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

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. PCR GATC) and, as a control, sense oligonucleotide (SE1: GATCCCATC- products were separated by electrophoresis on 2.5% agarose gels. CAGGGGA) according to the sequence of the p53AIP1 gene. AS1 (1 ␮M) was Detection of Apoptosis. For FACS analysis, adherent and detached cells transfected with Lipofectin reagent (Life Technologies, Inc.) for 4 h, and then were combined and fixed with 75% ethanol at 4°C. After two rinses with PBS, cells were treated with UV irradiation (50 J/m2) or incubated with 0.5 ␮M STS cells were incubated for 30 min with 1 ml of PBS containing 1 mg of boiled or 10 ng/ml TNF-␣. Apoptotic cells were analyzed by FACS analysis 36 h after RNase at 37°C. Cells were then stained in 1 ml of PBS containing 10 ␮gof treatment.

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-␣ damage for RT-PCR (A) and Western blotting (B). Expression of ␤2MG or ␤-actin served as a quality control. C, induction of apoptosis in MCF7 cells collected after continu- ous incubation with 0.5 ␮M STS for 36 h or 10 ng/ml TNF-␣ 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-oligonu- cleotides (SE; 1 ␮M 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 p53AIP1 expression 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 M) were transfected to MCF7 cells with Lipofectin reagent (Life Technologies, Inc.) for 4 h, and then cells were treated with UV, STS, or TNF-␣. The experiments were repeated at least three times in duplicate samples and the average scores are shown with error bars; bars, Ϯ SD.

