The neurogene BTG2TIS21/PC3 is transactivated by {Delta}Np73{alpha} via p53 specifically in neuroblastoma cells

David Goldschneider1, Karine Million1, Anne Meiller3, Hedi Haddada1, Alain Puisieux2, Jean Bénard1, Evelyne May3,* and Sétha Douc-Rasy1,{ddagger}

1 CNRS UMR 8126, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France
2 INSERM U590, Centre Leon Bérard, 28 rue Laënnec, 69008 Lyon, France
3 CEA-CNRS UMR217, PB6, 92265 Fontenay-aux-Roses, France

{ddagger} Author for correspondence (e-mail: sdouc{at}igr.fr)

Accepted 6 January 2005


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of {Delta}Np73{alpha}, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, {Delta}Np73{alpha} inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the {Delta}Np73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by {Delta}Np73{alpha} depends on both p53 status (as it is not observed in a p53–/– neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that {Delta}Np73{alpha} may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete {Delta}Np73{alpha} C-terminus sequence as it is not observed with {Delta}Np73ß, which lacks most of the C-terminal domain. We have previously shown that {Delta}Np73{alpha} is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that {Delta}Np73{alpha} not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Key words: p73, p53, BTG2TIS21/PC3, Transactivation, Neuroblastoma, Apoptosis, Differentiation, Development


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
The p73 gene and the p53 tumor suppressor gene share homology in terms of sequence and the structural organization of the three canonical functional domains, namely, the acidic N-terminal transactivating domain (TA), the core DNA binding domain (DBD) and the C-terminal oligomerization domain (OD) (Kaghad et al., 1997Go). The p73 gene encodes 636 amino acids generated from 14 exons and specifies two main N-terminal isoforms: the full-length TAp73 (TAp73{alpha}) initiated from the P1 promoter and the N-terminally truncated p73 isoforms. These TA-deficient p73 isoforms include isoforms initiated from P1 but that are lacking exon 2 as a result of alternate splicing (p73{Delta}exon2) and isoforms initiated from a second internal promoter, P2 ({Delta}Np73{alpha}) (Yang et al., 2002Go). Another particular feature of p73 may be the fact that its C-terminus contains three additional domains: a second transactivation domain TA2 (Takada et al., 1999Go), a SAM (`sterile alpha motif') protein-protein interaction domain (Chi et al., 1999Go) and a basic C-terminus sequence known as a transcriptional negative regulator (Ozaki, et al., 1999Go). Moreover, it is generally thought that the C-terminus domain negatively regulates the transcriptional ability of TAp73{alpha} (Yang et al., 2002Go).

Structural homology between p53 and p73 leads to functional homology. TAp73 mimics p53 in its ability to transactivate many p53-responsive genes (including the Mdm2, an E3 ubiquitin ligase and the cyclin-dependent kinase inhibitor p21) and to induce apoptosis (Jost et al., 1997Go). However, in sharp contrast with p53, Mdm2 cannot target p73 for ubiquitin-proteasome degradation, although it can block its transcriptional activity (Zeng et al., 1999Go). Initial studies on DNA-damaging agents report a lack of p73 induction upon actinomycin D treatment or UV irradiation. However, several reports show that endogenous p73 is a target of the non-receptor tyrosine kinase c-Abl in p53-deficient mouse embryo fibroblast cells in response to genotoxic stress such as cisplatin (Gong et al., 1999Go). Furthermore, p73 and c-Abl can associate with each other via the p73 PxxP motif and the c-Abl SH3 domain in {gamma}-irradiated cells to induce apoptosis or cell cycle arrest in the G1 phase (Agami et al., 1999Go).

In neuroblastoma cells, {Delta}Np73 expression is stimulated by overexpression of TAp73 or p53; moreover, induction or overexpression of {Delta}Np73 promotes cell survival by competition with TAp73 or wild-type (wt-) p53 (Nakagawa et al., 2002Go; Stiewe et al., 2002Go). In vitro, the spliced variant p73{Delta}exon2 also functions as a transdominant-negative inhibitor of TAp73 and wt-p53 as it interferes with the apoptotic function of p53 and is therefore one of the key regulatory elements involved in p53 signaling pathways (Fillippovich et al., 2001Go; Zaika et al., 2002Go).

