FIGURE 1.

Activation of Ras GTPases in OGG1-expressing cells by 8-oxoG base. A–F, parallel cell cultures were exposed to 1 μm (A, C, D, and F) or increasing concentration of 8-oxoG (B). Cell extracts were made at the times points indicated (in A) or 15 min after challenge (B, C, D, E, and F). Ras-GTP levels were determined by GST pulldown assays (15, 16). A and B, activation of Ras GTPases in a time-dependent (A) and dose-dependent manner (B) in MRC5 cells upon 8-oxoG exposure. C and D, siRNA-mediated OGG1 depletion causes decreased Ras activation. C and D, MRC5 (C) and HeLa-S (D) cells were transfected with siRNA to OGG1 (see “Experimental Procedures”) or control siRNA using two cycles of transfection and cell culture and then exposed to 8-oxoG for 15 min. Ras-GTP levels were determined as above. E, increase in Ras-GTP levels in murine lungs. BALB/c mice were challenged intranasally with 60 μl of 1 μm 8-oxoG (13), and the lungs were excised after 15 min. Ras-GTP levels in individual mouse lung extracts were determined as above. F, 8-oxoG base, but not 8-oxodG, FapyG, 8-oxoA, or guanine base increased Ras-GTP levels. 250 and 25 μg of cell extract per assay were used to determine Ras-GTP and Ras levels, respectively; n = 3–8.