2884

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 homog- enized 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 ϫ g for 5 min at 4°C to remove nuclei and debris. The supernatants were centrifuged at 17,000 ϫ g for 15 min at 4°C to collect mitochondria. The resulting supernatants were centrifuged at 100,000 ϫ g for 1hat4°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 ␮g/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 Involvement of p53AIP1 in the Mitochondrial Apoptotic Path- way. 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-␣, and UV irradiation. TNF-␣ 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 Fig. 2. Regulation of mitochondrial ⌬⌿m and release of cytochrome c by p53AIP1. A, membrane potential and triggering release of cytochrome c (35–37). H1299 and Saos-2 cells were infected with Ad-p53AIP1␣ or Ad-LacZ at an MOI of 100 As shown in Fig. 1A, expression of p53AIP1 increased significantly plaque-forming units/cell. The levels of mitochondrial ⌬⌿m (48 h after infection) and the population of apoptotic cells (96 h after infection) were examined by staining with during apoptosis induced by STS or UV irradiation but not in response Rhodamine 123 and FACS analysis, respectively (top). The expressions of exogenous to TNF-␣. The expression of p53AIP1 mRNA reached the peak at 0.1 p53AIP1 protein were shown by Western blot (bottom). B, release of cytochrome c from ␮M mitochondria by overexpression of p53AIP1. HCT116 cells were infected with Ad- of STS, in concert with Ser46-phosphorylation of p53 (Fig. 1B), p53AIP1␣ or Ad-LacZ at an MOI of 100 and collected at the indicated times. Cytosolic suggesting that the transcription of p53AIP1 might be regulated by the and mitochondrial fractions were each subjected to immunoblotting. The expression of modification of p53 as described previously (1). Bax and PIG3 also mitofilin or ␤-actin was examined as a quality control or a quantity control, respectively. 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 Immunoprecipitation and Western Blot Analysis. COS7 cells were elevated during apoptosis induced by STS and TNF-␣, whereas Noxa seeded (2 ϫ 106 cells/10-cm dish) before treatment and transfected with 8 ␮g of plasmid mixed with Fugene 6 reagent (Roche). Cells were collected 24 h was induced only when apoptosis was triggered by UV irradiation. In Ϫ Ϫ after transfection, lysed in NP40 based lysis buffer (0.5% NP40, 150 mM NaCl, contrast, when we used H1299 cells (p53 / ), none of the three 20 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride) for 1 h, and cleared of apoptosis stimulators induced expression of p53AIP1 despite induc- nuclear and cellular debris by centrifugation. Immunoprecipitations were per- tion of p53-independent apoptosis (data not shown). These results formed using rabbit anti-HA antibody (polyclonal) or mouse anti-bcl-2 anti- suggested that p53AIP1 is involved specifically in a mitochondrial body (monoclonal) plus protein G-Sepharose. The precipitates were washed apoptotic pathway and in a p53-dependent manner (Fig. 1C). four times in lysis buffer and proteins were eluted with Laemmli sample buffer To confirm the involvement of p53AIP1 in apoptotic processes, we (Bio Rad). Immunoblotting was performed as described previously (1). performed experiments using an AS to the p53AIP1 gene. Pretreat- Recombinant Adenoviruses and Infection. To construct Ad-p53AIP1 or ment of MCF7 cells with AS caused a remarkable reduction in the Ad-p53 virus, blunt-ended fragments of p53AIP1 or p53 cDNA were inserted level of p53AIP1 expression, as compared with sense-oligonucleotide 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 after UV irradiation (Fig. 1D). AS also reduced the apoptotic (sub-G1) regions. These procedures generated pAxCAp53AIP1 or pAxCAp53. The cell population from 28% to 16% after UV irradiation or from 35% to constructs were confirmed by sequencing. Recombinant adenoviruses were 27% after treatment with STS; however, AS treatment of MCF7 cells constructed by in vitro homologous recombination in the human embryonic did not inhibit cell death induced by TNF-␣ (Fig. 1E). kidney cell line 293 using pAxCAp53AIP1 or pAxCAp53 with the adenovirus p53AIP1 Regulates Mitochondrial ⌬⌿m and Release of Cyto- DNA terminal protein complex (Takara). Viruses were propagated in the chrome c from Mitochondria. Schuler et al. (38) showed that in- HEK293 cells and purified by two rounds of CsCl density centrifugation. Viral troduction of the p53 gene could induce down-regulation of mito- titers were measured in a limiting-dilution bioassay using the HEK293 cells. chondrial membrane potential and promote release of cytochrome c. Diluting viral stock to certain concentrations, adding viral solutions to cell Other p53 downstream genes such as Noxa, PUMA, and Bax, also monolayers, and incubating at 37°C for 60 min with brief agitation every 20 enhance release of cytochrome c from mitochondria (29, 31, 39). To min successfully infected all of the cell lines (33). Culture medium was added, and infected cells were incubated at 37°C. clarify the molecular mechanism of p53AIP1-inducible apoptosis, we Detection of Mitochondrial Membrane Potential. Cells were plated at examined the effect of overexpression of p53AIP1 on these phenom- densities of 5 ϫ 105 cells/6-cm dish and infected 24 h later with Ad-p53AIP1␣, ena. H1299 (lung carcinoma) and Saos-2 (osteosarcoma) cells were Ad-p53, or Ad-LacZ at an MOI of 100 plaque-forming units/cell. Trypsinized infected with Ad-p53AIP1␣, and then the levels of mitochondrial adherent and floating cells were collected 60 h after infection and washed ⌬⌿m were examined by staining with Rhodamine 123. Although the 2885

