Deregulation of Cdk2 causes Bim-mediated apoptosis in p53 ... - Nature

Oncogene (2008) 27, 4115–4121

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ORIGINAL ARTICLE

Deregulation of Cdk2 causes Bim-mediated apoptosis in p53-deficient tumors following actin damage HD Chae1,2, BM Kim1,2, UJ Yun1,2 and DY Shin1,2 1

National Research Laboratory, Department of Microbiology, Dankook University College of Medicine, Cheonan, Korea and Cancer Research Institute, Seoul National University, Seoul, Korea

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We previously reported that actin damage by treatment with an actin-depolymerizing agent including pectenotoxin-2 induces Bim-mediated apoptosis in p53-deficient human tumors. In this study, we investigated a molecular mechanism underlying Bim-mediated apoptosis of p53-deficient tumor cells following actin damage. We found that actin inhibitors increased the protein levels of p53 and p21 and thereby inactivated both Cdk2 and Cdc2 kinases. However, p53- or p21-knockout cells fail to induce p21 and hence kept both Cdk2 and Cdc2 kinases active even after treatment with actin inhibitor. The p53- or p21-knockout cells became multinucleate and polyploidy in association with induction of apoptosis. Expression of Bcl-xL resulted in accumulation of polyploid cells in association with inhibition of apoptosis. However, expression of a dominant negative mutant (Cdk2dn) and treatment with chemical inhibitors for Cdk2 suppressed not only accumulation of multinucleated cells, but also induction of Bim expression and apoptosis. Therefore, these results suggest that Bim-mediated apoptosis following actin damage due to deregulation of Cdk2 and the cell cycle by the absence of functional p53. Oncogene (2008) 27, 4115–4121; doi:10.1038/onc.2008.46; published online 17 March 2008 Keywords: actin; apoptosis; polyploidy; Bim; Cdk2

Introduction Cell cycle progression is regulated by checkpoint controls, which function to protect the integrity of the genome. Such controls act to prevent cell cycle progression until after completion of prior events (Hartwell and Weinert, 1989). The G1 checkpoint permits repair prior to replication, whereas arrest at the G2 checkpoint permits repair of the genome prior to its mitotic segregation. The p53 tumor suppressor gene, which is suppressed by mutation in approximately one-half of human tumors (Hollstein et al., 1991), has been shown to be integral to Correspondence: Professor DY Shin, Department of Microbiology, National Research Laboratory, Dankook University College of Medicine, Anseo 29, Cheonan 330-714, Korea. E-mail: [email protected] Received 22 August 2007; revised 22 January 2008; accepted 2 February 2008; published online 17 March 2008

both the G1 (Kuerbitz et al., 1992) and G2 (Bunz et al., 1998) DNA damage checkpoint machinery. In addition to DNA damage checkpoints, cells undergo a transient arrest at the metaphase–anaphase transition point upon spindle damage (Rudner and Murray, 1996; Amon, 1999). After a delay period, cells eventually escape from this block (a process termed ‘mitotic slippage’), and exit mitosis without a proper segregation of sister chromatids and cytokinesis (Jordan et al., 1991, 1996; Torres and Horwitz, 1998). Therefore, spindle-damaged cells arrests at G1-like state with an intact nucleus containing 4N DNA, but without ever completing mitosis (Minn et al., 1996; Lanni and Jacks, 1998). This is recently termed as tetraploid G1 state. Cells lacking wild-type p53 are still able to arrest transiently at mitosis, underscoring that the delay in mitosis is p53-independent (Minn et al., 1996; Lanni and Jacks, 1998). However, the p53-deficient cells are not prevented from re-entering the cell cycle and can reduplicate their DNA unchecked, leading to polyploidy and subsequent genomic instability (Di Leonardo et al., 1997; Khan and Wahl, 1998; Lanni and Jacks, 1998). In addition to microtubule inhibitors, dihydrocytochalasin B (DCB), which induces cleavage failure by depolymerizing actin filaments, also lead to tetraploid G1 arrest (Andreassen et al., 2001). Since DCB does not affect to spindle function and chromatid segregation, but only inhibits cytokinesis, it is likely that tetraploid G1 checkpoint seems to be a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest, and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. We previously reported that loss of p53 sensitizes tumor cells to actin damage induced by treatment with actin-depolymerizing or knotting agents (Chae et al., 2005). Upon actin damage, Bim expression was induced in tumor cells lacking functional p53, but not in cells with functional p53 (Chae et al., 2005). In this study, we found the deregulation of Cdk2 in p53-deficient tumor cells caused abrogation of tetraploid G1 checkpoint arrest and Bim-mediated apoptosis after actin damage. Results Actin inhibitors activate p53-signaling pathway We previously reported that actin damage induces Bimmediated apoptosis in p53-deficient tumors (Chae et al.,

