Title: TAp63α induces apoptosis by activating signaling via death receptors and mitochondria
Abstract: Article9 June 2005free access TAp63α induces apoptosis by activating signaling via death receptors and mitochondria Olav Gressner Olav Gressner Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Tobias Schilling Tobias Schilling Department of Internal Medicine I, Endocrinology, University Hospital, Heidelberg, Germany Search for more papers by this author Katja Lorenz Katja Lorenz Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Elisa Schulze Schleithoff Elisa Schulze Schleithoff Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Andreas Koch Andreas Koch Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Henning Schulze-Bergkamen Henning Schulze-Bergkamen Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Anna Maria Lena Anna Maria Lena Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Eleonora Candi Eleonora Candi Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Alessandro Terrinoni Alessandro Terrinoni Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Maria Valeria Catani Maria Valeria Catani Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Moshe Oren Moshe Oren Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel Search for more papers by this author Gerry Melino Gerry Melino Medical Research Council, Toxicology Unit, University of Leicester, Leicester, UK Search for more papers by this author Peter H Krammer Peter H Krammer Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Wolfgang Stremmel Wolfgang Stremmel Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Martina Müller Corresponding Author Martina Müller Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Olav Gressner Olav Gressner Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Tobias Schilling Tobias Schilling Department of Internal Medicine I, Endocrinology, University Hospital, Heidelberg, Germany Search for more papers by this author Katja Lorenz Katja Lorenz Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Elisa Schulze Schleithoff Elisa Schulze Schleithoff Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Andreas Koch Andreas Koch Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Henning Schulze-Bergkamen Henning Schulze-Bergkamen Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Anna Maria Lena Anna Maria Lena Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Eleonora Candi Eleonora Candi Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Alessandro Terrinoni Alessandro Terrinoni Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Maria Valeria Catani Maria Valeria Catani Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy Search for more papers by this author Moshe Oren Moshe Oren Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel Search for more papers by this author Gerry Melino Gerry Melino Medical Research Council, Toxicology Unit, University of Leicester, Leicester, UK Search for more papers by this author Peter H Krammer Peter H Krammer Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Wolfgang Stremmel Wolfgang Stremmel Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Martina Müller Corresponding Author Martina Müller Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany Search for more papers by this author Author Information Olav Gressner1, Tobias Schilling2, Katja Lorenz1, Elisa Schulze Schleithoff1, Andreas Koch1, Henning Schulze-Bergkamen3, Anna Maria Lena4, Eleonora Candi4, Alessandro Terrinoni4, Maria Valeria Catani4, Moshe Oren5, Gerry Melino6, Peter H Krammer3, Wolfgang Stremmel1 and Martina Müller 1 1Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany 2Department of Internal Medicine I, Endocrinology, University Hospital, Heidelberg, Germany 3Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany 4Department of Experimental Medicine, IDI-IRCCS, c/o University of Rome, Tor Vergata, Rome, Italy 5Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel 6Medical Research Council, Toxicology Unit, University of Leicester, Leicester, UK *Corresponding author. Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Tel.: +49 6221 5638795; Fax: +49 6221 564395; E-mail: [email protected] The EMBO Journal (2005)24:2458-2471https://doi.org/10.1038/sj.emboj.7600708 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TP63, an important epithelial developmental gene, has significant homology to p53. Unlike p53, the expression of p63 is regulated by two different promoters resulting in proteins with opposite functions: the full-length transcriptionally active TAp63 and the dominant-negative ΔNp63. We investigated the downstream mechanisms by which TAp63α elicits apoptosis. TAp63α directly transactivates the CD95 gene via the p53 binding site in the first intron resulting in upregulation of a functional CD95 death receptor. Stimulation and blocking experiments of the CD95, TNF-R and TRAIL-R death receptor systems revealed that TAp63α can trigger expression of each of these death receptors. Furthermore, our findings demonstrate a link between TAp63α and the mitochondrial apoptosis pathway. TAp63α upregulates expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of RAD9, DAP3 and APAF1. Of clinical relevance is the fact that TAp63α is induced by many chemotherapeutic drugs and that inhibiting TAp63 function leads to chemoresistance. Thus, beyond its importance in development and differentiation, we describe an important role for TAp63α in the induction of apoptosis and chemosensitivity. Introduction p63 and p73 give rise to proteins that have p53-agonistic as well as p53-antagonistic functions and new functions. One reason for this diversity in p53/p63/p73 function lies in their gene structure. p53 has a single promoter with three conserved domains, namely the transactivation domain (TA), the specific DNA-binding domain and the oligomerization domain. In contrast, p63 and p73 have two promoters, resulting in two different types of proteins with opposing functions: p53-like proteins containing the TA domain (TAp63 and TAp73), and inhibitory proteins lacking TA, called ΔNp63 and ΔNp73. These inhibitory proteins retain their DNA binding and tetramerization competence and, thus, can act as dominant-negative inhibitors of p53 and of themselves (Yang et al, 1999; Melino et al, 2002, 2003; Zaika et al, 2002; Moll and Slade, 2004). In addition, both of these genes undergo alternative splicing at the COOH-terminus producing three and nine different species of TAp63 and TAp73, respectively, named α, β, γ, δ, ε, etc., with α being the full length. Hence, p63 and p73 share some p53 functions, such as induction of cell cycle arrest and apoptosis (Osada et al, 1998; Vousden, 2000; Melino et al, 2003; Moll and Slade, 2004). However, there are many functional differences between p53, p63 and p73. Studies of p53-, p63- and p73-deficient mice established that the expression of p63 and p73 is more important for mouse development than the expression of p53. Knockout p63 mice are not viable and show severe structural deficiencies, such as the complete absence of skin, lack of limbs as well as other epithelial structures (Mills et al, 1999; Yang et al, 1999, 2002) and severe craniofacial dysplasia (Celli et al, 1999; Mills et al, 1999; Yang et al, 1999). The reason for these deficiencies lies in the lack of stem cells that are required for the development and differentiation of such complex epithelial structures (Yang et al, 1999, 2002). p63 is the only gene known to be of essential relevance for the survival of epithelial stem cells (Pellegrini et al, 2001), which is diametrically opposed to the function of p53, which again is strongly linked to cell cycle arrest and cell death. There is mounting evidence that p63 and p73 play an important role in human cancer (Casciano et al, 2002; Flores et al, 2002; Melino et al, 2003, 2004; Moll and Slade, 2004; Westfall and Pietenpol, 2004; Wu et al, 2005), although their precise roles in tumorigenesis remain to be clarified. There is also intense debate on whether and how p63 and p73 interact with p53 in apoptosis and tumor suppression (Benchimol, 2004). An example of cooperativity among the three p53 family members has