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Molecular and Cellular Biology, February 2004, p. 1341-1350, Vol. 24, No. 3
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.3.1341-1350.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

ASPP1 and ASPP2: Common Activators of p53 Family Members

Daniele Bergamaschi,1,{dagger} Yardena Samuels,1,{dagger} Boquan Jin,2 Sai Duraisingham,1 Tim Crook,1 and Xin Lu1*

Ludwig Institute for Cancer Research, Imperial College School of Medicine, St. Mary's Campus, London W2 1PG, United Kingdom,1 Department of Immunology, Fourth Military Medical University, Xian 710032, People's Republic of China2

Received 6 June 2003/ Returned for modification 31 July 2003/ Accepted 6 November 2003


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ABSTRACT
 
We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21WAF-1/CIP1. The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21WAF-1/CIP1. Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.


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INTRODUCTION
 
The p53 gene is mutated in around 35 to 40% of human tumors. Pathways that activate p53 are also disrupted in many other tumors. The p53 protein modulates cellular functions, such as gene transcription, DNA synthesis, DNA repair, cell cycle arrest, senescence, and apoptosis. Mutations of the gene may result in inhibited protein function, and it is this dysfunction that is linked to tumor progression and genetic instability. In response to a variety of cellular stresses, p53 is posttranslationally modified, and protein levels increase dramatically. Activation of the protein results in either arrest of the cell at G1 or commitment to death through apoptosis. Research has demonstrated the role of p53 transcriptional transactivation in cell cycle arrest through the up-regulation of the p21WAF-1/CIP1 cyclin-dependent kinase inhibitor (cdki). However, many reports have shown that p53 can induce apoptosis by both transcription-dependent and -independent mechanisms (19, 20).

p53 is a member of a family of three proteins: p53, p63, and p73. p63 and p73 have more than 60% amino acid identity within the DNA binding region of p53 (12, 13, 22). DNA binding specificity among p53 family members is very similar but not identical. As a result, a large number of p53 target genes are also transactivated by p63 and p73. Hence, p63 and p73 share some p53 functions, such as cell cycle arrest and apoptosis. However, there are many other structural and functional differences between p53, p63, and p73. For example, mutations in p63 and p73 are rare in human cancer. 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 and that the loss of p73 or p63 does not predispose mice to cancer (21). Cellular regulators of p53, such as mdm2, do not have the same effects on p63 and p73. While the binding of mdm2 to p53 inhibits the transactivation function of p53 and targets it for degradation (11, 14), it fails to target p63 and p73 for degradation (4, 8). In contrast, the binding of mdm2 to p63 stimulates the transactivation function of p63 by stabilizing the protein (6). Similarly, the CCAAT-binding transcription factor CTF2 binds to the DNA binding region of p53 and p73 but leads to different biological consequences. The binding of CTF2 to p53 enhances the DNA binding activity of p53, but the interaction of CTF2 with p73 inhibits the DNA binding activity of p73 (18). Moreover, unlike p53, p63 and p73 also do not interact with viral proteins, such as the large T antigen of simian virus 40, through their DNA binding domain (7, 8, 15). All these results suggested that an activator or inhibitor of p53 would not necessarily have similar physiological implications for family members p63 and p73. This may explain why no universal activator or inhibitor of the p53 family members has been identified so far.

We recently showed that the apoptotic function of p53 is significantly enhanced by two members of the ASPP family, ASPP1 and ASPP2 (16). Binding to the DNA binding domains of p53, ASPP1, and ASPP2 specifically stimulates the transactivation function of p53 on promoters of proapoptotic genes, such as Bax and PIG3, but not on promoters of p21WAF-1/CIP1 or mdm2. Since the DNA binding domain of p53 is the most homologous region among all p53 family members, we investigated whether ASPP1 and ASPP2 can also interact with the rest of the p53 family members, p63 and p73. The effects of ASPP1 and ASPP2 on the transactivation and apoptotic function of p63 and p73 were also studied.


