Figure 3. Effect of ionizing radiations on p53 stabilization.

Expression levels of TP53 and p53 protein evaluated by Real Time RT-PCR and Western blotting in H295R, SW-13 and SK-OV-3 cells 72 h after transfection with empty vector (mock) or p53-vector (WT). Samples were treated with IR at a dose of 6 Gy where indicated. (A) RT-PCR (top) and Real Time RT-PCR (bottom) analysis of TP53 expression in H295R cells. Samples transfected with pBABE-neo-p53 vector show a 5-fold increase in TP53 mRNA compared with mock. (B) Western blotting analysis of p53 in H295R cells (top) revealed a major band of ∼53 kDa (^wtp53) and a minor band of ∼44 kDa, corresponding to the truncated form of p53 (p53^Δ8–9). Densitometry shows that ^wtp53 is stabilized by IR treatment (bottom). (C) RT-PCR analysis of TP53 mRNA in SW-13 cells shows over-expression of the gene in samples transfected with pBABE-neo-p53 vector (top). Real Time RT-PCR analysis revealed a 3-fold increase in TP53 transcript in samples transfected with p53 vector. The expression of TP53 is not modulated by irradiation (bottom). (D) Levels of p53 protein in SW-13 cells significantly increase after IR treatment in cells transfected with pBABE-neo-p53 vector, while no change is detectable in samples transfected with empty vector. (E) In SK-OV-3 cells, no TP53 transcript was detectable by RT-PCR in samples transfected with empty vector, while in cells transfected with pBABE-neo-p53 vector a strong signal, corresponding to TP53 mRNA, is observable (top). Real Time RT-PCR analysis shows no modulation of TP53 expression after irradiation (bottom). (F) Western blotting analysis of p53 in SK-OV-3 cells revealed a detectable band only in samples transfected with pBABE-neo-p53 vector (top). Densitometry shows that p53 levels increase after irradiation, indicating the stabilization of the protein (bottom). Results are representative of at least three independent experiments. GAPDH and vinculin were used for normalization. (*, p<0.05; #, p<0.01).