Actin damage-induced apoptosis of p53-deficient tumors H-D Chae et al

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2005). In this study, we first examined whether actin inhibitors affect p53-signaling pathway. Western blot analysis showed that actin-depolymerizing agent, pectenotoxin-2 (PTX-2) increased the protein levels of p53 and p21 tumor suppressor proteins in HCT116 colon cancer cell lines (Figure 1a). Other actin inhibitors such as cytochalasin D and psychosine, which cause depolymerization and knotting, respectively, of actin filaments also increased expression of p53 and p21 (Figure 1a). PTX-2 also increased p53 and p21 in HepG2 cells that harbor wild-type p53, but not in Hep3B and H1299 lacking functional p53 (Figure 1b). We further examined alterations of cell cycle regulators upon PTX-2 treatment. Among cyclins and Cdks, cyclins A, B and Cdc2 were decreased, whereas cyclins D and E were increased by PTX-2 treatment in p53positive HCT116 cells (Figure 1c). However, the reduction of cyclins A, B and Cdc2 was not prominent in p53- and p21-knockout HCT116 cells, implying that p53 and p21 are required for decreases in protein levels of Cdc2, cyclins A and B (Figure 1c). immunoprecipitation (IP)-kinase assay showed that while both Cdk2 and Cdc2 were not detected in HCT116 cells, both kinases remain unchanged in p53- and p21-knockout cells following PTX-2 treatment (Figure 1c). Therefore, inactivation of Cdk2 and Cdc2 kinases in p53-positive cells should be due to p21-mediated inhibition, supporting our previous report that p21-mediated Cdk2 inhibition lead to Cdc2 inactivation (Yun et al., 2003; Chae et al., 2004). Absence of p53 causes polyploidy and multinucleation DNA content analysis revealed that parental HCT116 cells showed 4 or 8N DNA content, while the p53- and p21-knockout cells exhibited polyploidy with 16 and 32N DNA content after treatment with all three actin inhibitors (Figure 2a). In addition, the sub-G1 population

significantly increased in the p53- and p21-knockout cells, but not in their parental cells (Figure 2b), supporting previous report that p53-deficient tumor cells have the enhanced sensitivity to actin-damaging drugs (Chae et al., 2005). Because populations with 16 and 32N DNA content were not less increased as compared to reduction of cells with 4 and 8N DNA content, it is likely that polyploid cells move to sub-G1 populations. Reduction of populations with 4 and 8N and increases in sub-G1 populations were also observed in other p53-deficient tumor cell lines such as H1299 and Hep3B, but not in HepG2 that harbors functional p53 (Figure 2b). While staining with 40 ,6-diamidino-2-phenylindole (DAPI) showed that about 90% of parental HCT116 cells arrested with two nuclei, a majority of the p53- and p21-knockout cells became multinucleate, containing four or more nuclei after treatment with PTX-2 (Figures 3a and b). Since DNA replication uncoupled from mitosis causes polyploidy and multinucleation, we examined whether the p53- and p21-knockout cells treated with PTX-2 undergo DNA synthesis. 5-Bromo20 -deoxy-uridine (BrdU) incorporation was significantly decreased after PTX-2 treatment in parental HCT116 cells, but not in the p53- and p21-knockout cells, implying that p53 and p21 are essential to prevent DNA replication in PTX-2-treated cells (Figure 3c). We also examined the rate of mitosis entry after PTX-2 treatment using two-dimensional flow cytometry for both DNA content and the mitotic marker mitotic protein monoclonal 2 (MPM-2), in which mitotic content was quantitated from two-dimensional dot plots of DNA content versus MPM-2 stain by flow cytometry. p53- and p21-knockout cells entered mitosis at similar rate even after PTX-2 treatment as compared to nontreated cells (Figure 3d). These results suggest that p53 and p21 are required for preventing mitosis entry as well as DNA replication following PTX-2 treatment.

