Apoptosis induced by TAp63α involves activation of caspases. FACScan^® analysis of propidium iodide-stained nuclei (Nicoletti et al, 1991) of Hep3B cells, which were either transduced by rAd-GFP or rAd-TAp63α for 72 h. TAp63α-dependent apoptosis was blocked by the broad-spectrum caspase inhibitor ZVAD-FMK and by DEVD-FMK (C3Inh), Z-IETD-FMK (C8Inh) and Z-LEHD-FMK (C9Inh). Data obtained in two separate experiments were averaged. Presented is mean±s.d., n=6. ^*P<0.001, ^**P<0.05; Wilcoxon's test compared to rAd-TAp63α.

TAp63α induces the expression of proapoptotic genes in Tet-On Saos2 cells. (A, B) TAp63α induces upregulation of death receptors and caspases. Cells were analyzed by microarray technique. The data presented are mean±s.d., n=3. http://www.ebi.ac.uk/arrayexpress/; accession number E-MEXP-199. (C) Validation of targets identified in panels A and B by Western blot. Cells overexpressing ΔNp63α and dox-treated control cells (TET) were included to underline the TAp63α-specific upregulation of the target genes. Panel C shows representative results of two independent experiments.

TAp63α sensitizes hepatoma cells toward death receptor-mediated apoptosis. (A) Addition of the specific ligands following adenoviral expression of TAp63α sensitized Hep3B cells toward CD95-, TNF-R-, and TRAIL-R-mediated apoptosis. Following adenoviral transfer of TAp63α (48 h), specific ligands were added for further 24 h. This led to a significant increase of apoptosis mediated by the CD95, TNF and TRAIL death receptor systems. Addition of the specific blockers of these death receptors significantly reduced apoptosis triggered by TAp63α. Shown is one representative out of three experiments performed. Presented is mean±s.d., n=3. ^*P<0.05, Wilcoxon's test compared to TAp63α. (B) Transfer of rAd-TAp63α (72 h) increases cell surface expression of CD95, TNF-R1, TRAIL-R1 and TRAIL-R2. Assays were performed in triplicate, and three independent experiments were performed; a representative result is shown (mean±s.d., n=3). ^*P<0.05, Wilcoxon's test compared to GFP.

TAp63α sensitizes hepatoma cells toward chemotherapy. (A) TAp63α and chemotherapeutic agents synergize in the induction of apoptosis in Hep3B cells. Cells were treated either with bleomycin alone or in combination with rAd-TAp63α (10 moi) or rAd-GFP (10 moi) for 72 h. One out of four experiments performed in triplicate is shown. Mean±s.d., n=3. ^*P<0.01, ANOVA for between-subject effect (TAp63α versus GFP); ^**P<0.001, ANOVA for dose dependence of the effect of bleomycin on specific apoptosis. (B) The synergistic action of rAd-TAp63α and bleomycin is in part due to a cooperative effect on the transactivation of the CD95 gene. Presented is the fold rAd-TAp63α (10 moi)-dependent activation of the p1142CD95-luc reporter plasmid (1 μg), calculated relative to the value obtained with the same adenovirus in the absence of TAp63α or bleomycin. Assays were performed in triplicate in three independent experiments. One representative experiment is shown (mean±s.d., n=3). ^*P<0.05, Wilcoxon's test. (C) A set of death receptors is involved in the augmentation of TAp63α-mediated apoptosis by chemotherapeutic drugs. Blocking of the CD95, TNF or TRAIL receptor system cannot prevent the further enhancement of TAp63α-mediated apoptosis by chemotherapeutic drugs. Presented is one out of three independent experiments performed. Shown is mean±s.d., n=3. ^*P<0.05, Wilcoxon's test compared to the corresponding left column without bleomycin. (D) Following rAd-TAp63α transfer, mitochondrial membrane potential was significantly increased by addition of bleomycin (JC-1 staining, Hep3B cells, 72 h). Data obtained in two separate experiments, each performed in triplicate, were averaged. Presented is mean±s.d., n=6. ^*P<0.005, Wilcoxon's test. (E) The cooperative action of TAp63α transfer and chemotherapy leads to enhancement of Bax gene transactivation. Combined treatment with 3 μg/ml bleomycin led to a further increase of the transactivation of the Bax promoter. Treatment conditions are as detailed in Figure 7C. Shown is one representative out of three experiments performed, mean±s.d., n=3. ^*P<0.005, ANOVA, for time=24 h. (F) Western blot analysis confirms the cooperative induction of endogenous Bax protein by rAd-TAp63α and bleomycin. (G) TAp63α synergizes with different chemotherapeutic drugs in the induction of apoptosis. Cells were treated either with DNA-damaging agents alone or in combination with rAd-TAp63α (10 moi) or rAd-GFP (10 moi) for 72 h. One out of three experiments performed in triplicate is shown. Mean±s.d., n=3. ^*P<0.01, ANOVA for between-subject effect (TAp63α versus GFP), ^**P<0.001, ANOVA for dose -dependence of the effect of chemotherapy on specific apoptosis. (H) TAp63α cooperates with a variety of chemotherapeutic agents in the transactivation of CD95 and Bax genes. Treatment conditions are as detailed in Figures 5C and 7C. Shown is one representative out of three experiments performed, mean±s.d., n=3. ^*P<0.05, Wilcoxon's test.

