K-ras is mutated in many lung adenocarcinomas, but the nature of its contribution to the development and maintenance of these cancers is poorly understood. Generation of reactive oxygen species (ROS) is proposed to be part of its mechanism of action. We pursued this hypothesis in 12 lung adenocarcinoma cell lines expressing mutant K-ras and 8 lines with only wildtype K-ras. K-ras total protein showed a 14-fold variation in amount among the cell lines and was as high on average in cells with wildtype K-ras, as in those with mutant protein. As expected K-ras activity was much higher in lines with mutant K-ras, varied 6-fold among these, and correlated strongly with protein levels. Mn and CuZn superoxide dismutase (SOD) protein levels varied approximately 2-fold among the cell lines. Only MnSOD varied significantly with the parameters measured, and showed a weak positive correlation with levels of superoxide (as measured by reduction of nitroblue tetrazolium) and a strong negative correlation with K-ras activity. Superoxide correlated with DNA damage as indicated by the comet assay for single-strand breaks. In view of the complex relationships between superoxide, MnSOD and K-ras, K-ras activity relationships with superoxide and DNA damage were evaluated in a subset of cells with similar MnSOD levels. In these, K-ras activity correlated strongly and positively with superoxide levels (P = 0.0054) and also significantly with DNA single-strand breaks (P = 0.05). Peroxide levels were also measured in the cells by the DCF fluorescence assay, and were found to be correlated strongly and negatively with levels of the anti-oxidant enzymes peroxiredoxins (PRX) 1, 3, 4 and 6. In cell lines with mutant K-ras, K-ras activity also correlated negatively with these PRXs (P = 0.0065, 0.024, 0.0069, and 0.0065 for PRXs 1, 3, 4, and 6, respectively). There was a weak positive correlation between mutant K-ras activity and peroxide levels. In summary the data suggest that mutant K-ras could influence ROS levels and DNA damage in several ways in lung adenocarcinoma cells: by downregulation of MnSOD and of several PRX isoforms, and by increasing DNA-damaging superoxide. These results are consistent with the starting hypothesis and suggest that mutant K-ras may enhance ROS in lung adenocarcinoma cells through several pathways.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA