Elevated expression of 14-3-3γ correlates with mutations in p53. A. mRNA expression in NSCLC tumors. RNA was extracted from frozen specimens of normal and tumor tissues and the quantity of 14-3-3γ determined using quantitative RT-PCR. Fold changes were determined by 2^-ΔΔCt by comparing Ct values of 14-3-3γ with GAPDH. The box plot depicts 2^-ΔΔCt values for 14-3-3γ expression. p-values are for comparison of the levels of 14-3-3γ mRNA in tumor specimens with normal tissue, which is calculated using ΔCt values. B. Analysis of 14-3-3γ expression by Western blotting in NSCLC tumors. A Western blot showing expression of 14-3-3γ protein in 8 representative NSCLC tumors and protein loading was confirmed by β-actin. C. Gene copy number. Genomic DNA was extracted from normal and tumor tissues and the relative quantity of 14-3-3γ DNA determined using quantitative PCR from equal quantities of DNA. ΔCt values for 14-3-3γ were normalized against PI3KR1 (p85alpha regulatory subunit of PI3K) since the latter was found to be unaltered in NSCLCs. The fold changes were calculated by comparing normal and tumor tissues using 2^-ΔΔCt. Y-axis shows the fold change comparing normal and tumor tissues. D. 14-3-3γ expression and p53 mutations in NSCLC tumors. p53 mutational status was determined in the same lung tumors used in panels A and B using RT-PCR amplification of p53 mRNA followed by direct sequencing. Tumors were then segregated based on p53 mutation status, (wt-p53) or (mut-p53) and the 14-3-3γ mRNA expression determined as in panel A. p-values were determined by comparing normal tissue, tumors with wt-p53 and tumors with mut-p53.

Wt-p53 suppresses expression of 14-3-3γ. A. Expression of 14-3-3γ and p53 after γ-irradiation. Cultures of A549, H358 and H322 cells were either left untreated (A549, H358, H322) or exposed to 10 Gy of gamma radiation (A549-10 Gy, H358-10 Gy, H322-10 Gy). Total cell extracts prepared six hours after γ-irradiation and proteins fractionated on SDS-PAGE gels. Protein blots were probed for the presence of 14-3-3γ and p53 using the appropriate antibodies. The β-actin was used as a loading control. Typical results are shown. B. 14-3-3γ protein expression following Ad-p53 infection. A549 cells were grown and then either left untreated (A549) or infected at a multiplicity of infection (MOI) of 10 with a control adenovirus expressing GFP (A549-AdGFP) or with an adenovirus expressing a wt-p53-GFP (A549-Adp53). Seventy two hours after infection the cells were harvested and the presence of 14-3-3γ and p53 detected in total cell proteins by immunoblotting using the appropriate antibodies. β-actin was used as a loading control. C. 14-3-3γ mRNA expression following Ad-p53 infection. The relative levels of 14-3-3γ mRNA expression in samples of panel B were quantified using real-time RT-PCR. The bars depict % change compared with control samples from three different experiments ± SD. Bars showing a significant reduction in message level are marked with an asterisk (p < 0.01 versus Control or Ad-GFP infected). D. 14-3-3γ protein expression following endogenous p53 knockdown by siRNA. A549 cells were cultured and then transfected either with a control non-targeting siRNA (A549-Mock siRNA) or an anti-p53 siRNA (A549-p53siRNA) as described in Materials and Methods. The cells were harvested after 48 h, extracts prepared, and protein blots probed for 14-3-3γ and p53 using the appropriate antibodies. β-actin was used as a loading control.

p53 binds 14-3-3γ promoter and negatively regulates 14-3-3γ expression. A. Yeast one hybrid assays. The 600 and 1200 bp 14-3-3γ promoter sequences were subcloned into the GFP reporter vector as described in materials and methods. The p53 encoding plasmid was co-transformed with reporter vectors into Saccharomyces cerevisiae strain W303 and successful transformants were used for the GFP fluorescent assay in the presence of UV. The results were normalized to cells grown in the sucrose medium. Values in y-axis are represented as fold change. Experiments were repeated three times and p values calculated using student's t-test (*p < 0.05). B. A schematic diagram of the 1200 bp of the human 14-3-3γ gene promoter is shown with the position and sequence of the putative p53 binding motif identified. The p53 consensus binding motif is shown for comparison. Asterisks denote identity between the two sequences. This promoter fragment was cloned into PGL3 adjacent to the luciferase gene (shown at right). C. CHIP assays in lung cancer cell lines. A549, H358 and H322 cells were either left untreated (A549, H358, H322) or exposed to 10 Gy ionizing radiation (A549-IR, H358-IR, H322-IR). The cells were harvested eight hours after γ-irradiation and chromatin immunoprecipitation performed as described in the methods section. Reactions were performed either with a mouse IgG as a negative control (A549 -p53ab, H358 -p53ab, H322 -p53ab) or the anti-p53 DO1 antibody. PCR products were separated on agarose gels and visualized by staining with ethidium bromide and viewing with UV illumination. Three independent experiments were performed. Typical results are shown. D. CHIP assays in human colon cancer cell lines. HCT116p53^+/+ or HCT116p53^-/- cells were either left untreated or exposed to 10 Gy ionizing radiation and chromatin immunoprecipitation reactions performed as in panel C using either a mouse IgG as a negative control (-p53ab) or with the anti-p53 DO1 antibody. PCR products were visualized as in panel C. Reactions using input DNA are shown at the bottom of the panel. Three independent experiments were performed. A typical result is shown. E. 14-3-3γ promoter reporter assays in lung cancer cell lines following γ-irradiation. A549, H322, or H358 cells were transiently transfected either with empty vector (open bars) or with the reporter depicted in panel B (solid bars). Forty eight hours after transfection the cells were either left untreated (A549, H358, H322) or exposed to ionizing radiation (10 Gy). The cells were harvested eight hours after gamma-irradiation and luciferase activity measured and normalized relative to renilla luciferase to control for variations in transfection efficiency. The bars represent the average of three experiments ± SD. An asterisk denotes those values that are significantly reduced relative to unirradiated controls (*p < 0.01; control vs IR). F. 14-3-3γ promoter reporter assays in colon cancer cell lines following γ-irradiation. HCT116p53^+/+ or HCT116p53^-/- cells were treated in the same way as cells in panel E. Luciferase activity was determined in the non-irradiated and irradiated (IR) cells as described above. The bars depict the average of three independent experiments, ± SD. Open bars show values for cells transfected with the empty vector. Solid bars show values for cells transfected with the reporter vector shown in panel B. An asterisk marks those values that were significantly reduced relative to non-irradiated controls (*p < 0.01; control vs IR).

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^-/-).