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Cancer Biology, Department of Radiation Oncology, Emory. University ... Oncology, Winship Cancer Institute, Emory University School of Medicine, 1365C.
Volume 11 Number 10

October 2009

pp. 1012–1021

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www.neoplasia.com

A Nonhomologous End-joining Pathway Is Required for Protein Phosphatase 2A Promotion of DNA Double-Strand Break Repair1,2

Qinhong Wang*, Fengqin Gao*, Ton Wang*, ,† Tammy Flagg* and Xingming Deng* *UF Shands Cancer Center, Division of Hematology/ Oncology, Department of Medicine and Department of Anatomy & Cell Biology, University of Florida, Gainesville, FL 32610-3633, USA; †Winship Cancer Institute, Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA30322, USA

Abstract Protein phosphatase 2A (PP2A) functions as a potent tumor suppressor, but its mechanism(s) remains enigmatic. Specific disruption of PP2A by either expression of SV40 small tumor antigen or depletion of endogenous PP2A/C by RNA interference inhibits Ku DNA binding and DNA-PK activities, which results in suppression of DNA doublestrand break (DSB) repair and DNA end-joining in association with increased genetic instability (i.e., chromosomal and chromatid breaks). Overexpression of the PP2A catalytic subunit (PP2A/C) enhances Ku and DNA-PK activities with accelerated DSB repair. Camptothecin-induced DSBs promote PP2A to associate with Ku 70 and Ku 86. PP2A directly dephosphorylates Ku as well as the DNA-PK catalytic subunit (DNA-PKcs) in vitro and in vivo, which enhances the formation of a functional Ku/DNA-PKcs complex. Intriguingly, PP2A promotes DSB repair in wild type mouse embryonic fibroblast (MEF) cells but has no such effect in Ku-deficient MEF cells, suggesting that the Ku 70/86 heterodimer is required for PP2A promotion of DSB repair. Thus, PP2A promotion of DSB repair may occur in a novel mechanism by activating the nonhomologous end-joining pathway through direct dephosphorylation of Ku and DNA-PKcs, which may contribute to maintenance of genetic stability. Neoplasia (2009) 11, 1012–1021

Introduction Environmental factors and genotoxic products of endogenous metabolic processes pose a constant threat to genome integrity by generating hundreds of thousands of DNA modifications in each cell everyday [1]. Among these modifications, DNA double-strand breaks (DSBs) are particularly detrimental because both strands are damaged [2]. Inability to repair DSBs can lead to the accumulation of genomic rearrangements that potentially promote tumorigenesis [3,4]. DSBs are repaired by homologous recombination or by nonhomologous end-joining (NHEJ). Homologous recombination is the predominant DSB repair mechanism in prokaryotes, and in eukaryotes, it plays a principal role during the S and G2 phases of the cell cycle. In contrast, NHEJ, which simply pieces together the broken DNA ends, can function in all phases of the cell cycle [2,5]. In higher eukaryotes, the predominant recourse is the NHEJ DSB repair pathway. NHEJ is a versatile mechanism using the Ku heterodimer, DNA-PK catalytic subunit (DNA-PKcs), ligase IV/XRCC4, and a host of other proteins that juxtapose two free DNA ends for ligation [1]. The reversible phosphorylation of proteins, catalyzed by protein kinases and protein phosphatases, is a major mechanism for regulation

of many eukaryotic cellular processes including DSB repair [6]. DNAPK undergoes phosphorylation of all three of its protein components in vitro, and phosphorylation of the DNA-PK complex correlates with loss of protein kinase activity and disruption of DNA-PKcs from the Ku-DNA complex [7]. DNA-PKcs can be phosphorylated at multiple sites, and phosphorylation of DNA-PKcs at Ser 2056 inhibits Abbreviations: NHEJ, nonhomologous end-joining; DSB, DNA double-strand break; PP2A/C, protein phosphatase 2A catalytic subunit; small T, the SV40 small tumor antigen; PFGE, pulsed-field gel electrophoresis; HA, hemagglutinin; siRNA, small interfering RNA; RNAi, RNA interference Address all correspondence to: Xingming Deng, MD, PhD, Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, 1365C Clifton Rd NE, Room C3092, Atlanta, GA 30322. E-mail: [email protected] 1 This work was supported by National Cancer Institute, National Institutes of Health grant R01 CA112183, by an Award from Stop Children Cancer, Inc., and by a Flight Attendant Medical Research Institute Clinical Innovator Award (to X.D.). 2 This article refers to supplementary materials, which are designated by Figures W1 to W7 and are available online at www.neoplasia.com. Received 6 May 2009; Revised 25 June 2009; Accepted 25 June 2009 Copyright © 2009 Neoplasia Press, Inc. All rights reserved 1522-8002/09/$25.00 DOI 10.1593/neo.09720

