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Carcinogenesis Advance Access originally published online on November 28, 2007
Carcinogenesis 2008 29(2):273-281; doi:10.1093/carcin/bgm258
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with {Delta}Np73

A. Petitjean1,2,3,{dagger}, C. Ruptier1,2,{dagger}, V. Tribollet1,2,3, A. Hautefeuille3, F. Chardon3, C. Cavard4,5, A. Puisieux1,2, P. Hainaut3 and C. Caron de Fromentel1,2,*

1 INSERM UMR590, Unité d'Oncogenèse et de Progression Tumorale, Centre Léon Bérard, 28 rue Laënnec, F69008 Lyon, France
2 Université de Lyon 1, ISPB, Lyon, F-69008, France
3 International Agency for Research on Cancer, 150 cours A. Thomas, F69372 Lyon Cedex 08, France
4 INSERM U567, Paris, F-75014, France
5 CNRS UMR 8104, Paris, F-75014, France

* To whom correspondence should be addressed. Tel: +33 478 782 806; Fax: +33 478 782 720; Email: carondef{at}lyon.fnclcc.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
TP63, a member of the TP53 gene family, encodes two groups of three isoforms ({alpha}, β and {gamma}). The TAp63 isoforms act as transcription factors. The {Delta}Np63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as {Delta}Np63{alpha}, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was {Delta}Np63{gamma}, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63{gamma} being the most active form. In contrast, {Delta}Np63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63β and {gamma} isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of {Delta}Np73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents.

Abbreviations: DOX, doxorubicin; HCC, hepatocellular corcinoma; PCR, polymerase chain reactions; RT, reverse transcription; TA, transactivation


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
p63 protein exhibits high sequence and structural homology to the p53 tumor suppressor protein in its general transcription factor architecture and in the topology and recognition specificity of its DNA-binding domain. The TP63 gene is transcribed from two alternative promoters, producing TAp63 isoforms with an N-terminal transactivation (TA) domain, a core DNA-binding domain and a C-terminal oligomerization domain, or {Delta}Np63 isoforms lacking the TA domain (1). Nevertheless, several TA domains have been identified in {Delta}N isoforms (2). Both TA and {Delta}N transcripts are alternatively spliced at the 3' end to generate different C-terminal isoforms, called {alpha}, β and {gamma}. Only the {alpha} isoform contains a sterile alpha motif (3)—a protein–protein interaction domain involved in embryonic development—and a transcription inhibitory domain (4,5).

The p63 protein is expressed in a tissue- and cell type-specific manner and is essential for the development of stratified epithelia as well as for epidermal–mesenchymal interactions in bone morphogenesis, as demonstrated by the phenotype of the p63 knock-out mice (6). These mice are deficient for the development of limbs and for the complete morphogenesis of epithelial tissues such as skin, stratified and pseudostratified epithelia lining the upper aerodigestive tract, prostate, mammary gland and urothelium. In relation with this phenotype, it has been demonstrated that TAp63 isoforms regulate the expression of several genes involved in epithelial differentiation (7). Because of their premature death, p63 knock-out mice cannot be studied for their ability to form tumors. Nevertheless, this ability has been investigated with heterozygous p63+/– mice. One group reported that haploinsufficiency for TP63 leads to tumors in animals, mostly in tissues with stratified epithelia (8), whereas another group did not observed any tumor formation (9). In humans, germ line mutations in TP63 result in developmental syndromes with defects that recapitulate key aspects of the phenotype of p63–/– mice (10), but not in enhanced tumor susceptibility. Only very rare somatic mutations have been found in tumors. In contrast, deregulated expression of p63 isoforms with accumulation of {Delta}Np63 isoforms is observed in squamous cell carcinomas and sometimes associated with amplification of the 3q27–29 genomic region encompassing the TP63 gene (11,12). {Delta}N isoforms are thought to regulate the transcriptional activity of TA isoforms, either by the formation of inactive hetero-oligomers or by competition for specific responsive elements on DNA (1,13). This later property allows to {Delta}Np63 to act antagonistically toward p53, thus providing a theoretical mechanism of p53 functional inactivation during tumorigenesis. However, this hypothesis is a matter of debate since there is no clear evidence in favor of preferential amplification of TP63 in squamous cell carcinoma without TP53 mutation. {Delta}Np63 also exhibit other activities. It is able to transactivate HSP70 gene by binding on the CAAT boxes of its promoter (14) and to cooperate with HIF1{alpha} to regulate VEGF gene expression (15). More recently, by using a genome-wide approach, it has been demonstrated that {Delta}Np63 is involved in the regulation of cell adhesion, differentiation and cell proliferation pathways (16,17).

