Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

Abstract

We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.

Fig. 1.

A: endogenous PARC expression in U2OS, Phoenix and 10 human NB cell lines. HSP-70 was used for normalization. B: p53 coimmunoprecipitates with PARC. PARC was immunoprecipitated from LAN-5 cell lysate using an anti-PARC antibody. Control immunoprecipitation was performed using an anti-Akt antibody. Western blots were performed on immunoprecipitates to detect PARC, p53 and Akt.

Fig. 2.

A: dose-dependent uptake of the TAT-p53-C-ter peptide. Cells were treated for 1 hour with the indicated peptide amounts; intracellular TAT-p53-C-ter peptide stability in cells treated with medium containing 10 μg/ml peptide for 1 hour (0h) and in cells cultured for the indicated times in peptide-free medium. In A, peptides were detected with an anti-HA antibody. HSP-70 content was used for normalization. Blots are representative of two different experiments with similar results. B: representation of p53 peptides cloned in the pFN11A vector. NLS= Nuclear Localization Signal; TD= Tetramerization Domain; C1= C1 peptide. Numbers refer to the aminoacids of human p53 protein cloned in each plasmid. C: PARC/p53 binding assay. Phoenix cells were transfected with the reporter vector pGL4.31, pFN10A-CPH-PARC and the pFN11A plasmids containing the p53 peptides described in B. Thirty-six hours after transfection, luciferase activity was determined. Negative control was carried out by transfecting Phoenix cells with pGL4.31 and pACT and pBIND non-interacting vectors. D: Phoenix cells were transfected with pGL4.31, pFN10A-CPH-PARC and pFN11A-ΔC1 plasmids and treated for 6 hours with the indicated amounts of TAT-p53-C-ter peptide (administration every 2 hours) before luminometric detection. Binding activity of untreated cells was taken as 100. Negative control experiments were designed by transfecting Phoenix cells with pGL4.31 and pACT and pBIND non-interacting vectors. As positive control (right histogram), Phoenix cells were transfected with pGL4.31 and pACT-MyoD and pBIND-Id interacting vectors. Binding activity of positive control left untreated was taken as 100. Samples in C and D were run in triplicate. Values ± SD are reported. Asterisks indicate statistically significant differences (p<0.05) compared to negative control. Experiments were repeated twice with similar results.

Fig. 3.

A: 32D-BCR/ABL myeloid precursor cells stably transfected with reporter vector PG13 or MG15 (luciferase gene driven by a promoter with wt or mutated p53 binding sites respectively) were treated every 2 hours for 6 hours with 5 μg/ml of TAT-p53-C-ter peptide. As positive control, cells were treated for 16 hours with 0.2 μg/ml doxorubicin. B: human NB LAN-5 cells were co-transfected with reporter vectors PG13 or MG15 and expression vector pcDNA3-p53-C-ter-HA. Luciferase activity was determined 36 hours after transfection. As positive control, cells transfected with PG13 and empty vector pcDNA3 were treated for 16 hours with 2 μg/ml doxorubicin. To evaluate additive effects of the p53-C-ter peptide and doxorubicin, cells transfected with PG13 and pcDNA3-p53-C-ter-HA were treated for 16 hours with doxorubicin at the same concentration indicated above. Samples were run in triplicate. Values ± SD are reported. C: Cellular localization of endogenous p53 and TAT-p53-C-ter peptide. U2OS cells, untreated (a, b, c, d), or treated for 1 hour with 10 μg/ml TAT-p53-C-ter peptide (e, f, g and h), or for 1 hour with the peptide followed by 1 hour in peptide-free medium (i, j, k, l), were fixed and processed for immunodetection of endogenous p53 (b, f and j) or the TAT-p53-C-ter peptide (c, g and k). Nuclei were stained with DAPI (a, e and i). Merged fluorescences are shown in d, h and l. Inset in d represents a negative control carried out by treating the cells only with secondary antibodies.

Fig. 4.

p53 transcription targets are up-regulated by the TAT-p53-C-ter peptide. A: indicated cell lines were treated for 2 hours with 10 μg/ml TAT-p53-C-ter peptide added every hour to the medium. Immunodetection was carried out with the anti-HA (to detect TAT-p53-C-ter peptide), anti-p21, anti-Bax and anti-β-actin antibodies. B: total RNA was extracted from cells treated for 2 hours with 10 μg/ml TAT-p53-C-ter peptide. Quantitative real-time PCR was performed to detect MDM2 expression. Values are normalized for β-actin transcripts of each sample. Reactions were run in triplicate, values ± SD are reported. C: Imunodetection of p53 targets in U2OS cells transfected with p53-siRNA or control-siRNA, untreated or treated with TAT-p53-C-ter peptide (see text for details). D: Flow cytometric analysis of U2OS cells transfected with pcDNA3-p53-C-ter-HA, or control pcDNA3 and pCMVEGFP (4:1) vectors. 24 hours after transfection, cells were treated for 36 hours with 1 μM etoposide or left untreated before fixation and processing for cytometric evaluation. DNA content analyses were performed by gating 20 × 10⁴ green fluorescent cells. Experiments were repeated twice with similar results.