RUNX3 Modulates DNA Damage-mediated Phosphorylation of Tumor Suppressor p53 at Ser-15 and Acts as a Co-activator for p53*

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p ‫؍‬ 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3mediated tumor suppression.
RUNX3, which is mapped to human chromosome 1p36, is one of the RUNX family of transcription factors, including RUNX1-3 (1). The RUNX family contains the well conserved 128-amino acid region (Runt domain) and forms a stable complex with PEBP2␤/CBF␤ to exert its transactivation ability. Extensive studies demonstrated that RUNX1 plays an important role in the regulation of hematopoiesis (2,3), whereas RUNX2 contributes to the generation and maturation of osteoblasts (4,5). In contrast to RUNX1 and RUNX2, it has been shown that RUNX3 acts as a candidate tumor suppressor for human gastric cancers (6). According to their results, the RUNX3 gene was rarely mutated in primary gastric cancers; however, its expression levels were significantly down-regulated in primary gastric cancers and gastric cancer-derived cell lines, which might be due to the combination of its hemizygous deletion and the hypermethylation of its promoter region. Additionally, a mutation (R122C) found within the Runt domain of RUNX3 resulted in a complete lack of its tumor suppressive activity.
Subsequent studies revealed that the frequent reduction of RUNX3 expression levels is also observed in several human cancers such as lung cancer, breast cancer, colon cancer, pancreatic cancer, and prostate cancer, which might be attributed to promoter hypermethylation (7)(8)(9)(10)(11)(12)(13), indicating that the down-regulation of RUNX3 is not restricted to gastric cancer. Intriguingly, Yano et al. (15) demonstrated that, during transforming growth factor-␤-mediated apoptotic cell death, RUNX3 has an ability to transactivate pro-apoptotic Bim (Bcl-2-interacting mediator of cell death) (14) in gastric cancer-derived cell lines. Based on their observations, RUNX3 was induced to translocate into the cell nucleus in response to TGF-␤ 3 in association with a significant up-regulation of Bim, suggesting that RUNX3 has a critical role in the regulation of TGF-␤-mediated apoptotic cell death. In support of this notion, Yamamura et al. (16) described that RUNX3 cooperates with Forkhead transcription factor FoxO3a/FKHRL1 to induce apoptotic cell death through transcriptional activation of Bim. In addition, the gastric epithelium of RUNX3-knockout mice exhibited a hyperplasia, a reduced rate of apoptotic cell death, and a lower sensitivity to TGF-␤ (6). Recently, it has been shown that, in addition to the hemizygous deletions and promoter hypermethylation, protein mislocalization of RUNX3 to cytoplasm is an alternative molecular mechanism behind the dysfunction of RUNX3 in gastric and breast cancers (17,18). Jin et al. (19) found that p300 with histone acetyltransferase activity acetylates RUNX3 to protect its proteolytic degradation mediated by the E3 ubiquitin protein ligase Smurf.
p53 is a founding member of the p53 tumor suppressor family of sequence-specific nuclear transcription factors, including p53, p73, and p63 (20,21). In response to DNA damage, p53 is induced to stabilize and exert its pro-apoptotic function. DNA damage-induced post-translational modifications of p53, such as phosphorylation and acetylation, play a critical role in the regulation of p53. The activated form of p53 has an ability to transactivate its direct target genes implicated in cell cycle arrest and/or apoptotic cell death, including p21 WAF1 , BAX, PUMA, NOXA, and p53AIP1 (20). Thus, the sequence-specific transactivation activity of p53 is tightly linked to its pro-apoptotic function (22). In a sharp contrast to p73 and p63, p53 is frequently mutated within its sequence-specific DNA-binding domain in primary human cancers (23)(24)(25). Indeed, p53-deficient mice developed spontaneous tumors (26).
In this study, we found for the first time that there exists a functional relationship between RUNX3 and p53. Based on our present results, RUNX3 is closely involved in the regulation of DNA damage-mediated phosphorylation of p53 at Ser-15 and acts as its co-activator.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-African green monkey embryonic kidney COS7, human cervical carcinoma HeLa, human osteosarcoma U2OS, and SAOS-2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% of heat-inactivated fetal bovine serum (Invitrogen), penicillin (100 IU/ml), and streptomycin (100 g/ml). Human lung carcinoma H1299 cells were cultivated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotic mixture. Cells were grown at 37°C in a water-saturated atmosphere of 95% air and 5% CO 2 . Where indicated, cells were exposed to adriamycin (ADR). Transient transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions.