Model of TAp63α-induced apoptosis. Upon DNA damage, TAp63α activates multiple apoptosis pathways. In particular, both the death receptor (pathway 1) and the mitochondrial (pathway 2) cascades are induced by TAp63α.

TAp63α induces apoptosis of hepatoma cells. (A) Comparison of protein expression following rAd-p53, rAd-TAp63α or rAd-TAp73β expression in Hep3B cells. (B) Like rAd-p53 and rAd-TAp73β, rAd-TAp63α induces dose-dependent apoptosis of Hep3B cells within 72 h. Propidium iodide staining (Nicoletti et al, 1991) and FACScan^® analysis were performed. Three independent experiments were performed, and a representative result is shown, mean±s.d., n=3. ^*P<0.01, ^**P<0.001, multivariate analysis of variance (MANOVA), between-subject effect compared to rAd-GFP.

TAp63α induces apoptosis in Tet-On Saos2 cells. (A) Different clones were selected showing the ability to induce TAp63α under the control of exogenous dox. At 24 h, both p63 (HA tag) and p21 were upregulated in all Saos2 clones. (B) G1 cell cycle arrest and reduction of the relative portion of S and G2/M phases induced by TAp63α overexpression (dead cells were gated out). (C) Induction of apoptosis (sub-G1 hypodiploid peak by flow cytometry in propidium iodide-stained cells) by TAp63α. (D) Subcellular localization of TAp63α (HA tag) and p21. Both proteins colocalize in the nucleus. The expression of p21 is related to that of p63, both in dox-induced cells (middle row) and in noninduced cells (lower row), showing some degree of leakiness of the promoter used. (E) Subcellular localization of TAp63α (HA tag) and Ki67. The data presented in panel C are mean±s.d., n=3. Panels show representative results of four to six independent experiments.

TAp63α directly transactivates the CD95 gene via binding to the intronic p53 binding site and induces upregulation of the CD95 receptor. Map of the human CD95 gene. Exons 1–9 are numbered according to Wada et al (1995). The striped box indicates the intronic p53 binding site (p53-IBS) in the first intron of the CD95 gene (Müller et al, 1998). (A) Wild-type (wt) sequence of the CD95 p53-IBS (Müller et al, 1998). This sequence (top line) is compared with the consensus p53 binding site (bottom line) (El-Deiry et al, 1992). Missing vertical bars indicate deviations in the wt sequence from the consensus. R=purine, Y=pyrimidine, W=A or T. (B) In plasmid mt p1142CD95-luc, essential nucleotides for the binding of wt p53 protein have been mutated in the p53-IBS. (C) TAp63α, like p53, transactivated the CD95 gene. Mutation of the p53-IBS (mt p1142CD95-luc) completely abrogated transactivation by TAp63α. Hep3B cells were transfected with 1 μg of plasmid p1142CD95-luc together with 10 moi rAd-p53 or of rAd-p63 (1, 10, 20 or 50 moi). Presented is the fold p53- or TAp63α-dependent activation of the p1142CD95-luc reporter plasmid, calculated relative to the value obtained with the same adenovirus in the absence of p53 or TAp63α. Four independent experiments were performed, and a representative result is shown (mean±s.d., n=3). (D) ChIP of p63 on the CD95 gene. Crosslinked chromatin was extracted from dox-induced Saos2 cells and subjected to immunoprecipitation with specific (Sp-IP) and nonspecific (Unsp-IP) antibodies. DNA recovered from immunocomplexes and input material (Input) was PCR amplified using primers designed across the p53/p63 responsive element in intron 1 of the CD95 gene. A representative result of two independent experiments is shown (M: molecular weight marker). (E) Transfer of rAd-TAp63α (72 h) restores the ability of Hep3B cells to increase the CD95 receptor. Assays were performed in triplicate, and six independent experiments were performed; a representative result is shown (mean±s.d., n=3). ^*P<0.0001, ANOVA for effect of TAp63α-dependent upregulation of the CD95 receptor.