In human tumors, {Delta}Np73 and/or p73{Delta}exon2 transcript levels are found to be upregulated in about 80% of human cancers, including cancers of the ovary, endometrium, cervix, vulva, vagina, breast, kidney and colon (Zaika et al., 2002Go). In human neuroblastic tumors, p53 and p73 are very rarely mutated (Bénard et al., 2003Go) but p73 protein expression is clearly deregulated (strong accumulation in undifferentiated neuroblastic tumors and a total lack in Schwann cells). In addition, the {Delta}Np73{alpha} protein is the only isoform observed by western blotting in undifferentiated neuroblastic tumors (Douc-Rasy et al., 2002Go), which coincides with a poor outcome in these neuroblastoma patients (Casciano et al., 2002Go).

BTG2TIS21/PC3, the antiproliferative human homologue gene of PC3 (rat) or TIS-21 (mouse) belongs to the B-cell translocation gene family, initially found to be rapidly and transiently induced by nerve growth factor (NGF) and depolarization (Bradbury et al., 1991Go). It has been reported that BTG2TIS21/PC3 expression is induced through p53-dependent mechanisms and that the BGT2 function may play a role in cell cycle control and cellular response to DNA damage (Rouault et al., 1996Go). BTG2TIS21/PC3 has been found to be involved in cell growth, differentiation and DNA repair (Tirone, 2001Go). PC3 mRNA expression coincides both spatially and temporally with the pattern of CNS neurogenesis in the developing rat (Iacopetti et al., 1994Go). The human BTG2TIS21/PC3 was recently evaluated as a candidate tumor suppressor gene; the gene was cloned and its structural organization was determined (Duriez et al., 2002Go). In vitro studies have shown that BTG2TIS21/PC3 promotes neuronal differentiation and prevents apoptosis of terminally differentiated cells (el-Ghissassi et al., 2002Go). In normal human tissues, BTG2TIS21/PC3 is differentially expressed in several differentiated epithelial cells including the breast, lung, intestine and pancreas (Melamed et al., 2002Go). Physical interactions between BTG2TIS21/PC3 and other cell cycle regulator proteins, such as CCR4-CAF, have recently been demonstrated in HeLa cells (Morel et al., 2003Go). The BTG2TIS21/PC3, CAF1 and CCR4 complex has been shown to regulate the transcription activity mediated through the estrogen receptor {alpha}.

In recent studies involving p73 target genes in neuroblastoma cells using the adenoviral (Ad)-p73 recombinant approach we showed that both Ad-{Delta}Np73{alpha} and Ad-TAp73{alpha} induce accumulation and activation of the endogenous wt-p53 in wt-p53 expressing cells (Goldschneider et al., 2003Go; Miro-Mur et al., 2003Go; Goldschneider et al., 2004Go). Strikingly, {Delta}Np73 selectively regulates p53 target genes as the BTG2TIS21/PC3 transcript level was shown to be upregulated both in Ad-{Delta}Np73{alpha} and Ad-TAp73-infected cells, thus contrasting with other p53 target genes such as Waf1/p21Waf, which was completely repressed by {Delta}Np73 (Miro-Mur et al., 2003Go; Goldschneider et al., 2004Go). The present study was performed to determine whether: (1) BTG2TIS21/PC3 activation by {Delta}Np73 requires both BTG2TIS21/PC3 promoter sequences and p53; (2) the C-terminus plays a role in the {Delta}Np73-dependent transactivation of BTG2TIS21/PC3; and (3) the cellular context contributes to the {Delta}Np73{alpha}-dependent upregulation of the BTG2TIS21/PC3 gene initially observed in neuroblastoma cells.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
Plasmids and Recombinant p73-adenovirus constructs
pcDNA-TAp73{alpha}pcDNAp73ß and pcDNA-{Delta}Np73{alpha}/pcDNAp73ß plasmid constructs were donated by Mourad Kaghad (Sanofi Recherche, Labège). The recombinant adenoviral vector expressing full-length p73{alpha} (Ad-p73{alpha}) and {Delta}N-p73{alpha} (Ad-{Delta}Np73{alpha}) were produced by pcDNA-p73{alpha} and pcDNA-{Delta}N-p73{alpha} in vivo homologous recombination in 293 cells as previously described (Goldschneider et al., 2004Go). pDDm-TO were obtained by inserting the sequence that encodes the mouse p53DD truncated protein (amino acid residues 1-14 and 302-390) into the Invitrogen vector pcDNA/TO/B (Drané et al., 2002Go). pE1B-hWAF1 contains the p53RE of the Waf1/p21 promoter cloned ahead of the luciferase gene (Munsch et al., 2000Go). The pBTG2-Luc reporter constructs were obtained as previously described (Duriez et al., 2002Go).

