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J. Biol. Chem., Vol. 279, Issue 24, 25549-25561, June 11, 2004

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Polo-like Kinase 1 (Plk1) Inhibits p53 Function by Physical Interaction and Phosphorylation^*

Kiyohiro Ando¶, Toshinori Ozaki¶, Hideki Yamamoto, Kazushige Furuya, Mitsuchika Hosoda, Syunji Hayashi, Masahiro Fukuzawa, and Akira Nakagawara||

From the Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan and the First Department of Surgery, Nihon University School of Medicine, Tokyo 173-8610, Japan

Received for publication, December 26, 2003 , and in revised form, March 11, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21^WAF1, MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21^WAF1. In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The polo-like kinases (Plks)¹ are structurally and functionally related to the Drosophila polo serine/threonine (Ser/Thr) kinase (1), and are evolutionarily well conserved from yeast to mammals. A high degree of amino acid sequence similarity is detected within a catalytic domain and a unique noncatalytic domain (termed the polo-box) located at the NH₂- and COOH-terminal region, respectively (2). It has been shown that the polo-box is critical for the correct subcellular localization of Plks (3, 4), and the COOH-terminal region containing the polo-box serves to regulate its kinase activity (5). A growing body of evidence obtained in various experimental systems suggests that Plks play an important role in the regulation of a variety of M-phase-specific events including entry into and exit from mitosis (1, 6–8). In addition to their critical role during the G₂/M transition, Plks might be also required for G₁/S phase transition (9, 10).

In mammalian cells, there exist at least three Plk family members including Plk1, Plk2, and Plk3. Plk1 (also referred to as Plk) has been identified as a serine/threonine kinase that displays an extensive amino acid sequence homology to Drosophila polo (2, 9, 11–13), whereas Plk2 (alternatively named Snk) and Plk3 (alternatively termed as proliferation related kinase, Prk) have been originally shown to be transcriptionally induced in response to mitogens (14, 15). In mammalian cultured cells, the amounts of Plk1 mRNA and protein are regulated in a cell cycle-dependent manner, rising from a very low level in G₁ phase to a maximal level during G₂/M phase (11, 12). The kinase activity of Plk1 is regulated by its phosphorylation and peaks at M phase (16–18). Recently, it has been shown that the kinase activity of Plk1 is inhibited in response to DNA damage in mammalian cultured cells and this inhibition occurs in an ATM-dependent manner (19, 20). Plk1 phosphorylates various substrate proteins including cyclin B1 and Cdc25C. At the onset of mitosis, Plk1 phosphorylated cyclin B1 and promoted rapid nuclear translocation of an active Cdc2-cyclin B1 complex (21, 22). In addition, Toyoshima-Morimoto et al. (23) has found that, during G₂/M phase, Plk1 is capable of phosphorylating Cdc25C, which dephosphorylates and directly activates the Cdc2-cyclin B1 complex, and regulating the nuclear entry of Cdc25C. In contrast to Plk1, the expression level of Plk3 remains constant during the cell cycle progression and its kinase activity peaks during late S and G₂ phase (24, 25). Xie et al. (26) found that the kinase activity of Plk3 is rapidly increased in response to DNA damage in an ATM-dependent fashion.

In addition to the potential cell cycle-regulatory role, Plk1 has been implicated in the genesis and/or progression of tumors. Plk1 was overexpressed in rapidly proliferating cells as well as various human primary tumors (27), suggesting that the expression level of Plk1 is tightly linked to proliferation and could be used as a negative prognostic indicator for various tumors (28–30). Consistent with these observations, constitutive overexpression of Plk1 in NIH3T3 cells resulted in the oncogenic focus formation and induction of tumor growth in nude mice (10). Down-regulation of the endogenous Plk1 by using several antisense oligonucleotides targeted to Plk1 induced growth inhibition in certain cancerous cells (31). Additionally, treatment of the cells with small interfering RNA targeted against Plk1 caused the cell cycle arrest and apoptosis (32, 33). Of note, Liu and Erikson (33) reported that the tumor suppressor p53 might be involved in the Plk1 depletion-induced apoptosis. Recently, Plk1 has also been reported to have an ability to phosphorylate p53 in vitro, however, it is still unknown whether there exists a functional association between Plk1 and p53 (26). In sharp contrast to Plk1, the expression level of Plk3 was significantly down-regulated in several human primary tumors including lung carcinomas and head and neck squamous cell carcinomas, as compared with their corresponding normal tissues (34, 35). Overexpression of Plk3 in mammalian cultured cells inhibited proliferation and induced apoptosis (36). Furthermore, it has been demonstrated that Plk3 physically interacts with p53 and phosphorylates the Ser²⁰ of p53, which might result in the enhancement of its activity. These suggest that Plk1 and Plk3 play a differential role in regulating cell proliferation and oncogenesis, and that p53 participates in Plk3-dependent growth inhibition and/or apoptosis (25, 26, 36, 37).

In the present study, we examined the physical and functional interaction between Plk1 and p53. We found that Plk1 binds to the sequence-specific DNA-binding domain of p53, and inhibits the p53-dependent transcriptional activation as well as pro-apoptotic function. Intriguingly, overexpression of ATM abrogated the Plk1-mediated inhibitory effect on p53. These results suggest that the Plk1-mediated negative regulation of p53 might be a fundamental mechanism for the Plk1-induced oncogenesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tumor Samples—Surgically resected tumor tissues including three lung adenocarcinomas, two gastric adenocarcinomas, one uterus carcinoma, two bladder carcinomas, and their corresponding normal tissues used in this study were obtained as frozen specimens from the Tissue Bank in Chiba Cancer Center Hospital (Chiba, Japan). Six hepatoblastomas and their matched normal tissues were provided by the Japanese Study Group for Pediatric Liver Tumor.