In Vitro Pulldown Assay-Wild-type p53 and its deletion mutants were generated in vitro using a T7 Quick-coupled transcription/translation system (Promega, Madison, WI) in the presence of [ 35 S]methionine according to the manufacturer's recommendations. Cell lysates prepared from COS7 cells transfected with the expression plasmid encoding RUNX3 were mixed and incubated overnight at 4°C. Reaction mixtures were then immunoprecipitated with anti-RUNX3 antibody. The immunoprecipitates were washed extensively with the lysis buffer and resolved by SDS-PAGE. The gels were dried up and subjected to autoradiography.
Indirect Immunofluorescence Staining-U2OS cells were grown on coverslips and transiently transfected with the expression plasmid for RUNX3. Twenty four hours after transfection, cells were treated with 0.5 M of ADR or left untreated. Forty eight hours after ADR treatment, cells were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature, permeabilized in 0.2% Triton X-100 in PBS for 5 min at room temperature, and blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. After blocking, cells were then simultaneously incubated with polyclonal anti-RUNX3 and monoclonal anti-p53 antibodies followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG. Cell nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). Cells were imaged by Fluoview laser scanning confocal microscope (Olympus, Tokyo, Japan).
Subcellular Fractionation-U2OS cells were transiently transfected with the expression plasmid encoding RUNX3. Twenty eight hours after transfection, cells were treated with 0.5 M ADR or left untreated. Forty eight hours after ADR treatment, cells were washed in ice-cold PBS and lysed in lysis buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, and protease inhibitor mixture for 30 min at 4°C. Reaction mixture was centrifuged at 15,000 rpm at 4°C to separate the soluble fraction (cytoplasmic fraction) from the insoluble fraction (nuclear fraction). Equal amounts of cytoplasmic and nuclear fractions were separated by SDS-PAGE and immunoblotted with anti-RUNX3 antibody. Each fraction was analyzed by immunoblotting with monoclonal anti-lamin B (Ab-1, Oncogene Research Products) or with monoclonal anti-␣-tubulin antibody (Ab-2, NeoMarkers, Fremont, CA) to show the purity of each fraction.
Apoptotic Assay-U2OS and H1299 cells were transiently cotransfected with the constant amount of the expression plasmid for GFP together with or without the increasing amounts of RUNX3 expression plasmid. Forty eight hours after transfection, cells were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature and permeabilized in 0.2% Triton X-100 in PBS for 5 min at room temperature. The coverslips were mounted with DAPI-containing mounting medium (Vector Laboratories) and observed under a Fluoview laser scanning confocal microscope (Olympus, Tokyo, Japan). The number of GFP-positive cells with apoptotic nuclei was measured.
FACS Analysis-H1299, HeLa, U2OS, and SAOS-2 cells were transiently transfected with control siRNA or with siRNA against RUNX3. Twenty four hours after transfection, cells were exposed to the indicated concentrations of ADR. Forty eight hours after ADR treatment, floating and attached cells were collected, washed in PBS, and fixed in 70% ethanol at Ϫ20°C. Following incubation in PBS containing 40 g/ml propidium iodide and 200 g/ml of RNase A for 1 h at room temperature in the dark, stained nuclei were analyzed on a FACScan machine (BD Biosciences).
Luciferase Reporter Assay-H1299 cells were plated in a 12-well flat bottom plates on a day prior to transfection at a density of 50,000 cells/well. Cells were then co-transfected with 12.5 ng of p53 expression plasmid, 100 ng of p53-responsible luciferase reporter construct (p21 WAF1 or BAX), and 10 ng of pRL-TK Renilla luciferase cDNA together with or without the increasing amounts of the RUNX3 expression plasmid (100 and 300 ng). Total amount of plasmid DNA per transfection was kept constant (510 ng) with an empty plasmid pcDNA3 (Invitrogen). Forty eight hours after transfection, cells were lysed, and both firefly and Renilla luciferase activities were measured with Dual-Luciferase reporter assay system (Promega), according to the manufacturer's instructions. The firefly luminescence signal was normalized based on the Renilla luminescence signal.