Figure 1 Actin inhibitors activate the p53/p21 signaling pathway. (a) Expression of p53 and p21 following treatment with actin inhibitors. HCT116 cells were treated with pectenotoxin-2 (PTX-2) (100 ng ml1), cytochalasin D (10 mM) or psychosine (50 mM) for the indicated periods and were then analysed for p53 and p21 proteins. (b) Tumor cells lines that randomly selected were treated with PTX2 for 2 days and analysed for p53 and p21 proteins. (c) HCT116 and its p53- and p21-knockout derivatives were treated with PTX-2 (100 ng ml1) and were analysed for cell cycle-regulatory proteins. Histone H1 kinase activity was measured by the precipitates formed with the anti-Cdk2 and anti-Cdc2 antibodies in cells after PTX-2 treatment at 100 ng ml1 for the indicated periods. As shown is a representative of more than three independent experiments. Oncogene


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DNA content (Log scale) Figure 2 Cell cycle-regulatory proteins in pectenotoxin-2 (PTX-2) treated cells. (a) DNA content analysis of HCT116 and its p53- and p21-knockout derivatives after treatment with PTX-2 (100 ng ml1), cytochalasin D (10 mM) or psychosine 50 mM). The cells were fixed after 3 days and flow cytometrically analysed. (b) DNA content analysis of p53-positive (HepG2) and -deficient (Hep3B and H1299) cells treated with PTX-2 as in (a).

Inhibition of Bim-mediated apoptosis by Bcl-xL and Cdk inhibitors Since we previously reported that induction of Bim expression initiates apoptosis following actin inhibitor treatment, we examined the effects of Bcl-xL, which is a member of anti-apoptotic Bcl-2 family and antagonize Bax and Bak (Sedlak et al., 1995; Antignani and Youle, 2006). Enforced expression of Bcl-xL increased viability of p53-knockout cells following PTX-2 treatment (Figure 4a). However, flow cytometric analysis showed that Bcl-xL increased populations of polyploid cells in association with decrease in sub-G1 populations (Figure 4b), implying that Bcl-xL inhibits apoptosis of polyploid cells. In addition, Bcl-xL did not affect induction of Bim expression in p53-knockout cells upon treatment with PTX-2 (Figure 4c). To examine whether constitutive activation of Cdk2 in p53- or p21-knockout cells contribute to Bimmediated apoptosis, we treated cells with the Cdk2 inhibitors such as roscovitine and olomoucine as well as iso-olomoucine, an inactive analog of olomoucine, and examined their effects on apoptosis induction (Figure 5a). The inhibitors suppressed not only accumulation of multinucleated cells, but also apoptotic cell deaths following PTX-2 treatment (Figure 5a). Furthermore, treatment with roscovitine and olomoucine inhibited both Cdk2 and Cdc2 kinases and induction of Bim expression following PTX-2 treatment (Figure 5b). In addition, the protein levels of Bcl-2