Cell Culture—African green monkey kidney COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and penicillin (100 IU/ml)/streptomycin (100 µg/ml). Human neuroblastoma SH-SY5Y cells and human lung carcinoma H1299 cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and antibiotic mixture. Cultures were maintained at 37 °C in a water-saturated atmosphere of 5% CO₂ in air.

Transfection—COS7 cells were transfected with the indicated expression plasmids using FuGENE 6 transfection reagent (Roche Applied Science) in a 6-cm diameter culture dish in accordance with the manufacturer's specifications. Transfection of H1299 cells was conducted by lipofection with LipofectAMINE transfection reagent (Invitrogen) in a 12-well plate according to the manufacturer's instructions.

RT-PCR—Total RNA was prepared from SH-SY5Y cells exposed to cisplatin by using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol, and subjected to synthesis of the first strand cDNA with random primers and a SuperScript II reverse transcriptase (Invitrogen) at 42 °C for 1 h. When the reaction was complete, the cDNA was amplified in a final volume of 15 µl of reaction mixture containing 100 µM of each deoxynucleoside triphosphate, 1x PCR buffer, 1 µM of each primer, and 0.2 units of rTaq DNA polymerase (Takara, Ohtsu, Japan). The primers for p53 amplification were 5'-ATTTGATGCTGTCCCCGGACGATATTGAAC-3' and 5'-ACCCTTTTTGGACTTCAGGTGGCTGGAGTG-3'. The primers for p21^WAF1 amplification were 5'-ATGAAATTCACCCCCTTTCC-3' and 5'-CCCTAGGCTGTGCTCACTTC-3'. The primers for Plk1 amplification were 5'-ATCACCTGCCTGACCATTCCACCAAGG-3' and 5'-AATTGCGGAAATATTTAAGGAGGGTGATCT-3'. The primers for Plk3 amplification were 5'-GCGCGAGAAGATCCTAAATG-3' and 5'-GATCTGCCGCAGGTAGTAGC-3'. The primers for GAPDH amplification were 5'-ACCTGACCTGCCGTCTAGAA-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. The PCR-amplified products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide post-staining.

Generation of FLAG-tagged Expression Constructs—The FLAG-tagged human Plk1 construct was generated by PCR amplification using the cDNA derived from primary hepatoblastoma as a template. The forward and reverse primers used in the PCR were 5'-CCGCTCGAGAGTGCTGCAGTGACTGCAGGGAAG-3' and 5'-CTAGTCTAGATTAGGAGGCCTTGAGACGGTTGCT-3'. The underlined nucleotides represent the XhoI restriction sites in the forward primer and the XbaI restriction site in the reverse primer. The PCR product was subcloned into pGEM-T Easy (Promega Corp., Madison, WI), and its nucleotide sequence was verified by automated dideoxy terminator cycle sequencing. The PCR product was digested with XhoI and XbaI, and inserted between the XhoI to XbaI sites in the pcDNA3-FLAG expression plasmid in-frame to the downstream of the FLAG tag to give pcDNA3-FLAG-Plk1.

Construction of the Deletion Mutants of p53 and Plk1—The p53 deletion mutants, p53-(1–359), p53-(1–292), and p53-(1–101) were generated by using the forward primer 5'-CCCAAGCTTGGGATGGAGGAGCCGCAGTCAGATCCTAGCGTC-3' (1F) in combination with the reverse primers 5'-CCGGAATTCCGGTCATGGCTCCTTCCCAGCCTGGGCATCCTT-3' (359R), 5'-CCGGAATTCCGGTCATTTCTTGCGGAGATTCTCTTCCTCTGT-3' (292R) and 5'-CCGGAATTCCGGTCATTTCTGGGAAGGGACAGAAGATGACA-3' (101R), respectively. p53-(102–393) was amplified by using the forward primer 5'-CCCAAGCTTGGGATGACCTACCAGGGCAGCTACGGTTTCCGTCT-3' (102F) and the reverse primer 5'-CCGGAATTCCGGTCAGTCTGAGTCAGGCCCTTCTGTCTTGAACAT-3' (393R). Each of the forward and reverse primers contained the HindIII and EcoRI restriction sites to facilitate the subsequent cloning step. Underlined nucleotides in the oligonucleotides listed above were HindIII or EcoRI sites. Amplified fragments were digested with HindIII and EcoRI, and subcloned directly into the identical restriction sites of pcDNA3 to give pcDNA3-p53-(1–359), pcDNA3-p53-(1–292), pcDNA3-p53-(1–101), and pcDNA3-p53-(102–393). All of the constructs were confirmed by sequence analysis. For the construction of the deletion mutants of Plk1, pcDNA3-FLAG-Plk1 was digested with BamHI, BamHI and BstXI, or BamHI and NcoI. A restriction fragment encoding amino acid residues 1–401, 1–329, or 1–98 was purified from agarose gels, filled in the overhangs with Klenow, and then inserted in-frame into the enzymatically modified BamHI and XhoI sites of the pcDNA3-FLAG expression plasmid to give pcDNA3-FLAG-Plk1-(1–401), pcDNA3-FLAG-Plk1-(1–329), or pcDNA3-FLAG-Plk1-(1–98), respectively. DNA sequencing confirmed the authenticity of the expression plasmids prior to transfection.

