Figure 4
Deletion of the p53 binding motif in the 14-3-3γ promoter eliminates suppression by p53. A series of deletion mutants were constructed that successively deleted segments from the 5' end of the 14-3-3γ promoter. These are illustrated diagrammatically linked to a luciferase gene and are shown on the left side of panels A and B. The p53 binding motif is represented as a small striped box. Small boxes outlined with stippled lines show the position of p53 consensus sequences that were deleted. The numerical designation shown within the luciferase box indicates the length of the 14-3-3γ promoter sequence attached to the luciferase gene. A. Endogenous p53 repress wild-type 14-3-3γ promoter activity after γ-irradiation. Luciferase reporter vectors each containing either no 14-3-3γ promoter sequences (PGL3-LUC), 14-3-3γ promoter sequences of various lengths (PGL3-100 bp-LUC, PGL3-586 bp-LUC, PGL3-830 bp-LUC, PGL3-1200 bp-LUC), or 14-3-3γ promoter sequences that lacked either all or part of the p53 consensus binding sequence (PGL3-840 bp-LUC, PGL3-850 bp-LUC, PGL3-1200Δ850-840 or PGL3-1200Δ850-830) were transiently transfected into A549 cells together with a renilla luciferase vector. Forty-eight hours after transfection the cells were either left untreated (open bars) or irradiated with 10 GY ionizing radiation (solid bars). The cells were harvested 12 hours later, extracts prepared and luciferase activity quantified. The bars in the graph at the right of panel A represent the mean ± SD of three independent experiments. Values that were determined to be significantly reduced relative to the matched control are marked with an asterisk (p < 0.01; control vs IR). B. 14-3-3γ wild-type and mutant promoter reporter assays in A549 and HCT116 cells. The 14-3-3γ promoter deletion reporter vectors described in panel A were transfected into A549 cells that are infected with either GFP-expressing adenovirus (open bars) or with an adenovirus expressing a wt-p53 (solid bars). Forty-eight hours later the cells were harvested and luciferase activity quantified and normalized to renilla luciferase activity. Bars in the graph in the middle of panel B represent the mean of three independent experiments ± SD. Values that were found to be significantly reduced relative to the matched control are marked with an asterisk (p < 0.01; Ad-GFP vs Ad-p53). In a similar experiment, HCT116p53+/+ (open bars) and HCT116p53-/- (closed bars) with the same vectors described above and irradiated with 10 Gy ionizing radiation. Luciferase activity was determined and normalized to renilla luciferase and the values graphed. Results shown represent the mean of three independent experiments ± SD. Values determined to be significantly reduced relative to their matched controls are marked with an asterisk (p < 0.01; HCT116 p53+/+ vs HCT116 p53-/-).