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. REGULATION OF APOPTOSIS BY p53AIP1 expressions of exogenous p53AIP1 by infection of Ad-p53AIP1 were into COS7 cells, the harvested cells were double-stained with mouse observed in both cell lines, apoptosis was induced in H1299 cells by monoclonal anti-bcl-2 antibody and rabbit polyclonal anti-HA anti- Ad-p53AIP1␣, but Saos-2 cells were resistant to p53AIP1 gene trans- body. p53AIP1 was stained (green) in the cytoplasm as a granular fer; down-regulation of mitochondrial ⌬⌿m was observed only in pattern, as was bcl-2 (red); both signals were clearly overlaid in the H1299 cells 48 h after infection (Fig. 2A). This result indicated that double-color image as a yellow signal (Fig. 3B). Both proteins were the ability of p53AIP1 to induce apoptosis might depend on the cell confirmed to be located at mitochondria (data not shown). type as observed in case of other apoptotic regulators such as Bax. To Functional Interplay of p53AIP1 and bcl-2. As shown in Fig. 2, investigate the subcellular location of cytochrome c, subcellular frac- our experiments clearly indicated that p53AIP1 itself regulated mito- tions were collected at the times indicated in Fig. 2B, and cytosolic chondrial ⌬⌿m and triggered the release of cytochrome c. Bcl-2 is and mitochondrial fractions were subjected to Western blotting with well known as an important regulator for mitochondrial ⌬⌿m that anti-cytochrome c antibody. A remarkable translocation of cyto- also blocks the release of cytochrome c from mitochondria. These chrome c from mitochondria to cytosol was observed 48 h after facts and the interaction of p53AIP1 and bcl-2 at mitochondria infection with Ad-p53AIP1␣. prompted us to speculate that p53AIP1 itself might regulate mito- Potential Interaction of p53AIP1 and bcl-2. Overexpression of chondrial ⌬⌿m and trigger the release of cytochrome c by interacting bcl-2 can block p53-dependent dissipation of mitochondrial ⌬⌿m and with bcl-2. To test this hypothesis, we examined whether bcl-2 could apoptosis (40, 41). Because p53AIP1 and bcl-2 are mitochondrial inhibit p53AIP1-induced apoptosis and the change of mitochondrial components related to apoptosis, we speculated that these two proteins ⌬⌿m. A bcl-2 expression vector was transfected into HeLa cells, and might influence the apoptotic process by interacting. Expression vec- two independent cell lines (HeLa-bcl2-1 and HeLa-bcl2-2) that stably tors containing HA-p53AIP1␣ or bcl-2 were cotransfected into COS7 expressed bcl-2 were established. As shown in Fig. 4A, apoptosis was cells. Protein complexes containing p53AIP1␣ or bcl-2 were immu- inducible in HeLa-mock parental cells (i.e., integrated with the control noprecipitated from cell extracts with rabbit polyclonal anti-HA an- vector only, pcDNA3.1) by infection with Ad-p53AIP1␣ or Ad-p53 tibody or mouse monoclonal anti-bcl-2 antibody, respectively. West- but not Ad-LacZ. However, when two HeLa cell lines overexpressing ern blots indicated that the immune complex precipitated with either bcl-2 were infected with Ad-p53AIP1␣ or Ad-p53 the number of antibody included both bcl-2 and HA-p53AIP1␣ proteins (Fig. 3A). apoptotic cells decreased significantly, suggesting that bcl-2 was able Specific interaction between HA-p53AIP1␤ and bcl-2 was confirmed to inhibit the apoptotic pathway that involves p53AIP1 and p53. in the same manner (data not shown). Next we examined the mitochondrial ⌬⌿m itself. Induction of We then investigated the subcellular locations of p53AIP1 and exogenous p53AIP1␣ and p53 proteins but not exogenous LacZ bcl-2 proteins. After (24 h) the expression vectors were transfected down-regulated mitochondrial ⌬⌿m in HeLa-mock parental cells;

Fig. 3. Physical interaction of p53AIP1 and bcl-2. A, bcl-2 and HA-p53AIP1␣ expression vectors were cotransfected into COS7 cells; 24 h later cell extracts were isolated. The protein complex with bcl-2 or p53AIP1 was immunoprecipi- tated (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␣ 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.

2886

Ectopic expression of exogenous p53AIP1a by adenovirus-mediated gene transfer effectively caused apoptosis in six of the WTp53 cancer- cell lines (Fig. 5B). In fact, four of those six lines were killed more effectively by p53AIP1 than by p53.

