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

Abstract

A p53 C-terminal peptide (aa 361-382, p53p), fused at its Cterminus to the minimal carrier peptide of antennapedia (17 aa, Ant; p53p-Ant), induced rapid apoptosis in human cancer cells, via activation of the Fas pathway. We examined p53p-Ant mechanism of action, toxicity in various human normal, non-malignant, pre-malignant and malignant cancer cells and investigated its biophysical characteristics. p53p-Ant selectively induced cell death in only pre-malignant or malignant cells in a p53-dependent manner and was not toxic to normal and non-malignant cells. p53p-Ant was more toxic to the mutant p53 than wild-type p53 phenotype in H1299 lung cancer cells stably expressing human temperature-sensitive p53 mutant 143Ala. Surface plasmon resonance (BIACORE) analysis demonstrated that this peptide had higher binding affinity to mutant p53 as compared to wild-type p53. p53p-Ant induced-cell death had the classical morphological characteristics of apoptosis and had no features of necrosis. The The p53 tumor suppressor gene is one of the most commonly mutated genes found in human malignancies. 1 More than 50% of human tumors, including breast cancers, are associated with missense mutations or deletions of p53 and most of the missense mutations map to the DNA-binding domain of the protein. 2,3 p53 protein functions in the transcription of growth inhibiting genes, apoptosis, cell cycle arrest and DNA repair. 4 -6 p53 is a sequencespecific transcription factor that transactivates a number of genes whose products are involved in cell growth regulation. These include p21 (a cyclin-dependent kinase inhibitor known to arrest the cell cycle mainly in G1/S phase), GADD45 for DNA repair and Bax and Fas/APO-1 for apoptosis. 7-11 p53 induced Fas-FADD binding and transiently sensitized cells to Fas-induced apoptosis. 12 Programmed cell death depends on conserved structural moieties that transmit and regulate the death signal. This is most evident in death receptors of the tumor necrosis factor receptor (TNF-R) super-family. The intracellular regions of the Fas/CD95 and TNFR1 death receptors have a moderately well conserved NH2-region of about 80 residues that is required for death signalling and has been called the 'death domain' (DD). 13,14 Fas/CD95 induces rapid apoptosis upon binding to Fas ligand through the autocrine/paracrine signalling pathway. 15 When activated, the intracellular death domain in Fas/CD95 binds to FADD/MORT-1 at its C-terminus, which then recruits caspase-8 to FADD. A member of the TNF ligand family identified more recently is TNF-related apoptosis-inducing ligand (TRAIL). TRAIL shows high homology to Fas Ligand and binds to the TRAIL receptor family. TRAILinduced caspase activation shares many of same caspases involved in Fas/CD95 and TNF-induced cell death mechanisms. 16 FADD/ MORT-1 is a cytosolic adaptor protein, which is critical for signalling from Fas/APO-1 and other TNF-R family members. 17 Two protein interaction domains have been identified in FADD/ MORT-1. The C-terminal 'death domain' is needed for ...

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

“…To confirm that the observed plakoglobin-p53 interaction is not specific to SCC9-PG transfectants, we performed coimmunoprecipitation experiments using MCF-10-2A, a normal mammary epithelial cell line, MCF-7, a mammary carcinoma cell line, and A431, a vulvar carcinoma cell line, which all express plakoglobin and p53 (Setzer et al, 2004;Li et al, 2005;Kwok et al, 1994;Lam et al, 2009) (Fig. 2).…”

“…We further performed the same experiments in MCF-7 cells, which express wild-type p53 (Li et al, 2005). We argued that since SCC9-PG and A431 cells express mutant p53, these mutant proteins may be able to induce luciferase activity from both the wild-type and mutant p53 promoters.…”

“…To verify this, we first performed electrophoretic mobility shift assays (EMSA) using MCF-10-2A nuclear extracts and a radioactively labeled probe that corresponds to the p53 consensus sequence (GTAGCA-TTAGCCCAGACATGTCC) in the 14-3-3s gene promoter (Hermeking et al, 1997;Cai et al, 2009). MCF-10-2A cells were first used for these experiments because they express endogenous plakoglobin and wild-type p53 (Li et al, 2005;Lam et al, 2009). The results showed the formation of a distinct complex (Fig.…”

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…Conjugation of peptide-based drugs such as Shepherdin and p-53-derived peptides with CPPs and their efficient internalization into the cell can be a powerful strategy for cancer therapy and apoptosis studies (91,92).…”

Strategies for therapeutic targeting of the p53 pathway in cancer

“…[35][36][37][38][39] More recently, CDB3, a rationally designed 9-mer peptide derived from the p53-binding protein 53BP2 or ASPP, was shown to restore wild type conformation and DNA binding to mutant p53 in human tumor cells, followed by upregulation of p53 target genes. 40 In parallel, screening efforts for small molecules that reactivate mutant p53 have led to the discovery of several structurally unrelated lead compounds (reviewed in Bykov et al 41 ; Figures 1 and 2).…”