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MATERIALS AND METHODS
 
Cell culture, antibodies, and plasmids. Cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. DO-1 is a mouse anti-p53 antibody. The V5 epitope is recognized by the mouse monoclonal antibody V5. CD20Leu is a fluorescein isothiocyanate-conjugated monoclonal antibody specific for the cell surface marker CD20 (Becton Dickinson). The mouse and rabbit antibodies to ASPP1 and ASPP2 were described previously (16) (rabbit anti-ASPP1 polyclonal antibody ASPP1.88, rabbit anti-ASPP2 polyclonal antibody PB77, mouse monoclonal anti-ASPP2 antibody DX5410). The mouse monoclonal anti-ASPP1 antibody LX011 was made in the same way as YX.7 (16) and is specific to ASPP1 (data not shown). All expression plasmids used in this study were driven by the cytomegalovirus immediate-early promoter. ASPP1 was tagged with the V5 epitope (ASPP1-V5), while p73{alpha} was tagged with the hemagglutinin epitope. p63{gamma} was detected with the 4A4 mouse monoclonal antibody (Santa Cruz), and p73{alpha} was detected with ER-15 (Neomarker).

Transactivation assays. Saos-2 cells (5 x 105) were plated 24 h prior to transfection in 6-cm-diameter dishes. All transactivation assays contained 1 µg of reporter plasmid. Fifty nanograms of p53, 35 ng of p63{gamma}, 25 ng of p73{alpha}, and 4 µg of ASPP1 or ASPP2 expression plasmids were used as indicated. Cells were lysed in reporter lysis buffer 16 to 24 h after transfection and assayed using the luciferase assay kit (Promega, Madison, Wis.). The factor of activation of a particular reporter was determined by the activity of the transfected plasmid divided by the activity of vector alone.

Flow cytometry. Cells (106) were plated 24 to 48 h prior to transfection in 10-cm-diameter plates. All cells were transfected with 2 µg of a plasmid expressing CD20 as a transfection marker. The cells in Fig. 1A and B were transfected with increasing amounts of ASPP1 and ASPP2 (7.5, 15, and 25 µg). The cells in Fig. 6 were transfected with 1 µg of human p53, 1 or 2.5 µg of p63{gamma} or p73{alpha}, or 10 µg of ASPP1 and ASPP2 plasmid as indicated in the figure. The cells in Fig. 7 were cotransfected with 25 µg of ASPP1 or ASPP2 or 3 µg of p63 or p73 and with 10 µg of pSuper plasmids containing p63, p73, or p53 RNA interference (RNAi) as indicated in the figure. Thirty-six hours after the transfection, both attached and floating cells were harvested and analyzed as described (16).



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  		FIG. 1. ASPP1 and ASPP2 induce apoptosis independently of p53 in Saos-2 (A) and H1299 (B) cells. In response to treatment with cisplatin (3.5 µg/ml for 16 h), the number of apoptotic cells induced by the expression of ASPP1 and ASPP2 was increased further (C and D). The transfected cells were gated based on the expression of CD20 (16). The percentage of apoptotic cells was measured by the accumulation of cells with a sub-G1 DNA content. The bar graphs show the percentage of apoptotic cells 36 h after transfection and were derived from at least two independent experiments. (E) Expression levels of endogenous p63{gamma}, p73{alpha}, ASPP1, and ASPP2 in response to cisplatin treatment (+) in Saos-2 and H1299 cells. IVT, in vitro translation.



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  		FIG. 6. Fluorescence-activated cell sorting analysis showing that expression of ASPP1 and ASPP2 stimulate the apoptotic function of p53, p63{gamma}, and p73{alpha}. The transfected cells were gated and analyzed as described in the legend to Fig. 1. The percentage of apoptotic cells 36 h after transfection is shown. The values were derived from two independent experiments.



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  		FIG.7. Saos-2 and H1299 cells were transfected with plasmid expressing a cell surface marker CD20 together with p53, p63{gamma}, or p73{alpha} in the presence or absence of ASPP1, ASPP2, p63 RNAi, p73 RNAi, or p53 RNAi as indicated (A, C, and D). The ability of p63 and p73 RNAi to inhibit the expression of p63 and p73 is shown in panel B. The vector used to express RNAi of p63, p73, or p53 is pSuper, and cisplatin treatment was performed as described in the legend to Fig. 1. The bar graph represents the percentage of apoptotic cells 36 h after transfection and was derived from two independent experiments. cmv, cytomegalovirus; IVT, in vitro translation.

Protein biochemistry. For Western blotting, cells were lysed in either Nonidet P-40 (NP-40) lysis buffer or luciferase reporter lysis buffer. Between 15 and 100 µg of protein extract was loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gels. For immunoprecipitation, cells were lysed in NP-40 lysis buffer and precleared with protein G beads for 1 h at 4°C. The protein concentration was determined, and then 1 to 2 mg of the extract was incubated with antibody prebound to protein G beads for 4 h or overnight at 4°C. The beads were washed twice in NP-40 lysis buffer and twice in 100 mM NaCl-1 mM EDTA-10 mM Tris (pH 8). The immunoprecipitation beads were mixed with 5x sample buffer and loaded onto an SDS-polyacrylamide gel. The gels were transferred (wet) to Protran nitrocellulose membranes, and the resulting blots were incubated first with primary antibody and then with the appropriate secondary horseradish peroxidase-conjugated antibody (Dako). The blot was exposed to hyperfilm following the use of enhanced chemiluminescence substrate solution (Amersham Life Science).

In vitro translation and in vitro immunoprecipitation. p53, p63{gamma}, and p73{alpha} were in vitro translated and labeled with [35S]methionine, and ASPP1-V5 and ASPP2-V5 were in vitro translated with cold methionine (all translations done with the TNT T7 Quick coupled transcription-translation system (Promega). The lysates containing indicated proteins were incubated at 30°C for 1 h. The anti-V5 antibody immobilized on protein G-agarose beads was added to the binding reaction mixtures and incubated on a rotating wheel at 4°C for 16 h. The beads were then washed with phosphate-buffered saline. The bound proteins were released in SDS gel sample buffer and analyzed by SDS-10% polyacrylamide gel electrophoresis. Results were visualized by autoradiography. ASPP1-V5 and ASPP2-V5 were detected by Western blotting with anti-V5 antibodies.

Construction of short interfering RNA of p63 and p73. Oligonucleotides (19 bp) derived from p63 and p73 were ligated into pSuper expression plasmids as described previously (5). The plasmids containing correct 19-bp oligonucleotides of p63 and p73 were confirmed by sequencing. The sequences of p63 and p73 sense and antisense oligonucleotides used in this study are as follows (lowercase indicates the vector sequence from pSuper; uppercase indicates the target sequence for the RNAi): for p63, 5'gatccccTGAATTCCTCAGTCCAGAGGttcaagagaCCTCTGGACTGAGGAATTCAtttttggaaa (sense) and 5'agcttttccaaaaaTGAATTCCTCAGTCCAGAGGtctcttgaaCCTCTGGACTGAGGAATTCAggg (antisense); for p73, 5'gatccccGCCGGGGGAATAATGAGGTttcaagagaACCTCATTATTCCCCCGGCttttggaaa3' (sense) and 5'agcttttccaaaaaGCCGGGGGAATAATGAGGTtctcttgaaACCTCATTATTCCCCCGGCggg3' (antisense).

Northern blots. ASPP1 and ASPP2 expression plasmids or vector-only control pcDNA3 plasmids were introduced into subconfluent Saos-2 cells using Lipofectamine, according to the manufacturer's instructions (Gibco/Invitrogen). Twenty-four hours after transfection, cells were harvested for RNA isolation using RNAzol B. For Northern blot analysis, total RNA (15 µg) was resolved on 1.4% agarose-formaldehyde gels, transferred to nylon, and probed with a 32P-labeled PUMA or PIG3 cDNA. Equal loading was confirmed by stripping and reprobing the blots with glyceraldehyde-3-phosphate dehydrogenase cDNA.


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RESULTS
 
High-level expression of ASPP1 and ASPP2 induces p53-independent apoptosis. In our previous study of ASPP family proteins, we noticed that expression of ASPP1 or ASPP2 induced small but detectable amounts of apoptosis in the p53 null cell line, Saos-2 (16). High-level expression of ASPP2 (140-fold above the endogenous ASPP2 level) also caused apoptosis in 293 cells, where wild-type p53 was inactivated by an adenovirus protein E1B, suggesting that ASPP may induce apoptosis independently of p53 when expressed at high levels (2). To demonstrate that ASPP1 and ASPP2 can induce apoptosis independently of p53, increasing amounts of plasmids expressing ASPP1 or ASPP2 were introduced into two p53 null cell lines, Saos-2 and H1299. In Saos-2 cells, the expression of ASPP1 or ASPP2 caused a two- to threefold increase in apoptosis (Fig. 1A), while in H1299 cells, the number of apoptotic cells detected in ASPP1- or ASPP2-expressing cells was three- to sevenfold higher than that of cells transfected with vector alone (Fig. 1B). Moreover, the proapoptotic function of ASPP1 and ASPP2 was significantly increased in both Saos-2 and H1299 cells exposed to cisplatin (Fig. 1C and D). Hence, high-level expression of ASPP1 or ASPP2 plays an important role in inducing apoptosis independently of p53. This property of ASPP1 and ASPP2 is more pronounced in response to DNA damage and is partly due to the ability of cisplatin to induce the expression levels of endogenous p63 and p73 but not ASPP1 and ASPP2 (Fig. 1E).

ASPP1 and ASPP2 interact with p63 and p73. ASPP1 and ASPP2 interact with the DNA binding domain of p53 and stimulate its apoptotic function (16). As the most homologous region among all p53 family members is their DNA binding domain, ASPP could also interact with p63 and p73 and influence their apoptotic function. Consistent with this, five of nine p53 residues reported to bind the C terminus of ASPP2, 53BP2 (10) are present in p63 and p73 (Fig. 2A), suggesting that ASPP1 and ASPP2 may interact with p63 and p73. Saos-2 and H1299 cells express the p53 family members p63 and p73, both of which are known to induce apoptosis. Thus, at least part of the proapoptotic property of ASPP1 and ASPP2 seen in Saos-2 and H1299 cells could be mediated by p63 and p73. This hypothesis was first tested using in vitro-translated ASPP1, ASPP2, and p53 family members p63 and p73. We chose the transcriptionally active isoforms of p63 and p73, p63{gamma} and p73{alpha}, to represent the family members. As shown in Fig. 2B, p53, p63{gamma}, or p73{alpha} were coimmunoprecipitated by antibodies specific to ASPP1 or ASPP2, suggesting that ASPP interacts with p63{gamma} or p73{alpha} in vitro (Fig. 2B and C). However, less p73{alpha} was in complex with ASPP1 and ASPP2 than that seen with p53 and p63{gamma}. The background band seen in Fig. 2B and C could be caused by the presence of ASPP in reticulocyte lysate.



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  		FIG. 2. ASPP1 and ASPP2 interact with p63 and p73 in vitro and in vivo. (A) Sequence comparison of the DNA binding domains (DBDs) of p53, p63, and p73 reveals that the majority of the residues involved in ASPP binding are conserved. p53, p63, and p73 sequences were obtained from GenBank and aligned using CLUSTAL W. The ASPP contact residues of human p53 (Hp53) are indicated with numbered arrows as follows: 1, H178; 2, H179; 3, R181; 4, S183; 5, S241; 6, M243; 7, N247; 8, R248; and 9, R273. ASPP1 and ASPP2 interact with p53 and its family members in vitro (B and C). p53, p63{gamma}, and p73{alpha} were in vitro translated and labeled with [35S]methionine. V5-tagged ASPP1 and ASPP2 proteins were in vitro translated with cold methionine and immunoprecipitated with anti-V5 antibody. The immunoprecipitates were fractionated on SDS-10% polyacrylamide gels. The presence of radiolabeled p53, p63{gamma}, or p73{alpha} complexed with ASPP1 or ASPP2 was detected by autoradiography, and the amount of ASPP1 and ASPP2 immunoprecipitated was detected using anti-V5 antibody by Western blotting.

Interaction between ASPP and p63{gamma} and p73{alpha} was further studied in vivo in H1299 and Saos-2 cells. Endogenous ASPP1 and ASPP2 were immunoprecipitated by antibodies specific to ASPP1 or ASPP2 (Fig. 3). Consistent with our in vitro observation, the anti-ASPP1 (Fig. 3A and B) and anti-ASPP2 (Fig. 3C and D) antibodies were able to coimmunoprecipitate endogenous p63{gamma} and p73{alpha} in both H1299 and Saos-2 cells. Under the same conditions, the control antibody Gal4 failed to coimmunoprecipitate p63{gamma} or p73{alpha}. All these results suggested that ASPP1 and ASPP2 can interact with p63 and p73 in vitro and in vivo.