Construction of the Kinase-deficient Mutant Form of Plk1—The K82M mutation was introduced into wild-type Plk1 by the PCR-based strategy using PfuUltra^TM High Fidelity DNA polymerase (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The following oligonucleotides were used: 5'-ATGATTGTGCCTAAGTCTCTGCTGCTCAAGCCGCA-3' (underlined segment encodes Met at amino acid position 82) and 5'-GCCCGCGAACACCTCCTTGGTGTCCGCGTCCGAGA-3'. Nucleic acid sequencing was performed to verify the presence of the desired mutation and absence of random mutations. The amplified fragment that contains the K82M mutation was then digested with HindIII and NcoI, gel-purified, and ligated with the NcoI/XbaI restriction fragment containing the 3'-portion of the wild-type Plk1 cDNA. The resulting entire cDNA encoding the full-length Plk1 carrying the amino acid substitution at position 82 was inserted in-frame into the HindIII and XbaI sites of the pcDNA3-FLAG expression plasmid to give pcDNA3-FLAG-Plk1(K82M).

Construction of the Expression Plasmid for Antisense Plk1—A full-length human Plk1 cDNA was ligated into the pcDNA3 expression plasmid in a reverse orientation to give As-Plk1, and the product was evaluated by restriction digestion. To assess the effect of As-Plk1 on the endogenous Plk1, whole cell lysates prepared from H1299 cells transfected with As-Plk1 were analyzed for Plk1 by immunoblotting.

GST Pull-down Assay—Whole cell lysates prepared from COS7 cells expressing FLAG-Plk1 were incubated with 1 µg of GST or GST-p53 (Santa Cruz Biotechnologies, Santa Cruz, CA) immobilized on glutathione-Sepharose beads for 2 h at 4 °C. The beads were washed extensively with NETN buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, and 1 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride. The bound proteins were eluted with 2x SDS sample buffer by boiling for 5 min, and separated by 10% SDS-polyacrylamide gel electrophoresis followed by immunoblotting.

Immunofluorescent Labeling and Confocal Microscopy—COS7 cells, cultured onto glass coverslips, were transiently transfected with the expression plasmid for FLAG-Plk1. Transfected cells were washed twice with 1x PBS and then fixed with 1x PBS containing 3.7% formaldehyde for 30 min at room temperature. After washing with 1x PBS, cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature and blocked for 1 h in 1x PBS containing 3% bovine serum albumin. Cells were then incubated with polyclonal anti-p53 antibody (Cell Signaling Technology, Inc., Beverly, MA) and monoclonal anti-FLAG antibody (M2, Sigma) for 1 h at room temperature. After incubation with primary antibodies, cells were washed twice with 1x PBS and incubated with fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies (Invitrogen) diluted 1:200 for 1 h at room temperature. Cell nuclei were stained with 4,6-diamidino-2-phenylindole at a final concentration of 1 µg/ml (Sigma). Cells were finally washed with 1x PBS, the coverslips were removed from the dishes, mounted onto slides, and observed under Fluoview laser scanning confocal microscope (Olympus, Tokyo, Japan).

Western Blot Analysis—Cells were transfected with the indicated combinations of the expression plasmids. Forty-eight hours after transfection, cells were extracted directly with the lysis buffer containing 25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture (Sigma) and the whole cell lysates were sonicated for 10 s followed by centrifugation at 15,000 rpm for 10 min at 4 °C to remove insoluble materials. The protein concentrations were determined using the Bradford protein assay according to the instructions of the vendor (Bio-Rad). Equal amounts of the whole cell lysates (50 µg of protein) were boiled in an SDS sample buffer consisting of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 2% -mercaptoethanol, and 0.01% bromphenol blue and subjected to 10% SDS-polyacrylamide gel electrophoresis under reducing conditions and then electrotransferred onto Immobilon-P membranes (Millipore, Bedford, MA) at room temperature for 1 h. The membranes were blocked with TBS-T (50 mM Tris-Cl, pH 7.6, 100 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk at room temperature for 1 h, and subsequently incubated for 1 h with monoclonal anti-Plk1 (PL2 and PL6, Zymed Laboratories, Inc., San Francisco, CA), monoclonal anti-p53 (DO-1, Oncogene Research Products, Cambridge, MA), monoclonal anti-FLAG antibody (M2, Sigma), monoclonal anti-Plk3 antibody (B37–2, BD Pharmingen), polyclonal antibody specific for p53 phosphorylated at Ser¹⁵ (Cell Signaling, Beverly, MA), polyclonal anti-p21^WAF1 antibody (H-164, Santa Cruz Biotechnologies), or polyclonal anti-actin antibody (20–33, Sigma) in TBS-T, followed by an incubation with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted at 1:2,000 for 1 h at room temperature. The membranes were washed extensively with TBS-T and protein bands were visualized by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham Biosciences).

Subcellular Fractionation—Cells were fractionated into cytosolic and nuclear fractions as described previously (38). Briefly, cells were washed twice with ice-cold 1x PBS and lysed in lysis buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, and a protease inhibitor mixture (Sigma) for 30 min at 4 °C. Cell lysates were centrifuged at 15,000 x g for 10 min to collect the soluble fraction as cytosolic extracts. Insoluble materials were washed with the lysis buffer and further dissolved in 1x SDS sample buffer to collect the nuclear fraction. The nuclear and cytoplasmic fractions were subjected to immunoblot analysis using the anti-FLAG, monoclonal anti-lamin B (Ab-1, Oncogene Research Products), or monoclonal anti-Ras (RASK-3, Seikagaku Co., Tokyo, Japan) antibody.