We previously showed that p63 isoforms may represent a component of responses to genotoxic stress in several hepatocellular carcinoma (HCC) cell lines (18). However, little is known on the capacity of p63 isoforms to transactivate genes involved in growth suppression after stress, as well as on the posttranslational modifications and stability of p63 in stressed and non-stressed cells. Phosphorylations on Ser/Thr residues have been reported that result in the stabilization of exogenous TAp63{alpha} and {gamma} isoforms upon genotoxic treatment (19,20) and, at the opposite, in the accelerated degradation of {Delta}Np63{alpha} (21). Also, the addition of SUMO protein to the p63{alpha} isoforms leads to their proteasome-dependent degradation (22). Moreover, structural features present only in specific isoforms are involved in protein stability. In particular, the FWL motif in the N-terminal end of TAp63 appears critical for their degradation (5). This could explain why {Delta}Np63{gamma} appears to be more stable than TAp63{gamma} (20).

To better understand the specific properties of the p63 isoforms and their capacity to contribute to genotoxic responses, we have expressed each of the p63 isoform, either alone or in combination, in the p53-null HCC cell line Hep3B. So far, only two reports have studied all the six isoforms in parallel (4,22) and none has analyzed their modulation in response to genotoxic stress. We report the characterization of some of their stability and transcriptional activity, after transient or stable transfection, in response or not to the genotoxic agent doxorubicin (DOX), a topoisomerase II inhibitor commonly used in chemotherapy.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Plasmids
TAp73{alpha} and TAp73β expression vectors were obtained from Dr W. Kaelin (Harvard Medical School, Boston, Massachussets), {Delta}Np63{alpha} from Dr M. Oren (The Weizmann Institute, Rehovot, Israel). The cDNA of TAp63{alpha} was amplified by polymerase chain reaction (PCR) and cloned into the pcDNA3 vector (Invitrogen, Cergy Pontoise, France). TAp63β-, TAp63{gamma}-, {Delta}Np63β- and {Delta}Np63{gamma}-expressing vectors were generated by replacing the C-terminus of the TA and {Delta}Np63{alpha} cDNA with the PCR-amplified β and {gamma} C-termini. All plasmids were verified by sequencing. Primers and PCR conditions are described on supplementary Table 1 (available at Carcinogenesis Online).

pWWP-luc (23) and pGL3p73N (24) reporter plasmids contain the firefly luciferase cDNA under the control of WAF1 and {Delta}Np73 promoters, respectively. pGL3p73N was obtained from Prof. M. Dobbelstein (Institute for Medical Biology, Odense, Denmark).

Cells and transfection
The Hep3B cell line (ATCC HB-8064) is derived from a hepatocellular carcinoma. These cells lack p53 protein expression due to homozygous deletion (25). Cells were cultured at 37°C, 5% CO2, in eagle minimum essential medium (Cambrex, Lonza, Emerainville, France) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin, 1% non-essential amino acids (Gibco, Invitrogen, Cergy Pontoise, France) and 1 mM sodium pyruvate (Gibco BRL). Transfection was performed with 5 µg plasmid by using Exgen 500 (Euromedex, Mundolsheim, France), in a 1:5 ratio of DNA (µg)/Exgen 500 (µl). At different times after transfection, cells were harvested and used for RNA and protein analyses. To generate polyclonal populations, cells were divided 24 h after transfection at 1/10e, selected by addition of 400 µg/ml G418 (Geneticin; Gibco®, Invitrogen) 24 h later and maintained under selection during 2 weeks. Then, selected cells were pooled, amplified and used in experiments.

Twenty-four-hour-transfected Hep3B or polyclonal populations were treated with 20 µM MG132 (Calbiochem® VWR, Fontenay sous Bois, France) for 6 h or with 10 µg/ml cycloheximide (Sigma, Saint Quentin Fallavier, France) or 1 µM DOX (doxorubicin hydrochloride, Teva® Classics, Paris, France 0,2%) for various times.

For the luciferase assay, cells were plated in 12-well plates (0.4 x 105 cells per well) and 24 h later transfected with 0.3 µg of expression vectors and 1 µg of luciferase reporter plasmid by using LipofectamineTM 2000 (Invitrogen), in a 1:2.5 ratio of DNA (µg)/LipofectamineTM 2000 (µl). Twenty-four hours after transfection, cells were analyzed with the Dual-Luciferase® Reporter 1000 Assay System kit (Promega, Charbonnières, France). For testing the inhibition of TA by {Delta}Np63{alpha}, 0.3–0.6 µg of {Delta}Np63{alpha}-pcDNA3 was used in addition to TA isoform expression vectors.

