A Chimeric p53 Evades Mutant p53 Transdominant Inhibition
in Cancer Cells
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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