been reported in E1A-expressing mouse embryo fibroblasts and in primary neuronal cultures (Flores et al, 2002). However, results of a recent study indicate that at least in thymocytes, p53-dependent apoptosis occurs independently of p63 and p73 (Senoo et al, 2004). To further define the interactions between the three p53 family members in human cancer, it is essential to investigate common/distinct targets in the apoptosis pathways triggered by p63, p73 and p53. The aim of our study has been to provide insight into the molecular mechanisms accounting for the role of TAp63 in cancer and its involvement in cell death induced by chemotherapeutic drugs. We have analyzed the downstream mechanisms of TAp63α-induced apoptosis in different cellular systems. Here we report that TAp63α, like p53, activates major apoptosis pathways by triggering signaling via death receptors and mitochondria and thus sensitizes cancer cells toward chemotherapy. Of note, we found that endogenous TAp63α is induced by many chemotherapeutic agents and that blocking TAp63α function confers chemoresistance. Results TAp63α-mediated apoptosis involves activation of caspases Adenoviral transfer of the TAp63α gene into Hep3B cells induced apoptosis in a dose- and time-dependent manner (Figure 1). Figure 1.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. Download figure Download PowerPoint TAp63α-mediated apoptosis was strongly inhibited by the caspase inhibitors ZVAD-FMK, DEVD-FMK, Z-IETD-FMK and Z-LEHD-FMK (Figure 2). This confirms the involvement of caspases in TAp63α-mediated apoptosis. Figure 2.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. Download figure Download PowerPoint Microarray analysis of TAp63α-mediated apoptosis Following adenoviral TAp63α expression in Hep3B cells, the genes encoding for the death receptors CD95, TNF-R1, TRAIL-R1 and TRAIL-R2 were found to be upregulated (Table I). Induction of TNF, TRAF (TNF receptor-associated factor)-interacting protein (TRIP) and DAP3 (death-associated protein-3) provides further evidence for the involvement of receptor-mediated signaling. Table 1. List of selected TAp63α-regulated genes in Hep3B cells, according to the MIAME criteria (Brazma et al, 2001; The Tumor Analysis Best Practices Working Group, 2004) Feature Reporter Composite sequence Fold activation Coordinates on arrays Reporter ID (RZPD defined) Biosequence type Clone ID GenBank access. no. Reporter usage Control type Comp. ID Designation Related gene symbol Database entry 24 h 48 h 72 h X-Pos Y-Pos Spot plate Row Column Death receptor-mediated apoptosis 34 27 1 A 13 1048421 cDNA clone IMAGp998E031157 X65019 Exp — 005-B03 Caspase-1 CASP1 LocusID834 +4.08 43 12 1 M 10 49729 cDNA clone IMAGp998G22281 U13737 Exp — 002-G05 Caspase-3 CASP3 LocusID836 +5.33 1 15 1 L 24 302539 cDNA clone IMAGp998C20678 U28014 Exp — 004-F12 Caspase-4 CASP4 LocusID837 +1.61 +1.46 14 18 3 K 20 341763 cDNA clone IMAGp998F04780 U28015 Exp — 006-F10 Caspase-5 CASP5 LocusID838 +1.37 47 15 3 L 9 338776 cDNA clone IMAGp998I17772 X98175 Exp — 009-D12 Caspase-8 CASP8 LocusID841 +1.12 37 12 1 M 12 121693 cDNA clone IMAGp998D14114 U60521 Exp — 002-G06 Caspase-9 CASP9 LocusID842 +3.80 +3.04 42 20 2 J 11 1693595 cDNA clone IMAGp998L124300 M10988 Exp — 007-E06 Tumor necro- sis factor TNF LocusID7124 +3.00 +0.38 6 38 2 D 23 2521744 cDNA clone IMAGp998F176279 M33294 Exp — 007-B12 Tumor necro- sis factor receptor superfamily- member 1A TNFRSF1A/TNF-R1 LocusID7132 +2.07 +2.25 18 26 2 H 19 309671 cDNA clone IMAGp998L24696 M67454 Exp — 007-D10 Tumor necro- sis factor receptor superfamily-member 6 TNFRSF6/CD95 LocusID355 +2.43 +1.83 +1.54 60 26 2 H 5 526788 cDNA clone IMAGp998C131262 U90875 Exp — 007-D03 Tumor necro- sis factor receptor superfamily- member 10A TNFRSF10A/TRAIL-R1 LocusID8797 +2.81 +0.25 72 14 2 L 1 3948060 cDNA clone IMAGp958L13810 AF016268 Exp — 007-F01 Tumor necro- sis factor receptor superfamily-member 10b TNFRSF10B/TRAIL-R2 LocusID8795 +3.16 19 12 1 M 18 27476 cDNA clone IMAGp998M21142 U77845 Exp — 002-G09 TRAF (TNF receptor- associated factor) interacting protein TRIP LocusID10293 +4.65 Mitochondrial genes 37 18 1 K 12 293401 cDNA clone IMAGp998G02654 AF013263 Exp — 002-F06 Apoptotic protease- activating factor APAF1 LocusID317 +1.64 +3.13 43 30 1 G 10 300194 cDNA clone IMAGp998B03672 AF032458 Exp — 002-D05 BCL2-like protein 11 BCL2L11 LocusID10018 +1.71 +10.41 54 2 2 P 7 812290 cDNA clone IMAGp998K112005 U18321 Exp — 007-H04 Death- associated protein-3 DAP3 LocusID7818 +2.80 35 3 3 p 13 713617 cDNA clone IMAGp998L021748 U53174 Exp — 011-H07 RAD9 (S. pombe) homolog (nuclear- and mito- chondrial- localized apoptosis inducer) RAD9 LocusID5883 +1.71 Control clones 32 9 3 N 14 148920 cDNA clone IMAGpC01228 H13168 Control Pos — Hemoglobin, alpha 1 HBA1 LocusID3039 Nan Nan Nan 20 33 3 F 18 203166 cDNA clone IMAGpG07392 R44290 Control Pos — Actin, beta actin1 LocusID51164 Nan Nan Nan 44 9 3 N 10 201154 cDNA clone IMAGpC11387 R17745 Control Pos — Transferrin TF LocusID7018 Nan Nan Nan Shown is the fold activation of TAp63α-dependent genes relative to GFP (=1) as evaluated by SAM (six independent hybridizations). Nan=not a number. http://www.ebi.ac.uk/arrayexpress/; accession number E-MEXP-199. Caspase-1, -3, -4, -5, -8 and -9 were induced by rAd-TAp63α (Table I and Figures 2, 4B and C). Furthermore, we identified the genes encoding the proapoptotic Bcl-2 family member BCL2L11 and the genes encoding RAD9 and APAF1 as targets for transcriptional upregulation by TAp63α (Table I). Thus, microarray analyses provide evidence that TAp63α stimulates both, genes that regulate the extrinsic apoptosis pathways initiated by ligation of death receptors and genes that regulate the intrinsic/mitochondrial apoptosis pathway. Figure 3.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. Download figure Download PowerPoint Induction of apoptosis in TAp63α-inducible p53-negative cells In order to generalize these results, we performed similar experiments using a completely different cellular system. We generated Tet-On-inducible osteosarcoma cells overexpressing TAp63α. Saos2 cells are p53 negative, and show no detectable levels of p63 and p73 at either mRNA or protein level. The expression of TAp63α protein was induced in a time-dependent manner following treatment with 2.5 μg/ml doxycycline (dox; Figure 3A). The expression of TAp63α was functional, as shown by the ability to induce expression of p21. Following the induction of p21, the cells showed a G1 cell cycle arrest, with a significant reduction in S and G2/M phases, as indicated in Figure 3B. Consistent with the induction of TAp63α, Saos2 cells underwent apoptosis, as measured by flow cytometric analysis of sub-G1 events (Figure 3C). Figure 4.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. Download figure Download PowerPoint TAp63α is localized only in the nucleus, and is strictly correlated to the expression of p21 both in induced and noninduced leaky cells (Figure 3D), and it is independent of the expression of Ki67 (Figure 3E). We performed microarray analyses in this second model of apoptosis. Similarly to the results obtained in Hep3B cells, TAp63α was able to induce expression of several proapoptotic genes (Figure 4A and B) and their corresponding proteins: CD95, APAF1, RAD9, caspase-1, caspase-3 and caspase-9 (Figure 4C). TAp63α is a transcriptional activator of the CD95 gene We have previously shown that the CD95 gene is a transcriptional target of wild-type (wt) p53, whose expression is induced through binding of wt p53 to a regulatory region within its first intron (Müller et al, 1997, 1998). Based on these observations and on our microarray data, we investigated the possibility that TAp63α transactivates the CD95 gene (Figure 5A). This was carried out by transient transfection assays, employing a plasmid in which the expression of a luciferase reporter gene is driven by regulatory DNA elements from the CD95 gene. These regulatory sequences include the physiological sequence of the CD95 gene, the CD95 promoter, exon 1 and a region from intron 1 encompassing the p53-responsive element (p1142CD95-luc). Figure 5C shows that cotransfection of TAp63α, like cotransfection of p53 (Müller et al, 1997, 1998), significantly increased p1142CD95-luc activity. The TAp63α-dependent transactivation strictly depends on the intronic p53 binding site of the CD95 gene, as it is totally abrogated when using a CD95 luciferase construct with a mutated intronic p53 binding site (Figure 5B and C). This strongly argues in favor of the conclusion that the CD95 gene is a direct transcriptional target for TAp63α. A direct evidence has been found by chromatin immunoprecipitation (ChIP). Figure 5D shows the ability of p63 protein to bind directly the p53/p63 binding site in the first intron of the CD95 gene. Figure 5.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. Download figure Download PowerPoint TAp63α induces upregulation of the CD95 receptor and sensitizes toward CD95-mediated apoptosis Importantly, FACS analysis revealed that overexpression of TAp63α also led to an increase in the amount of CD95 death receptors displayed on the cell surface (dose-dependent, P<0.001; Figure 5E). Next we tested whether the induction of CD95 resulted in a sensitization toward CD95-mediated apoptosis. In fact, the agonistic antibody anti-APO-1 triggered cell death in TAp63α-overexpressing Hep3B cells (Figure 6). These data indicate that the CD95 death receptor induced by TAp63α is functional. Figure 6.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. Download figure Download PowerPoint TAp63α sensitizes hepatoma cells toward TNF-R- and TRAIL-R-mediated apoptosis To further dissect the death receptor pathways involved in the mediation of TAp63α-induced apoptosis, we performed stimulation and blocking experiments of CD95, TNF-R and TRAIL-R. As shown for the CD95 death receptor system, addition of the specific ligands (TNFα or human LZ-TRAIL) led to a further increase of TAp63α-mediated apoptosis. Addition of the specific blockers of these death receptors, F(ab′)2-anti-APO-1, human TNF-R1-Fc and TRAIL-R2-Fc, significantly reduced apoptosis triggered by rAd-TAp63α but not by rAd-GFP (green fluorescent protein) transfer (Figure 6A). Flow cytometry analysis confirmed upregulation of CD95, TNF-R1, TRAIL-R1 and TRAIL-R2 following rAd-TAp63α transfer (Figure 6B). Thus, it is evident that TAp63α-induced apoptosis is not solely mediated by the CD95 system, but by a set of death receptors including the TNF and TRAIL receptor system. TAp63α induces the mitochondrial apoptosis pathway In order to further characterize the molecular mechanisms of TAp63α-mediated apoptosis, we investigated the influence of TAp63α on mitochondrial apoptosis. FACScan® analysis revealed an alteration of the mitochondrial membrane potential following adenoviral TAp63α transfer in Hep3B cells (Figure 7A). To investigate the possible involvement of Bax in TAp63α-induced apoptosis, we performed transient transfection assays using a reporter plasmid (Figure 7B), containing the full-length Bax promoter placed upstream of a luciferase cDNA. Figure 7C shows that cotransfection of TAp63α significantly increased Bax promoter activity. Western blot analysis confirmed induction of endogenous Bax protein following rAd-TAp63α transfer (Figure 7D). In addition, as shown above by microarray and immunoblot analyses, BCL2L11, APAF1, caspase-9, RAD9 and DAP3 were induced. Thus, TAp63α contributes to apoptosis by inducing the expression of several proapoptotic proteins acting on mitochondria. Figure 7.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. Download figure Download PowerPoint TAp63α sensitizes hepatoma cells toward chemotherapy p53 is frequently mutated in hepatocellular carcinoma. This has been implicated in resistance toward anticancer therapy. We investigated whether TAp63α could restore response of hepatoma cells toward chemotherapeutic drugs. Figure 8A shows that rAd-TAp63α enhances cell killing by bleomycin. Transfection assays revealed that the additive action of anticancer drugs and TAp63α is partially due to a cooperative effect on the transactivation of the CD95 gene (Figure 8B). Figure 8.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