Small Interfering RNA-U2OS cells were transiently transfected with a SMART pool siRNA specifically designed against RUNX3 or a control nontargeting siRNA (Dharmacon, Chicago) by using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. Forty eight hours after transfection, total RNA was prepared and subjected to RT-PCR. For knocking down of the endogenous p53, U2OS cells were transiently transfected with the empty plasmid (pSUPER, OligoEngine, Seattle, WA) or with pSUPER expression plasmid encoding siRNA against p53 (pSUPER-siRNA-p53) by using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instruction.
Colony Formation Assay-H1299 cells were seeded at a final density of 200,000 cells/6-well plate and allowed to attach overnight. Cells were then co-transfected with the indicated combinations of the expression plasmids. Total amount of plasmid DNA per transfection was kept constant (2 g) with pcDNA3. Forty eight hours after transfection, cells were transferred to the fresh medium containing G418 (800 g/ml). After 14 days, viable colonies were washed in PBS and stained with Giemsa solution.

siRNA-mediated Knockdown of RUNX3 Decreases the Drug
Sensitivity in a p53-dependent Manner-To address whether RUNX3 could be involved in p53-mediated DNA damage response, RUNX3 was knocked down in human osteosarcomaderived U2OS, human lung carcinoma-derived A549, human lung carcinoma-derived H1299, human cervical carcinoma-derived HeLa, and human osteosarcoma-derived SAOS-2 cells (Fig. 1A). U2OS and A549 cells carry wild-type p53. On the other hand, H1299 and SAOS-2 cells are p53-null and HeLa RUNX3 Acts as a Co-activator for p53 MAY (27,28). Knocked down cells were then exposed to the indicated concentrations of DNA-damaging reagent, adriamycin (ADR). As shown in Fig. 1B, silencing of RUNX3 had undetectable effects on ADR-induced apoptotic cell death in H1299, HeLa, and SAOS-2 cells, whereas ADR-mediated apoptotic cell death was significantly inhibited in U2OS and A549 cells. Similar results were also obtained in cells treated with other DNAdamaging drugs, including etoposide and camptothecin, but not with paclitaxel (supplemental Fig. S1). These results suggest a presence of functional association between RUNX3 and p53 in response to DNA damage.
We next examined whether RUNX3 could be induced in response to DNA damage. U2OS cells treated with ADR at a final concentration of 1 M induced the accumulation of p53 and its phosphorylation at Ser-15 as well as expression of its target genes such as BAX, p21 WAF1 , PUMA, and p53AIP1 ( Fig.  1, C and D). Under our experimental conditions, RUNX3 was significantly up-regulated at both mRNA (Fig. 1D) and protein (Fig. 1E) levels. These observations indicate that RUNX3 is one of the DNA damage-response genes. In addition, we could not detect ADR-mediated phosphorylation of p53 at Ser-20 and Ser-46 under our experimental conditions (data not shown).
These results prompted us to examine the functional relationship between RUNX3 and p53 in response to DNA damage. To this end, U2OS cells were transiently transfected with the control siRNA or with siRNA against RUNX3 and then exposed to ADR. As shown in Fig. 1F, the knocking down of RUNX3 had a negligible effect on ADR-dependent accumulation of p53, whereas ADR-dependent phosphorylation of p53 at Ser-15 was markedly abrogated. Consistent with these results, ADR-mediated induction of p53 target gene products was strongly inhibited in RUNX3-knocked down cells. Additionally, ADR-dependent cleavage of PARP was undetectable in RUNX3-knocked down cells. Thus, our present findings imply that RUNX3 contributes to the regulation of DNA damage-induced phosphory- Forty eight hours after transfection, total RNA was prepared and subjected to RT-PCR. B, FACS analysis. Transfected cells were treated with the indicated concentrations of ADR or left untreated. Forty eight hours after ADR treatment, floating and attached cells were collected, and the percentage of cells with sub-G 1 DNA content was analyzed by FACS. C-E, ADR-mediated induction of RUNX3. U2OS cells were exposed to ADR at a final concentration of 1 M. At the indicated times after ADR treatment, cell lysates and total RNA were prepared and processed for immunoblotting (IB) (C) and RT-PCR (D), respectively. Actin and GAPDH were used as loading and internal controls, respectively. For the detection of RUNX3, cell lysates were immunoprecipitated (IP) with anti-RUNX3 antibody followed by immunoblotting with anti-RUNX3 antibody (E). F, RUNX3 affects the phosphorylation status of p53 in response to ADR. U2OS cells were transiently transfected with control siRNA or with siRNA targeting RUNX3 and treated with ADR (at a final concentration of 1 M). At the indicated time points after ADR treatment, cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. Actin was used as a loading control.
