Conformational and Molecular Basis for Induction of Apoptosis by a p53 C-terminal Peptide in Human Cancer Cells

Abstract

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/ APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.

Selective induction of apoptosis through the FADD/caspase‐8 pathway by a p53 c‐terminal peptide in human pre‐malignant and malignant cells

“…27,38 In this study, we showed that this peptide was not toxic to normal colon and normal, as well as, non-malignant breast cells with endogenous wt p53. The peptide selectively induced apoptosis in pre-malignant colon cells with mutant p53 (Fig.…”

“…27 All peptides were HPLC purified to Ͼ95% pure. Peptide stocks (4 mM) were prepared in sterile distilled water and stored in aliquots at Ϫ80°C.…”

“…26 Using conformational energy calculations to compute the lowest energy conformations for the C-and N-terminal regulatory domains, we found that these 2 domains form a unique low energy complex, suggesting that a direct interaction can potentially exist between them. 27 Previous studies demonstrated that the addition of a chemically modified p53 C-terminal peptide (aa 363-393) restored in vitro sequence-specific DNA binding function to mutant p53-273 (Arg to His). Furthermore, intranuclear microinjection of this peptide into human colon cancer cells SW480 carrying an endogenous p53-273 His mutant restored transcriptional activation of a p53-responsive reporter construct.…”

“…29 Our previous studies indicated that the C-terminal p53 peptide (aa 361-382)-Antennapedia peptide (p53p-Ant) induced selective and rapid apoptosis in human breast cancer cells associated with increased Fas and Fas ligand expression. 27 The mechanism and components of the Fas pathway needed for apoptosis by p53p-Ant were not elucidated. In addition, using human null p53 lung cancer cells H1299 and prostate cancer cells PC-3 stably transfected with the human p53 temperature-sensitive (ts) mutant 143Ala (Val-Ala) as a model, we found Fas-mediated apoptosis occurred only at the wt p53 phenotype at 32.5°C.…”

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…A synthetic peptide derived from the p53 C-terminal domain (peptide 46) was shown to restore the specific DNA binding and transcriptional transactivation function of several hot-spot mutant p53 proteins and rescue the function of endogenous mutant p53 proteins resulting in growth inhibition and cell death by apoptosis (Selivanova et al, 1997;Kim et al, 1999). The observation that C-terminal peptides can restore the DNA-binding activity to isolated core domain proteins indicated that it might directly affect the p53 domain where tumor-derived mutations are clustered.…”