and Mcl-1 were not changed by PTX-2 treatment (Figure 5b). To further analyse a role of deregulated Cdk2 kinase, we expressed a dominant negative mutant form of Cdk2, Cdk2dn in p53-knockout cells (Figure 6). Expression of Cdk2dn, like Cdk2 inhibitors (Figure 5), inhibited cell death and suppressed accumulation of multinucleated cells following PTX-2 treatment (Figure 6a). Protein analysis showed that Cdk2dn inhibited Bim expression, Bax activation, a release of cytochrome c and Smac/ Diablo into the cytosol and activation of caspase-9 and caspase-3 (Figure 6b). Therefore, these results suggest that deregulation of Cdk2 causes Bim-mediated apoptosis in p53-deficient tumor cells following actin damage (Figure 7). Discussion The changes in PTX-2-treated p53-positive cells closely resemble those in normal cells treated with spindle inhibitors, which enter into tetraploid G1 state (Lanni and Jacks, 1998). Cyclins D and E were increased, but cyclins A and B were decreased following PTX-2 treatment (Figure 2a), implying that those cells arrested at G1 checkpoint. However, while normal human diploid fibroblast cells arrested at G1 with 4N DNA content, majority of HCT116 and HepG2 contained 4 and 8N DNA (Figure 2). Thus, those tumor cells, Oncogene


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Figure 3 p53-Deficient cells reduplicate and enter mitosis after pectenotoxin-2 (PTX-2) treatment. (a) HCT116 cells and its p53- and p21-knockout derivatives were treated with PTX-2 at 100 ng ml1 for 3 days and were then 4 0 ,6-diamidino-2-phenylindole (DAPI) stained. The number of nuclei in each cell was counted. Data represent the percentage of cells with different number of nuclei as the mean±s.e.m., n ¼ 3. (b) DAPI staining of HCT116 cells and its derivatives treated with PTX-2 as in (a). (c) 5-Bromo-20 -deoxy-uridine (BrdU) incorporation assay. Parental HCT116 cells and their derivatives, p53/ and p21/, were treated with PTX-2 at 100 ng ml1 for the indicated periods and were then subjected to the BrdU incorporation assay as described in the Materials and methods. The percentage of BrdU-positive cells is shown as the mean±s.e.m., n ¼ 3. (d) Flow cytometric analysis of asynchronous HCT116 and HCT116 p53/ cultures either untreated or treated with 100 ng ml1 PTX-2 for 2 days. Cells were labeled with the mitotic protein monoclonal 2 (MPM-2) antibody and propidium iodide (PI). MPM-2-positive mitotic cells were identified by flow cytometry, as described in the Materials and methods. A set of data representative of three independent experiments is shown. Percentages of MPM-2-positive cells were presented.

Figure 4 Bcl-xL inhibits p53-independent apoptosis. (a) Bcl-xL inhibits pectenotoxin-2 (PTX2)-induced apoptosis. HCT116 p53/ cells stably harboring either an empty vector or Bcl-xL were treated with PTX-2 (100 ng ml1) for 3 days. The cell viability was determined by Trypan blue exclusion. Data represent the percentage of dead cells as the mean±s.e.m., n ¼ 3. (b) Bcl-xL enhances polyploidy. The cells treated as in (a) were propidium iodide (PI)-stained and then flow cytometrically analysed. (c) Western blot analysis for Bim and Bcl-xL in the cells was treated as in (a). Oncogene


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Figure 5 Cdk2 inhibitors block Bim-mediated apoptosis. (a) Inhibition of cell death and multinucleation by Cdk2 inhibitors. HCT116 p53/ cells were treated with roscovitine (15 mM), olomoucine (30 mM), iso-olomoucine (30 mM) or the vehicle control with or without PTX-2 (100 ng ml1) for 3 days. Then the viability and numbers of nucleus were determined by Trypan blue exclusion and 40 ,6-diamidino-2-phenylindole (DAPI) staining respectively. (b) Western blot for Bcl-2 family proteins and immunoprecipitation (IP)-kinase assay for Cdk2 and Cdc2 kinases. HCT116 p53/ cells were treated with roscovitine (15 mM), olomoucine (30 mM), iso-olomoucine (30 mM) or the vehicle control with or without PTX-2 (100 ng ml1) for 36 h. Whole cell (WCE) and mitochondrial (Mito) fractions were prepared.