Neoplasia Vol. 11, No. 10, 2009 DNA end processing [8]. Ku 70 can be phosphorylated by DNA-PK at Ser 6 and Ser 51 [9,10]. Ku 86 is phosphorylated at Ser 577, Ser 580, and Thr 715 [9]. However, the functional role of Ku 70/86 phosphorylation is not fully understood. Protein phosphatase 2A (i.e., PP2A) is one of the major Ser/Thr phosphatases implicated in the regulation of many cellular processes including cell cycle progression, apoptotic cell death, DNA replication, gene transcription, and DSB repair [11–13]. The AC catalytic complex alone has phosphatase activity, whereas the distinct B subunits can recruit PP2A/C to distinct subcellular locations and then define a specific substrate target [14–16]. The A and C subunits are evolutionary conserved and ubiquitously expressed [17]. These two subunits form a catalytic complex (PP2A/AC) that interacts with at least three families of regulatory subunits (B, B′, and B″) and tumor antigens (i.e., SV40 small tumor antigen [18]). The B subunits determine the substrate specificity of PP2A [14]. PP2A has recently been reported to dephosphorylate γ-H2AX leading to facilitation of DSB repair [11]. A previous report indicates that PP2A can directly dephosphorylate Ku and DNA-PKcs in vitro, which leads to increased DNA-PK protein kinase activity in vitro [6]. However, it is not clear whether the effect of PP2A on DSB repair results from a direct activation of the NHEJ factors (i.e., Ku and DNA-PKcs) in vivo. Here, we provide strong evidence that PP2A facilitates DNA end-joining and DSB repair in a novel mechanism by activating Ku and DNA-PKcs, which contributes to the suppression of genetic instability.

Materials and Methods

Materials PP2A/C, Ku 70, Ku 86, DNA-PKcs, and tubulin antibodies as well as PP2A/C small interfering RNA (siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Small Tantibody was purchased from Research Diagnostics, Inc (Concord, MA). Anti–γ-H2AX antibody was purchased from Upstate Biotech (Charlottesville, VA). Ku 70/Ku 86 DNA Repair Kit was obtained from Active Motif (Carlsbad, CA). The SignaTECT DNA-PK Assay Kit was purchased from Promega (Madison, WI). Alexa Fluor 594–conjugated goat antimouse immunoglobulin G antibody and Alexa Fluor 488–conjugated goat antirabbit immunoglobulin G antibody were obtained from Invitrogen (Carlsbad, CA). Telomere PNA FISH Kit/Cy3 was obtained from DakoCytomation (DK-2600; Glostrup, Denmark). Purified PP2A was obtained from Calbiochem (San Diego, CA). Purified Ku 70 and Ku 86 proteins were purchased from GenWay Biotech, Inc (San Diego, CA). Camptothecin (CPT) was obtained from SigmaAldrich (St Louis, MO). The hemagglutinin (HA)-tagged PP2A/C/ pcDNA3 construct was generously provided by Dr. Brain Law (University of Florida). Wild type small T antigen complementary DNA (cDNA) in pCMV5 was kindly provided by Dr. Marc Mumby (University of Texas Southwestern Medical Center, Dallas, TX). Wild type and Ku 86−/− mouse embryonic fibroblast (MEF) cells were kindly provided by Dr. David J. Chen (University of Texas Southwestern Medical Center, Dallas, TX). All of the reagents used were obtained from commercial sources unless otherwise stated.

Cell Lines, Plasmids, and Transfections H69, H82, H157, H1299, and H460 cells were maintained in RPMI 1640 with 10% fetal bovine serum (FBS). A549 cells were main-

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tained in F-12K medium with 10% FBS. Wild type and Ku 86−/− MEF cells were maintained in Dulbecco’s modified Eagle medium with 10% FBS and 4 mM L-glutamine. The HA-tagged PP2A/C/ pcDNA3 or small T/pCMV5 were transfected into H1299 cells using LipofectAMINE 2000 (Invitrogen Life Technology, Carlsbad, CA) according to the manufacturer’s instructions.