lation of p53 at Ser-15 and thereby acting as a co-activator for p53 with undetectable effect on its stability. This may give some clues to understand the previous observations as described by Ashcroft et al. (29) showing that DNA damageinduced stabilization of p53 is detectable irrespective of its phosphorylation status, although both phenomena usually occur in parallel (20).
RUNX3 Is Not a Direct Transcriptional Target of p53-Consistent with the previous observations (30), our present results strongly suggest that U2OS cells undergo apoptotic cell death in a p53-dependent manner. To further confirm this issue, we have performed siRNA-mediated knockdown of the endogenous p53 in U2OS cells. U2OS cells were transiently transfected with the empty plasmid or with the expression plasmid for siRNA against p53. Forty eight hours after transfection, total RNA was prepared and subjected to RT-PCR. As clearly shown in Fig. 2A, the amounts of the endogenous p53 significantly reduced in the presence of siRNA against p53 under our experimental conditions. Next, the knocked down cells were treated with ADR or left untreated. Forty eight hours after ADR treatment, whole cell lysates were prepared and analyzed by immu-noblotting. As shown in Fig. 2B, siRNA-mediated knocking down of the endogenous p53 resulted in a remarkable inhibition of ADR-dependent accumulation of p53 as well as ADRdependent phosphorylation of p53 at Ser-15. In accordance with these results, knocking down of the endogenous p53 led to a significant reduction in number of dead cells in the presence of ADR as compared with the control cells exposed to ADR (Fig.  2C). Intriguingly, a substantial number of dead cells was detectable in ADR-treated knockdown cells, which might be due to the pro-apoptotic effect of the remaining p53 and/or the other p53 family members such as p73 (31). Taken together, U2OS cells underwent apoptotic cell death at least in part in a p53-dependent manner.
Because there existed a clear correlation between ADR-dependent phosphorylation of p53 at Ser-15 and transcriptional induction of RUNX3, it is likely that RUNX3 could be one of the direct target genes of p53. To address this issue, U2OS cells were transiently transfected with the empty plasmid or with the expression plasmid for p53. As shown in Fig. 2D, forced expression of p53 resulted in a significant induction of BAX, whereas the expression levels of the endogenous RUNX3 remained

. U2OS cells undergo apoptotic cell death in a p53-dependent manner.