Figure 6 Cdk2dn suppressed Bim-mediated apoptosis. (a) Suppression of PTX-2-induced cell death and multinucleation by expression of Cdk2dn. p53- Knockout cells were infected with a recombinant adenovirus encoding Cdk2dn (Ad-Cdk2dn) or its control virus (Ad-DE1). One day after virus infection, the cells were treated with PTX-2 (100 ng ml1) for 3 days. Then the viability and numbers of nucleus was determined. The percentage of dead cells (left panel) or multinucleate (right panel) is shown as the mean±s.e.m., n ¼ 3. (b) Cdk2dn inhibited Bim expression following PTX-2 treatment. Total, cytosol and mitochondrial fractions were prepared from lysates of p53-deficient cells treated as in (a) and were analysed for Bcl-2 family proteins, active caspase-3 (17 of 19 kDa), active caspase-9 (37 kDa), XIAP, Smac and cytochrome c. The representative data from more than three independent experiments are shown.

although they harbor wild-type p53, may have other genetic defects involved in tetraploid G1 arrest and genome instability. However, unlike tetraploid G1 arrest induced by spindle inhibitors, p53- and p21-knockout cells treated

with actin inhibitors became multinucleate (Figure 3). Since those cells maintained active Cdc2 and entered mitosis even after actin damage, multinucleation seems due to mitosis entry without cytokinesis. Thus, a p53/ p21-dependent backup mechanism is critical not only to Oncogene


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genomic instability by preventing both p53-dependent and -independent apoptosis. Materials and methods Cell culture and transfection HCT116, a human colorectal cancer cell line, and its p53- and p21-knockout derivatives (denoted p53/ and p21/, respectively) were kindly provided by Dr Bert Vogelstein (John Hopkins Oncology Center, Baltimore, MD, USA). DNA transfections were performed using the CaPO4 coprecipitation procedure (Graham and van der Eb, 1973). HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies Inc., Grand Island, NY, USA) and penicillin–streptomycin (50 U ml1). To select stable cell lines expressing Bcl-xL, we transfected p53-knockout cells with pPuro-Bcl-xL. A recombinant adenovirus encoding Cdk2dn was constructed as previously described (Chae et al., 2004).

Figure 7 Schematic presentation of Bim-mediated apoptosis in p53-deficient tumor cells. Actin inhibitors that block cytokinesis activate p53-signaling pathway and hence arrests the cell cycle at G1 checkpoint. However, tumor cells lacking p53 fail to inhibit DNA replication and mitosis entry by keeping both Cdk2 and Cdc2 kinases active, and hence become multinucleate and polyploidy.

sustain tetraploid G1 arrest, but also inhibit mitosis entry following actin damage. Cdk2dn and chemical inhibitors such as roscovitine and olomoucine suppressed accumulation of multinucleated cells by inhibiting both Cdk2 and Cdc2 kinases (Figures 5 and 6). Cdk2 inhibition in p53knockout cells also caused inhibition of Bim-mediated apoptosis (Figures 5 and 6). However, it remains unclear whether Bim expression is affected directly by Cdk2 or merely a result of uncoupling between mitosis and DNA replication. It has been shown that FOXO1/3 can activate transcription of Bim (Stahl et al., 2002; Gilley et al., 2003; Barreyro et al., 2007). However, actin damage did not increase phosphorylation and expression of FOXO1/3 after PTX-2 treatment (data not shown). Recent study reported that CHOP-C/EBPa can activate transcription of FOXO in a response to Endoplasmic reticulum stress (Puthalakath et al., 2007), implying that multiple transcription factors can activate FOXO transcription in a response to environmental signals. Thus, it remains to be elucidated which mediates Bim expression in a response to actin damage. Cells with overexpression of Bcl-xL have an increased rate of spontaneous tetraploidization (Minn et al., 1996; Castedo et al., 2006), suggesting that apoptosis may play an important role in eliminating cells that fail to complete mitosis properly. We showed that Bcl-xL suppressed the apoptosis of p53-deficient cells by PTX2. Instead of apoptotic death, p53-deficient cells with Bcl-xL became polyploidy (Figure 5b). Therefore, these results support that Bcl-xL increases aneuploidy and Oncogene