Western blot
Cell pellets were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid and 1% NP40) with protease inhibitors (complete mini, ethylenediaminetetraacetic acid free, Roche, Meylan, France), 30 min at 4°C. Protein lysate was isolated by a 15 min centrifugation at 15 700g at 4°C. Then, proteins were separated by electrophoresis in a 7.5% sodium dodecyl sulfate–polyacrylamide (30:0.8) gel, transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Marnes la Coquette, France) and probed with antibodies against all p63 isoforms (4A4 clone, Santa Cruz Biotechnology, Tebu-Bio, Le Perray en Yvelines, France 1/300e), {Delta}Np63 isoforms (anti-p40, Calbiochem, 1/1000e), p63 phosphorylated at residues Ser 160–162 (TA forms) or Ser 66–68 ({Delta}N forms) [#4981, Cell Signaling Technology, Ozyme, Saint Quentin en Yvelines, France 1/1000e—for note, phosphorylated residues are actually Ser 121–123 and Ser 27–29 in TA and {Delta}N forms, respectively, according to p63 sequences reported under accession numbers AF075430 [GenBank] and AF075431 [GenBank] by Yang et al. (1)] or p53 (DO7, PharMingen, BD Biosciences, Rungis, France 1/1000e). Membranes were then incubated with a peroxydase-conjugated secondary antibody against mouse or rabbit immunoglobulins (Jackson, Beckman Coulter, Roissy, France). The reaction was revealed by chemiluminescence with the enhanced chemiluminescence kit (Santa Cruz Biotechnology). The antibody against Ku80 (Serotec, Cergy Saint Christophe, France 1/40 000e) was used as control of loading.

Immunofluorescence
Twenty-four hours after transfection, cells were trypsinized and 3 x 105 cells per well were plated on a glass coverslip in 12-well plates. Twelve hours later, cells were fixed and permeabilized for 5 min on ice with cold 50% methanol–50% acetone. Immunofluorescence was carried out using 4A4 (1/100e) or anti-p40 (1/250e) antibody in 90 µl Dako antibody diluent (Dako, Trappes, France) for 1 h at room temperature. Then, fluorescein isothiocyanate-conjugated secondary antibody against mouse or rabbit immunoglobulin (Jackson, 1/1500e) was added for 30 min at room temperature in the dark in 90 µl Dako antibody diluent. Finally, coverslips were mounted on glass slides in Dako fluorescent mounting medium (Dako) with 0.5 µg/ml propidium iodide and were analyzed with a fluorescence microscope.

Reverse transcription–PCR
Total RNA was extracted with TriREAGENT (Sigma) according to the manufacturer's conditions. RNA (2 µg) was retrotranscribed with Superscript II enzyme (Invitrogen) and amplified by PCR as described previously (18). Primers and PCR conditions are described on Supplementary Table 2 (available at Carcinogenesis Online).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Transient expression of p63 isoforms
Expression vectors for each p63 isoform were transiently transfected into Hep3B cells, which express only low levels of p63. Total proteins were analyzed by western blot, using an antibody that recognizes all isoforms. All exogenous p63 proteins were expressed and detectable, but at variable level for each isoform (Figure 1A and B), although no significant difference in transfection efficiency was observed, as evaluated by immunofluorescence (Figure 1D) and fluorescent-activated cell sorting analysis (data not shown). The apparent molecular weight of p63 proteins was higher than the theoretical one: 85 kDa for TAp63{alpha}, 68 kDa for TAp63β, 58 kDa for TAp63{gamma}, 65–72 kDa for {Delta}Np63{alpha}, 55–62 kDa for {Delta}Np63β and 47–53 kDa for {Delta}Np63{gamma}. Moreover, the {Delta}N isoforms migrated as a doublet, with a difference of 6–7 kDa. Only the higher species was detected with the p40 antibody that recognizes the amino acids 5–17 of the N-terminal domain of {Delta}Np63 (Figure 1C), indicating that the lower species may not contain the extreme N-terminus. Finally, all p63 isoforms showed expected nuclear, non-nucleolar localization, as determined by immunofluorescence (Figure 1D).


Figure 1
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  		Fig. 1. Exogenous expression of p63 isoforms in the Hep3B cell line. One hundred micrograms of total proteins from 24-h-transfected Hep3B cells were analyzed by western blot. (A and B) p63 proteins were detected with the 4A4 antibody at two times of exposition or (C) with the anti-p40 antibody. According to molecular weight marker, the species detected with anti-p40 antibody correspond to the upper band of the {Delta}Np63 doublets. Ku-80 was used for standardizing the loading. C, empty vector; molecular weight marker is indicated (in kDa). (D) The subcellular localization of p63 isoforms was studied by immunofluorescence with 4A4 antibody (for TA isoforms) or anti-p40 antibody (for {Delta}N isoforms). Nuclei were stained with propidium iodide.