DISCUSSION In earlier studies we had isolated the p53AIP1 gene through at- tempts 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 func- tioned 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-␣. Inhibition of p53AIP1 expression by AS confirmed the im- portance of p53AIP1 accumulation in mitochondria in STS- and Fig. 4. Functional interaction between p53AIP1 and bcl-2. A, inhibition by bcl-2 of UV-induced apoptosis. At present two separate apoptotic pathways apoptosis induced by Ad-p53 or Ad-p53AIP1␣. Ad-p53AIP1␣, 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, Ϯ SD. B, down-regulation of mitochondrial ⌬⌿mby Ad-p53AIP1␣ and Ad-p53. Ad-p53AIP1␣, 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. however, this phenomenon was blocked in both of the bcl-2 overexpressing HeLa cell lines (Fig. 4B). This result indicated that bcl-2 can inhibit the down-regulation of mitochondrial ⌬⌿m induced by either p53AIP1␣ or p53, and supported the notion that interaction between bcl-2 and p53AIP1 might regulate mitochondrial ⌬⌿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␣ infection, in a time-dependent manner (Fig. 5A). To eval- uate the ability of p53AIP1 to induce apoptosis of cancer cells derived from a variety of tissues, we infected Ad-p53AIP1␣ 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␣ 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 Fig. 5. Antitumor effect of p53AIP1 on diverse cancer-cell lines in vitro. A, expression ␣ ␣ p53 gene on nine cancer cell lines that carry WTp53: HCT116 (colon of exogenous p53AIP1 in lung-carcinoma cells by infection with Ad-p53AIP1 . H1299 cells were collected at the indicated hours after treatment, and cell lysates were subjected cancer), LS174T (colon cancer), A549 (lung cancer), MKN45 (gastric to immunoblotting with anti-p53AIP1 antibody. B, apoptosis induced in nine cancer-cell cancer), MCF7 (breast cancer), TERA2 (teratoma), DBTRG05MG lines (p53 wild type) by infection with various doses (MOI) of Ad-p53 or Ad-p53AIP1␣. Apoptotic cells were analyzed by FACScan 96 h after infection. The experiments were (glioma), HepG2 (Hepatocarcinoma), and Lu99A (lung cancer). All repeated at least three times in duplicate samples and the average scores are shown with nine of these cell lines revealed 100% infectivity of Ad-LacZ vector. error bars; bars, Ϯ SD. 2887

