FIGURE 4.

TDG overexpression decreases cell proliferation, increases S phase population, and increases DNA breaks. A, Western blot analysis of doxycycline-induced TDG expression in stably transfected HeLa cell lines. B, top, doxycycline-induced TDG overexpression reduces cell growth. Cell growth was assessed by an MTT assay. Bottom, TDG(ΔKR) is not as toxic as TDG(ΔPIP). Cells grown in the absence of doxycycline still express some TDG from leaky expression (Western blot). C, TDG-overexpressing cells accumulate in S phase. Cells were treated with 50 ng/ml of doxycycline for 3 days to induce TDG(WT), TDG(ΔPIP), or TDG(ΔKR). Cell cycle profile was analyzed by flow cytometry of propidium iodide-stained cells. Left, changes in the percentage of S phase cells induced by doxycycline, relative to cells not treated with doxycycline, are plotted (mean ± S.D. (error bars) of >3 measurements). Right, percentage of cells in each phase of the cell cycle in three independent experiments. D, increase of TDG increases DNA breaks. Cells (transfected with TDG(WT) or mock-transfected) were treated with 100 ng/ml doxycycline for 3 days. DNA breaks were analyzed by an alkaline comet assay. A box and whiskers plot (median and interquartile range) shows the yield of strand breaks as evaluated from the tail moment (n > 100 cells).