Cell culture and adeno-recombinant infection
The parental human neuroblastoma cell lines, SH-SY5Y and IMR-32, were purchased from the European Collection of Cell Cultures (ECACC, Wiltshire, UK) and express wt-p53. The neuroblastoma LAN-1 cell line (gift of Nicole Gross, Pediatric Oncology Research, Lausanne, Switzerland) does not express p53. The MCF-7 cell line, purchased from American Type Culture Collection (ATCC, Manassas, VA) expresses wt-p53. The human IGR-N-91 cell line was established in our laboratory from the bone marrow of a patient with metastatic neuroblastoma after unsuccessful adriamycin-vincristine chemotherapy (Ferrandis et al., 1994Go). The IGR-N-91 p53 gene is mutated by an in-frame duplication of exons 7-8-9 (Goldschneider et al., 2004Go). The human breast cancer MCF-7 and the neuroblastoma cell lines were grown under standard conditions in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine and 10% calf fetal serum. Cells were infected with the recombinant adenovirus vector expressing either p73 full-length (Ad-p73{alpha} or ß) or N-terminal truncated p73 (Ad-{Delta}Np73{alpha} or ß) at an MOI of 15. The cells were harvested after 48 hours' infection.

Western blotting
Western blotting was performed as previously described (Goldschneider et al., 2004Go). Briefly, the cells were directly lysed in Laemmli buffer and the total protein extracts (50 µg to detect endogenous protein or 1 µg to detect protein in infected cells) were loaded onto SDS-PAGE (7.5%). After electrophoresis, the proteins were electrotransferred to nitrocellulose filters and the filters were probed with either polyclonal p73 antibody to reveal p73, or DO-7 monoclonal anti-p53 antibody (DAKO) to reveal p53, or with anti-p73ß (clone GC15 from Upstate Biotechnology) to reveal p73ß or anti-p63 (clone 4A4 from Oncogene Research) to reveal p63. The dilution for each antibody was 1:1000. Protein detection was carried out using an ECL kit (Amersham Pharmacia Biotech, France). Controls were performed using ß-actin antibody MAB 1501 (Chemicon).

Real-time quantitative PCR (RTQ-PCR)
BTG2 and Waf1/p21 mRNA were quantified using the Applied Biosystem apparatus, Abi Prism 7000 Sequence Detection system. Briefly, total RNA was extracted from non-infected or infected cells using RNeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. Total RNA (1 µg) was reversed transcribed using SuperScript II (Invitrogen). RTQ-PCR was performed in a final volume of 25 µl containing 25 ng of each total RNA template, 10 pmoles of each primer and 12.5 µl of a SYBR®-Green master mix. The primers were designed using the Oligo 6 Primer Analysis Software (Molecular Biology Insights). Three sets of primers were used: GAPDH-F, 5'-AGCTCACTGGCATGGCCTTC-3'; GAPDH-R, 5'-ACGCCTGCTTCACCACCTTC-3'; p21-F, 5'-GGACCTGTCACTGTCTTGTA-3'; p21-R, 5'-GGCTTCCTCTTGGAGAAGAT-3'; BTG2-F, 5'-CGAGCAGAGGCTTAAGGTCTTC-3'; BTG2-R, 5'-CTGGCTGAGTCCGATCTGG-3'. Quantification was carried out using the comparative CT method and water was used as the negative control. An arbitrary threshold was chosen on the basis of the variability of the baseline. Threshold cycle (CT) values were calculated by determining the point at which the fluorescence exceeded the threshold limit. CT was reported as the cycle number at this point. The average of target gene was normalized to GAPDH as endogenous housekeeping gene and relative to the non-infected condition as control and was given by 2{Delta}{Delta}CT where {Delta}{Delta}CT ={Delta}CT (sample) – {Delta}CT (control); {Delta}CT = CT (target gene) – CT (GAPDH).

