Mutant K-rasV12 increases COX-2, peroxides and DNA damage in lung cells.
K-ras is frequently mutated in lung adenocarcinomas. Recent discovery that wild-type K-ras is tumor suppressive in the lung raises a question: how is mutant K-ras aggressively oncogenic? We hypothesized that mutant K-ras might lead to generation of reactive oxygen species (ROS) and DNA damage, contributing to malignant transformation. We stably transfected human mutant K-ras(V12) into non-transformed peripheral mouse lung epithelial cells (E10 line). Constitutively active mutant K-ras(V12) in E10 cells led to a highly significant (P < 0.001) increased level of peroxides, and a corresponding increase in the amount of DNA strand-break damage, compared with the parental line E10 and the vector control. Levels of superoxide were not increased, suggesting a direct source of peroxides, such as cyclooxygenase-2 (COX-2). COX-2 protein and activity measured as prostaglandin E(2) level were up-regulated in cells expressing mutant K-ras(V12); COX-2 activity correlated with K-ras activity (K-ras p21-GTP). Both peroxide generation and DNA single strand breaks were significantly reduced by pre-treatment with COX-2-specific inhibitor SC 58125, confirming COX-2 as the source of the ROS. COX-2 has been repeatedly implicated in lung cancer, and is known to be regulated by ras and to release ROS. Our data suggest that up-regulation of COX-2, with a consequent increase in peroxides and DNA damage, contributes to the dominant oncogenicity of mutant K-ras.
Maciag
Anna
A
Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Inc., Frederick, MD 21702, USA. amaciag@mail.ncifcrf.gov
Sithanandam
Gunamani
G
Anderson
Lucy M
LM
D000818:Animals; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D004249:DNA Damage; D005260:Female; D011905:Genes, ras; D005822:Genetic Vectors; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010545:Peroxides / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013481:Superoxides / Q000378:metabolism
D008565:Membrane Proteins; D010545:Peroxides; D017382:Reactive Oxygen Species; D013481:Superoxides; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human