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. REGULATION OF APOPTOSIS BY p53AIP1 are known; one is the mitochondrial pathway triggered by DNA ment of cancer than p53 itself. In fact, in our experiments p53AIP1 damage or STS and the other is the death-receptor pathway triggered induced apoptosis effectively in both p53-sensitive and p53-resistant by members of the TNF family or Fas ligand (47). p53 is activated in cancer-cell lines (Fig. 5B). Those results implied that p53AIP1 trig- both pathways, and it can induce apoptosis by regulating its down- gers apoptosis directly, whereas p53 induces growth suppression by stream genes (3, 48). Among p53-downstream genes, Bax, Noxa, and regulating its downstream genes. Hence p53AIP1 in adenoviral vec- PUMA are mediators of the mitochondrial pathway, whereas Fas and tors may become an attractive agent for gene therapy to kill cancer killer/DR5 mediate the death-receptor pathway. Apoptotic stimuli cells, especially p53-resistant cells that carry WTp53. However, in such as UV, STS, and TNF-␣ caused induction of p21WAF1 in our vivo research is still needed to define the role of p53AIP1 in human study, but p53AIP1 was induced only after UV or STS damage. So the gene therapy. 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 REFERENCES at mitochondria. This evidence partly explains the molecular mecha- 1. Oda, K., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, nism of p53AIP1-inducible apoptosis, because bcl-2 can block apo- H., Tamai, K., Tokino, T., Nakamura, Y., and Taya, Y. p53AIP1, a potential mediator ptotic processes induced by diverse cytotoxic insults such as ␥ or UV of p53-dependent apoptosis, and its regulation by ser-46-phosphorylated p53. Cell, 102: 849–862, 2000. irradiation, treatment with dexamethasone, STS, or cytotoxic antican- 2. el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., and Vogelstein, B. cer drugs, or by withdrawal of cytokines (49). Hence, bcl-2 functions Definition of a consensus binding site for TP53. Nat. Genet., 1: 45–49, 1992. as a negative regulator of apoptosis. Indeed, bcl-2 may be able to 3. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323–331, 1997. block p53-dependent apoptosis (40, 41). The fact that bcl-2 interacts 4. Ko, L. J., and Prives, C. p53: puzzle and paradigm. Genes Dev., 10: 1054–1072, with p53AIP1, a bona-fide direct target of p53, as well as with Bax, 1996. Noxa, and PUMA, is solid evidence for a functional association 5. Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. A ribonucleotide reductase gene involved in a p53-dependent between bcl-2 and p53 in apoptotic regulation. Although bcl-2 is an cell-cycle check point for DNA damage. Nature (Lond.), 404: 42–49, 2000. antiapoptotic effector against a broad spectrum of apoptosis stimula- 6. Fiscella, M., Ullrich, S. J., Zambrano, N., Shields, M. T., Lin, D., Lees-Miller, S. P., Anderson, C. W., Mercer, W. E., and Appella, E. Mutation of the serine 15 phos- tors, its inhibitory effect on apoptosis through the death-receptor phorylation site of human p53 reduces the ability of p53 to inhibit cell cycle pathway is small (50–52); therefore, the death-receptor pathway is progression. Oncogene, 8: 1519–1528, 1993. likely to bypass the apoptotic pathway controlled by bcl-2. These facts 7. Walker, K. K., and Levine, A. J. Identification of a novel p53 functional domain that is necessary for efficient growth suppression. Proc. Natl. Acad. Sci. USA, 93: are consistent with our observation that p53AIP1 is not involved in 15335–15340, 1996. TNF-␣-induced apoptosis; i.e., in the death-receptor pathway. 8. Zhu, J., Zhou, W., Jiang. J., and Chen, X. Identification of a novel p53 functional Bcl-2 directly or indirectly prevents the release of cytochrome c domain that is necessary for mediating apoptosis. J. Biol. Chem., 273: 13030–13036, 1998. from mitochondria. Cytochrome c forms an essential part of the 9. Vaux, D. L., Cory, S., and Adams, J. M. Bcl-2 gene promotes haemopoietic cell apoptosome composed of Apaf-1 and procaspase-9 (53). Bcl-2 func- survival and cooperates with c-myc to immortalize pre-B cells. Nature (Lond.), 335: 440–442, 1988. tions as an antioxidant, preventing apoptosis by decreasing lipid 10. Tsujimoto, Y. Stress-resistance conferred by high level of bcl-2 ␣ protein in human peroxidation and increasing the resistance of the cell to reactive B lymphoblastoid cells. Oncogene, 4: 1331–1336, 1989. oxygen species (54). Bcl-2 blocks reactive oxygen species production 11. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, O., and Korsmeyer, S. J. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. by preventing the permeability-transition pores on mitochondrial Cell, 67: 879–888, 1991. membranes from opening, an early step in the signaling cascade of 12. Strasser, A., Harris A. W., and Cory, S. bcl-2 transgene inhibits T cell death and apoptosis (55), and then stabilizes the mitochondrial ⌬⌿m. In our perturbs thymic self-censorship. Cell, 67: 889–899, 1991. 13. Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, P. C., and Croce, C. M. Cloning of study, p53AIP1 actually induced down-regulation of the mitochon- the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome drial ⌬⌿m and promoted release of cytochrome c, whereas bcl-2 translocation. Science (Wash. DC), 226: 1097–1099, 1984. blocked the proapoptotic activity of both p53AIP1 and p53. Our 14. Bakhshi, A., Jensen, J. P., Goldman, P., Wright, J. J., McBride, O. W., Epstein, A. L., and Korsmeyer, S. J. Cloning the chromosomal breakpoint of t(14;18) human lym- results indicate that regulation of mitochondrial membrane potential phomas: clustering around JH on chromosome 14 and near a transcriptional unit on might be essential for p53- and p53AIP1-inducible apoptosis, and that 18. Cell, 41: 899–906, 1985. 15. Cleary, M. L., and Sklar, J. Nucleotide sequence of a t(14;18) chromosomal break- this regulation might be determined by the physical and functional point in follicular lymphoma and demonstration of a breakpoint-cluster region near a interaction between bcl-2 and p53AIP1. transcriptionally active locus on chromosome 18. Proc. Natl. Acad. Sci. USA, 82: Introduction of the WTp53 gene into cancer cells by adenoviral 7439–7443, 1985. 16. Ellis, H. M., and Horvitz, H. R. Genetic control of programmed cell death in the vectors is a potential therapeutic approach for human cancers, and in nematode C. elegans. Cell, 44: 817–829, 1986. fact gene therapy using p53 has been undergoing clinical evaluation in 17. Hengartner, M. O., and Horvitz, H. R. Programmed cell death in Caenorhabditis patients with various types of cancer (33, 56–58). However, although elegans. Curr. Opin. Genet. Dev., 4: 581–586, 1994. 18. Hengartner, M. O., and Horvitz, H. R. C. elegans cell survival gene ced-9 encodes a transfer of WTp53 kills cancer cells in some cases it does not do so in functional homolog of the mammalian proto-oncogene bcl-2. Cell, 76: 665–676, others, and the factors determining this variability in effectiveness 1994. 19. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai. J., Peng, T-I., Jones, have not been identified. Some clues might be found in recent obser- D. P., and Wang, X. Prevention of apoptosis by Bcl-2: release of cytochrome c from vations that ectopic expression of p53 can result in apoptosis in some mitochondria blocked. Science (Wash. DC), 275: 1129–1132, 1997. cases and growth arrest in others (59), although the mechanisms 20. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. accounting for these differences remain largely unknown. The results Science (Wash. DC), 275: 1132–1136, 1997. of our earlier experiments implied that the “decision” about the fate of 21. Shimizu, S., Narita, M., and Tsujimoto, Y. Bcl-2 family proteins regulate the release a cell, i.e., “arrest” or “death,” partly depends on the selection of of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature (Lond.), 399: 483–487, 1999. downstream target genes to be activated through specific binding to 22. Reed, J. C. Bcl-2 family proteins. Oncogene, 17: 3225–3236, 1998. p53-target sequences, and moreover that the type of modification 23. Miyashita, T., and Reed, J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 80: 293–299, 1995. (phosphorylation or acetylation) of p53 protein is likely to be associ- 24. Polyak, K., Xia, Y., Zweler, J. L., Klinzler, K. W., and Vogelstein, B. A model for ated with this selectivity (1, 7, 8, 60, 61). However, because p53AIP1 p53-induced apoptosis. Nature (Lond.), 389: 300–305, 1997. is a direct mediator of apoptosis, as indicated by the fact that p53AIP1 25. Venot, C., Maratrat, M., Dureuil, C., Conseiller, E., Bracco, L., and Debussche, L. The requirement for the p53 proline-rich functional domain for mediation of apoptosis can induce apoptosis of various cancer cells regardless of their p53 is correlated with specific PIG3 gene transactivation and with transcriptional repres- status, the p53AIP1 gene might be more widely applicable for treat- sion. EMBO J., 17: 4668–4679, 1998. 2888