A, siRNA-mediated knockdown of the endogenous p53. U2OS cells were transiently transfected with the empty plasmid (pSUPER) or with the expression plasmid for siRNA against p53 (pSUPER-siRNA-p53). Forty eight hours after transfection, total RNA was prepared and subjected to RT-PCR. GAPDH was used as an internal control. B, siRNA-mediated knockdown of p53 reduces its accumulation and phosphorylation in response to ADR. U2OS cells were transiently transfected with the empty plasmid or with the expression plasmid for siRNA targeting p53. Twenty four hours after transfection, cells were treated with 1 M ADR or left untreated. Forty eight hours after ADR treatment, whole cell lysates were prepared and processed for immunoblotting with anti-p53 antibody or with antibody against phospho-p53 at Ser-15. Actin was used as a loading control. C, trypan blue exclusion assay. U2OS cells were transiently transfected as in B. Forty eight hours after ADR treatment, floating and attached cells were collected and stained with 0.4% trypan blue. After trypan blue staining, a number of trypan blue-positive cells were measured. D and E, RUNX3 is not a direct transcriptional target of p53. U2OS cells were transiently transfected with the empty plasmid or with the expression plasmid for p53. Forty eight hours after transfection, total RNA was prepared and subjected to RT-PCR. BAX and GAPDH were used as a positive and an internal control, respectively (D). Similarly, U2OS cells were transiently transfected with the empty plasmid or with the expression plasmid for siRNA against p53. Forty eight hours after transfection, total RNA was prepared and processed for RT-PCR. BAX and GAPDH were used as a positive and an internal control, respectively (E). MAY 28, 2010 • VOLUME 285 • NUMBER 22 unchanged even in the presence of the exogenously expressed p53. To further confirm these results, we performed siRNA-mediated knockdown of the endogenous p53. As seen in Fig. 2E, a significant down-regulation of BAX was detectable in p53-knocked down cells, whereas siRNA-mediated knocking down of p53 had an undetectable effect on the expression levels of the endogenous RUNX3. Thus, it is conceivable that RUNX3 might not be a direct target gene of p53. Indeed, we failed to find out the putative p53-responsive element within the 5Ј-upstream region (ϳ1 kb) of human RUNX3 gene. At present, the precise molecular mechanisms behind ADR-mediated up-regulation of RUNX3 remained to be elusive.

RUNX3 Acts as a Co-activator for p53
Interaction between RUNX3 and p53 in Cells-It has been shown that nuclear translocation of RUNX3 plays a critical role in the regulation of apoptotic cell death (16,17). Our indirect immunofluorescence staining and biochemical fractionation experiments revealed that RUNX3 is induced to translocate from the cytoplasm into the cell nucleus in response to ADR and co-localizes with p53 (Fig. 3, A and B), suggesting that RUNX3 might form a complex with p53 in cells.
To further confirm this issue, we performed co-immunoprecipitation analysis using cell lysates prepared from U2OS cells exposed to ADR. As shown in Fig.  4A, the endogenous RUNX3 was coimmunoprecipitated with stress-induced p53 in U2OS cells. In accordance with these results, the anti-RUNX3 immunoprecipitates contained the endogenous p53. Similar results were also obtained in overexpression systems and in vitro pulldown assay (supplemental Fig. S2). Interestingly, RUNX3 was associated with another p53 family member p73 but not with p63 as examined by co-immunoprecipitation analysis (supplemental Fig. S3).
To identify essential region(s) of p53 required for interaction with RUNX3, we carried out in vitro pulldown assay using the indicated deletion mutants of p53 (Fig. 4B). Wildtype p53 and p53(102-393) were efficiently co-immunoprecipitated with RUNX3, whereas p53(1-353), p53(1-292), and p53(1-101) were not, suggesting that its extreme COOH-terminal region is required for the interaction with RUNX3. We also defined region(s) of RUNX3 essential for the interaction with p53 (Fig. 4C). Wild-type RUNX3 but not RUNX3  and RUNX3(1-67) was co-immunoprecipitated with p53, indicating that COOH-terminal region of RUNX3 is required for the complex formation with p53.
RUNX3 Acts as a Co-activator for p53-To further examine the functional significance of the interaction between RUNX3 and p53, we performed luciferase reporter assays. As shown in Fig. 5A, RUNX3 had an ability to enhance p53-mediated luciferase activities driven by p21 WAF1 and BAX promoters in a dose-dependent manner. In a good agreement with these observations, RUNX3 increased p53-dependent expression of the endogenous p21 WAF1 , PUMA, and BAX as examined by RT-PCR (Fig. 5B), indicating that RUNX3 acts as a co-activator for p53. We then asked whether RUNX3 could affect p53-dependent apoptotic cell death. Co-expression of p53 with RUNX3 resulted in a remarkable reduction in number of drug-resistant colonies as compared with cells expressing p53 alone (Fig. 5C). Furthermore, a number of GFP-positive cells with apoptotic nuclei increased in U2OS cells expressing RUNX3 relative to control transfectants, whereas forced expression of RUNX3 increased the number of apoptotic cells to a significantly lesser degree in p53-deficient H1299 cells (Fig. 5D). Thus, it is likely that RUNX3 enhances both transcriptional and pro-apoptotic activities of p53.