 
The difference of p63 protein level after transient transfection (Figure 1A and B) led us to study the stability of each isoform by addition of cycloheximide, which blocks translation. TA isoforms were rapidly degraded, with a half-life of ~1 h, the most rapidly degraded form being TAp63{alpha}. Among {Delta}N isoforms, the {alpha} one showed also a rapid turnover, whereas {Delta}Np63{gamma} had a half-life of at least 8 h. The {Delta}Np63β isoform had a very peculiar pattern of degradation, with variations that prevented us to assess its precise turnover (Figure 2A). Treatment with MG132, a proteasome inhibitor, stabilized all isoforms, indicating that their degradation was dependent on proteasome (Figure 2B).


Figure 2
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  		Fig. 2. Stability of p63 isoforms after transient transfection. Twenty-four-hour-transfected Hep3B cells were treated with (A) cycloheximide (CHX) for the time indicated or (B) MG132 for 6 h. Total proteins were analyzed by western blot. Ku-80 was used for standardizing the loading. Arrows indicate specific bands and asterisks, non-specific bands.

 
TA activity of exogenous p63 isoforms toward p53 target genes
In order to determine whether p63 isoforms may functionally overlap with p53 in Hep3B cells, we studied their transcriptional activities toward several p53 target genes. First, we studied their TA activity in vitro on the WAF1 promoter (Figure 3A). In parallel, the intracellular level of transfected proteins was assessed by western blot (Figure 3B). As expected, {Delta}Np63{alpha} isoform, as well as {Delta}Np63β and {gamma} (data not shown), did not show any TA activity on WAF1 promoter. TAp63{alpha} exhibited a weak TA activity as compared with TAp63β and TAp63{gamma}. TAp63{gamma} had the strongest transcriptional activity toward this promoter and, by taking into account the protein levels, the activity of TAp63{gamma} appeared to be at least comparable with the one of p53 (Figure 3A and B).


Figure 3
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  		Fig. 3. TA activity of p63 isoforms in transient transfection experiments. Luciferase assay on the WAF1 promoter in presence of TAp63{alpha}, TAp63β, TAp63{gamma} and {Delta}Np63{alpha} (A). p53 was used as positive control. The amount of proteins expressed in luciferase assays was evaluated by western blot (B). Expression of some endogenous p53 target genes in cells transfected with the p63 isoforms, studied by RT–PCR. β-ACTIN was used for standardizing PCRs (C). Luciferase assay on the {Delta}Np73 promoter (D). TA activity of p53, TAp63{alpha}, TAp63β and TAp63{gamma}, in presence of increasing quantities of {Delta}Np63{alpha}, studied by luciferase assay on the WAF1 promoter (E). At least three independent experiments were performed for each condition. y-axis represents the induction according to the empty vector. C, empty vector.

 
The TA activity of p63 isoforms was also checked in vivo, by studying the expression of endogenous p53 target genes by semi-quantitative reverse transcription (RT)–PCR in Hep3B cells transfected with the p63 expression plasmids (Figure 3C). Whereas the expression of HDM2, BAX and GADD45A genes was not modulated whatever the isoform studied, the expression of the proapoptotic gene PIG3 appeared to be inhibited by {Delta}Np63{alpha} and {Delta}Np63{gamma} (Figure 3C). TAp63{gamma} increased the expression of the WAF1 and 14-3-3{sigma} genes, both involved in cell-cycle arrest. TAp63{alpha} was also able to increase the expression of WAF1 in a weak, but reproducible, manner. Moreover, the expression of CD95/FAS, a member of the death receptor family, and {Delta}Np73 genes also increased upon the expression of all TAp63 isoforms. {Delta}Np73 is the {Delta}N counterpart of TP73, another TP53 family gene, and is involved in the regulation of TA forms of the p53 family (24,26). These results were confirmed by quantitative RT–PCR for WAF1, GADD45A and {Delta}Np73 (data not shown). Moreover, the ability of TAp63 isoforms to regulate {Delta}Np73 promoter in Hep3B was further demonstrated using a luciferase assay (Figure 3D). One should be noted that the induction of WAF1 and {Delta}Np73 by TA isoforms was not restricted to the Hep3B cell line since it was also observed in HeLa cells and in two esophageal squamous cell carcinoma cell lines (data not shown).

As {Delta}N forms of the p53 family have previously been described as being dominant negative toward the TA forms (1,24), we studied this property on the WAF1 or {Delta}Np73 promoter–luciferase constructs by cotransfecting Hep3B cells with p53 or TAp63 forms and increasing quantities of {Delta}Np63{alpha} (Figure 3E and data not shown). {Delta}Np63{alpha} decreased the TA of both WAF1 and {Delta}Np73 promoters by TAp63 isoforms, in a dose-dependent manner. However, its effect on p53 activity was dependent on the promoter studied, i.e. weak on WAF1 and stronger on {Delta}Np73. {Delta}Np63β and {gamma} also decreased TAp63 activity on both promoters, but p53 activity on {Delta}Np73 promoter only (data not shown). All these results confirmed that {Delta}Np63 isoforms are able to inhibit the activity of TAp63 isoforms and to play a role in the regulation of p53 activity.