Immunoprecipitation and Western Blot Analysis—For the immunoprecipitation of Plk1 and p53, COS7 cells were transiently transfected with 2 µg of the expression plasmid for FLAG-Plk1 using FuGENE 6 transfection reagent. Forty-eight hours post-transfection, cells were harvested and lysed by incubation with mixing in 400 µl of the EBC buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Whole cell lysates were then subjected to centrifugation at 15,000 x g for 20 min at 4 °C to remove insoluble materials. Equal amounts of whole cell lysates were precleared with 30 µl of a 50% slurry of protein A-Sepharose (Amersham Biosciences). After centrifugation, the supernatant was incubated with the normal mouse serum (NMS), monoclonal anti-FLAG, or monoclonal anti-p53 antibody at 4 °C for 2 h. The immunocomplexes were precipitated with the protein A-Sepharose beads at 4 °C for 30 min, which were then pelleted by centrifugation at 15,000 x g for 5 min. The precipitates were washed with the lysis buffer three times at 4 °C, resuspended in 30 µl of the SDS sample buffer, and treated at 100 °C for 5 min. Proteins were then resolved by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto the Immobilon-P membranes. The protein complex was detected by Western blot analysis using the monoclonal anti-FLAG or monoclonal anti-p53 antibodies.

Luciferase Reporter Assays—p53-deficient H1299 cells were seeded in a 12-well tissue culture dish at a density of 5 x 10⁴ cells/well. Cells were transfected with 100 ng of the p53-responsive luciferase reporter plasmid (p21, MDM2, or BAX), 10 ng of pRL-TK Renilla luciferase cDNA, and 25 ng of the expression plasmid for p53 together with or without increasing amounts of FLAG-Plk1 expression plasmid. The total amount of DNA was kept constant (510 ng) with pcDNA3 (Invitrogen) per transfection. Forty-eight hours post-transfection, transfected cells were washed twice with 1x PBS, and resuspended in passive lysis buffer (Promega Corp.). Both firefly and Renilla luciferase activities were assayed with the dual-luciferase reporter assay system (Promega Corp.) according to the manufacturer's instructions. The fluorescent light emission was determined by TD-20 luminometer (Turner Design, Sunnylvale, CA). The firefly luminescence signal was normalized based on the Renilla luminescence signal. The results were obtained from at least three sets of transfection and were presented as the mean ± S.D.

Cell Survival Assays—Cell viability was determined by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, SH-SY5Y cells were seeded in 96-well microtiter plates (5 x 10³ cell/well) with 100 µl of complete medium and allowed to attach. The next day, the medium were changed and cells were treated with cisplatin for 24 h. For the MTT assay, 10 µl of MTT solution was added to each well for 3 h at 37 °C. The absorbance readings for each well were carried out at 570 nm using the microplate reader (model 450, Bio-Rad).

Apoptotic Analysis—H1299 cells were transfected with a constant amount of the expression plasmid for green fluorescence protein (GFP) and p53 expression plasmid together with or without the expression plasmid encoding FLAG-Plk1. Forty-eight hours after transfection, transfected cells were identified by the presence of green fluorescence. To verify apoptosis, cell nucleus was stained with propidium iodide to reveal nuclear condensation and fragmentation. The number of GFP-positive cells with fragmented nuclei was scored, and presented as a percentage of the total number of fluorescent cells.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Plk1 and Plk3 in Paired Tumors and Adjacent Normal Tissues—It has been shown that the expression level of Plk1 is increased in human tumors of various origins as compared with that of their corresponding normal tissues, suggesting that Plk1 contributes to the genesis and/or progression of tumors (13, 27–29). Recently, we have also identified Plk1 as one of the genes whose expression level is markedly elevated in human hepatoblastomas.² In contrast, Plk3 expression is down-regulated in certain human tumors including lung carcinomas and head and neck squamous cell carcinomas (34, 35). To confirm the differential expression of both Plk1 and Plk3 in the same tissue samples, we examined their expression patterns among the indicated various paired cancer-normal tissues by RT-PCR. The levels of GAPDH mRNA were comparable between these paired samples. Consistent with the previous results, without exceptions, the expression levels of Plk1 mRNA were significantly higher in cancerous tissues than those of their adjacent normal tissues (Fig. 1). On the other hand, Plk3 was expressed at low levels in all the lung, uterus, and bladder carcinomas that we examined, as compared with their corresponding normal tissues, whereas a significant decrease in Plk3 expression level in tumor tissues was undetectable in 2 of 6 hepatoblastomas and in 1 of 2 gastric carcinomas (Fig. 1). Thus, deregulated overexpression of Plk1 is detected in all various types of tumors, whereas down-regulation of Plk3 expression may be restricted to certain tumors.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Expression of Plk1 and Plk3 mRNA in various human primary tumors and their corresponding normal tissues. Total RNA (5 µg) prepared from the indicated tumor (T)-normal (N) paired samples was subjected to RT-PCR analysis for Plk1 and Plk3 mRNA expression using the specific primers as shown under "Experimental Procedures." The PCR-amplified products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Amplification of GAPDH was used as an internal control.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Down-regulation of Plk1 during the cisplatin-induced apoptosis. A, cell survival assays of SH-SY5Y cells treated with cisplatin. SH-SY5Y cells were exposed to cisplatin at a final concentration of 20 µM. At the indicated time periods after treatment with cisplatin, cell viability was determined by MTT assay. Data are presented as the mean ± S.D. of three independent experiments. B, RT-PCR analysis. Human neuroblastoma-derived SH-SY5Y cells were treated with or without cisplatin at a final concentration of 20 µM. At the indicated time periods after treatment with cisplatin, total RNA was prepared and subjected to RT-PCR analysis for the expression of Plk1 (1st panel), Plk3 (2nd panel), p53 (3rd panel), and p21WAF1 (4th panel). Amplification of GAPDH serves as an internal control (5th panel). The PCR products were resolved in 1.5% agarose gels and visualized by ethidium bromide staining. C, Western blot analysis. Whole cell lysates were prepared from SH-SY5Y cells exposed to cisplatin for 24 h (at a final concentration of 20 µM) or left untreated and immunoblotted against anti-Plk1 (1st panel), anti-Plk3 (2nd panel), anti-p53 (3rd panel), antibody specific for p53 phosphorylated at Ser15 (4th panel), or with the anti-p21WAF1 antibody (5th panel). Immunoblotting for actin is shown as control for protein loading (6th panel).

View larger version (23K): [in this window] [in a new window]   FIG. 3. Co-immunoprecipitation and nuclear co-localization of Plk1 and p53. A, complex formation between Plk1 and p53 in mammalian cultured cells. COS7 cells were transiently transfected with the expression plasmid for FLAG-tagged Plk1. Forty-eight hours after transfection, whole cell lysates were prepared and immunoprecipitated (IP) with NMS or with monoclonal anti-p53 antibodies. The immunocomplexes were resolved by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted (IB) with monoclonal anti-FLAG antibody. Whole cell lysates were immunoblotted with monoclonal anti-p53, or with monoclonal anti-FLAG antibody to show the expression of endogenous p53, or FLAG-Plk1, respectively. The p53 blot was reprobed for actin to ensure equal loading. B, a similar immunoprecipitation assay was performed with NMS or monoclonal anti-FLAG antibody, followed by immunoblotting with monoclonal anti-p53 antibody. Whole cell lysates were monitored on immunoblot for the expression of endogenous p53 or FLAG-Plk1. The p53 blot was reprobed for actin to ensure equal loading. C, GST pull-down assay. Whole cell lysates prepared from COS7 cells expressing FLAG-Plk1 were incubated with GST or GST-p53 immobilized on glutathione-Sepharose beads. The bound proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, and subjected to immunoblotting with the anti-FLAG antibody. D, association between endogenous p53 and Plk1. Cell lysates prepared from U2OS cells were immunoprecipitated with NMS or with monoclonal anti-Plk1 antibody, and the anti-Plk1 immunoprecipitates were immunoblotted with monoclonal anti-p53 antibody. E, subcellular localization of Plk1. COS7 cells were transiently transfected with the expression plasmid encoding FLAG-Plk1. Forty-eight hours after transfection, cells were fractionated into nuclear (N) and cytosolic (C) fractions as described under "Experimental Procedures." Equal amounts of each fraction were resolved by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with monoclonal anti-FLAG antibody (top panel). These extracts were also immunoblotted with monoclonal antibody specific for lamin B (middle panel) or Ras (RASK-3) (bottom panel) to show the validity of our fractionation technique. F, nuclear co-localization of Plk1 and p53. COS7 cells were transiently transfected with the FLAG-Plk1 expression plasmid. Following transfection, cells were fixed and incubated with polyclonal anti-p53 and monoclonal anti-FLAG antibodies that were revealed by fluorescein isothiocyanate-conjugated anti-rabbit IgG (green) and rhodamine-conjugated anti-mouse IgG (red), respectively. Merge analysis (yellow) showed the nuclear co-localization of Plk1 and p53.

The Sequence-specific DNA-binding Region of p53 Is Required for the Interaction with Plk1—To assess regions of p53 involved in the interaction with Plk1, we constructed a series of p53 deletion mutants including p53-(1–359) (lacking an extreme COOH-terminal region), p53-(1–292) (lacking the most COOH-terminal region including an oligomerization domain), p53-(1–101) (retaining only an NH₂-terminal transactivation domain), and p53-(102–393) (lacking an NH₂-terminal transactivation domain) (Fig. 4A). We first examined their subcellular localization by indirect immunofluorescent staining. To this end, p53-deficient human lung carcinoma H1299 cells (43) were transiently transfected with each expression plasmid. Forty-eight hours after transfection, cells were fixed and stained with the appropriate monoclonal anti-p53 antibody. As described previously (44, 45), there exist three potential nuclear localization signals (NLS I, II, and III) in the COOH-terminal region of p53, and NLS I alone has an ability to translocate the pyruvate kinase fusion protein to the nucleus. As shown in Fig. 4B, wild-type p53 and p53-(102–393), which retain the intact COOH-terminal region, accumulated in the nucleus. In addition, p53-(1–359), which lacks the NLS II and III but retains the NLS I, localized largely in the nucleus. On the other hand, p53-(1–292) and p53-(1–101), which lack three potential NLSs, were detected both in the nucleus and the cytoplasm. We then examined their abilities to interact with Plk1. H1299 cells were transiently co-transfected with the FLAG-Plk1 expression plasmid along with the expression plasmid for wild-type p53, p53-(1–359), p53-(1–292), or p53-(1–101), and the anti-FLAG immunoprecipitates were analyzed for the presence of wild-type p53 and these truncated forms of p53. As shown in Fig. 4C, wild-type p53 as well as p53 deletion mutants including p53-(1–359) and p53-(1–292), were detected in the anti-FLAG immunoprecipitates, whereas p53-(1–101) has lost the ability to bind to Plk1, indicating that the extreme COOH-terminal region, the oligomerization domain, and the NH₂-terminal transactivation domain of p53 are not involved in the interaction with Plk1. Similar immunoprecipitation analyses revealed that p53-(102–393) co-precipitates with FLAG-Plk1 (Fig. 4D). Thus, the region between amino acid residues 102 and 292 of p53, which includes the sequence-specific DNA-binding domain, appears to be required and sufficient for the interaction with Plk1.

