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. 2009 Mar 25;100(7):1275–1283. doi: 10.1111/j.1349-7006.2009.01166.x

Figure 3.

Figure 3

Reactive oxygen species (ROS) levels in cancer cell lines in response to p21 expression levels. (a) ROS levels were evaluated by FACS analysis after staining HeLa, LoVo and HCT116 cells with the aminophenyl fluorescein (APF) fluorescent probe. Relative ratio of the geometric mean that is the average of the logarithm of the linear value for events expressed as the anti‐log in Ad‐p21C‐ (40 MOI) and Ad‐p21F (20 MOI or 40 MOI)‐infected cells as compared to the control (20 MOI). Generated ROS levels were significantly higher in the cancer cells infected with 20 MOI Ad‐p21F than in controls and 40–50 MOI Ad‐p21C‐infected cells, *P < 0.05. In LoVo and HCT116 cells, generated ROS levels were significantly higher in the cancer cells infected with 40–50 MOI Ad‐p21F than those in 20 MOI Ad‐p21F‐infected cells, **P < 0.05. Apoptosis was induced in the former and senescence in the latter. (b) Senescence induced by p21‐overexpression was inhibited by N‐acetyl‐L‐cystein (NAC) (ROS scavenger). LoVo and HCT116 cells were cultured in 10 mM NAC and were infected with 20 MOI Ad‐p21F. The ratio of senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive cells after 74 h of the infection was significantly decreased in the presence of NAC. Results represent mean values of three experiments, and the error bar shows the SD. (c) Induction of apoptosis with 50 MOI Ad‐p21F was also inhibited by the addition of NAC in LoVo and HCT116 cells, as indicated by the significant decrease of the subG1 fraction. Results represent mean values of three experiments, and the error bars show the SD. (d) p53‐siRNA inhibited apoptotis induction by p21. Western blot analysis of p53 expression in lysates from Ad‐p21F (40 MOI)‐infected HCT116 cells in the absence or presence of control‐siRNA and p53‐siRNA is shown. Induction of apoptosis by p21 was suppressed by p53‐siRNA in HCT116 cells, as indicated by the significant decrease in the subG1 fraction (upper graph). The shift to the right due to increased fluorescence corresponds to an increase in the intracellular levels of ROS. The black line indicates the mean fluorescence values in the control siRNA treated cells (lower left figure). The ROS level was significantly decreased in the presence of p53‐siRNA (P < 0.05) (lower right graph). Results represent mean values of three experiments, and the error bar shows the SD.