Luciferase reporter assays
Cells were seeded in triplicate onto six-well plates at a density of 2x104 cells per cm2 and transfected with 0.5 µg (2.5 µg/ml) of either pGL3 firefly luciferase reporter gene plasmids containing a 2700 bp fragment (BTG2 2700) or a 266 bp fragment of the BTG2 (BTG2 266) or the Waf1/p21 luciferase reporter plasmid, pE1B-hWAF1 (WAF-1) using the calcium phosphate method as previously described (Drané et al., 2001Go). To evaluate the transactivation effect of p73, cells were co-transfected with 1 µg (5 µg/ml) of either TAp73{alpha}- or {Delta}Np73 ({alpha} or ß)-expressing vectors or control plasmid. At 24 hours after transfection, cells were lysed with 200 µl/well of passive lysis buffer provided with the Luciferase assay kit (Promega). Luciferase activity was measured according to the manufacturer's protocol with the Microlumat LB96P luminometer (EG&G Berthold).


    Results and Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
p53-dependent activation of BTG2TIS21/PC3 by {Delta}N-p73{alpha} through the BTG2TIS21/PC3 promoter sequence
In previous studies we showed that in wt-p53 SH-SY5Y cells TAp73 proteins were able to upregulate the p53 target gene Waf1/p21 involved in cell cycle arrest, the proapoptotic genes PUMA and BAX, and the BTG2TIS21/PC3 gene. On the other hand, and as expected, {Delta}Np73{alpha}, a dominant-negative isoform of both p53 and TAp73, efficiently inhibited the conventional p53 responsive genes such as MDM2 BAX or Waf1/p21. Surprisingly, however, the expression of BTG2TIS21/PC3 was upregulated by {Delta}Np73 and this effect occurred specifically in wt-p53 SH-SY5Y cells but not in mutated-p53 IGR-N-91 cells (Goldschneider et al., 2004Go).

To further define the mechanism by which {Delta}Np73 activates BTG2TIS21/PC3 transcription, we performed parallel co-transfection assays with recombinants expressing the various p73 isoforms and reporter constructs containing either long (2700 bp) or short (266 bp) BTG2TIS21/PC3 promoter sequences (Duriez et al., 2002Go) linked to the luciferase gene. The BTG2-p53 binding site (p53BS) is located at position –119 from the initiation site (Fig. 1A).



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Fig. 1. BTG2TIS21/PC3 promoter activation by TA- and {Delta}N-p73{alpha} in wt-p53 neuroblastoma cell lines but not in wt-p53 breast cancer line MCF-7. (A) Schematic representation of pGL3-reporter constructs containing p53 binding site (BS) at –119, as previously described (Duriez et al., 2002Go); long BTG2TIS21/PC3 promoter sequence fragments of 2700 bp (–2700) and short promoter fragment (–266). (B) SH-SY5Y, (C) IMR-32, (D) LAN-1 and (E) MCF-7 cells were transiently co-transfected with pBTG2-Luc plasmid carrying long (2700) or short (266) promoter sequences in the presence of either TA- or {Delta}Np73 expressing vectors or empty control plasmids. Transfection techniques and luciferase activity measurements were carried out as described in the Materials and Methods. Error bars represent standard deviation calculated from three independent experiments. Cells were transfected with empty vector, TAp73{alpha}, {Delta}Np73{alpha}, p53DD, TAp73{alpha} + p53, {Delta}Np73{alpha} + p53 as indicated.

 

Results obtained with SH-SY5Y are presented in Fig. 1B. Luciferase activity was significantly higher when the reporter gene was driven by short rather than long BTG2TIS21/PC3 promoter sequences (Fig. 1B, empty vector). Promoter activity was inhibited by the p53 dominant-negative form, p53DD, in both cases, demonstrating that expression through the BTG2TIS21/PC3 promoter depends on endogenous p53. More interestingly, co-transfection of {Delta}Np73 increased transcriptional activity to the same level as TAp73, strongly suggesting that an upregulation of BTG2 promoter activity may account for our previous published data showing an increase of endogenous BTG2 mRNA levels in SH-SY5Y overexpressing {Delta}Np73 (Goldschneider et al., 2004Go).

To determine whether BTG2TIS21/PC3 activation by {Delta}Np73 occurs in other neuroblastoma cell lines expressing wt-p53, the same experiments were performed with IMR-32 cells (Fig. 1C). Again, the BTG2TIS21/PC3 promoter was consistently activated by {Delta}Np73 as well as by TAp73 and again, the short promoter sequence was more efficient than the long sequence. As with SH-SY5Y, p53DD interfered negatively with the activity of the BTG2TIS21/PC3 promoter in IMR-32 cells.

