P53aip1 Regulates the Mitochondrial Apoptotic Pathway1

[CANCER RESEARCH 62, 2883–2889, May 15, 2002] p53AIP1 Regulates the Mitochondrial Apoptotic Pathway1 Koichi Matsuda, Koji Yoshida, Yoichi Taya, Kozo Nakamura, Yusuke Nakamura,2 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 family have been reported as anti- or proapoptotic players; all of them possess at least one of the four domains. We identified recently the p53AIP1 gene, a novel p53 target that Although p53-dependent apoptosis is thought to be the most im- mediates p53-dependent apoptosis. In the experiments reported here, portant feature of tumor suppression by p53, a large part of that ectopic expression of p53AIP1 induced down-regulation of mitochondrial ⌬⌿m and release of cytochrome c from mitochondria in human cells. mechanism remains to be explained. Several target genes have been Immunoprecipitation and immunostaining experiments indicated interac- isolated as attractive candidates for p53-dependent apoptosis, includ- tion between p53AIP1 and bcl-2 proteins at mitochondria. Overexpression ing bax (23), PIG3 (24, 25), Killer/DR5 (26), Fas (27, 28), Noxa (29), of bcl-2 blocked the down-regulation of mitochondrial ⌬⌿m and the PERP (30), and PUMA (31, 32). Bax, Noxa, and PUMA are mito- proapoptotic activity of p53AIP1. Our results implicate p53AIP1 as a chondrial proteins and members of the bcl-2 family, because they pivotal mediator of the p53-dependent mitochondrial apoptotic pathway. contain the BH-3 domain. PIG3 is homologous to TED2, a plant NADPH oxidoreductase, which is involved in the apoptotic process INTRODUCTION necessary for formation of plant meristems. Killer/DR5 and Fas are receptors that mediate external death signals. PERP is a cellular The p53AIP1 gene, a novel target for the p53 tumor suppressor, plasma-membrane protein, and its overproduction induces apoptosis. ␣ ␤ ␥ generates three transcripts ( , , and ) by alternative splicing, Each of these known targets is an attractive candidate for mediating encoding peptides of 124, 86, and 108 amino acids, respectively (1). p53-dependent apoptosis, but none by itself can clearly account for ␣ ␤ Because p53AIP1 and p53AIP1 are localized at mitochondria, they exactly how p53 induces the apoptotic process. are likely to regulate mitochondrial membrane potential. Expression To clarify the molecular mechanism of p53-dependent apoptosis, of this gene is inducible by Ser-46-phosphorylated p53 in response to we examined the role of p53AIP1 further. We report here that severe DNA damage, and evidence gathered to date suggests that p53AIP1 induces release of cytochrome c from mitochondria and that p53AIP1 is indispensable for p53-dependent apoptosis to occur (1). it interacts with bcl-2, affecting p53AIP1-mediated apoptosis through The p53 gene is mutated more frequently than any other gene in regulation of the mitochondrial membrane potential. Moreover, our cancers of various types. It encodes a transcription factor that binds to experiments provide evidence to suggest that gene therapy involving specific DNA sequences in its target genes and transactivates their p53AIP1 may be more effective for the treatment of some cancers transcription (2). Cell-cycle arrest and induction of apoptosis gener- than the use of p53 itself. ally have been considered the two most important functions of the p53 gene product (3, 4). However, we isolated recently a new p53 target, MATERIALS AND METHODS p53R2, which is involved in DNA repair (5); that discovery provided solid evidence that p53 plays another important role, that of main- Cell Culture. Human cancer cell lines SW480 (colorectal adenocarcino- taining integrity of the genome. Therefore, p53 might determine cell ma), H1299 (lung carcinoma), MCF7 (breast carcinoma), HeLa (cervical fates by selecting its target genes, and specific modifications of p53 carcinoma), Saos-2 (osteosarcoma), HCT116 (colorectal adenocarcinoma), protein might be essential for this phenomenon to occur (6–8). We LS174T (colorectal adenocarcinoma), A549 (lung carcinoma), MKN45 (gas- have already shown that phosphorylation of p53 at the Ser-46 residue tric carcinoma), HEPG2 (hepatoblastoma), TERA2 (malignant embryonal car- is important for induction of p53AIP1 and for p53-dependent cinoma), and DBTRG-05MG (glioblastoma) were purchased from American apoptosis (1). Type Culture Collection. T98G (glioblastoma) and LU99A (lung carcinoma) were purchased from the Human Science Research Resource Bank (Osaka, Bcl-2, a mitochondrial protein, inhibits the apoptotic process and Japan). All of the cells were cultured under conditions recommended by their promotes cell survival (9–12). Initially it was isolated as an oncogene respective depositors. that was activated by chromosomal translocation in human follicular Apoptosis-inducing Treatments. Cells seeded 24 