View larger version (32K): [in this window] [in a new window]   FIG. 4. DNA-binding domain of p53 is required for interaction with Plk1. A, schematic drawing of full-length p53 and various deletion mutants used in this study. TA, transactivation domain; DB, sequence-specific DNA-binding domain; OD, oligomerization domain. Numbers indicate amino acid position. B, subcellular localization of various deletion mutants of p53. p53-deficient H1299 cells were transiently transfected with the indicated expression plasmids. Forty-eight hours after transfection, cells were fixed and incubated with monoclonal anti-p53 antibody (DO-1 or PAb 421). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (blue). Expression of p53 derivatives was visualized with rhodamine-conjugated secondary antibody (red). C and D, Plk1 interacts with the DNA-binding domain of p53. H1299 cells were transiently co-transfected with the indicated combinations of the expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and subjected to immunoprecipitation with monoclonal anti-FLAG antibody followed by immunoblotting with monoclonal anti-p53 antibody (upper panel). The lower panels show the direct immunoblot analyses of whole cell lysates performed with monoclonal anti-p53, monoclonal anti-FLAG, or with polyclonal anti-actin antibody (C). Whole cell lysates from H1299 cells overexpressing FLAG-Plk1 and p53-(102–393) were immunoprecipitated with monoclonal anti-p53 antibody (PAb421) or with NMS followed by immunoblotting with monoclonal anti-FLAG antibody. Expression levels of p53-(102–393), FLAG-Plk1, and actin were examined by immunoblotting (D).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Mapping of the region of Plk1 required for interaction with p53. A, schematic representation of Plk1 deletion mutants. KD, kinase domain; Polo, polo-box. Numbers indicate amino acid position. B, immunofluorescent studies of Plk1 deletion mutants. Transfected COS7 cells were fixed in 3.7% formaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked in PBS containing 3% bovine serum albumin for 1 h. Cells were then incubated with monoclonal anti-FLAG antibody followed by incubation with rhodamine-conjugated secondary antibody (red), and analyzed by confocal microscopy. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (blue). C and D, interaction between various Plk1 deletion mutants and endogenous p53. Whole cell lysates from COS7 cells transfected with the indicated expression plasmids were immunoprecipitated with monoclonal anti-FLAG antibody, and immunoblotted with monoclonal anti-p53 antibody to observe the interaction between Plk1 deletion mutants and p53. Immunoprecipitation with NMS was used as a negative control. Equal amounts of protein derived from cell lysates were immunoblotted with monoclonal anti-p53, monoclonal anti-FLAG, or polyclonal anti-actin antibody.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Plk1 abrogates the p53-mediated transcriptional activation. p53-deficient H1299 cells (5 x 104 cells/well) were transiently co-transfected with 25 ng of the expression plasmid for p53 together with 100 ng of the luciferase reporter construct that carries the p53-responsive element derived from p21WAF1 (A), BAX (B), or MDM2 (C) promoter and 10 ng of the Renilla luciferase plasmid (pRL-TK) in the presence or absence of increasing amounts of pcDNA3-FLAG-Plk1 (50, 100, or 200 ng). The total amount of plasmid DNA per transfection was kept constant (510 ng) with pcDNA3. All transfections were performed in triplicate. Forty-eight hours after transfection, cells were lysed, and analyzed for their luciferase activities. Firefly luminescence signal was normalized based on the Renilla luminescence signal. Results are shown as -fold induction of the firefly luciferase activity compared with control cells transfected with pcDNA3 alone. D, immunoblot analysis. H1299 cells were transiently co-transfected with the indicated combinations of expression plasmids. Whole cell lysates were prepared 48 h post-transfection, and analyzed for the expression of FLAG-Plk1 (1st panel), p53 (2nd panel), or p21WAF1 (3rd panel) by immunoblot analysis with monoclonal anti-FLAG, monoclonal anti-p53, or polyclonal anti-p21WAF1 antibody, respectively. Total protein levels were controlled with polyclonal anti-actin antibody (4th panel).

View larger version (16K): [in this window] [in a new window]   FIG. 7. Antisense Plk1 increases the transcriptional activity of p53. A, antisense Plk1 expression in H1299 cells results in a reduction of endogenous Plk1. H1299 cells were transfected with 2 µg of antisense Plk1 expression plasmid (As-Plk1). Whole cell lysates prepared from transfected cells were subjected to immunoblotting with the anti-Plk1 antibody (top panel). Western blotting for actin is shown as a control for protein loading (bottom panel). B–D, luciferase reporter analysis. H1299 cells were transiently co-transfected with 12.5 ng of the p53 expression plasmid along with 100 ng of the indicated luciferase reporter construct in the presence or absence of increasing amounts of As-Plk1 (200 and 400 ng). Determination and calculation of the luciferase activities are described in the legend to Fig. 6.

View larger version (13K): [in this window] [in a new window]   FIG. 8. Plk1 inhibits the pro-apoptotic activity of p53. A, H1299 cells were transiently co-transfected with 0.6 µg of the expression plasmid for p53 together with or without 1.2 µg of the FLAG-Plk1 expression plasmid. The total amount of plasmid DNA was kept constant (2 µg) with the empty plasmid. At 48 h after transfection, cell viability was determined by MTT cell survival assays. The graph (mean ± S.D. of three independent experiments) represents relative viability based on the percent of viable cells compared with the control transfection (pcDNA3). The percentage of viable cells expressing p53 alone is significantly different from that of viable cells expressing p53 and FLAG-Plk1 (p < 0.0001). B, H1299 cells were transiently co-transfected with the indicated combinations of the expression plasmids. A constant amount of the GFP expression plasmid (200 ng) was included in all combinations, and the total amount of plasmid DNA was kept constant (2 µg) by including an appropriate amount of empty plasmid. Forty-eight hours after transfection, transfected cells were identified by the presence of green fluorescence. Cell nucleus was stained with propidium iodide to reveal nuclear condensation and fragmentation. The number of GFP-positive cells with condensed and fragmented nuclei was scored, and the percentage of apoptotic cells shown in each column represents the mean of three independent experiments.

