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[Cancer Research 62, 2883-2889, May 15, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

p53AIP1 Regulates the Mitochondrial Apoptotic Pathway1

Koichi Matsuda, Koji Yoshida, Yoichi Taya, Kozo Nakamura, Yusuke Nakamura2 and Hirofumi Arakawa

Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 [K. M., K. Y., Y. N., H. A.], and Department of Orthopedic Surgery, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033 [K. M., K. N.], and National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045 [Y.T.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We identified recently the p53AIP1 gene, a novel p53 target that mediates p53-dependent apoptosis. In the experiments reported here, ectopic expression of p53AIP1 induced down-regulation of mitochondrial {Delta}{Psi}m and release of cytochrome c from mitochondria in human cells. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and bcl-2 proteins at mitochondria. Overexpression of bcl-2 blocked the down-regulation of mitochondrial {Delta}{Psi}m and the proapoptotic activity of p53AIP1. Our results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p53AIP1 gene, a novel target for the p53 tumor suppressor, generates three transcripts ({alpha}, ß, and {gamma}) by alternative splicing, encoding peptides of 124, 86, and 108 amino acids, respectively (1) . Because p53AIP1{alpha} and p53AIP1ß are localized at mitochondria, they are likely to regulate mitochondrial membrane potential. Expression of this gene is inducible by Ser-46-phosphorylated p53 in response to severe DNA damage, and evidence gathered to date suggests that p53AIP1 is indispensable for p53-dependent apoptosis to occur (1) .

The p53 gene is mutated more frequently than any other gene in cancers of various types. It encodes a transcription factor that binds to specific DNA sequences in its target genes and transactivates their transcription (2) . Cell-cycle arrest and induction of apoptosis generally have been considered the two most important functions of the p53 gene product (3 , 4) . However, we isolated recently a new p53 target, p53R2, which is involved in DNA repair (5) ; that discovery provided solid evidence that p53 plays another important role, that of maintaining integrity of the genome. Therefore, p53 might determine cell fates by selecting its target genes, and specific modifications of p53 protein might be essential for this phenomenon to occur (6, 7, 8) . We have already shown that phosphorylation of p53 at the Ser-46 residue is important for induction of p53AIP1 and for p53-dependent apoptosis (1) .

Bcl-2, a mitochondrial protein, inhibits the apoptotic process and promotes cell survival (9, 10, 11, 12) . Initially it was isolated as an oncogene that was activated by chromosomal translocation in human follicular lymphomas (13, 14, 15) . In the nematode Caenorhabditis elegans, ced-3 and ced-4 are essential for apoptosis during development, and ced-9 prevents their action (16 , 17) . Because bcl-2 is the functional and structural human homologue of ced-9 (18) , this mechanism of apoptosis appears to be remarkably well conserved. Although the mechanism of bcl-2 action is largely unknown, the gene product may, directly or indirectly, prevent the release of cytochrome c from mitochondria (19, 20, 21) . Bcl-2 contains four major functional domains, BH1, BH2, BH3, and BH4 (22) . More than 17 members of the bcl-2 family have been reported as anti- or proapoptotic players; all of them possess at least one of the four domains.

Although p53-dependent apoptosis is thought to be the most important feature of tumor suppression by p53, a large part of that mechanism remains to be explained. Several target genes have been isolated as attractive candidates for p53-dependent apoptosis, including bax (23) , PIG3 (24 , 25) , Killer/DR5 (26) , Fas (27 , 28) , Noxa (29) , PERP (30) , and PUMA (31 , 32) . Bax, Noxa, and PUMA are mitochondrial proteins and members of the bcl-2 family, because they contain the BH-3 domain. PIG3 is homologous to TED2, a plant NADPH oxidoreductase, which is involved in the apoptotic process necessary for formation of plant meristems. Killer/DR5 and Fas are receptors that mediate external death signals. PERP is a cellular plasma-membrane protein, and its overproduction induces apoptosis. Each of these known targets is an attractive candidate for mediating p53-dependent apoptosis, but none by itself can clearly account for exactly how p53 induces the apoptotic process.