Based on our present results, it is likely that the regulation of DNA damage-mediated phosphorylation of p53 at Ser-15 is a major role of RUNX3. To gain molecular insights into understanding how RUNX3 could affect DNA damage-mediated phosphorylation of p53 at Ser-15, we examined whether ATM could have functional relevance to RUNX3. As shown in Fig. 6A, RUNX3 induced proteolytic cleavage of PARP and phosphorylation of p53 at Ser-15 in U2OS cells, indicating that RUNX3 might have an ability to promote phosphorylation of p53 at Ser-15, and thereby enhancing the transcriptional and pro-apoptotic activities of p53. Next, we have examined whether RUNX3 could interact with phosphorylated forms of ATM. To this end, HeLa cells were transiently transfected with the expression plasmid for RUNX3 and exposed to ADR. As shown in Fig. 6B, the anti-RUNX3 immunoprecipitates contained phosphorylated forms of ATM. These results imply that RUNX3 forms a complex with phosphorylated forms of ATM in response to ADR. To further investigate the functional relationship between RUNX3 and ATM, we employed wild-type and ATM-null A-T cells. As shown in Fig. 6C, the exogenous RUNX3 enhanced the phosphorylation of p53 at Ser-15, which was further augmented in wild-type cells but not in A-T cells exposed to ADR. Of note, the exogenous RUNX3 had an ability to enhance the ADR-mediated phosphorylation level of p53 at Ser-15. These results suggest that RUNX3 recruits phosphorylated forms of ATM onto p53 and thereby induces ATM-dependent phosphorylation of p53 at Ser-15 after DNA damage.
These findings prompted us to ask whether RUNX3 could affect the extent of ADR-mediated DNA damage. For this purpose, U2OS cells were transiently transfected with control siRNA or with siRNA against RUNX3 and exposed to ADR or left untreated. Twenty four hours after the treatment, total RNA and nuclear lysates were prepared and subjected to RT-PCR and immunoblotting, respectively. As shown in Fig. 7, ADR-mediated up-regulation of the endogenous RUNX3 in cells transfected with control siRNA was detectable, and siRNA-mediated knocking down of the endogenous RUNX3 was successful under our experimental conditions. Consistent with the previous observations (32), immunoblot analysis revealed that the expression levels of total ATM remain unchanged regardless of DNA damage. Of note, knocking down of the endogenous RUNX3 had a marginal effect on the phosphorylation levels of ATM in response to ADR; however, we did not observe a significant difference of ADR-dependent accumulation of ␥H2AX (the phosphorylated form of histone H2AX) between control cells and RUNX3-knocked down cells,  MAY 28, 2010 • VOLUME 285 • NUMBER 22

RUNX3 Acts as a Co-activator for p53
suggesting that RUNX3 does not affect the extent of DNA damage.
Clinical Significance-We next examined whether p53 inactivation and decreased expression of RUNX3 could affect clinical prognosis of human cancer. For this purpose, we selected 105 patients with lung adenocarcinoma, in which p53 mutations and its transcriptional silencing have been described (33). As shown in Fig. 8, long term cumulative survival rates were significantly decreased in the combination of p53 mutation and low expression of RUNX3 (p ϭ 0.0203). Notably, the combined p53 mutation and decreased expression of RUNX3 were found in 25 patients in stages II to IV cancer (93%) compared with only 2 in stage I cancer (7%) (Fig. 7, p ϭ 0.0018). These results suggest that functional interaction between RUNX3 and p53 might be associated with the clinical outcome of patients with lung adenocarcinoma.

DISCUSSION
To our knowledge, it remained unclear whether and how RUNX3 could be involved in the regulation of DNA damage response. In this study, we have found for the first time that RUNX3 modulates DNA damage-induced phosphorylation of tumor suppressor p53 at Ser-15 and thereby acting as a coactivator for p53. Thus, our present findings provide a novel insight into understanding the molecular mechanisms behind p53-dependent apoptotic cell death in response to DNA damage.