Regulation of p63 isoforms upon DOX treatment
We previously showed that the expression of endogenous TAp63{alpha} was induced in Hep3B cells treated with DOX (18), suggesting that p63 isoforms may be responsive to genotoxic stress in a manner that resembles p53. Here, we studied the regulation of all p63 isoforms in response to DOX (Figure 4). The intracellular level of all p63 proteins rapidly increased from 4 to 8 h until at least 24 h of treatment (Figure 4A) similarly to p53 (data not shown). This increase did not result from messenger RNA upregulation since no significant variation was observed (data not shown). However, it was accompanied by a dramatic increase in the half-life of the proteins (Figure 4C), indicating that DOX treatment induced their stabilization, except for {Delta}Np63{gamma} (data not shown), which was very stable in basal conditions.


Figure 4
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  		Fig. 4. p63 expression in response to a genotoxic stress. Hep3B cells were transiently transfected with p63-encoding plasmids for 24 h and then treated with 1 µM DOX for 4, 8, 12, 18, 24, 36, 48 or 72 h. (A) Eighty micrograms of total proteins were analyzed by western blot with 4A4 antibody or with an antibody recognizing phosphorylated Ser 160–162 (TA forms) or Ser 66–68 ({Delta}N forms) residues (P-Ser panel). (B) Upon DOX treatment, species with a higher molecular weight appear (arrows). Molecular weight marker is indicated (in kDa). (C) Twenty-four hours-transfected Hep3B cells were treated with 1 µM DOX for 24 h, and then with cycloheximide (CHX) for the time indicated. Total proteins were analyzed by western blot. Ku-80 was used for standardizing the loading.

 
After treatment with DOX, all isoforms were strongly reactive with an antibody specifically recognizing p63 proteins phosphorylated on serine residues 160–162 for TA forms, corresponding to serine 66–68 for {Delta}N forms (21) (Figure 4A, P-Ser panels). For {alpha} and β forms, the signal obtained for phosphorylated species after 8–16 or 24 h of treatment was stronger than those obtained for total protein, showing that treatment induced an increase not only in protein level but also in phosphorylation. Moreover, upon treatment, additional species with a higher molecular weight were observed for all the TA forms (enlarged in Figure 4B). The migration of TAp63{alpha} and TAp63β was particularly affected, with an apparent molecular weight of 100 and 75 kDa, respectively. This may correspond to a change in protein charge induced by phosphorylation.

Stable expression of p63 isoforms
To assess the long-term expression of p63 isoforms in Hep3B cells, we generated stable polyclonal populations of p63-expressing cells. These populations showed a weak or even no detectable expression of the exogenous p63, as seen by western blot (Figure 5A, lanes -). Only TAp63{alpha}, {Delta}Np63{alpha} and {Delta}Np63{gamma} isoforms could be detected, even when large amounts of proteins were loaded onto gel electrophoresis (data not shown). By immunofluorescence, only {Delta}Np63{gamma}-expressing cells exhibited a weak but uniform labeling and the same nuclear localization as in transient transfection (data not shown). MG132 treatment stabilized the six isoforms (Figure 5B) and consequently TAp63β, TAp63{gamma} and {Delta}Np63β became detectable. This showed that p63 degradation was still dependent on proteasome pathway in these polyclonal cell populations. This also demonstrated that the expression vectors had not been lost during the generation of polyclonal populations.


Figure 5
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  		Fig. 5. p63 expression and stability in polyclonal populations. (A) One hundred micrograms of total proteins from p63-expressing polyclonal populations, treated (+) or not (–) with 1 µM DOX for 24 h, were analyzed by western blot with 4A4 antibody. (B) The stability of the proteins was studied by using MG132 for 6 h. p63 proteins are detected with the 4A4 antibody; molecular weight marker is indicated (in kDa). (C) p63 phosphorylation, after (+) or not (–) 1 µM DOX treatment for 24 h, was also analyzed by using an antibody recognizing phosphorylated Ser 160–162 (TA forms) or Ser 66–68 ({Delta}N forms) residues. Ku-80 was used for standardizing the loading.

 
Interestingly, all cell populations exhibited a similar proliferation rate, independently of the isoform initially transfected (supplementary Figure 1, available at Carcinogenesis Online). Moreover, the expression of almost all p53 target genes studied by RT–PCR did not vary, as compared with control population (Figure 6 and data not shown). In particular, WAF1 and 14-3-3{sigma} gene expression was unchanged, in contrast to results obtained after transient transfection. Only the expression of CD95/FAS was increased in all TAp63-expressing cells and {Delta}Np73 in the TAp63β- and TAp63{gamma}-expressing cells, although both these latter isoforms were undetectable by western blot. The ability of TAp63β and TAp63{gamma} to induce the expression of CD95/FAS and {Delta}Np73, despite their undetectable protein level, probably reflects their strong TA activity compared with TAp63{alpha}. This is in accordance with the respective TA capacity of the TAp63 isoforms as seen in Figure 3.


Figure 6
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  		Fig. 6. TA activity of p63 isoforms in polyclonal populations. The expression of some endogenous p53 target genes in p63-expressing polyclonal populations was studied by RT–PCR, after (+) or not (–) 1 µM DOX treatment for 24 h. C, empty vector. β-ACTIN was used for standardizing PCRs.

 
Activity of stable exogenous p63 isoforms in response to DOX
We next assessed whether exposure to DOX may also induces stably expressed p63. Twenty-four hours of treatment of polyclonal populations with 1 µM DOX resulted in changes compatible with those detected in transient transfection experiments. The intracellular level of all isoforms was increased (Figure 5A). In particular, the expression of TAp63β, TAp63{gamma} and {Delta}Np63β isoforms, undetectable without treatment, appeared in treated samples. Even though the level of messenger RNA was not determined in stable populations, we assume that p63 isoforms accumulate essentially through posttranslational protein modification. Increased expression of TAp63 isoforms was also associated with a slowdown in their migration, whereas the apparent molecular weight of {Delta}N isoforms, still present as a doublet, did not appear to be modified (Figure 5A). Like in transient expression experiments, the {alpha} and β isoforms were phosphorylated on serine residues after DOX treatment (Figure 5C). No significant difference of TA activity toward p53 target genes was detectable in DOX-treated cells as compared with untreated cells (Figure 6 and data not shown). Indeed, among the p53 target genes studied, only the expression of {Delta}Np73 gene was clearly increased in TAp63-expressing polyclonal populations. The slight variation observed for some other genes (WAF1 and CD95/FAS) seems to be independent of p63 isoforms (Figure 6, lane C). RT–quantitativePCR confirmed results for WAF1. In contrast, {Delta}Np73 induction by TAp63{gamma} did not appear significant (data not shown). To summarize, in colonies of Hep3B cells stably expressing p63 isoforms, the proteins appear to be downregulated, perhaps because high levels of the protein may be incompatible for long-term cell survival in culture. However, upon treatment with DOX, p63 levels increase. Finally, among the target genes tested, only {Delta}Np73 appears to be specifically upregulated.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
TP63 plays critical roles in epidermal–mesenchyme interactions and acts as a key regulator in the differentiation and morphogenesis of various epithelial tissues, in particular stratified epithelia. In addition, there is evidence that p63 may participate in several aspects of cellular response to stress. In a previous study, we have shown that both TAp63 and {Delta}Np63 isoforms were expressed in cultured liver cells. Furthermore, TAp63 was induced in response to genotoxic stress (18). As liver is an organ that does not seem primarily dependent upon p63 for its morphogenesis (10), this observation led us to speculate that p63 may take over some of the growth suppressive functions of wild-type p53 in selected tissues such as liver, an organ with special metabolic and detoxification properties in which p53 activation in response to DNA damage proceeds with different kinetics and dose responses than in many other tissues (27,28). In the present study, we have used a p53-null HCC cell line to ectopically express each of the six isoforms of the p63 protein and we have assessed their expression, stability and capacity to transactivate p53 target genes involved in growth suppression. Our results confirm that only TAp63 isoforms exert transcriptional activity toward such genes and that {Delta}N isoforms act as dominant-negative inhibitors on this TA. Moreover, the three TA isoforms strongly differ in their transcriptional capacities, TAp63{gamma} being the most potent as well as the closest to p53 in its profile of transcriptional activities. All isoforms had a relatively short half-life due to proteasome-mediated degradation, with the exception of the {Delta}Np63{gamma} isoforms, which is stable over 8 h. In response to DNA-damaging stress by the cytotoxic agent DOX, all isoforms undergo transient stabilization, with changes in molecular mass and immunoreactivity compatible with phosphorylation. Induction by DOX leads to a strong transcriptional activation of {Delta}Np73 whose expression product has the capacity to counteract the transcriptional activities of p53 family members. Overall, these results show that p63 isoforms appear as genotoxic stress-regulated proteins. Furthermore, the complexity of the expression pattern of p63 isoforms, as well as the capacity of p63 to activate the dominant-negative form {Delta}Np73, suggests the existence of several complementary regulatory loops that provide tight control over p63 activities.