To clarify the molecular mechanism of p53-dependent apoptosis, we examined the role of p53AIP1 further. We report here that p53AIP1 induces release of cytochrome c from mitochondria and that it interacts with bcl-2, affecting p53AIP1-mediated apoptosis through regulation of the mitochondrial membrane potential. Moreover, our experiments provide evidence to suggest that gene therapy involving p53AIP1 may be more effective for the treatment of some cancers than the use of p53 itself.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Human cancer cell lines SW480 (colorectal adenocarcinoma), H1299 (lung carcinoma), MCF7 (breast carcinoma), HeLa (cervical carcinoma), Saos-2 (osteosarcoma), HCT116 (colorectal adenocarcinoma), LS174T (colorectal adenocarcinoma), A549 (lung carcinoma), MKN45 (gastric carcinoma), HEPG2 (hepatoblastoma), TERA2 (malignant embryonal carcinoma), and DBTRG-05MG (glioblastoma) were purchased from American Type Culture Collection. T98G (glioblastoma) and LU99A (lung carcinoma) were purchased from the Human Science Research Resource Bank (Osaka, Japan). All of the cells were cultured under conditions recommended by their respective depositors.

Apoptosis-inducing Treatments.
Cells seeded 24 h before treatment were 60–70% confluent at the time of treatment. To examine the expression of p53AIP1 in response to apoptotic stresses, MCF7 cells were continuously incubated with STS3 or TNF-{alpha}, or treated with UV at selected dosages (J/m2) using a UV cross-linker (Stratagene). Floating and adherent cells were collected for Western blotting, FACS analysis and RT-PCR. Incubation times and dosages were as follows: for RT-PCR and Western blotting, STS 36 h, TNF-{alpha} and UV 48 h. For FACS and terminal deoxynucleotidyl transferase-mediated nick end labeling assay, STS (0.5 µM) and UV (50 J/m2) 36 h, TNF-{alpha} (10 ng/ml) 72 h.

Antibodies.
Antibodies used in the experiments included rabbit polyclonal antibody to HA (Medical & Biological Laboratories; 561), mouse monoclonal antibody to bcl-2 (Santa Cruz; sc-509), mouse monoclonal antibody to p21WAF1 (Calbiochem; OP64), mouse monoclonal antibody to p53 (Calbiochem; OP43), rabbit polyclonal antibody to phosphorylated p53-serine 46 (1) , mouse monoclonal antibody to mitochondria mitofilin (Calbiochem: NB11L), and mouse monoclonal antibody to cytochrome c (Santa Cruz: sc-7159).

Semiquantitative RT-PCR Analysis.
Total RNA was isolated from cells using RNeasy spin-column kits (Qiagen) according to the manufacturer’s instructions. cDNAs were synthesized from 5 µg total RNAs with the SuperScript Preamplification System (Life Technologies, Inc.). The RT-PCR exponential phase was determined on 15–30 cycles to allow semiquantitative comparisons among cDNAs developed from identical reactions. Each PCR regime involved a 2-min initial denaturation step at 94°C, followed by 33 cycles (for p53AIP1), 24 cycles (for Noxa and PIG3), 25 cycles (for KILLER/DR5), 21 cycles (for Bax), or 18 cycles (for ß2MG) at 94°C for 30 s, 55–59°C for 30 s, and 72°C for 1 min, on a Gene Amp PCR system 9600 (Perkin-Elmer). Primer sequences were, for p53AIP1: F, CCA AGT TCT CTG CTT TC and R, AGC TGA GCT CAA ATG CTG AC; for PIG3: F, GCA GCT GCT GGA TTC AAT TAC and R, GCC TAT GTT CTT GTT GGC CTC; for Noxa: F, AGG ACT GTT CGT GTT CAG CTC and R, GTG CAC CTC CTG AGA AAA CTC; for KILLER/DR5: F, CCA ACA GGT GTC AAC ATG TTG and R, CAA TCT TCT GCT TGG CAA GTC; and for Bax: F, GGA GCT GCA GAG GAT GAT TG and R, CCA CAA AGA TGG TCA CGG TC. PCR products were separated by electrophoresis on 2.5% agarose gels.

Detection of Apoptosis.
For FACS analysis, adherent and detached cells were combined and fixed with 75% ethanol at 4°C. After two rinses with PBS, cells were incubated for 30 min with 1 ml of PBS containing 1 mg of boiled RNase at 37°C. Cells were then stained in 1 ml of PBS containing 10 µg of propidium iodide. The percentages of sub-G1 nuclei in the population were determined from at least 2 x 104 cells in a flow cytometer (FACScalibur; Becton Dickinson).

