terminus. K-ras 4B is usually the predominant isoform expressed, though increased K-ras 4A has been reported in cancer cells. Secondly, mutant ras isoforms have been shown to cause an increase in reactive oxygen species in several models, including lung cells. Mutant K-ras caused increased superoxide generation in transformed normal rat kidney (NRK) cells. In our prior studies in lung adenocarcinoma cell lines with K-ras mutations, cellular total K-ras protein levels correlated positively with superoxide production, but the relationship was not significant (p = 0.28). We considered that the K-ras 4A isoform, which has been shown to act synergistically with K-ras 4B to increase lung tumor size and number in genetically modified mice, might act independently as an inducer of superoxide production. Methods: K-ras 4A levels in lysates from 20 different human adenocarcinoma cell lines were measured by Western blotting, normalized to levels in cell line H441 included with each blot. The specific antibody used was not cross-reactive with K-ras 4B. Superoxide levels were determined on the same cell lines using a nitroblue tetrazolium assay. Results: In cell lines with mutant K-ras (n=11), relative K-ras 4A protein expression was strongly and positively correlated with superoxide production by linear regression (p<0.0001). The level of K-ras 4A protein expression in 5 cell lines with wild-type K-ras 4A did not correlate with superoxide production (p = 0.61). Discussion: These results support a possible role for mutant K-ras 4A protein in the production of superoxide in human lung adenocarcinoma cell lines. Previously we demonstrated that superoxide correlated positively with DNA damage (comet assay) in these cell lines (Free Rad. Biol. Med. 43:1145, 2007) and hence was implicated in maintenance of the transformed phenotype. Whether superoxide production is a direct result of the presence of mutant K-ras 4A in lung adenocarcinoma cells is currently under study. If so, then level of mutant K-ras 4A could be an important prognostic marker and possible therapeutic target in this type of malignancy. Funded in part by NO1CO-2008-00001
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 437.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO