A chimeric p53 evades mutant p53 transdominant inhibition in cancer cells

Abstract

Because of the dominant negative effect of mutant p53, there has been limited success with wild-type (wt) p53 cancer gene therapy. Therefore, an alternative oligomerization domain for p53 was investigated to enhance the utility of p53 for gene therapy. The tetramerization domain of p53 was substituted with the coiled-coil (CC) domain from Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains proper nuclear localization in breast cancer cells detected via fluorescence microscopy and shows a similar expression profile of p53 target genes as wt-p53. Additionally, similar tumor suppressor activities of p53-CC and wt-p53 were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD), and colony-forming assays. Furthermore, p53-CC was found to cause apoptosis in four different cancer cell lines, regardless of endogenous p53 status. Interestingly, the transcriptional activity of p53-CC was higher than wt-p53 in 3 different reporter gene assays. We hypothesized that the higher transcriptional activity of p53-CC over wt-p53 was due to the sequestration of wt-p53 by endogenous mutant p53 found in cancer cells. Co-immunoprecipitation revealed that wt-p53 does indeed interact with endogenous mutant p53 via its tetramerization domain, while p53-CC escapes this interaction. Therefore, we investigated the impact of the presence of a transdominant mutant p53 on tumor suppressor activities of wt-p53 and p53-CC. Overexpression of a potent mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53, p53-CC retains the same level of tumor suppressor activity. Finally, viral transduction of wt-p53 and p53-CC into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53.

Figure 1.

Proposed mechanism of p53-CC activity. Left side of figure: exogenously added wt-p53 can still form heterotetramers with mutant p53 due to the presence of the TD and becomes inactivated. Right side of figure: p53-CC can bypass transdominant inhibition by mutant p53 in cancer cells and still exhibit tumor suppressor activity.

Figure 2.

(A) Schematic representation of the experimental constructs and controls. Full length p53 (wt-p53) contains a MDM2 binding domain (MBD), a transactivation domain (TA) in the amino terminus, a proline-rich domain (PRD), a DNA binding domain (DBD), a strong nuclear localization signal (NLS), a tetramerization domain (TD) that also contains a nuclear export signal (E), and a carboxy terminus (C-terminus) that includes two weak NLS’s. For p53-CC, the TD and C-terminus were replaced by the coiled-coil (CC) from Bcr. p53-ΔTDC lacks both the TD and the C-terminus. All constructs were fused to EGFP on the N-terminus (not shown in diagram). (B) Representative fluorescence microscopy images of 1471.1 cells confirm exclusive nuclear accumulation of EGFP-p53-CC similar to EGFP-wt-p53. EGFP fluorescence, nuclear staining with H33342, and phase contrast images are shown, left to right. 1471.1 breast cancer cells were chosen for this study due to their optimal microscopy characteristics (elongated morphology and distinguishable subcellular compartments). White scale bars on the top left corners are 10 μm.

Figure 3.

p53-CC is capable of transactivating several p53 target genes. (A) Scatter plot representation of mRNA levels of 84 p53 target genes in T47D cells transfected with wt-p53 or p53-CC. Each dot represents one of the 84 genes assayed in this PCR array. The two magenta lines represent a boundary of 2-fold upregulation or downregulation in mRNA levels. Cells treated with wt-p53 or p53-CC showed similar levels of mRNA for all 84 genes except for one, p53AIP1, which is circled on the scatter plot. (B) Representative cropped Western blots of T47D cell lysates 24 h post transfection with wt-p53, p53-CC, p53-ΔTDC, or CC. Similar levels of Bax and p21/WAF1 protein expression were detected from cells treated with wt-p53 or p53-CC. Each Western blot was repeated at least three times.

Figure 4.

Apoptotic and cell proliferation assays were performed in T47D cells 48 h after transfection. (A) TUNEL assay shows similar apoptotic activity of p53-CC compared to wt-p53. Both p53-CC and wt-p53 demonstrate a significantly higher activity compared to CC negative control. Similar results were obtained from (B) annexin V staining and (C) 7-AAD staining. (D) The colony forming assay shows the transformative ability of T47D cells post treatment with wt-p53, p53-CC, and CC. Cells treated with wt-p53 and p53-CC show significant reduction in transformative ability (oncogenic potential) of T47D cells compared to untreated cells or cells treated with CC. Mean values were analyzed using one-way ANOVA with Bonferroni’s post test; * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars represent standard deviations from at least three independent experiments (n = 3).

Figure 5.

7-AAD assay conducted in four different cell lines with varying p53 status: (A) Hela, (B) MDA-MB-231, (C) MCF-7, and (D) H1373. In all four cases, p53-CC is capable of inducing cell death in a similar fashion compared to wt-p53, regardless of the endogenous p53 status or the cancer cell line used. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post test; **p < 0.01 and ***p < 0.001.

Figure 6.

Relative luminescence representing the activation of (A) the p53-cis reporter, (B) the p21/WAF1 reporter, and (C) the PUMA reporter in T47D cells. The ability of p53-CC to transactivate these promoters is higher than wt-p53. In all three cases, 3.5 μg of construct (wt-p53, p53-CC, CC, or EGFP) was cotransfected with 0.35 μg of pRL-SV40 plasmid encoding for Renilla luciferase to normalize for transfection efficiency. In addition to Renilla luciferase, constructs were cotransfected with 3.5 μg of p53-Luc cis-reporter, p21/WAF1 reporter, or PUMA reporter encoding for firefly luciferase. Mean values were analyzed using one-way ANOVA with Bonferroni’s post test; **p < 0.01 and ***p < 0.001. Error bars represent standard deviations from three independent experiments (n = 3). (D) Interaction of endogenous p53 with exogenous wt-p53 or p53-CC was investigated in T47D via co-IP. A representative cropped Western blot of protein complexes coimmunoprecipitated using anti-GFP antibody is shown. Left lane, endogenous p53 (53 kDa) coimmunoprecipitates with exogenous EGFP-wt-p53 (70 kDa). Right lane, endogenous p53 fails to coimmunoprecipitate with exogenous EGFP-p53-CC (71 kDa).

Figure 7.

p53-CC circumvention of transdominant inhibition by mutant p53. (A) Overexpression of mutant p53 reduces the activity of exogenous wt-p53 but has no influence on exogenous p53-CC activity. H1373 cells were chosen for this experiment since they are p53 null and hence there will be no additional p53 activity from the cells due to lack of endogenous p53. (B) 7-AAD assay was conducted 48 h post transducing MDA-MB-468 cells, which harbor a potent transdominant mutant p53 (R273H), with adenoviral vectors expressing either wt-p53 or p53-CC with a multiplicity of infection (MOI) of 200. As expected, exogenous wt-p53 (Ad-p53) activity is limited in this cell line due to the presence of endogenous transdominant tumor derived p53. (C) 7-AAD assay was also performed 48 h post transduction of 4T1 cells (MOI 250). Interestingly, p53-CC is more active than wt-p53 in this particular cell line. The adenoviral vector alone was used as a negative control. Mean values were analyzed using one-way ANOVA with Bonferroni’s post test; **p < 0.01 and ***p < 0.001. Error bars represent standard deviations from three independent experiments (n = 3).