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  		FIG. 3. ASPP1 and ASPP2 interact with p63{gamma} and p73{alpha} in vivo. Samples (2 mg) of H1299 or Saos-2 cell lysates were immunoprecipitated with rabbit anti-ASPP1 ASPP1.88 and anti-ASPP2 BP77 antibodies, respectively. The immunoprecipitates were separated on SDS-polyacrylamide gels, and the presence of p63{gamma} and p73{alpha} on the immunoblots was detected by mouse anti-p63 4A4 and anti-p73 ER-15 monoclonal antibodies, respectively. The presence of ASPP1 or ASPP2 was detected with mouse monoclonal antibody LX011 or DX5410, respectively. In vitro-translated (IVT) p63{gamma} and p73{alpha} lysates were used as positive controls for the detection of antibody. IP, immunoprecipitation; Ab, antibody.

ASPP1 and ASPP2 specifically stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 and p21WAF-1/CIP1. Binding of ASPP1 and ASPP2 to p53 stimulates the transactivation function of p53 on promoters of proapoptotic genes, such as Bax, PIG3 and PUMA. Therefore, we investigated whether the binding of ASPP1 or ASPP2 could also increase the transactivation function of p63 and p73. Bax, PIG3, PUMA, mdm2, and p21WAF-1/CIP1 promoters were used to measure the transactivation function of p53, p63{gamma}, or p73{alpha}. As shown in Fig. 4, the expression of ASPP1 and ASPP2 clearly enhanced the ability of p63 to transactivate the promoters of Bax (data not shown) and PIG3 (Fig. 4A and B). However, under the same conditions, the ability of ASPP1 and ASPP2 to enhance the transactivation function of p73 is much less profound. To compare the degree of activation of different p53 family members by ASPP1 and ASPP2, we divided the luciferase counts derived from each p53 family member plus ASPP by that of the p53 family member alone. This calculation showed that the ability of ASPP1 and ASPP2 to stimulate the transactivation function of p53 is greater than that seen with p63{gamma} and p73{alpha} (Fig. 4C). Expression of ASPP1 stimulated the transactivation function of p53 by ca. sevenfold, and it stimulated the transactivation function of p63{gamma} and p73{alpha} by five- and threefold, respectively, on both Bax and PIG3 promoters. Coexpression of ASPP2 with p53 enhanced the transactivation function of p53 on the promoters of Bax and PIG3 20- and 10-fold. On the Bax promoter, however, the expression of ASPP2 enhanced the transcriptional activity of p63g and p73{alpha} by only seven- and sixfold, respectively. The ability of ASPP2 to enhance the transactivation function of p63 and p73 on the PIG3 promoter is less pronounced than that seen on BAX promoter. Coexpression of ASPP1 and ASPP2 failed to stimulate the transactivation function of p53, p63{gamma}, and p73{alpha} on the promoters of mdm2 and p21WAF-1/CIP1 (Fig. 4C). This is consistent with our previous observation that ASPP1 and ASPP2 specifically stimulate the transactivation function of p53 on the promoters of Bax and PIG3 but not mdm2 and p21WAF-1/CIP1 (16).



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  		FIG.4. ASPP1 and ASPP2 can specifically stimulate the transactivation function of p53 family members on the promoters of proapoptotic genes, such as Bax, PIG3, and PUMA but not mdm2 and p21WAF-1/CIP1. (A and B) The bar graphs show the effects of ASPP1 and ASPP2 on the transactivation (TA) function of p53, p63{gamma}, or p73{alpha} on the PIG3 promoter. luc, luciferase. (C) The fold increase in p53, p63{gamma}, or p73{alpha} transactivation activity by either ASPP1 or ASPP2 on five luciferase (luc) reporters of p53 target genes, Bax, PIG3, PUMA, mdm2, and p21WAF-1/CIP1, is shown. The fold increase was calculated as follows: (activity of p53 family members plus ASPP)/(activity of p53 family members alone). The expression levels of various transfected proteins, ASPP1 (V5 tagged), ASPP2, p53, p63, and p73, were detected using 40 µg of the respective lysates using antibodies V5, DX.5410, DO1, 4A4, and ER-15, respectively. The results were derived from at least three independent experiments.