This study is the first one to assess the expression of the six isoforms of p63 in a stress-response context. The use of Hep3B as a model cell line is justified by the fact that, based on our previous results, p63 may exert some antiproliferative activities in liver cells. Moreover, this cell line lacks p53 expression and expresses only small amount of endogenous p63 that is not detectable in the conditions used for analyzing exogenous p63 expression (18), allowing us to assess the activities of p63 isoforms toward p53 target genes without interference. All exogenous p63 isoforms were detectable after transient transfection, with an apparent molecular weight higher than their theoretic one. This might be due to the high number of proline residues present in the proteins (8.5% of the total amino acids for TAp63{alpha}), as already reported for p53 (29). The subcellular localization of all p63 isoforms was nuclear, in agreement with the TA activity of TA isoforms and the dominant-negative effect of {Delta}N isoforms.

In order to have long-term and more homogeneous p63-expressing cell populations, stable transfection was performed on Hep3B cell line. Whatever the isoform expressed in cells, the proliferation rate of the resulting polyclonal populations was similar to the population transfected with the empty vector (supplementary Figure 1, available at Carcinogenesis Online). However, the expression of p63 was different between each one and only TAp63{alpha}, {Delta}Np63{alpha} and {Delta}Np63{gamma} were slightly detectable by western blot. The reasons of these low levels, such as an alleviation of expression from the CMV promoter or changes in the mechanisms of protein degradation have not been characterized. Nevertheless, the weak or undetectable expression of p63 isoforms suggests that their overexpression may be incompatible with cell survival and proliferation. This hypothesis is supported by the decrease in colony number observed with p63-expressing cells compared with control cells (supplementary Figure 2, available at Carcinogenesis Online). A similar result was obtained with TAp73{alpha} and β (data not shown) and {Delta}Np73 (24), whereas ectopic expression of wild-type p53 in Hep3B is lethal (supplementary Figure 2, available at Carcinogenesis Online).

As described previously for TAp63{alpha}, TAp63{gamma} and {Delta}Np63{gamma}, the stability of all p63 isoforms was proteasome dependent (20,30,31). {Delta}N isoforms were expressed at consistently higher intracellular levels compared with TA isoforms. This effect is not due to intrinsic differences in the expression of the p63 constructs since they all depend on the same promoter or to differences in the stability of messenger RNA (data not shown). Thus, we conclude that {Delta}N proteins have greater stability than their TA counterparts, even if only {Delta}Np63{gamma} showed a significantly higher half-life in cycloheximide transfection experiments. This difference in stability between TA and {Delta}N isoforms was previously reported for some isoforms (20,30), leading some authors to suggest that TA isoforms could induce the expression of genes involved in their own degradation (31).

Whereas the TA isoforms were expressed under a single species, the {Delta}N isoforms appeared as a doublet and only the higher band was detected with the p40 antibody that reacts with the proximal end of the {Delta}N proteins, indicating that the lower band did not possess the extreme N-terminus. These two bands were also observed using a pCI-Neo vector, excluding the generation of a splicing event in the recombinant pcDNA3 vector (data not shown), and have been detected by others as well (21). The lower species could be initiated from a second AUG, located 76 nucleotides downstream the first one and surrounded by a sequence that differs from the Kozak consensus sequence by only two nucleotides (32). The use of this second AUG would remove the 25 first amino acids of {Delta}N proteins, corresponding to an expected, theoretical decrease of molecular weight of ~3 kDa, compatible with our observations. Therefore, these lower bands seem to have a double N-terminal truncation as compared with TAp63 and may be identified as {Delta}{Delta}Np63. The production of truncated forms from the use of an internal AUG has already been reported for p73 (26) and p53 (31,33).

In order to check for the functionality of the exogenous p63 proteins, the TA activity of transiently expressed TA isoforms has been studied in vitro on the WAF1 promoter, a well-defined p53 family target gene. The three exogenous TAp63 isoforms were functional and showed relative TA activities similar to those observed in other cell types (1,4). As expected, the TA activity of TAp63{alpha} was dramatically lower than that of β and {gamma} isoforms due to the presence of the inhibitory post-sterile alpha motif domain in {alpha} isoforms (5). Transiently expressed TAp63 forms were also able to transactivate some endogenous p53 target genes involved in the cell-cycle arrest such as WAF1, confirming our in vitro results, and 14-3-3{sigma}. Again, TAp63{alpha} showed a low activity on WAF1 and no activity on 14-3-3{sigma}. In contrast, TAp63{gamma} was as efficient as p53 (data not shown). This feature has been previously reported in several models, making TAp63{gamma} a p53-like protein (8). In polyclonal populations, no significant variation was observed for any of the genes involved in cell-cycle arrest that we studied. Among the proapoptotic p53 target genes that we tested, only CD95/FAS expression increased after overexpression of all TAp63 in both transient and stable experiments. Gressner et al. (34) described the ability of TAp63{alpha} to induce the expression of CD95/FAS and BAX genes involved in death receptor- and mitochondrial-mediated apoptosis, in Hep3B cells. BAX expression was also observed in TAp63{gamma}-induced apoptosis of neurons (35). However, we did not observe any upregulation of BAX expression in our experiments. This discrepancy may be due to different methods for expressing p63, i.e. infection versus transfection, achieving different levels of expression. Overall, our results and those of others (34,35) are concordant in identifying p63 as a potential inducer of proapoptotic responses.