Under our experimental conditions, siRNA-mediated silencing of the endogenous RUNX3 strongly inhibited apoptotic cell FIGURE 5. RUNX3 acts as a co-activator for p53. A and B, RUNX3 enhances p53-mediated transcriptional activation. p53-deficient human lung carcinomaderived H1299 cells were transiently co-transfected with a constant amount of p53 expression plasmid, the luciferase reporter construct bearing p53responsive element derived from human p21 WAF1 or BAX promoter and Renilla luciferase plasmid together with or without the increasing amounts of RUNX3 expression plasmid. Forty eight hours after transfection, cells were lysed, and their luciferase activities were measured A. H1299 cells were transiently transfected with the constant amount of the expression plasmid for p53 along with or without the increasing amounts of the expression plasmid encoding RUNX3. Forty eight hours after transfection, total RNA was prepared and processed for RT-PCR. GAPDH was used as an internal control. C and D, RUNX3 enhances pro-apoptotic activity of p53. H1299 cells were transfected with the indicated combinations of expression plasmids. Forty eight hours after transfection, cells were transferred into fresh medium containing G418 at a final concentration of 800 g/ml. Two weeks after the selection with G418, drug-resistant colonies were stained with Giemsa solution (C). U2OS and H1299 cells were transiently transfected with a constant amount of GFP expression plasmid together with or without the increasing amounts of RUNX3 expression plasmid. Forty eight hours after transfection, a number of GFP-positive cells with apoptotic nuclei was measured (D).
death in response to ADR in cells bearing wild-type p53 but not in p53-deficient cells. Of note, knocking down of the endogenous RUNX3 led to a significant inhibition of ADR-mediated phosphorylation of p53 at Ser-15 in association with a massive inhibition of p53-dependent up-regulation of several p53-target genes as well as proteolytic cleavage of PARP. Furthermore, RUNX3 was induced and translocated from the cytoplasm into the cell nucleus in response to ADR. Consistent with these observations, forced expression of RUNX3 promoted phosphorylation of p53 at Ser-15 and proteolytic cleavage of PARP, suggesting that RUNX3 contributes to the regulation of DNA damage-induced phosphorylation of p53 at Ser- 15. It has been established that DNA damage-mediated phosphorylation of ATM is an early event in transducing DNA damage signal (34). After the phosphorylation of ATM, the phosphorylated form of histone H2AX (␥H2AX), which is mediated by phospho-ATM, marks a chromatin region at or near the sites of DNA damage (35). According to our present results, RUNX3 had a marginal effect on ADR-mediated phosphorylation level of ATM, whereas RUNX3 had an undetectable effect on the amounts of ␥H2AX in response to ADR, indicating that RUNX3 is not involved in the initial step of DNA damage response and also does not affect the extent of DNA damage. It is worth noting that phosphorylated forms of ATM are associated with RUNX3 as examined by immunoprecipitation experiments. Thus, it is likely that RUNX3 assists DNA damage-induced phosphorylation of p53 at Ser-15 through complex formation with phosphorylated forms of ATM and enhances transcriptional as well as pro-apoptotic activity of p53, although Khanna et al. (36) reported that ATM directly binds to and phosphorylates p53 at Ser-15.
As described previously (15,37), RUNX3 had an ability to increase the expression levels of p21 WAF1 and pro-apoptotic Bim in collaboration with the activated Smad proteins during TGF-␤-mediated apoptotic cell death. In addition, p300-mediated acetylation of RUNX3 resulted in an increase in its stability (15). So far, RUNX3 has been implicated in carcinogenesis as a nuclear effecter of TGF-␤ tumor suppressor pathway (14,15) and as an attenuator of oncogenic Wnt signaling pathway (21). We found the third RUNX3mediated pro-apoptotic pathway that includes functional interaction between RUNX3 and p53. These pathways might be chosen under certain conditions in the tissue-or lineagespecific progenitor or stem cells to suppress their malignant transformation.