View larger version (21K): [in this window] [in a new window]   FIG. 9. Kinase-deficient Plk1(K82M) fails to reduce the activity of p53. A, Plk1(K82M) retains an ability to interact with p53. COS7 cells were transiently transfected with the expression plasmid for FLAG-Plk1 or FLAG-Plk1(K82M). Forty-eight hours after transfection, cell lysates were prepared and subjected to anti-FLAG immunoprecipitation followed by immunoblotting with monoclonal anti-p53 antibody. Immunoprecipitation with NMS was used as a negative control. Equal amounts of protein derived from cell lysates were immunoblotted with monoclonal anti-p53, monoclonal anti-FLAG, or with polyclonal anti-actin antibody. B–D, Plk1(K82M) has an undetectable effect on the transcriptional activity of p53. H1299 cells were transiently co-transfected with a fixed amount of the p53 expression plasmid (25 ng) and the p53-responsive luciferase reporter construct carrying the p21WAF1 (B), BAX (C), or MDM2 (D) promoter (100 ng) in the presence or absence of increasing amounts of the expression plasmid encoding FLAG-Plk1(K82M) (100 or 200 ng). The total amount of plasmid DNA per transfection was kept constant (510 ng) with pcDNA3. Determination and calculation of the luciferase activities are described in the legend to Fig. 6. E, Plk1(K82M) is unable to inhibit the pro-apoptotic function of p53. H1299 cells were transiently co-transfected with the p53 expression plasmid (0.6 µg) together with or without the expression plasmid for FLAG-Plk1(K82M) (1.2 µg). Forty-eight hours after transfection, their viability was measured by MTT cell survival assays as described in the legend to Fig. 8.

ATM Antagonizes the Inhibitory Effect of Plk1 on p53—Plk1 kinase activity has been shown to be inhibited in an ATM-dependent manner in response to DNA damage (19, 20). The kinase activity of ATM was significantly increased after DNA damage, and ATM was able to phosphorylate p53 at the NH₂ terminus on serine 15 to enhance its stability as well as its transactivation activity (48–50). To examine whether ATM could affect the Plk1-mediated inhibition of p53, we transiently co-transfected H1299 cells with expression plasmids for p53 and FLAG-Plk1 together with or without increasing amounts of ATM expression plasmid, and the ability of p53 to drive transcription from the p21^WAF1 reporter was measured. As expected, co-expression of p53 with ATM resulted in an increase in the transcriptional activity of p53 as compared with that of cells expressing p53 alone (Fig. 10). Increasing amounts of ATM largely abrogated the Plk1-mediated inhibition of the p53-dependent transcriptional activation. It thus appears that ATM could inhibit the activity of Plk1 and thereby restore the transcriptional activity of p53.

View larger version (11K): [in this window] [in a new window]   FIG. 10. ATM antagonizes the inhibitory effect of Plk1 on the p53-dependent transactivation. H1299 cells were transiently co-transfected with the expression plasmids encoding p53 (25 ng) and FLAG-Plk1 (200 ng) along with the luciferase reporter construct containing the p53-responsive element from the p21WAF1 promoter in the presence or absence of increasing amounts of ATM expression plasmid (50 or 100 ng). pcDNA3 was used to equalize the amount of plasmid in each transfection, and the Renilla luciferase plasmid was included in the transfection mixture to normalize the transfection efficiency. Determination and calculation of the luciferase activities are described in the legend to Fig. 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression of Plk1 was significantly down-regulated in response to cisplatin treatment. Recently, Ree et al. (51) found that ionizing radiation leads to the suppression of Plk1 mRNA expression. It is of interest to examine whether genotoxic stresses other than cisplatin and ionizing radiation could also repress the expression of Plk1. On the other hand, Plk1 mRNA expression is significantly induced in various human primary tumors (27). It is necessary to identify the promoter region as well as the transcription factor(s) required for the transcriptional regulation of Plk1 in cancerous cells. In good agreement with the previous observations (52), Uchiumi et al. (53) have identified the regulatory regions responsible for the activation of the human Plk1 promoter, which include a consensus Sp1-binding site and a CCAAT box. It has been shown that the transcription factor NF-Y, a heterotrimeric complex consisting of NF-YA, NF-YB, and NF-YC, recognizes and binds to the CCAAT box (54). Indeed, the electrophoretic mobility shift assay revealed that NF-Y binds to the CCAAT box present within the human Plk1 promoter region, however, it remains to be determined whether NF-Y and/or Sp1 could actually transactivate the Plk1 promoter in tumor cells (53). Recently, Lee and Pedersen (55) have reported that there exist 6 GC boxes and the CCAAT box within the type II hexokinase (HKII) promoter, and that NF-Y and Sp family members including Sp1 might contribute to up-regulation of the HKII gene in tumor cells. Further studies regarding the transcriptional regulation of the Plk1 gene are necessary to clarify the molecular mechanisms of Plk1-dependent tumorigenesis.

During the DNA damage response, the activity of ATM is significantly increased and is responsible for the rapid phosphorylation of p53 at Ser¹⁵ (48–50). This ATM-dependent phosphorylation contributes to the increased stability and activity of p53 by facilitating its dissociation from MDM2 (56). In addition, phosphorylation of p53 at Ser¹⁵ induces its binding to the transcriptional co-activator p300 (57). Recently, it has been shown that Plk1 activity is inhibited in response to DNA damage, and this inhibition occurs in an ATM-dependent manner (19, 20). Our present data demonstrate that the Plk1-mediated inhibition of p53 activity is rescued by the co-expression of ATM, suggesting that, in addition to the ATM-dependent phosphorylation of p53, the activity of p53 may be enhanced at least in part by the ATM-dependent inhibition of Plk1. Intriguingly, Liu and Erikson (33) reported that p53 is significantly stabilized in Plk1-depleted cells. In accordance with their findings, we have shown that the exposure of SH-SY5Y cells to cisplatin leads to a remarkable accumulation of p53, which is strongly associated with a significant down-regulation of the endogenous Plk1 both at mRNA and protein levels, suggesting that Plk1 is closely involved in the regulation of p53 stability and thereby modulates its activity. Under our experimental conditions, however, overexpression of FLAG-Plk1 did not affect the amounts of the endogenous as well as the ectopically expressed p53.