It seems that ASPP1 and ASPP2 had very little effect on the transactivation function of p73 on the promoters of Bax and PIG3. Interestingly, however, the expression of ASPP1 and ASPP2 had the biggest impact in stimulating the transactivation function of p73 on the PUMA promoter (Fig. 4C). Moreover, under the same conditions, ASPP1 and ASPP2 hardly enhanced the transactivation function of p53. This is in contrast to that seen on the promoters of Bax and PIG3. All these observations suggested that ASPP1 and ASPP2 are common activators of the p53 family members. Furthermore, cellular regulators of the p53 family members, such as ASPP1 and ASPP2, play an important role in determining the selectivity of individual p53 family members in controlling the expression of their target genes in vivo.

ASPP1 and ASPP2 induce the expression of endogenous PIG3 and PUMA. The ability of ASPP1 and ASPP2 to induce expression of endogenous target genes of p53 family members, such as PIG3 or PUMA, was investigated in Saos-2 cells. As shown in Fig. 5A, expression of ASPP1 or ASPP2 induced expression of endogenous PIG3 and PUMA mRNA. Increased expression of ASPP1 and ASPP2 also enhanced expression of PIG3 protein in both Saos-2 and H1299 cells (Fig. 5B). Under the same conditions, the expression of other p53 target genes, mdm2 and p21WAF-1/CIP1, was not changed. All these observations demonstrated that ASPP1 and ASPP2 could specifically enhance expression of apoptosis-related p53 target genes, such as PUMA and PIG3, independently of p53. This property of ASPP1 and ASPP2 is likely to be mediated by p63 and p73, as ASPP1 and ASPP2 interact with and stimulate the transcriptional activities of p63 and p73.



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  		FIG. 5. Increasing amounts of ASPP1 and ASPP2 induce the expression of two endogenous p53 target genes, PIG3 and PUMA, at both RNA (A) and protein (B) levels in Saos-2 and 1299 cells independently of p53. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ASPP1 and ASPP2 enhance the apoptotic function of p63 and p73. Since expression of ASPP1 or ASPP2 stimulated the transactivation function of p63 and p73 on promoters of proapoptotic genes, such as Bax, PIG3, and PUMA, it is likely that ASPP1 and ASPP2 also stimulate the apoptotic function of p63 and p73. This issue was addressed in Saos-2 cells using transfected p63{gamma} and p73{alpha}. Increasing amounts of p63{gamma} and p73{alpha} were transfected into Saos-2 cells to induce apoptosis to an extent similar to that seen with p53. Coexpression of ASPP1 and ASPP2 clearly enhanced the apoptotic function of p63{gamma} and p73{alpha} (Fig. 6). Interestingly, the extent of increase in the apoptotic function of p63{gamma} and p73{alpha} is lower than that seen with p53. This is in agreement with the results shown in Fig. 4, where ASPP1 and ASPP2 tend to stimulate the transactivation function of p53 better than p63{gamma} and p73{alpha} on the promoters of Bax and PIG3 genes.

The p53-independent apoptotic function of ASPP1 and ASPP2 is mediated by p63 and p73. Being activators of p63 and p73, perhaps the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated by p63 and p73. This hypothesis was tested using an RNA interference approach to inhibit the activity of endogenous p63 and p73 in Saos2 and H1299 cells. The effectiveness and specificity of p63 and p73 RNAi were first tested. As shown in Fig. 7A, coexpression of p63 and p73 RNAi specifically inhibited the apoptosis induced by p63{gamma} or p73{alpha}, respectively, in both Saos-2 and H1299 cells. Under the same conditions, p53 RNAi did not have any effects on the apoptotic function of p63 and p73. The ability of p63 and p73 RNAi to reduce the protein expression of p63 and p73 is also evident (Fig. 7B). Cotransfection of p63 or p73 RNAi to reduce the expression of endogenous p63 or p73 significantly reduced the apoptotic function of ASPP1 and ASPP2 in both Saos-2 and H1299 cells, demonstrating that in the absence of p53, ASPP1 and ASPP2 induce apoptosis via endogenous p63 and p73 (Fig. 7C). When p63 and p73 RNAi were coexpressed together, almost 80% of the apoptotic function of ASPP1 and ASPP2 was inhibited. Finally, the ability of ASPP1 and ASPP2 to enhance the apoptotic function of p63 and p73 in response to DNA damage is also evident, since the expression of RNAi of p63 or p73 but not p53 significantly reduced the number of apoptotic cells (Fig. 7D). These findings illustrated that ASPP1 and ASPP2 are common activators of all p53 family members and that most of the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated by p63 and p73.