Based on our present results, RUNX3 was able to enhance the transactivation and pro-apoptotic activities of p53. Indeed, RUNX3 enhanced the p53-mediated up-regulation of p21 WAF1 , BAX, and PUMA. Our extensive immunoprecipitation and in vitro pulldown assays revealed that the extreme COOH-terminal portion of p53 is required for the interaction with RUNX3. Initial studies suggest that the COOH-terminal region of p53 acts as a negative regulator and might lock its DNA-binding domain in a latent conformation (38,39). However, this model has been challenged by the findings showing that stress-induced acetylation of COOH-terminal Lys-373 and Lys-382 of Lysates prepared from HeLa cells exposed to ADR at a final concentration of 1 M were immunoprecipitated (IP) with normal rabbit serum (NRS) or with polyclonal anti-RUNX3 antibody followed by immunoblotting with anti-phospho-ATM antibody. C, RUNX3 enhances ADR-mediated phosphorylation of p53 at Ser-15. Wild-type (WT) and ATM-deficient A-T cells were transiently transfected with or without RUNX3 expression plasmid in the presence or absence of ADR at a final concentration of 1 M. Lysates were subjected to immunoblotting with the indicated antibodies. p53 is required for the transcriptional activity of p53 (40). Additionally, this COOH-terminal acetylation resulted in an inhibition of p53 ubiquitination (40). Furthermore, McKinney et al. (41) described that the COOH-terminal region of p53 might facilitate the search for specific target sequences in the context of a complex genome. These results indicate that the COOHterminal region of p53 is a positive regulator of DNA binding and transactivation. In support of this notion, COOH-terminally truncated splice variant, p53␤, lacked the transactivation function (42,43). It remains unclear how RUNX3 could contribute to the activation of p53 through its binding to the COOH-terminal region of p53. Further studies should be required to address this issue.
Another important finding of this study was that RUNX3 affects the phosphorylation status of p53, whereas RUNX3 has undetectable effect on its stability. Based on our present results, siRNA-mediated knockdown of the endogenous RUNX3 and forced expression of RUNX3 had a negligible effect on the stability of p53. However, RUNX3 enhanced the p53-mediated transcriptional and pro-apoptotic activities. Although the accumulating evidence strongly suggests that DNA damageinduced accumulation and phosphorylation of p53 correlates with a significant increase in its activity (20), it is conceivable that DNA damage-induced stabilization of p53 might be a distinct phenomenon from DNA damage-mediated phosphorylation at Ser-15 and activation of p53 in certain experimental systems. In support of this notion, Ashcroft et al. (29) demonstrated that DNA damage-induced stabilization of p53 is observed irrespective of its phosphorylation status.
As described previously (15), TGF-␤ treatment led to a nuclear translocation of RUNX3 and induced apoptotic cell death through the transactivation of pro-apoptotic Bim. In a good agreement with these observations, RUNX3 was inactivated through its mislocalization to cytoplasm (17,18). Under our experimental conditions, exogenously expressed RUNX3 was localized both in the cytoplasm and cell nucleus in the absence of DNA damage as examined by indirect immunofluorescence staining and immunoblotting experiments. It was worth noting that ADR treatment induces a significant nuclear translocation of RUNX3 and RUNX3 is co-localized with p53 in cell nucleus. Collectively, our present study strongly suggests that RUNX3 contributes at least in part to p53-mediated apoptotic cell death in response to DNA damage through the regulation of p53 phosphorylation at Ser-15.
In addition, our clinical study using primary human lung adenocarcinoma tissues suggested that patients with low expression of RUNX3 and p53 mutation display the poor prognosis. Thus, it is likely that the RUNX3/p53 pro-apoptotic pathway might be disrupted in the advanced stages of lung adenocarci- FIGURE 7. Effect of silencing of RUNX3 on ADR-mediated phosphorylation of ATM and histone H2AX. U2OS cells were transiently transfected with control siRNA or with siRNA targeting RUNX3. Twenty four hours after transfection, cells were treated with 1 M ADR or left untreated. Twenty four hours after ADR treatment, nuclear lysates and total RNA were subjected to immunoblotting and RT-PCR, respectively. For immunoblotting, lamin B was used as a loading control. For RT-PCR, GAPDH was used as an internal control. noma. Further precise understandings of molecular mechanisms behind the collaboration of RUNX3 with p53 may provide a clue for developing novel diagnostic tools and therapeutic strategies against high risk cancers.