Immunocytochemistry.
Cos-7 cells were seeded 24 h before transfection. Cells were cotransfected with pcDNA3.1-bcl-2 and pCAGGS-HA-p53AIP1 with Fugene 6 Reagent (Roche). Later (24 h), cells were fixed with 100% methanol for 10 min, washed once with PBS, and covered with blocking solution (3% BSA in 0.05% Tween 20 in TBS) for 60 min at room temperature to block nonspecific binding sites. Then the cells were incubated with rabbit anti-HA antibody and mouse anti-bcl-2 antibody for 1 h at room temperature. The antibodies were stained with a goat antirabbit secondary antibody conjugated to FITC or a goat antimouse secondary antibody conjugated to Texas Red, and viewed with an ECLIPSE 600 microscope (Nikon).

ASs.
To inhibit expression of endogenous p53AIP1, we prepared high-performance liquid chromatography-purified AS (AS1: TCCCCTGGATGGGATC) and, as a control, sense oligonucleotide (SE1: GATCCCATCCAGGGGA) according to the sequence of the p53AIP1 gene. AS1 (1 µM) was transfected with Lipofectin reagent (Life Technologies, Inc.) for 4 h, and then cells were treated with UV irradiation (50 J/m2) or incubated with 0.5 µM STS or 10 ng/ml TNF-{alpha}. Apoptotic cells were analyzed by FACS analysis 36 h after treatment.

Immunoprecipitation and Western Blot Analysis.
COS7 cells were seeded (2 x 106 cells/10-cm dish) before treatment and transfected with 8 µg of plasmid mixed with Fugene 6 reagent (Roche). Cells were collected 24 h after transfection, lysed in NP40 based lysis buffer (0.5% NP40, 150 mM NaCl, 20 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride) for 1 h, and cleared of nuclear and cellular debris by centrifugation. Immunoprecipitations were performed using rabbit anti-HA antibody (polyclonal) or mouse anti-bcl-2 antibody (monoclonal) plus protein G-Sepharose. The precipitates were washed four times in lysis buffer and proteins were eluted with Laemmli sample buffer (Bio Rad). Immunoblotting was performed as described previously (1) .

Recombinant Adenoviruses and Infection.
To construct Ad-p53AIP1 or Ad-p53 virus, blunt-ended fragments of p53AIP1 or p53 cDNA were inserted into the Swa1 site of the cosmid pAxCAwt (Takara), which contains the CAG promoter and the entire genome of type 5 adenovirus except for the E1 and E3 regions. These procedures generated pAxCAp53AIP1 or pAxCAp53. The constructs were confirmed by sequencing. Recombinant adenoviruses were constructed by in vitro homologous recombination in the human embryonic kidney cell line 293 using pAxCAp53AIP1 or pAxCAp53 with the adenovirus DNA terminal protein complex (Takara). Viruses were propagated in the HEK293 cells and purified by two rounds of CsCl density centrifugation. Viral titers were measured in a limiting-dilution bioassay using the HEK293 cells. Diluting viral stock to certain concentrations, adding viral solutions to cell monolayers, and incubating at 37°C for 60 min with brief agitation every 20 min successfully infected all of the cell lines (33) . Culture medium was added, and infected cells were incubated at 37°C.

Detection of Mitochondrial Membrane Potential.
Cells were plated at densities of 5 x 105 cells/6-cm dish and infected 24 h later with Ad-p53AIP1{alpha}, Ad-p53, or Ad-LacZ at an MOI of 100 plaque-forming units/cell. Trypsinized adherent and floating cells were collected 60 h after infection and washed twice with cold PBS. Rhodamine 123 in PBS (10 nM) was added, and the cells were incubated for 30 min at 37°C. Fluorescence was measured by flow cytometry.

Preparation of Subcellular Fractions.
Cells were washed twice with PBS, and each pellet was suspended in 1.1 ml of hypotonic buffer [10 mM NaCl, 1.5 mM MgCl2, and 10 mM Tris-HCl (pH 7.5)]. The cells were homogenized by 10 strokes in a 21-gauge needle. Then 0.8 ml of 2.5X MS Buffer [25 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl (pH 7.5), and 2.5 mM EDTA (pH 7.5)] was added to each lysate. The homogenates were centrifuged three times at 1,300 x g for 5 min at 4°C to remove nuclei and debris. The supernatants were centrifuged at 17,000 x g for 15 min at 4°C to collect mitochondria. The resulting supernatants were centrifuged at 100,000 x g for 1 h at 4°C to prepare the final supernatants referred to as cytosolic fractions.

Establishment of HeLa Cells Stably Overexpressing bcl-2.
HeLa cells were cotransfected with pcDNA3.1-bcl-2 and pCMV-puromycin at a ratio of 5:1 with Fugene 6 Reagent (Roche). Transfected cells were selected in medium containing 1.25 µg/ml puromycin for 20 days, and bcl-2 expression was determined by immunocytochemistry and immunoblotting. Cells stably expressing bcl-2 were referred to as HeLa-bcl-2 cells.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Involvement of p53AIP1 in the Mitochondrial Apoptotic Pathway.
To explore whether p53AIP1 is a common mediator for different apoptotic pathways, we examined its function with regard to three different apoptosis-stimulators, STS, TNF-{alpha}, and UV irradiation. TNF-{alpha} stimulates apoptosis via the death-receptor pathway independent of the mitochondrial pathway (34) . UV and STS induce apoptosis through the mitochondrial pathway, down-regulating mitochondrial membrane potential and triggering release of cytochrome c (35, 36, 37) . As shown in Fig. 1ACitation , expression of p53AIP1 increased significantly during apoptosis induced by STS or UV irradiation but not in response to TNF-{alpha}. The expression of p53AIP1 mRNA reached the peak at 0.1 µM of STS, in concert with Ser46-phosphorylation of p53 (Fig. 1B)Citation , suggesting that the transcription of p53AIP1 might be regulated by the modification of p53 as described previously (1) . Bax and PIG3 also were induced by STS and UV damage, although a dose dependency of those expressions in response to UV irradiation were not observed in contrast to that of p53AIP1. Expression of Killer/DR5 mRNA was elevated during apoptosis induced by STS and TNF-{alpha}, whereas Noxa was induced only when apoptosis was triggered by UV irradiation. In contrast, when we used H1299 cells (p53-/-), none of the three apoptosis stimulators induced expression of p53AIP1despite induction of p53-independent apoptosis (data not shown). These results suggested that p53AIP1 is involved specifically in a mitochondrial apoptotic pathway and in a p53-dependent manner (Fig. 1C)Citation .



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  		Fig. 1. Expression of p53AIP1 via different apoptotic pathways. MCF7 cells were collected 36 h after STS damage and 48 h after UV or TNF-{alpha} damage for RT-PCR (A) and Western blotting (B). Expression of ß2MG or ß-actin served as a quality control. C, induction of apoptosis in MCF7 cells collected after continuous incubation with 0.5 µM STS for 36 h or 10 ng/ml TNF-{alpha} for 72 h, or 36 h after a 50 J/m2 dose of UV irradiation. The apoptotic cells were indicated as the sub-G1 fraction in FACS analysis. D, inhibition of p53AIP1 expression by AS. AS or sense-oligonucleotides (SE; 1 µM each) were transfected to MCF7 cells with Lipofectin reagent (Life Technologies, Inc.) for 4 h, then cells were damaged by UV (50 J/m2). After transfection (36 h), expression of p53AIP1 was examined by RT-PCR. Control indicated the basal level of p53AIP1expression in MCF7 cells without DNA damage and the treatment of antisense- or sense-oligonucleotide. E, inhibition of p53AIP1 expression by AS block STS- and UV-dependent apoptosis, as measured by FACScan (sub-G1 fraction). AS or SE (1 µM) were transfected to MCF7 cells with Lipofectin reagent (Life Technologies, Inc.) for 4 h, and then cells were treated with UV, STS, or TNF-{alpha}. The experiments were repeated at least three times in duplicate samples and the average scores are shown with error bars; bars, ± SD.

 
To confirm the involvement of p53AIP1 in apoptotic processes, we performed experiments using an AS to the p53AIP1 gene. Pretreatment of MCF7 cells with AS caused a remarkable reduction in the level of p53AIP1 expression, as compared with sense-oligonucleotide after UV irradiation (Fig. 1D)Citation . AS also reduced the apoptotic (sub-G1) cell population from 28% to 16% after UV irradiation or from 35% to 27% after treatment with STS; however, AS treatment of MCF7 cells did not inhibit cell death induced by TNF-{alpha} (Fig. 1E)Citation .

p53AIP1 Regulates Mitochondrial {Delta}{Psi}m and Release of Cytochrome c from Mitochondria.
Schuler et al. (38) showed that introduction of the p53 gene could induce down-regulation of mitochondrial membrane potential and promote release of cytochrome c. Other p53 downstream genes such as Noxa, PUMA, and Bax, also enhance release of cytochrome c from mitochondria (29 , 31 , 39) . To clarify the molecular mechanism of p53AIP1-inducible apoptosis, we examined the effect of overexpression of p53AIP1 on these phenomena. H1299 (lung carcinoma) and Saos-2 (osteosarcoma) cells were infected with Ad-p53AIP1{alpha}, and then the levels of mitochondrial {Delta}{Psi}m were examined by staining with Rhodamine 123. Although the expressions of exogenous p53AIP1 by infection of Ad-p53AIP1 were observed in both cell lines, apoptosis was induced in H1299 cells by Ad-p53AIP1{alpha}, but Saos-2 cells were resistant to p53AIP1 gene transfer; down-regulation of mitochondrial {Delta}{Psi}m was observed only in H1299 cells 48 h after infection (Fig. 2A)Citation . This result indicated that the ability of p53AIP1 to induce apoptosis might depend on the cell type as observed in case of other apoptotic regulators such as Bax. To investigate the subcellular location of cytochrome c, subcellular fractions were collected at the times indicated in Fig. 2BCitation , and cytosolic and mitochondrial fractions were subjected to Western blotting with anti-cytochrome c antibody. A remarkable translocation of cytochrome c from mitochondria to cytosol was observed 48 h after infection with Ad-p53AIP1{alpha}.



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  		Fig. 2. Regulation of mitochondrial {Delta}{Psi}m and release of cytochrome c by p53AIP1. A, H1299 and Saos-2 cells were infected with Ad-p53AIP1{alpha} or Ad-LacZ at an MOI of 100 plaque-forming units/cell. The levels of mitochondrial {Delta}{Psi}m (48 h after infection) and the population of apoptotic cells (96 h after infection) were examined by staining with Rhodamine 123 and FACS analysis, respectively (top). The expressions of exogenous p53AIP1 protein were shown by Western blot (bottom). B, release of cytochrome c from mitochondria by overexpression of p53AIP1. HCT116 cells were infected with Ad-p53AIP1{alpha} or Ad-LacZ at an MOI of 100 and collected at the indicated times. Cytosolic and mitochondrial fractions were each subjected to immunoblotting. The expression of mitofilin or ß-actin was examined as a quality control or a quantity control, respectively.

 
Potential Interaction of p53AIP1 and bcl-2.
Overexpression of bcl-2 can block p53-dependent dissipation of mitochondrial {Delta}{Psi}m and apoptosis (40 , 41) . Because p53AIP1 and bcl-2 are mitochondrial components related to apoptosis, we speculated that these two proteins might influence the apoptotic process by interacting. Expression vectors containing HA-p53AIP1{alpha} or bcl-2 were cotransfected into COS7 cells. Protein complexes containing p53AIP1{alpha} or bcl-2 were immunoprecipitated from cell extracts with rabbit polyclonal anti-HA antibody or mouse monoclonal anti-bcl-2 antibody, respectively. Western blots indicated that the immune complex precipitated with either antibody included both bcl-2 and HA-p53AIP1{alpha} proteins (Fig. 3A)Citation . Specific interaction between HA-p53AIP1ß and bcl-2 was confirmed in the same manner (data not shown).



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  		Fig. 3. Physical interaction of p53AIP1 and bcl-2. A, bcl-2 and HA-p53AIP1{alpha} expression vectors were cotransfected into COS7 cells; 24 h later cell extracts were isolated. The protein complex with bcl-2 or p53AIP1 was immunoprecipitated (IP) with anti-bcl-2 antibody or anti-HA antibody and analyzed on immunoblots (IB). WCL, whole cell lysate. B, colocalization of bcl-2 and p53AIP1. bcl-2 and HA-p53AIP1{alpha} expression vectors were cotransfected into COS7 cells; 24 h later the cells were double-stained with mouse monoclonal anti-bcl-2 antibody (red) and rabbit polyclonal anti-HA antibody (green). Both signals are overlaid in the double-color image as a yellow signal.

 
We then investigated the subcellular locations of p53AIP1 and bcl-2 proteins. After (24 h) the expression vectors were transfected into COS7 cells, the harvested cells were double-stained with mouse monoclonal anti-bcl-2 antibody and rabbit polyclonal anti-HA antibody. p53AIP1 was stained (green) in the cytoplasm as a granular pattern, as was bcl-2 (red); both signals were clearly overlaid in the double-color image as a yellow signal (Fig. 3B)Citation . Both proteins were confirmed to be located at mitochondria (data not shown).

Functional Interplay of p53AIP1 and bcl-2.
As shown in Fig. 2Citation , our experiments clearly indicated that p53AIP1 itself regulated mitochondrial {Delta}{Psi}m and triggered the release of cytochrome c. Bcl-2 is well known as an important regulator for mitochondrial {Delta}{Psi}m that also blocks the release of cytochrome c from mitochondria. These facts and the interaction of p53AIP1 and bcl-2 at mitochondria prompted us to speculate that p53AIP1 itself might regulate mitochondrial {Delta}{Psi}m and trigger the release of cytochrome c by interacting with bcl-2. To test this hypothesis, we examined whether bcl-2 could inhibit p53AIP1-induced apoptosis and the change of mitochondrial {Delta}{Psi}m. A bcl-2 expression vector was transfected into HeLa cells, and two independent cell lines (HeLa-bcl2-1 and HeLa-bcl2-2) that stably expressed bcl-2 were established. As shown in Fig. 4ACitation , apoptosis was inducible in HeLa-mock parental cells (i.e., integrated with the control vector only, pcDNA3.1) by infection with Ad-p53AIP1{alpha} or Ad-p53 but not Ad-LacZ. However, when two HeLa cell lines overexpressing bcl-2 were infected with Ad-p53AIP1{alpha} or Ad-p53 the number of apoptotic cells decreased significantly, suggesting that bcl-2 was able to inhibit the apoptotic pathway that involves p53AIP1 and p53.



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  		Fig. 4. Functional interaction between p53AIP1 and bcl-2. A, inhibition by bcl-2 of apoptosis induced by Ad-p53 or Ad-p53AIP1{alpha}. Ad-p53AIP1{alpha}, Ad-p53, or Ad-LacZ were infected to three different cell lines, HeLa-pcDNA, HeLa-bcl-2(1), and HeLa-bcl-2(2), and apoptosis (sub-G1 fraction) was evaluated by FACS analysis 72 h after infection. The experiments were repeated at least three times in duplicate samples, and the average scores are shown with error bars; bars, ± SD. B, down-regulation of mitochondrial {Delta}{Psi}m by Ad-p53AIP1{alpha} and Ad-p53. Ad-p53AIP1{alpha}, Ad-p53, or Ad-LacZ were infected to HeLa-pcDNA, HeLa-bcl-2(1), and HeLa-bcl-2(2) cell lines. The cells were labeled 60 h after infection by Rhodamine 123, and fluorescence was measured by FACScan.

 
Next we examined the mitochondrial {Delta}{Psi}m itself. Induction of exogenous p53AIP1{alpha} and p53 proteins but not exogenous LacZ down-regulated mitochondrial {Delta}{Psi}m in HeLa-mock parental cells; however, this phenomenon was blocked in both of the bcl-2 overexpressing HeLa cell lines (Fig. 4B)Citation . This result indicated that bcl-2 can inhibit the down-regulation of mitochondrial {Delta}{Psi}m induced by either p53AIP1{alpha} or p53, and supported the notion that interaction between bcl-2 and p53AIP1 might regulate mitochondrial {Delta}{Psi}m by balancing positive and negative effects.

Antitumor Effect of p53AIP1 on Diverse Cancer Cell Lines in Vitro.
p53AIP1 was strikingly expressed in H1299 cells after Ad-p53AIP1{alpha} infection, in a time-dependent manner (Fig. 5A)Citation . To evaluate the ability of p53AIP1 to induce apoptosis of cancer cells derived from a variety of tissues, we infected Ad-p53AIP1{alpha} into six diverse cancer cell lines: HeLa, in which p53 is inactivated by E1A; T98G (p53 mutant); H1299 (p53 null); SW480 (p53 mutant); HCT116 (p53 wild-type); and MCF7 (p53 wild-type). Apoptosis was inducible by infection with Ad-p53AIP1{alpha} in all six of the cell lines regardless of their p53 status, although the extent of apoptosis varied from one line to another (data not shown). Cancer cells containing WTp53 protein are relatively resistant to p53 gene therapy (42 , 43) . Thus, we compared the antitumor effect of the p53AIP1 gene in vitro with that of the p53 gene on nine cancer cell lines that carry WTp53: HCT116 (colon cancer), LS174T (colon cancer), A549 (lung cancer), MKN45 (gastric cancer), MCF7 (breast cancer), TERA2 (teratoma), DBTRG05MG (glioma), HepG2 (Hepatocarcinoma), and Lu99A (lung cancer). All nine of these cell lines revealed 100% infectivity of Ad-LacZ vector. Ectopic expression of exogenous p53AIP1a by adenovirus-mediated gene transfer effectively caused apoptosis in six of the WTp53 cancer-cell lines (Fig. 5B)Citation . In fact, four of those six lines were killed more effectively by p53AIP1 than by p53.



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  		Fig. 5. Antitumor effect of p53AIP1 on diverse cancer-cell lines in vitro. A, expression of exogenous p53AIP1{alpha} in lung-carcinoma cells by infection with Ad-p53AIP1{alpha}. H1299 cells were collected at the indicated hours after treatment, and cell lysates were subjected to immunoblotting with anti-p53AIP1 antibody. B, apoptosis induced in nine cancer-cell lines (p53 wild type) by infection with various doses (MOI) of Ad-p53 or Ad-p53AIP1{alpha}. Apoptotic cells were analyzed by FACScan 96 h after infection. The experiments were repeated at least three times in duplicate samples and the average scores are shown with error bars; bars, ± SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In earlier studies we had isolated the p53AIP1 gene through attempts to clone p53-binding sequences directly from the whole human genome (44) . We found that when cells were severely damaged, reaching a critical condition by different genotoxic stresses, p53AIP1 mRNA was strongly induced in a p53-dependent manner and functioned to eliminate dangerous cells. We demonstrated that ectopic expression of p53AIP1 using adenovirus-mediated gene transfer could induce apoptosis to a remarkable degree in various types of cancer cells. Because high levels of p53AIP1 seemed to be cytotoxic for cells under normal conditions, we speculated that its expression must be strictly controlled by p53 protein. As we reported previously, expression of p53AIP1 was induced only by p53 that had been phosphorylated at the Ser-46 residue (1) . Those experiments suggested that the type of modification occurring in p53 protein may determine the fate of a cell, survival or death, by inducing a specific set of p53-target gene(s), e.g., p53R2 and p21 for cell survival or p53AIP1 for cell death (1 , 5 , 45 , 46) .

The discovery of p53AIP1 had provided several clues toward understanding how p53 induces apoptosis. We have demonstrated here that p53AIP1 is induced and then accumulates in mitochondria in response to UV irradiation and STS treatment but not in response to TNF-{alpha}. Inhibition of p53AIP1 expression by AS confirmed the importance of p53AIP1 accumulation in mitochondria in STS- and UV-induced apoptosis. At present two separate apoptotic pathways are known; one is the mitochondrial pathway triggered by DNA damage or STS and the other is the death-receptor pathway triggered by members of the TNF family or Fas ligand (47) . p53 is activated in both pathways, and it can induce apoptosis by regulating its downstream genes (3 , 48) . Among p53-downstream genes, Bax, Noxa, and PUMA are mediators of the mitochondrial pathway, whereas Fas and killer/DR5 mediate the death-receptor pathway. Apoptotic stimuli such as UV, STS, and TNF-{alpha} caused induction of p21WAF1 in our study, but p53AIP1 was induced only after UV or STS damage. So the expression of p53AIP1 protein is closely linked only to the first of these two pathways.