TAp63α engages mitochondrial apoptosis pathways. (A) FACScan^® analysis of Hep3B cells following JC-1 staining (Zuliani et al, 2003) showed alteration of the mitochondrial membrane potential and a significant increase in J-aggregates due to TAp63α (72 h). Data obtained in two separate experiments, each performed in triplicate, were averaged. Presented is mean±s.d., n=6. ^*P<0.005, Wilcoxon's test. (B) Overview of the Bax-luciferase gene construct used in panel C, containing a luciferase construct of the Bax promoter with its four p53 promoter binding sites (white boxes) (Miyashita and Reed, 1995). (C) TAp63α transactivates the Bax gene. Hep3B cells were transfected with 1 μg of the reporter plasmid presented in panel B together with 100 ng of a TAp63α plasmid. Shown is the fold TAp63α-dependent activation of the Bax reporter plasmid, calculated relative to the value obtained with the same reporter in the absence of TAp63α. Shown is one representative out of three experiments performed. Presented is mean±s.d., n=3. (D) Immunoblot of endogenous Bax expression following rAd-TAp63α transfer.

Blocking TAp63 function confers chemoresistance. (A) Endogenous TAp63α is induced by chemotherapy. Anti-p63α and anti-β-actin immunoblot of Hep3B cells treated with different chemotherapeutic drugs. Saos2 cells transduced by rAd-TAp63α were used as positive control. (B) Anti-p63 and anti-tubulin immunoblots of HEK293 and Hep3B cells cotransfected with pcDNA-TAp63α and siRNA directed against TAp63 at 1:1, 1:2 and 1:4 ratios. In parallel, cells were cotransfected with annealing buffer only (mock), scrambled TAp63 siRNA or vector alone. Transfection of siRNA TAp63 but not siRNA scramble led to decreased TAp63α in response to DNA damage induced by bleomycin treatment (lower right panel). Positive control is as detailed in panel A. (C) Blocking TAp63 function with siRNA inhibits chemotherapy-induced apoptosis. Hep3B cells transfected with the indicated siRNAs were treated with different anticancer drugs for 72 h. FACScan^® analysis of propidium iodide-stained nuclei was performed. Data obtained in two separate experiments were averaged. Shown is mean±s.d., n=6. ^*P<0.05, Wilcoxon's test compared to the corresponding left column (siRNA scramble). (D) Hep3B cells transfected with the indicated siRNAs were treated with bleomycin, and caspase-3, -8 or -9 activation assays were performed 48 h later. Fold activation represents the caspase-3, -8 or -9 activity measured in cells transfected with siRNA scramble or siRNA TAp63 compared to untreated cells. Data obtained in two separate experiments were averaged. Shown is mean±s.d., n=6. ^*P<0.05, ^**P<0.005, Wilcoxon's test. (E) Blocking TAp63 diminishes alteration of the mitochondrial membrane potential. Hep3B cells transfected with the indicated siRNAs were treated with bleomycin and mitochondrial membrane potential was assessed by flow cytometry (JC-1 staining, 72 h). Data obtained in two separate experiments were averaged. Shown is mean±s.d., n=6. ^*P<0.05, Wilcoxon's test. (C–E) Results similar to transfection with scrambled siRNA were seen with mock transfection or transfection with the empty pSuperRetro vector alone (data not shown).