Phosphorylation/Dephosphorylation Assay Cells were cultured with 0.5% FBS overnight. The cells were washed with phosphate-free RPMI medium and metabolically labeled with [32P]orthophosphoric acid for 60 minutes. After agonist or inhibitor addition, the cells were washed with ice-cold phosphatebuffered saline and lysed in detergent buffer. Ku 70, Ku 86, or DNAPKcs was then immunoprecipitated. Phosphorylation of Ku 70, Ku 86 or DNA-PKcs was determined by autoradiography. For dephosphorylation, 32P-labeled Ku 70, Ku 86, or DNA-PKcs was immunoprecipitated as described. The beads were washed and resuspended in 60 μl of phosphatase assay buffer containing 50 mM Tris-HCl, pH 7.0, 20 mM β-mercaptoethanol, 2 mM MnCl2, and 0.1% BSA. Purified PP2A was added, and the samples were incubated at 30°C for 10 minutes as described [19]. The samples were boiled for 5 minutes before loading onto SDS-PAGE. Phosphorylation of Ku 70, Ku 86, or DNA-PKcs was determined by autoradiography.

Preparation of Cell Lysates Cells were washed with 1× PBS and resuspended in ice-cold EBC buffer (0.5% NP-40, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, and 1 mM β-mercaptoethanol) with a cocktail of protease inhibitors (EMD Biosciences, Gibbstown, NJ). Cells were lysed by sonication and centrifuged at 14,000g for 10 minutes at 4°C. The resulting supernatant was collected as the total cell lysate.

Pulsed-Field Gel Electrophoresis Pulsed-field gel electrophoresis (PFGE) was performed as described [20,21]. Briefly, cells were harvested and resuspended in ice-cold buffer L (0.1 M Na2-EDTA, 0.01 M Tris, 0.02 M NaCl, pH 8.0) at a concentration of 5 × 106 cells per milliliter and mixed with an equal volume of 1% low–melting point agarose (Beckman, Fullerton, CA) at 42°C. The mixture was pipetted into a small length of Tygon tubing, clamped tight at both ends, and chilled to 0°C. The solidified agarose “snake” was extruded from the tubing, added to 10× volume of buffer L containing 1 mg/ml proteinase K and 1% sarkosyl, and incubated for 16 hours at 50°C. After lysis, the agarose snake was washed four times with Tris/EDTA buffer and then cut into 0.5-cm plugs. The plugs were inserted into the wells of a precooled 1% low– melting point agarose gel (4°C). PFGE (200-second pulse time, 150 V, 15 hours at 14°C) was performed using the clamped, homogenous electric fields Mapper (Bio-Rad, Hercules, CA). After electrophoresis, the gel was stained with ethidium bromide for photography.

Immunofluorescence The cells were washed with 1× PBS, fixed with methanol and acetone (1:1) for 5 minutes, and then blocked with 10% normal mouse or rabbit serum for 20 minutes at room temperature. Cells were incubated with a mouse or rabbit primary antibody for 90 minutes. After washing, samples were incubated with Alexa Fluor 488 (green)– conjugated antimouse or Alexa Fluor 594 (red)–conjugated antirabbit

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secondary antibodies or 4′-6-diamidino-2-phenylindole (DAPI) for 60 minutes. Cells were washed with 1× PBS and observed under a fluorescent microscope (Zeiss, Thornwood, NY).

Telomere Fluorescence In Situ Hybridization Analysis Telomere fluorescence in situ hybridization (T-FISH) was performed using the Telomere PNA FISH Kit/Cy3 (DakoCytomation) as described [21,22]. Briefly, cells were incubated with colcemid (KaryoMAX; Gibco, Carlsbad, CA) at 100 ng/ml for 1 hour and then harvested by trypsinization. Cells were swollen in prewarmed 30 mM sodium citrate for 30 minutes at 37°C, fixed in methanol/acetic acid (3:1), and air-dried on slides overnight. After pepsin digestion, slides were denatured at 80°C for 5 minutes, hybridized with Cy3-labeled peptide nucleic acid (PNA) telomeric probe (Cy3-[TTAGGG]3) in 70% formamide at room temperature for 3 to 4 hours, washed, dehydrated, and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Metaphase images were captured using a fluorescent microscope (Zeiss). At least 30 metaphases of each cell line were scored for chromosomal aberrations.

RNA Interference H1299 cells were transfected with PP2A/C siRNA using LipofectAMINE 2000. A control siRNA (nonhomologous to any known gene sequence) was used as a negative control. The levels of PP2A/C expression were analyzed by Western blot. Specific silencing of the targeted PP2A/C gene was confirmed by at least three independent experiments.

Cell Viability Assay The apoptotic and viable cells were detected using an ApoAlert Annexin-V Kit from Clontech (Palo Alto, CA) according to the manufacturer’s instructions. Cell viability was determined by analyzing annexin-V binding on FACS.