The pro-apoptotic function of p53 involves its ability to act as a transcription factor in transactivating downstream target gene promoters. The majority of missense mutations of p53 detected in human tumors occur within its sequence-specific DNA-binding domain, and these mutations cause the loss of p53 activity (58). Thus, the structural integrity of this domain is required for p53 function. On the other hand, several viral and cellular proteins inactivate p53 through a variety of different mechanisms (58). MDM2 and Pirh2 promote ubiquitination and degradation of p53 (59–62). Sir2 interacts with p53 and induces its deacetylation (63). In addition, S100B calcium-binding protein prevents the oligomerization of p53 to inhibit its function (64). Based on our systematic immunoprecipitation analysis, Plk1 binds to p53 through the sequence-specific DNA-binding domain of p53. Intriguingly, SV40 large T antigen binds to the sequence-specific DNA-binding domain of p53, and abrogates DNA binding as well as the transactivation function of p53 (65, 66). It is thus likely that, like SV40 large T antigen, Plk1 might mask this domain of p53 by direct binding, and thereby inhibit its sequence-specific transcriptional activity. Further study is required to identify the detailed molecular mechanism.

In sharp contrast to Plk1, Xie et al. (26) found that the kinase activity of Plk3 is rapidly enhanced in response to DNA damage in an ATM-dependent fashion. They also described that Plk3 has the ability to interact directly with p53 and phosphorylate p53 at Ser²⁰. Moreover, a kinase-defective mutant form of Plk3 fails to phosphorylate p53, and abrogates the p53-mediated transcriptional activation as well as growth suppression, indicating that Plk3 might enhance the p53 activity through Ser²⁰ phosphorylation of p53 (26). In addition to Ser¹⁵ phosphorylation of p53, DNA damage-induced phosphorylation of p53 at Ser²⁰ prevents its association with MDM2 and results in its stabilization (67). Of note, it has been shown that Plk1 phosphorylates p53 in vitro but on residues that might be different from that mediated by Plk3 (26). According to their phosphopeptide mapping analysis, at least three unique radiolabeled tryptic peptides derived from recombinant p53 were detected in the presence of Plk1. Recently, Nakajima et al. (68) identified a sequence (D/E)X(S/T)X(D/E) (X, any amino acid; , a hydrophobic amino acid) as a consensus motif for Plk1-dependent phosphorylation (68). During the search for a putative phosphorylation site(s) targeted by Plk1 within the amino acid sequence of p53, we found a related motif (²⁵⁴IITLED²⁵⁹) present within the sequence-specific DNA-binding domain of p53, suggesting that this motif could be one of the putative phosphorylation sites of p53 targeted by Plk1, although there is no direct evidence for this possibility. According to our present results, the kinase-deficient mutant form of Plk1 that retained an ability to associate with p53, failed to reduce the transcriptional as well as apoptosis-inducing activity of p53, suggesting that the kinase activity of Plk1 is critical for the Plk1-dependent inhibition of p53. Thus, identification of the major phosphorylation site(s) of p53 by Plk1 is required to establish the functional significance of the Plk1-mediated phosphorylation of p53. In contrast, Liu and Erikson (33) reported that, like wild-type mouse Plk1, co-expression of the kinase-defective (K82M) mouse Plk1 partially rescued the apoptotic phenotype induced by the depletion of Plk1, indicating that the kinase activity is not necessary for its anti-apoptotic activity. They also described that their kinase-defective mouse Plk1 has 15–20% of wild-type kinase activity, raising a possibility that the residual kinase activity of their mouse Plk1(K82M) might be enough to inhibit the Plk1 depletion-induced apoptosis.

Fig. 11 shows a model that incorporates our present findings, and illustrates various interactions in response to DNA damage. Given the fact that the differential expression of Plk1 and Plk3 during the cisplatin-induced apoptosis, and their differential effects on p53, it is conceivable that the balance between intracellular expression levels of Plk1 with oncogenic potential and pro-apoptotic Plk3 is at least in part responsible for the determination of the cell fate via the physical and functional interaction with p53.

View larger version (19K): [in this window] [in a new window]   FIG. 11. Schematic representation of interactions among ATM, Plk1, Plk3, and p53 in response to DNA damage.

	FOOTNOTES

^¶ Both authors contributed equally to this work.

^|| To whom correspondence should be addressed: Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuohku, Chiba 260-8717, Japan. Tel.: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: akiranak{at}chiba-ccri.chuo.chiba.jp.

¹ The abbreviations used are: Plk, polo-like kinase; ATM, ataxia telangiectasia mutated; GFP, green fluorescence protein; GST, glutathione S-transferase; NLS, nuclear localization signal; NMS, normal mouse serum; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TK, thymidine kinase; RT, reverse transcriptase.

² Yamada, S., Ohira, M., Horie, H., Ando, K., Takayasu, H., Suzuki, Y., Sugano, S., Hirata, T., Goto, T., Matsunaga, T., Hiyama, E., Hayashi, Y., Ando, H., Suita, S., Kaneko, M., Sakaki, F., Hashizume, K., Ohnuma, N., and Nakagawara, A. (2004) Oncogene, in press.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Y. Shiloh and the Japanese Study Group for Pediatric Liver Tumor for kindly providing the ATM expression plasmid and hepatoblastoma tissues, respectively. We thank Dr. S. Sakiyama and members of our laboratory for helpful discussions. We also thank Y. Nakamura and M. Kikawa for excellent technical assistance.

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