In addition, we have now shown that p53AIP1 interacts with bcl-2 at mitochondria. This evidence partly explains the molecular mechanism of p53AIP1-inducible apoptosis, because bcl-2 can block apoptotic processes induced by diverse cytotoxic insults such as {gamma} or UV irradiation, treatment with dexamethasone, STS, or cytotoxic anticancer drugs, or by withdrawal of cytokines (49) . Hence, bcl-2 functions as a negative regulator of apoptosis. Indeed, bcl-2 may be able to block p53-dependent apoptosis (40 , 41) . The fact that bcl-2 interacts with p53AIP1, a bona-fide direct target of p53, as well as with Bax, Noxa, and PUMA, is solid evidence for a functional association between bcl-2 and p53 in apoptotic regulation. Although bcl-2 is an antiapoptotic effector against a broad spectrum of apoptosis stimulators, its inhibitory effect on apoptosis through the death-receptor pathway is small (50, 51, 52) ; therefore, the death-receptor pathway is likely to bypass the apoptotic pathway controlled by bcl-2. These facts are consistent with our observation that p53AIP1 is not involved in TNF-{alpha}-induced apoptosis; i.e., in the death-receptor pathway.

Bcl-2 directly or indirectly prevents the release of cytochrome c from mitochondria. Cytochrome c forms an essential part of the apoptosome composed of Apaf-1 and procaspase-9 (53) . Bcl-2 functions as an antioxidant, preventing apoptosis by decreasing lipid peroxidation and increasing the resistance of the cell to reactive oxygen species (54) . Bcl-2 blocks reactive oxygen species production by preventing the permeability-transition pores on mitochondrial membranes from opening, an early step in the signaling cascade of apoptosis (55) , and then stabilizes the mitochondrial {Delta}{Psi}m. In our study, p53AIP1 actually induced down-regulation of the mitochondrial {Delta}{Psi}m and promoted release of cytochrome c, whereas bcl-2 blocked the proapoptotic activity of both p53AIP1 and p53. Our results indicate that regulation of mitochondrial membrane potential might be essential for p53- and p53AIP1-inducible apoptosis, and that this regulation might be determined by the physical and functional interaction between bcl-2 and p53AIP1.

Introduction of the WTp53 gene into cancer cells by adenoviral vectors is a potential therapeutic approach for human cancers, and in fact gene therapy using p53 has been undergoing clinical evaluation in patients with various types of cancer (33 , 56, 57, 58) . However, although transfer of WTp53 kills cancer cells in some cases it does not do so in others, and the factors determining this variability in effectiveness have not been identified. Some clues might be found in recent observations that ectopic expression of p53 can result in apoptosis in some cases and growth arrest in others (59) , although the mechanisms accounting for these differences remain largely unknown. The results of our earlier experiments implied that the "decision" about the fate of a cell, i.e., "arrest" or "death," partly depends on the selection of downstream target genes to be activated through specific binding to p53-target sequences, and moreover that the type of modification (phosphorylation or acetylation) of p53 protein is likely to be associated with this selectivity (1 , 7 , 8 , 60 , 61) . However, because p53AIP1 is a direct mediator of apoptosis, as indicated by the fact that p53AIP1 can induce apoptosis of various cancer cells regardless of their p53 status, the p53AIP1 gene might be more widely applicable for treatment of cancer than p53 itself. In fact, in our experiments p53AIP1 induced apoptosis effectively in both p53-sensitive and p53-resistant cancer-cell lines (Fig. 5B)Citation . Those results implied that p53AIP1 triggers apoptosis directly, whereas p53 induces growth suppression by regulating its downstream genes. Hence p53AIP1 in adenoviral vectors may become an attractive agent for gene therapy to kill cancer cells, especially p53-resistant cells that carry WTp53. However, in vivo research is still needed to define the role of p53AIP1 in human gene therapy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Grant #13216031 from the Ministry of Education, Culture, Sports, Science and Technology (to H. A.), and in part by Research for the Future Program Grant #00L01402 from The Japan Society for the Promotion of Science (to Y. N.). Back

2 To whom requests for reprints should be addressed, at Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639 Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: yusuke{at}ims.u-tokyo.ac.jp Back

3 The abbreviations used are: STS, staurosporine; TNF, tumor necrosis factor; FACS, fluorescence-activated cell sorter; RT-PCR, reverse transcription-PCR; MOI, multiplicity of infection; AS, antisense oligonucleotide; WTp53, wild-type p53. Back

Received 6/ 7/01. Accepted 3/19/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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