To ascertain the involvement of wt-p53, luciferase assays were repeated using LAN-1, a p53-deficient (null-p53) neuroblastoma cell line. Cells were co-transfected with pluc-BTG2 and a vector encoding a p73 isoform and/or a vector encoding wt-p53. In the absence of exogenous p53 expression, neither TA- nor {Delta}Np73{alpha} was able to activate the BTG2TIS21/PC3 promoter (Fig. 1D). Interestingly, in these cells, an ectopic expression of wt-p53 had no effect on BTG2TIS21/PC3 promoter activity, but the co-expression of wt-p53 and TA- or {Delta}Np73{alpha} significantly increased the luciferase activity, supporting a synergic effect of p53 and p73 at the level of BTG2TIS21/PC3 promoter activation. It is worthwhile to note that the canonical p53 responsive element of Waf1/p21 gene is upregulated by an ectopic expression of p53 in these cells (D. G., unpublished data).

The cellular context may interfere with the {Delta}Np73{alpha}-mediated BTG2 transactivation
We next addressed the question of whether BTG2TIS21/PC3 can be activated by the {Delta}Np73 isoform in wt-p53-expressing cells derived from other tissue. The same experiments were thus performed in MCF-7, a human breast carcinoma cell line that expresses wt-p53. Like SH-SY5Y and LAN-1, the MCF-7 cells expressed an endogenous p73{alpha} protein as detected by western blot analysis (Fig. 2). P73{alpha} was also expressed in IMR-32 cells, but to a lesser extent, as the protein could only be detected when the blot was overexposed (30 minutes compared to 0.5 minutes). In contrast, it was not possible to detect either the p73ß or the p63{alpha} protein by western blotting in any of these cell lines.



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Fig. 2. Western blot showing p73, p63 and p53 endogenous protein expression in SH-SY5Y, IMR-32, LAN-1 and MCF-7 cell lines. Total protein extracts were obtained as previously described (Goldschneider et al., 2004Go) and 30 µg total lysate was loaded for western blotting. The blot on the right was overexposed for 30 minutes rather than 0.5 minutes as for the blots on the left.

 

Surprisingly, in the MCF-7 cellular context neither TA- nor {Delta}Np73{alpha} ectopic expression was able to stimulate luciferase expression from the BTG2TIS21/PC3 promoter sequences (Fig. 1E). In contrast, the activity of these sequences was diminished. These results indicate that the stimulatory effect of both p73{alpha} and {Delta}Np73{alpha} on the p53-dependent BTG2TIS21/PC3 transcription is cell specific. As neuroblastoma is a peripheral primitive neuroectodermal tumor, derived from neural crests cells, an important issue will be to determine whether or not BTG2TIS21/PC3 transactivation by {Delta}Np73{alpha} also occurs in other wt-p53-expressing cells originating from the same precursor.

Transactivation by {Delta}Np73 is restricted to BTG2TIS21/PC3
It is well established that TAp73 proteins like p53 are able to upregulate the Cdk inhibitor Waf1/p21 (De Laurenzi et al., 1998Go), whereas {Delta}Np73 exerts a dominant-negative effect on p53 and TA transcriptional activity (Kartasheva et al., 2002Go; Vossio et al., 2002Go). This coincides with our previous results, which showed that an overexpression of {Delta}Np73{alpha} in SH-SY5Y neuroblastoma cells downregulated Bax, PUMA and Waf1/p21 genes although p53 was accumulated (Goldschneider et al., 2004Go). However, surprisingly, we found that an overexpression of {Delta}Np73{alpha} upregulated BTG2TIS21/PC3 expression. This unexpected result has now been confirmed at the promoter level. To ascertain that BTG2TIS21/PC3 promoter activation was not an artifact of the system analysis, the same experiment was performed using a plasmid encoding the luciferase reporter gene under the control of the Waf1/p21 p53-responsive element. The co-transfection of {Delta}Np73{alpha} dramatically decreased luciferase activity in both SH-SY5Y and MCF7 cells, two cell lines expressing wt-p53 (Fig. 3A,B). These results led us to postulate that {Delta}Np73 is able to cooperate with p53 to activate some specific p53 target genes (BTG2TIS21/PC3 in the present study) depending on both the promoter sequences and the cellular context.



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Fig. 3. Transcriptional activity of Waf1/p21 p53BS is inhibited by {Delta}Np73. The mean relative luciferase activity±s.e.m. is shown for wt-p53 SH-SY5Y (A) and MCF-7 (B) cells co-transfected with 0.5 µg pE1-hWAF1 and 1 µg of plasmid encoding either {Delta}Np73{alpha} or {Delta}Np73ß. The p53DD effect is also shown.