Cell cycle analysis For each DNA content analysis, 1 106 cells were harvested by trypsinization and fixed by rapid submersion in 1 ml cold 70% ethanol. After fixation at 20 1C for at least 1 h, cells were pelleted and subsequently resuspended in 1 ml staining solution (50 mg ml1 propidium iodide, 50 mg ml1 RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8) and analysed with Coulter EPICS XL (Coulter Electronics, Hialeah, FL, USA). For the determination of mitotic cells, labeling with MPM-2 antibody (Upstate Biotechnology, Charlottesville, VA, USA) was used: Cells were harvested at specific time points, and fixed with ice-cold 90% methanol. After washing with phosphatebuffered saline (PBS), the fixed cells were labeled with MPM-2 antibody (final concentration: 10 mg MPM-2 per ml) for 3 h at room temperature. The labeled cells were washed with PBS, incubated first with a fluorescein isothiocyanate-conjugated donkey anti-mouse immunoglobulin G secondary antibody and then with 50 mg ml1 propidium iodide as described above. The cells staining positive for MPM-2 (circled) show increased fluorescence. The presented data are representative of more than three experiments. Nuclear staining and the BrdU incorporation assay To assess nuclear morphology, we seeded HCT116, p53/ and p21/ cells on cover glasses, and after allowing them to grow for 36 h, treated them with PTX-2 (10 ng ml1) for 3 days and finally fixed them in Cannoid fixative (methanol/acetic acid (3:1)). After staining the nuclei with DAPI (0.2 mg ml1, Sigma, St Louis, MO, USA), the distribution of cells with 1, 2 and X4 nuclei was determined for each group by observation of at least 600 cells. Proliferation assay was performed using a 5-Bromo-20 -deoxy-uridine Labeling and Detection Kit II (Roche, Mannheim, Germany). HCT116, p53- and p21knockout cells were seeded on cover glasses, grown for 36 h before PTX-2 treatment (100 ng ml1). After 1, 2 and 3 days, cells were labeled for 1 h with 10 mM BrdU and BrdUincorporated cells were detected with the manufacturer’s instructions. Western blot analyses Protein (20 mg) was subjected to SDS–polyacrylamide gel electrophoresis and transferred to PolyScreen membranes (NEN, Boston, MA, USA). The membranes were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline Tween-20 (Jung et al., 2001) and probed with antibodies.


4121 The following antibodies were used in this study: antibodies against cleaved caspase-9 and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA); p21 (Oncogene, Boston, MA, USA); cyclins A, B, D, E, Cdk2, Cdk4, actin, Bax, p53 and HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were detected with a horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit or donkey anti-goat secondary antibody with enhanced chemiluminescence detection (Amersham, Buckinghamshire, UK). In vitro kinase assay The specific activities of Cdk–cyclin complexes were determined by 32P incorporation into substrates. For IP-kinase assay, cells were washed with cold PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 5 mM NaCl, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 10 mM Na3VO4, 1 mg ml1 aprotinin, 1 mg ml1 leupeptin, 1 mg ml1 pepstatin A, 0.1 mM PMSF, 1 mM DTT). Extracts (200 mg total protein) were incubated for 12 h at 4 1C with 2 mg of the anti-Cdk2 (SC-163), anti-Cdc2 (SC-54, Santa Cruz Biotechnology) antibody. The immunoprecipitates were immobilized on protein A-agarose

beads (Roche, IN, USA) by incubation for 4 h at 4 1C. The beads were washed twice with 1 ml RIPA buffer and twice more with kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM DTT). After the final wash, the immune complexes were suspended in 50 ml of the corresponding kinase buffer containing 20 mM ATP, 5 mCi [g-32P]ATP and 2 mg histone H1 (Boehringer Mannheim). The reactions were allowed to proceed for 30 min at 30 1C. Phosphorylated proteins were separated on a 12% SDS–PAGE and visualized by autoradiography. Acknowledgements pCMV-Cdk2D145N (Cdk2dn) was given from Dr van den Heuvel (MGH cancer center, MA, USA). This research was supported by Korea Science & Engineering Foundation through the NRL Program, the Aging-Tumorigenesis Research program, and the New Drug target discovery Program, and by Korean Ministry of Health and Welfare through the Cancer Control Program and the Molecular Aging Research Center.

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