Results

Disruption of PP2A Activity by Expression of Small T Antigen Downregulates Ku/DNA-PK Activity in Association with Suppression of DNA End-joining and DSB Repair Leading to Increased Genetic Instability Ku 70, Ku 86, and DNA-PKcs are the major components of the NHEJ machinery required for DSB repair and are widely expressed in both small cell lung cancer and non–small cell lung cancer cells (Figure W1). PP2A has recently been reported to promote DSB repair, but the mechanism(s) involved is not fully understood [11]. It is well known that the SV40 small tumor antigen (small T) can interact with the 36-kDa catalytic C and the 63-kDa A subunits of PP2A, which can specifically disrupt PP2A but not PP1’s activity [23]. To specifically test whether PP2A can regulate NHEJ activity, the pCMV5/small T antigen construct was transfected into H1299 cells. Ku DNA binding activity or DNA-PK activity was assessed using a Ku 70/86 DNA Repair Kit or a SignaTECT DNA-PK Assay Kit, respectively. Intriguingly, decreased levels of Ku or DNA-PK activity were observed in small T–expressing cells compared with vector-only control cells (Figure 1A and W2A), indicating that specific inhibition of PP2A by small T downregulates Ku/DNA-PK activity. CPT is a potent agent for induction of cellular DSBs [11]. To further

Neoplasia Vol. 11, No. 10, 2009 test whether disruption of PP2A activity affects DSB repair, H1299 cells expressing small T or vector-only control were treated with CPT (5 μM) for 1 hour, then washed and incubated in regular culture medium for various times up to 24 hours. A PFGE under neutral conditions (a direct method for DSB measurement) was performed as described [20]. After CPT treatment, a significant amount of genomic DNA from all cells ran out of the wells and migrated into the gel, which indicates DNA fragmentation (i.e., DSBs). By 24 hours after washing, most genomic DNA from vector-only control cells was retained in the well, indicating that most DSBs had been repaired (Figure 1B). In contrast, significant DNA fragmentation can still be observed at the 24-hour time point in cells expressing small T (Figure 1B). Formation of a chromatin-associated γ-H2AX focus is considered to be a sensitive and selective signal for the existence of DSBs [24,25]. γ-H2AX foci were also observed in both vector-only control and small T–expressing cells after exposure of cells to CPT (Figure W2B). The intensity and number of γ-H2AX foci per cell as well as the percentage of foci-positive cells are significantly reduced by 12 to 24 hours in vector-only control cells. Intriguingly, more γ-H2AX foci with stronger intensity per cell and a higher percentage of foci-positive cells are observed in cells expressing small T compared with vector-only control after 12 to 24 hours (Figure W2B). Western blot analysis using γ-H2AX provides further evidence that expression of small T significantly prolongs the persistence of γH2AX in cells after CPT treatment (Figure W2C ). These findings reveal that specific inhibition of PP2A activity by small T results in suppression of DSB repair. DNA end-joining is the major mechanism for repairing DSBs [26,27]. Ku/DNA binding and DNA-PK activity are essential for rejoining of the broken DNA ends [28,29]. Because expression of small T not only impedes Ku/DNA binding but also suppresses DNA-PK activity (Figure W2A), inhibition of PP2A by small T may affect DNA end-joining capacity. DNA nonhomologous endjoining activity was measured using the linearized pGL3 plasmid as described in Supplemental Methods. Results indicate that expression of small T significantly inhibits DNA end-joining activity compared to vector-only control (Figure W2D), indicating that PP2A activity is critical for DNA end-joining. To test the effect of small T on chromosomal aberrations, we used a FISH assay that combines DAPI staining with a telomere-specific PNA probe (T-FISH) to assess metaphase chromosomes for instability as described [22]. Increased levels of cytogenetic abnormalities, including chromosomal and chromatid breaks, were observed in small T–expressing cells compared with vector-only control cells (Figure 1, C and D). These findings reveal that small T–mediated inhibition of PP2A and DSB repair facilitates genetic instability, which may potentially contribute to carcinogenesis. Similar experiments were also performed using other cell lines (i.e., MEF cells), and similar results were obtained (data not shown). This suggests that the inhibitory effect of small T on DSB repair is a general reaction and not a cell type–specific phenomenon.