 

{Delta}Np73{alpha} overexpression induces an increase of endogenous BTG2TIS21/PC3 mRNA transcript in SH-SY5Y but not MCF-7 cells
To confirm the results obtained with reporter plasmids, the endogenous BTG2TIS21/PC3 mRNA levels were compared to that of Waf1/p21 in SH-SY5Y cells infected with either Ad-TAp73{alpha} or Ad-{Delta}Np73{alpha}. The results obtained using SYBR®-Green RTQ-PCR are presented in Fig. 4. As expected, Waf1/p21 expression was strongly upregulated by TAp73{alpha} and significantly repressed by the dominant-negative mutant {Delta}Np73{alpha} (Fig. 4A). Very interestingly, however, unlike Waf1/p21, BTG2TIS21/PC3 endogenous transcript levels were comparably higher in both the TAp73{alpha} and {Delta}Np73{alpha}-infected cells (more than tenfold when compared to empty vector infected cells) (Fig. 4B). These results, combined with those obtained by transfection, strongly suggest that the upregulation of the BTG2TIS21/PC3 promoter could account for the BTG2TIS21/PC3 mRNA increase in Ad-{Delta}Np73{alpha}-infected SH-SY5Ycells.



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Fig. 4. Effect of ectopic expression of either TA- or {Delta}Np73 on the endogenous expression of Waf1/p21 and BTG2TIS21/PC3 in wt-p53-expressing SH-SY5Y and MCF-7 cell lines. The transcript levels of Waf1/p21 (A,C) and BTG2TIS21/PC3 (B,D) were estimated by SYBR®-Green RTQ-PCR from total RNA extracted from SH-SY5Y (A,B) or MCF-7 (C,D) cells infected with either TA- or {Delta}Np73{alpha}-recombinant adenovirus. Results are mean expression levels±s.e.m. compared with those obtained from either uninfected cells or cells infected with an empty vector

 

Parallel experiments were performed in the MCF-7 breast cancer cell line. Comparable results were obtained for Waf1/p21 with a marked increase in Waf1/p21 mRNA levels in response to Ad-p73{alpha} infection but not to Ad-{Delta}Np73{alpha} infection (Fig. 4C). However, consistent with the luciferase results shown in Fig. 1E, a relatively low increase in the level of BTG2TIS21/PC3 mRNA was found in Ad-p73{alpha}-infected MCF-7 cells and no significant increase was found in parallel cells infected with Ad-{Delta}Np73{alpha} (Fig. 4D).

{Delta}Np73-mediated stimulation of p53-dependent BTG2TIS21/PC3 transcription required the p73{alpha} C-terminus
It is well known that p73ß is more active in transcription than p73{alpha} owing to a repressive effect of the C-terminus. We wondered whether the C-terminal domain had any effect on BTG2TIS21/PC3 activation by {Delta}Np73. To this end, we performed luciferase assays using long and short promoter sequences in cells transfected by {Delta}Np73ß lacking the SAM domain, and compared these results to results obtained from the co-transfection of the p53 dominant-negative mutant, p53DD. Unlike {Delta}Np73{alpha}, {Delta}Np73ß isoforms did not stimulate the activity of the BTG2TIS21/PC3 promoter sequences in two wt-p53-expressing neuroblastoma cell lines, SH-SY5Y and IMR-32 (Fig. 5A,B). Interestingly, the decrease in luciferase activity is much more pronounced in cells expressing p53DD than in those expressing {Delta}Np73ß. One hypothesis to explain these results would be that, in these particular cells, the affinity of p53 for the BTG2 p53-responsive element is higher than that of {Delta}Np73ß. This is consistent with the fact that {Delta}Np73{alpha} does not act as a dominant-negative mutant of p53 in either of these two cell lines. In contrast, the decrease in luciferase activity in MCF-7 cells is even more pronounced with {Delta}Np73ß than with p53DD (Fig. 5C), suggesting once again that the BTG2TIS21/PC3 promoter response to p53 and p73 expression is cell specific. The same observation was noted when BTG2TIS2/PC3 mRNA levels were analyzed by RTQ-PCR in cells infected with {Delta}Np73ß. As already shown in Fig. 4, BTG2TIS2/PC3 upregulation was observed in SH-SY5Y cells infected with Ad-{Delta}Np73{alpha} but not in MCF-7 cells (Fig. 4B and 4D respectively). To confirm that the presence of C-terminus sequences are necessary for BTG2TIS2/PC3 activation, BTG2 mRNA levels were quantified by RTQ-PCR in SH-SY5Y and MCF-7 infected with either Ad-{Delta}Np73ß or Ad-{Delta}Np73{alpha}. There was six times less mRNA in SH-SY5Y cells infected with the ß isoform than in those infected with the {alpha} isoform, which is in contrast to MCF-7 (Fig. 6A,B). This is consistent with the luciferase results. Nevertheless, p53 accumulation in SH-SY5Y was observed in both the Ad-{Delta}Np73ß and Ad-{Delta}Np73{alpha}-infected cells (Fig. 7), indicating that the presence of wt-p53 is somewhat necessary but not sufficient for BTG2TIS2/PC3 upregulation, and suggesting that p73 C-terminus sequences might enhance p53 binding with the BTG2 p53-responsive element. When combined, our findings show that BTG2TIS2/PC3 expression is upregulated in wt-p53 neuroblastoma cells by {Delta}Np73{alpha} but not by {Delta}Np73ß. The role of p73ß, which is known to be transcriptionally more active than the {alpha} isoform, may not be systematic and should perhaps be revisited. {Delta}Np73{alpha}, thus, may play a role in upregulating certain genes like BTG2TIS2/PC3 in addition to its known dominant-negative role.