h before treatment were lymphomas (13–15). In the nematode Caenorhabditis elegans, ced-3 60–70% confluent at the time of treatment. To examine the expression of and ced-4 are essential for apoptosis during development, and ced-9 p53AIP1 in response to apoptotic stresses, MCF7 cells were continuously prevents their action (16, 17). Because bcl-2 is the functional and incubated with STS3 or TNF-␣, or treated with UV at selected dosages (J/m2) structural human homologue of ced-9 (18), this mechanism of apo- using a UV cross-linker (Stratagene). Floating and adherent cells were col- ptosis appears to be remarkably well conserved. Although the mech- lected for Western blotting, FACS analysis and RT-PCR. Incubation times and anism of bcl-2 action is largely unknown, the gene product may, dosages were as follows: for RT-PCR and Western blotting, STS 36 h, TNF-␣ directly or indirectly, prevent the release of cytochrome c from mi- and UV 48 h. For FACS and terminal deoxynucleotidyl transferase-mediated nick end labeling assay, STS (0.5 ␮M) and UV (50 J/m2) 36 h, TNF-␣ (10 tochondria (19–21). Bcl-2 contains four major functional domains, ng/ml) 72 h. BH1, BH2, BH3, and BH4 (22). More than 17 members of the bcl-2 Antibodies. Antibodies used in the experiments included rabbit polyclonal antibody to HA (Medical & Biological Laboratories; 561), mouse monoclonal Received 6/7/01; accepted 3/19/02. antibody to bcl-2 (Santa Cruz; sc-509), mouse monoclonal antibody to The costs of publication of this article were defrayed in part by the payment of page p21WAF1 (Calbiochem; OP64), mouse monoclonal antibody to p53 (Calbio- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chem; OP43), rabbit polyclonal antibody to phosphorylated p53-serine 46 (1), 1 Supported in part by Grant #13216031 from the Ministry of Education, Culture, mouse monoclonal antibody to mitochondria mitofilin (Calbiochem: NB11L), Sports, Science and Technology (to H. A.), and in part by Research for the Future Program and mouse monoclonal antibody to cytochrome c (Santa Cruz: sc-7159). Grant #00L01402 from The Japan Society for the Promotion of Science (to Y. N.). 2 To whom requests for reprints should be addressed, at Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, 3 The abbreviations used are: STS, staurosporine; TNF, tumor necrosis factor; FACS, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639 Japan. Phone: 81-3-5449-5372; Fax: fluorescence-activated cell sorter; RT-PCR, reverse transcription-PCR; MOI, multiplicity 81-3-5449-5433; E-mail: [email protected]. of infection; AS, antisense oligonucleotide; WTp53, wild-type p53. 2883 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. REGULATION OF APOPTOSIS BY p53AIP1 Semiquantitative RT-PCR Analysis. Total RNA was isolated from cells propidium iodide. The percentages of sub-G1 nuclei in the population were using RNeasy spin-column kits (Qiagen) according to the manufacturer’s determined from at least 2 ϫ 104 cells in a flow cytometer (FACScalibur; instructions. cDNAs were synthesized from 5 ␮g total RNAs with the Super- Becton Dickinson). Script Preamplification System (Life Technologies, Inc.). The RT-PCR expo- Immunocytochemistry. Cos-7 cells were seeded 24 h before transfection. nential phase was determined on 15–30 cycles to allow semiquantitative Cells were cotransfected with pcDNA3.1-bcl-2 and pCAGGS-HA-p53AIP1 comparisons among cDNAs developed from identical reactions. Each PCR with Fugene 6 Reagent (Roche). Later (24 h), cells were fixed with 100% regime involved a 2-min initial denaturation step at 94°C, followed by 33 methanol for 10 min, washed once with PBS, and covered with blocking cycles (for p53AIP1), 24 cycles (for Noxa and PIG3), 25 cycles (for KILLER/ solution (3% BSA in 0.05% Tween 20 in TBS) for 60 min at room temperature ␤ DR5), 21 cycles (for Bax), or 18 cycles (for 2MG)at94°C for 30 s, 55–59°C to block nonspecific binding sites. Then the cells were incubated with rabbit for 30 s, and 72°C for 1 min, on a Gene Amp PCR system 9600 (Perkin- anti-HA antibody and mouse anti-bcl-2 antibody for1hatroom temperature. Elmer). Primer sequences were, for p53AIP1: F, CCA AGT TCT CTG CTT The antibodies were stained with a goat antirabbit secondary antibody conju- TC and R, AGC TGA GCT CAA ATG CTG AC; for PIG3: F, GCA GCT GCT gated to FITC or a goat antimouse secondary antibody conjugated to Texas GGA TTC AAT TAC and R, GCC TAT GTT CTT GTT GGC CTC; for Noxa: Red, and viewed with an ECLIPSE 600 microscope (Nikon). 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 ASs. To inhibit expression of endogenous p53AIP1, we prepared high- R, CAA TCT TCT GCT TGG CAA GTC; and for Bax: F, GGA GCT GCA performance liquid chromatography-purified AS (AS1: TCCCCTGGATGG- GAG GAT GAT TG and R, CCA CAA AGA TGG TCA CGG TC.

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