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DISCUSSION
 
We show here that ASPP1 and ASPP2 are common activators of p53 family members. By binding to the most conserved and homologous region of the p53 family members, the DNA binding domain, ASPP1 and ASPP2 specifically stimulate the transactivation function of p53 family members on the promoter of Bax but not of mdm2. Consequently, they increase the apoptotic function of p53 family members. To our knowledge, ASPP1 and ASPP2 are the first two activators identified so far that have similar biological impacts on all p53 family members. This is particularly important, as the effects of many known cellular regulators of p53, such as p300/CBP, WT1, and c-Abl were shown only on p73 (1, 3, 9, 17, 23, 24). Importantly, a number of p53 regulators, including mdm2 and mdmx, have different or opposite effects on the activities of p53 family members (4, 8) (6). Even proteins like simian virus 40 large T antigen that bind to the most homologous region of p53 family members, the DNA binding region of p53, failed to bind and regulate the activity of p73 (7, 8, 15). All these findings reinforce the biological differences of the individual p53 family members and illustrate the complexity of cellular regulation of p53 family members. However, the demonstration that ASPP1 and ASPP2 are common activators of all p53 family members provides the first evidence that some cellular regulation of p53 can apply to all the family members.

Nevertheless, the ability of ASPP1 and ASPP2 to increase these functions of p53 family members is not identical. The ability of ASPP1 and ASPP2 to stimulate the transactivation and apoptotic functions of p53 is slightly higher than that seen on p63{gamma} and p73{alpha}. Interestingly, however, while ASPP1 and ASPP2 have very little effect on the transactivation function of p73 on the promoters of Bax and PIG3, they both had the most impact stimulating the transactivation function of p73 on the PUMA promoter (Fig. 4C). Moreover, under the same conditions, ASPP1 and ASPP2 hardly enhanced the transactivation function of p53. This suggested that p53 and p73 might not use an identical set of target genes to induce apoptosis. This observation is particularly important, since very little is known about the specific targets of p63 and p73 that are critical in inducing apoptosis. Perhaps cellular regulators of p53 family members, such as ASPP1 and ASPP2, play an important role in determining the selectivity of individual p53 family members in controlling the expression of their target genes in vivo.

Although we do not yet know the molecular mechanism of how ASPP1 and ASPP2 control the promoter specificity of individual p53 family members, it is clear that ASPP1 and ASPP2 can stimulate the apoptotic function of all members of the p53 family. This is extremely important, as in the absence of p53, ASPP1 and ASPP2 could act as activators of p63 and p73 to induce apoptosis. Hence, the expression of ASPP1 and ASPP2 could suppress tumor growth and confer cellular sensitivity to cancer treatments, even in tumors expressing mutant p53. The results also provide a molecular explanation for why reduced expression of ASPP1 and ASPP2 was observed in 23% of human breast tumors containing mutant p53, although down-regulation of ASPP1 and ASPP2 was seen in 60% of human breast tumors expressing wild-type p53 (16). Deciphering the interactions of the various p53 family members with ASPP family members is certain to offer important insights into tumorigenesis and cancer treatments.


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ACKNOWLEDGMENTS
 
We thank Gerry Melino and Frank McKeon for providing expression plasmids for p63 and p73 and Paul Farrell for reading the manuscript.

The work was mainly supported by the Ludwig Institute for Cancer Research. D.B. was supported by a grant from the Association of International Cancer Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Ludwig Institute for Cancer Research, Imperial College School of Medicine, St. Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom. Phone: 442075637710. Fax: 442077248586. E-mail: x.lu{at}ic.ac.uk. Back

{dagger} D.B. and Y.S. contributed equally to this study. Back


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Molecular and Cellular Biology, February 2004, p. 1341-1350, Vol. 24, No. 3
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.3.1341-1350.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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