Superoxide generation in v-Ha-ras-transduced human keratinocyte HaCaT cells

The oncogenic ras protein controls signal-transduction pathways that are cr itical for cell proliferation and tumorigenesis. Here, we demonstrate that v-Ha-ras-transduced human keratinocyte HaCaT cells produced significantly l arger amounts of superoxide than did control cell lines. The superoxide gen eration was mediated by the transduced ras protein, because superoxide gene ration was modified by an inhibitor, lovastatin, that inhibits ras farnesyl ation during ras protein maturation. Superoxide generation was also inhibit ed by diphenylene iodonium, an inhibitor of flavoproteins, including NADPH oxidase, but not by rotenone, an inhibitor of the respiratory chain of the mitochondria. Those observations suggested that a phagocytic-like NADPH oxi dase exists in keratinocytes that could be activated by the dominant activa ted v-Ha-ras and produce superoxide. Overexpression of manganese-containing superoxide dismutase and copper and zinc-containing superoxide dismutase c DNA via adenovirus infection also attenuated superoxide generation. Previou s work has demonstrated that extracellular superoxide dismutase (SOD) can l ower superoxide generation; this is the first report that intracellular SOD could also modify the amount of superoxide production from the cells. This report implies that superoxide radical may act as a second messenger molec ule of oncogenic ras. (C) 1999 Wiley-Liss, Inc.