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Fig. 5. BTG2 transactivation is not induced by p73ß isoforms. Transcriptional activity of BTG2TIS21/PC3 was analyzed by luciferase assays in wt-p53 SH-SY5Y (A), IMR-32 (B) and MCF-7 (C) cells co-transfected with 0.5 µg of luciferase reporter plasmid (BTG2 2700 or BTG2 266) and 1 µg of either {Delta}Np73ß or p53DD expression plasmids. The values represent mean relative luciferase activity±s.e.m.

 


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Fig. 6. No accumulation of BTG2 endogenous mRNA in Ad-p73ß-infected SH-SY5Y cells. Comparative RTQ-PCR analyses of BTG2TIS21/PC3 mRNA levels in SH-SY5Y (A) and MCF-7 cells (B) infected with either p73{alpha} or p73ß isoforms.

 


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Fig. 7. Western blot showing an accumulation of p53 protein in response to an ectopic expression of either the {alpha} or ß isoforms of TA- and {Delta}Np73 proteins.

 

A second transactivating domain called (TA2), which is different from the well-known N-terminus TAD domain, has been identified in the C-terminus of the p73 protein (Takada et al., 1999Go). However, this TA2 domain is present both in p73{alpha} and ß, and cannot, therefore, account for the stimulation of BTG2 promoter activity by the {Delta}Np73{alpha} protein. It has been demonstrated more recently that a stable expression of inducible {Delta}Np73ß was able to suppress cell growth and induce apoptosis, even at a physiological level (Liu et al., 2004Go). The authors attributed this transactivating activity to the N-terminal part of the {Delta}Np73ß protein. Interestingly, {Delta}Np73{alpha} harbors the same N-terminus as the ß isoform but, according to their experiments, it was unable to transactivate promoters, possibly owing to the established repressor role of the p73{alpha} C-terminus. So, once again, such a model fails to explain our results. Furthermore, the {Delta}Np73ß transactivating potential reported in Liu's study (Liu et al., 2004Go), was found to be p53-independent as it can be induced in p53-null H1299 cells as well as in wt-p53-expressing MCF-7 cells. Our results showed that BTG2TIS21/PC3 activation by {Delta}Np73{alpha} is p53-dependent.