26. Wu, G. S., Burns, T. F., McDonald, E. R., III, Jiang, W., Meng, R., Krantz, I. D., Kao, transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer G., Gan, D. D., Zhou, J. Y., Muschel, R., Hamilton, S. R., Spinner, N. B., Markowitz, Gene Ther., 3: 121–130, 1996. S., Wu, G., and el-Deiry, W. S. KILLER/DR5 is a DNA damage-inducible p53- 44. Tokino, T., Thiagalingam, S., el-Deiry, W. S., Waldman, T., Kinzler, K. W., and regulated death receptor gene. Nat. Genet., 17: 141–143, 1997. Vogelstein, B. TP53 tagged sites from human genomic DNA. Hum. Mol. Genet., 3: 27. Owen-Schaub, L. B., Zhang, W., Cusack, J. C., Angelo, L. S., Santee, S. M., 1537–1542, 1994. Fujiwara, T., Roth, J. A., Deisseroth, A. B., Zhang, W. W., Kruzel, E., and Radinsky, 45. Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C. W., Chessa, L., Smorodinsky, R. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 N. I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. Enhanced phosphorylation of p53 expression. Mol. Cell. Biol., 15: 3032–3040, 1995. by ATM in response to DNA damage. Science (Wash. DC), 281: 1674–1677, 1998. 28. Muller, M., Wilder, S., Bannasch, D., Israeli, D., Lehlbach, K., Li-Weber, M., 46. Canman, C. E., Lim, D. S., Cimprich, K. A., Taya, Y., Tamai, K., Sakaguchi, K., Friedman, S. L., Galle, P. R., Stremmel, W., Oren, M., and Krammer, P. H. p53 Appella, E., Kastan, M. B., and Siliciano, J. D. Activation of the ATM kinase by activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer ionizing radiation and phosphorylation of p53. Science (Wash. DC), 281: 1677–1679, drugs. J. Exp. Med., 188: 2033–2045, 1998. 1998. 29. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., 47. Hengartner, M. O. The biochemistry of apoptosis. Nature (Lond.), 407: 770–776, Taniguchi, T., and Tanaka, N. Noxa, a BH3-only member of the bcl-2 family and 2000. candidate mediator of p53-induced apoptosis. Science (Wash. DC), 288: 1053–1058, 48. Donato, N. J., and Perez, M. Tumor necrosis factor-induced apoptosis stimulates p53 2000. accumulation and p21WAF1 proteolysis in ME-180 cells. J. Biol. Chem., 273: 30. Attardi, L. D., Reczek, E. E., Cosmas, C., Demicco, E. G., McCurrach, M. E., Lowe, 5067–5072, 1998. S. W., and Jacks, T. PERP, an apoptosis-associated target of p53, is a novel member 49. Green, D. R., and Reed, J. C. Mitochondria and apoptosis. Science (Wash. DC), 281: of the PMP-22/gas3 family. Genes Dev., 14: 704–718, 2000. 1309–1312, 1998. 31. Nakano, K., and Vousden, K. H. PUMA, a novel proapoptotic gene, is induced by 50. Vanhaesebroeck, B., Reed, J. C., De Valck, D., Grooten, J., Miyashita, T., Tanaka, S., p53. Mol. Cell, 7: 683–694, 2001. Beyaert, R., Van Roy, F., and Fiers, W. Effect of bcl-2 proto-oncogene expression on 32. Yu, J., Zhang, L., Hwang P. M., Kinzler, K. W., and Vogelstein, B. PUMA induces cellular sensitivity to tumor necrosis factor-mediated cytotoxicity. Oncogene, 8: the rapid apoptosis of colorectal cancer cells. Mol. Cell, 7: 673–682, 2001. 1075–1081, 1993. 33. Wills, K. N., Maneval, D. C., Menzel, P., Harris, M. P., Sutjipto, S., Vaillancourt, 51. Strasser, A., Harris, A. W., Huang, D. C., Krammer, P. H., and Cory, S. Bcl-2 and M. T., Huang, W. M., Johnson, D. E., Anderson, S. C., Wen, S. F., Bookstein, R., Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J., 14: 6136– Shepard, H. M., and Gregory, R. J. Development and characterization of recombinant 6147, 1995. adenoviruses encoding human p53 for gene therapy of cancer. Hum. Gene Ther., 5: 52. Vaux, D. L., and Strasser, A. The molecular biology of apoptosis. Proc. Natl. Acad. 1079–1088, 1994. Sci. USA, 93: 2239–2244, 1996. 34. Li, K., Li, Y., Shelton, J. M., Richardson, J. A., Spencer, E., Chen, Z. J., Wang, X., 53. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Williams, R. S. Cytochrome c deficiency causes embryonic lethality and atten- and Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 uates stress-induced apoptosis. Cell, 101: 389–399, 2000. complex initiates an apoptotic protease cascade. Cell, 91: 479–489, 1997. 35. Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., and Reed J. C. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation 54. Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., and Korsmeyer, S. J. during apoptosis. Nat. Cell Biol., 2: 318–325, 2000. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell, 75: 241–251, 36. Scarlett, J. L., Sheard, P. W., Hughes, G., Ledgerwood, E. C., Ku, H. H., and Murphy, 1993. M. P. Changes in mitochondrial membrane potential during staurosporine-induced 55. Kroemer, G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. apoptosis in Jurkat cells. FEBS Lett., 475: 267–272, 2000. Med., 3: 614–620, 1997. 37. Shimizu, S., Matsuoka, Y., Shinohara, Y., Yoneda, Y., Tsujimoto, Y. Essential role 56. Roth, J. A., Nguyen, D., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Ferson, of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. D. Z., Hong, W. K., Komaki, R., Lee, J. J., Nesbitt, J. C., Pisters, K. M., Putnam, J. B., J. Cell Biol., 152: 237–250, 2001. Schea, R., Shin, D. M., Walsh, G. L., Dolormente, M. M., Han, C. I., Martin, F. D., 38. Schuler, M., Bossy-Wetzel, E., Goldstein, J. C., Fitzgerald, P., and Green, D. R. p53 Yen, N., Xu, K., Stephens, L. C., McDonnell, T. J., Mukhopadhyay, T., and Cai, D. induces apoptosis by caspase activation through mitochondrial cytochrome c release. Retrovirus-mediated wildtype p53 gene transfer to tumors of patients with lung J. Biol. Chem., 275: 7337–7342, 2000. cancer. Nat. Med., 2: 985–991, 1996. 39. Jurgensmeier, J. M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., and Reed, J. C. 57. Habib, N. A., Hodgson, H. J., Lemoine, N., and Pignatelli, M. A phase I/II study of Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumors. Acad. Sci. USA, 95: 4997–5002, 1998. Hum. Gene Ther., 10: 2019–2034, 1999. 40. Wang, Y., Szekely, L., Okan, I., Klein, G., and Wiman, K. G. Wild-type p53- 58. Weill, D., Mack, M., Roth, J., Swisher, S., Proksch, S., Merritt, J., and Nemunaitis, triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line. J. Adenoviral-mediated p53 gene transfer to non-small cell lung cancer through Oncogene, 8: 3427–3431, 1993. endobronchial injection. Chest, 118: 966–970, 2000. 41. Chiou, S. K., Rao, L., and White, E. Bcl-2 blocks p53-dependent apoptosis. Mol. Cell. 59. Polyak, K., Waldman, T., He, T. C., Kinzler, K. W., and Vogelstein, B. Genetic Biol., 14: 2556–2563, 1994. determinants of p53-induced apoptosis and growth arrest. Genes Dev., 10: 1945– 42. Gomez-Manzano, C., Fueyo, J., Kyritsis, A. P., Steck, P. A., Roth, J. A., McDonnell, 1952, 1996. T. J., Steck, K. D., Levin, V. A., and Yung, W. K. Adenovirus-mediated transfer of 60. Gu, W., and Roeder, R. G. Activation of p53 sequence-specific DNA binding by the p53 gene produces rapid and generalized death of human glioma cells via acetylation of the p53 C-terminal domain. Cell, 90: 595–606, 1997. apoptosis. Cancer Res., 56: 694–699, 1996. 61. Sakaguchi, K., Herrera, J. E., Saito, S., Miki, T., Bustin, M., Vassilev, A., Anderson, 43. Harris, M. P., Sutjipto, S., Wills, K. N., Hancock, W., Cornell, D., Johnson, D. E., C. W., and Appella, E. DNA damage activates p53 through a phosphorylation- Gregory, R. J., Shepard, H. M., and Maneval, D. C. Adenovirus-mediated p53 gene acetylation cascade. Genes Dev., 12: 2831–2841, 1998.

2889

Koichi Matsuda, Koji Yoshida, Yoichi Taya, et al.

Cancer Res 2002;62:2883-2889.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/62/10/2883

Cited articles This article cites 61 articles, 24 of which you can access for free at: http://cancerres.aacrjournals.org/content/62/10/2883.full#ref-list-1

Citing articles This article has been cited by 10 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/62/10/2883.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/62/10/2883. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.