In luciferase assays, {Delta}Np63 isoforms appeared to be able to dramatically decrease the TA activity of TAp63 isoforms in a dose-dependent manner. This suggests that {Delta}Np63 could form transcriptionally inactive hetero-oligomers with TAp63, as previously reported (1). The negative regulation of {Delta}Np63 isoforms toward p53 activity appeared to be promoter specific. On the other hand, {Delta}Np63 isoforms did not induce the expression of any genes in our conditions, in contrast to reports in H1299 and MCF-7 cell lines (36).

Finally, {Delta}Np73 expression was induced by all TAp63 isoforms in transient transfection assays or by either TAp63β or {gamma} in stable transfection assays. {Delta}Np73 has been demonstrated to be a transcriptional target of TAp63{alpha} and {gamma}, in addition of p53 and TAp73 (24,37). {Delta}Np73 is a known negative regulator of p53 and TAp73 activity (24,26). Kartasheva et al. (24) demonstrated that {Delta}Np73 can also negatively regulate TAp63{gamma} TA activity. Therefore, the induction of {Delta}Np73 expression by TAp63 isoforms may contribute to the fine balance of the transcriptional activities of p63. In agreement with this hypothesis, {Delta}Np63{alpha} has been described as an inhibitor of TAp73-dependent apoptosis (38,39). Taken together, these data underline the relationship between the members of the p53 family and suggest that the balance between TA and {Delta}N forms of the family drives cell survival. Overall, the levels of expression achieved in stable colonies, as well as the effects on the expression of {Delta}Np73, may represent the selection of cells that have achieved equilibrium between pro- and antisuppressive activities that is compatible with proliferation and survival in culture.

We have described previously an increase in the expression of endogenous TAp63 isoforms in hepato cellular carcinoma cell lines in response to DOX (18). Here, we show that all exogenous p63 isoforms are stabilized after DOX treatment, in a manner comparable with p53. Upon DOX treatment, TAp63-expressing cells present additional species with higher molecular weight, which may result from posttranslational modifications, such as phosphorylation as described in other cell lines (21,22). Indeed, all TAp63 isoforms in transient transfection experiments, as well as {alpha} and β forms in stable populations, appeared detectable with a phospho-p63-specific antibody. We do not exclude that additional post-translational modifications, such as sumoylation, may also occur. {Delta}Np63 are phosphorylated on the same residue as TA forms. This phosphorylation has been described only for {Delta}Np63{alpha} so far, resulting in the proteasome-dependent degradation of this isoform in ultraviolet-treated keratinocytes (21). In Hep3B, this phosphorylation is associated with the accumulation of the proteins rather than their degradation.

In conclusion, expression of TAp63 isoforms in Hep3B cells leads to the overexpression of some p53 target genes involved in cell-cycle arrest (WAF1, 14.3.3{sigma}) and apoptosis (CD95/FAS), as well as of {Delta}Np73. The expression of TAp63 isoforms is reduced in polyclonal populations as compared with transiently transfected cells, suggesting that TAp63 may be harmful for cell survival. Moreover, p63 isoforms accumulate in response to genotoxic stress through a phosphorylation-dependent, proteasome degradation escape mechanism. These observations provide support that p63 may contribute to DNA damage responses in liver cells, as companion to p53, or as a palliative mechanism in cells that lack wild-type p53 activity. Consistent with this hypothesis, Gressner et al. (34) showed that TAp63{alpha} confers to Hep3B cell line an increased chemosensitivity via the induction of apoptosis. Therefore, further studies should be developed to determine whether modulation of p63 activities may represent an additional target to increase the chemosensitivity in organs such as liver, which are notoriously poorly responsive to classical chemotherapy.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables 1 and 2 and Figures 1 and 2 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
This work was funded by the Institut National de la Santé Et de la Recherche Médicale and the Association pour la Recherche contre le Cancer (3117); Ministère de l'Enseignement et de la Recherche to A.P. (Petitjean); comité départemental de l'Allier of the French Ligue Nationale Contre le Cancer to C.R.; International Agency for Research on Cancer to V.T.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
The authors thank M. Dobbelstein, W. Kaelin and M. Oren for the generous gift of pGL3p73N, TAp73{alpha} and β and DNp63{alpha}, respectively, and I. Durand for FACS analysis.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received June 28, 2007; revised November 7, 2007; accepted November 8, 2007.


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