BTG2TIS21/PC3 transactivation may result from the unusual orientation of the pentamers in the p53 responsive element
This study shows that the {Delta}Np73{alpha} isoform activates BTG2TIS21/PC3 through its promoter in a p53-dependent manner in neuroblastoma wt-p53 cells. Transcriptional activation by p53 requires the fixation of p53 to its consensus binding site (p53BS). The p53BS consists of a canonical sequence comprising two decamers `PuPuPuC(A/T)(A/T)GpyPyPy' separated by 0 to 13 base pairs. The two pentamers in each decamer are arranged head to head (HH) (Fig. 8a). The Waf1/p21 promoter is the paradigm of this kind of base arrangement (El-Deiry, 1998Go). Alternately, p53 can repress the transcription of other genes such as MDR1. The p53BS harbored by the MDR1 promoter presents a particular orientation, with a head-to-tail (HT) pentamer arrangement (Fig. 8b) (Johnson et al., 2001Go). The authors proposed that this orientation could affect p53 protein conformation and lead to an inactive p53. For BTG2TIS21/PC3, the pentamer organization of the p53BS, with a tail-to-tail (TT) arrangement, is again different from that of the canonical p53BS (HH) and MDR1 promoter (HT) (Fig. 8c). This specific arrangement could account for the particular response of the BTG2TIS21/PC3 promoter to the {Delta}Np73{alpha} protein. One possibility could be that {Delta}Np73{alpha} and p53 form heterotetramers which are transcriptionally inactive on the canonical p53BS, and active (through the adoption of a different conformation) on the BTG2TIS21/PC3 promoter p53BS. Such a conformational change could possibly be due to the p73 C-terminus domain. However, such a model is unlikely according to Davison and colleagues (Davison et al., 1999Go), who demonstrated that p73 and p53 proteins only interact weakly. A second possibility, therefore, could be that p73{alpha} is unable to bind the non-canonical p53BS of BTG2TIS21/PC3, which means that {Delta}Np73{alpha} would not be able to compete with p53 in terms of binding to its responsive element and could, therefore, not repress the p53-dependent transactivation of the BTG2TIS21/PC3 promoter.



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Fig. 8. Schematic representation of the p53BS of three p53-target genes showing the pentamer orientation in each decamer. (a) Head-to-head (HH) for the canonical 20-bp p53-binding site, as in the Waf1/p21 promoter; (b) in tandem, head-to-tail (HT), as in the MDR-1 promoter; and (c) tail-to-tail (TT) as in the BTG2TIS21/PC3 promoter. Pu, purine; Py, pyrimidine.

 

Earlier studies of ours showed that both {Delta}Np73{alpha} and TAp73{alpha} were able to induce accumulation and activation of the endogenous wt-p53 in various cell lines (Miro-Mur et al., 2003Go; Goldschneider et al., 2004Go). The present data indicate that {Delta}Np73{alpha} stimulates the p53-dependent activation of BTG2TIS21/PC3 expression at the promoter level. The molecular mechanism by which BTG2TIS21/PC3 sequences might be transactivated by {Delta}Np73{alpha} remains to be elucidated. Two possible mechanisms can be proposed. The first model involves a cooperative effect of {Delta}Np73{alpha} and p53 acting on BTG2-p53BS but not on a canonical p53BS such as that of Waf1/p21. Such a model could be instrumental in explaining that {Delta}Np73{alpha} not only does not inhibit BTG2TIS21/PC3 but that it also activates it. The second model excludes {Delta}Np73{alpha}-BTG2 promoter binding because of the BTG2-p53BS TT orientation, which means that it could not inhibit BTG2TIS21/PC3 activation as it could for Waf1/p21, where the p53BS is HT orientated. In this alternative, the stimulation of BTG2TIS21/PC3 gene expression could result from the p73{alpha}-dependent activation of p53. Indeed, we have already published that an ectopic expression of p73{alpha} activates the transcriptional activity of the endogenous wt-p53 expressed in several transformed cell lines, independently of its own transcriptional activity (Miro-Mur et al., 2003Go). We are currently looking for the mechanism that could account for p53 activation by p73{alpha}.


    Conclusion
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
Our current findings, which demonstrate that it is the {Delta}Np73{alpha} isoform and not the {Delta}Np73ß isoform that activates the BTG2TIS21/PC3, are particularly interesting in terms of gene regulation and the physiology of neuroblastoma cells. They suggest that, by activating the BTG2TIS21/PC3, the {Delta}Np73{alpha} isoform might function, not only as an anti-apoptotic mediator in the neuronal system in vivo or as a transdominant negative in human tumors as previously reported, but may also play an antiproliferative and/or differentiating role in neural development and in neuroblastoma.


    Acknowledgments
 
We would like to express our sincerest gratitude to Mourad Kaghad for his kind donation of the pcDNA-p73 plasmid constructs and to Eric Le Cam for his helpful contributions. This work was supported by Ligue Contre le Cancer, Comité du Cher, l'Association pour la Recherche sur le Cancer (ARC) and Bonus Qualité Recherche (BQR) from University Paris-Sud. Edited by English Booster.


    Footnotes
 
* Present address: CNRS UPR9045, Institut André Lwoff, 7 rue Guy Moquet, 94801 Villejuif, France Back


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
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