A Chimeric p53 evades mutant p53 transdominant inhibition in cancer cells

摘要

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. Coimmunoprecipitation 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.