Overexpression of PP2A/C Upregulates Ku and DNA-PK Activities in Association with Acceleration of DSB Repair To directly test the effect of PP2A on Ku activity and DSB repair, HA-tagged PP2A/C was transfected into H1299 cells. Intriguingly, overexpression of HA-PP2A/C significantly not only enhances the Ku DNA binding and DNA-PK activities but also accelerates DSB repair (Figures 2 and W3). These findings provide strong evidence

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Figure 1. Disruption of PP2A activity by small T downregulates Ku and DNA-PK activity in association with suppression of DSB repair leading to increased genetic instability. (A) Small T antigen cDNA/pCMV5 constructs were transfected into H1299 cells. Expression levels of small T protein were analyzed by Western blot using a small T antibody. (B) H1299 cells expressing small T or vector-only control were treated with CPT (5 μM) for 1 hour. Then, cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSB was determined by PFGE. (C and D) Cytogenetic abnormalities were analyzed by T-FISH in H1299 cells expressing small T or vector-only control. DAPI-stained chromosomes are blue. Red dots come from telomere signals. Color-coded arrowheads indicate a normal chromosome and different kinds of cytogenetic abnormalities (white indicates normal chromosome with four telomere signals; green, chromosomal break with two telomere signals; yellow, chromatid break with three telomere signals). The percentage of abnormal metaphases and frequency of cytogenetic abnormality per metaphase from small T–expressing cells or vectoronly control cells were quantified by T-FISH analysis. At least 30 metaphases per culture were analyzed. Each experiment was repeated three times and data represent the mean ± SD of three separate determinations.

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Figure 2. Overexpression of exogenous HA-tagged PP2A/C accelerates DSB repair. (A) HA-tagged PP2A/C/pcDNA3 construct was transfected into H1299 cells. Expression of HA-PP2A/C was determined by Western blot using anti-HA antibody. (B and C) H1299 cells overexpressing HA-PP2A/C or vector-only control were treated with CPT (5 μM) for 1 hour. Cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSB was determined by PFGE or analysis of γ-H2AX by Western blot using a γ-H2AX antibody.

that PP2A promotion of DSB repair may occur in a mechanism involving activation of the NHEJ pathway.

CPT-Induced DSBs Promote PP2A/C to Interact with Ku Proteins The Ku protein mainly localizes and functions in nucleus [30]. In contrast, PP2A can be localized in both cytoplasm and nucleus [11,31]. A previous report has demonstrated that CPT-induced DSB signal promotes PP2A accumulation in the nucleus [11], suggesting a potential mechanism by which PP2A regulates DSB repair. Here, we found that PP2A/C localizes in both the cytoplasm and the nucleus, which partially overlap with areas showing expression of either Ku 70 or Ku 86 in nucleus (Figure 3A). Coimmunoprecipitaion (coIP) reveals that treatment of H1299 cells with CPT enhances PP2A/C to directly associate with Ku 70 or Ku 86 in the nucleus in a dose-dependent manner (Figure 3B). A direct interaction between PP2A and Ku was also confirmed by coIP using purified PP2A/C and purified Ku 70 or Ku 86 proteins in vitro (data not shown). Because PP2A is a serine/threonine protein phosphatase, a direct binding of PP2A to Ku may facilitate Ku dephosphorylation.

PP2A-Induced Dephosphorylation Enhances Ku/DNA-PKcs Binding PP2A is the most abundant known serine/threonine–specific protein phosphatase expressed in mammalian cells [32]. To test whether

PP2A can directly dephosphorylate Ku and DNA-PKcs, phosphorylated Ku 70, Ku 86, or DNA-PKcs was incubated with increasing concentrations of purified, active PP2A at 30°C for 10 minutes. Results show that purified PP2A can directly dephosphorylate Ku 70, Ku 86, and DNA-PKcs in a dose-dependent manner (Figure 4A). Importantly, overexpression of HA-PP2A/C results in decreased phosphorylation levels of Ku 70, Ku 86, and DNA-PKcs (Figure 4B). In contrast, inhibition of PP2A activity by expression of small T enhances phosphorylation levels of Ku 70, Ku 86, and DNA-PKcs (Figure W4A). These findings reveal that PP2A functions as a physiological phosphatase for both Ku and DNA-PKcs. DNA-PK is a large functional complex consisting of Ku and DNA-PKcs, which are required for DSB repair by the process of NHEJ [33]. To test the effect of phosphorylation/dephosphorylation on Ku/DNA-PKcs binding, levels of the Ku/DNA-PKcs complex were measured in H1299 cells expressing small T, HA-PP2A/C, or vector-only control. Compared with vector-only control cells, decreased levels of Ku/DNA-PKcs binding were observed in small T–expressing cells and increased levels of Ku/DNA-PKcs complex were detected in HA-PP2A/C–overexpressing cells (Figures 4C and W4B), indicating that phosphorylation dissociates the Ku/DNAPKcs complex and that dephosphorylation promotes Ku to interact with DNA-PKcs. These findings help solve the puzzle why PP2Amediated dephosphorylation of Ku and DNA-PK enhances their activities with acceleration of DSB repair.

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Figure 3. PP2A/C colocalizes and interacts with Ku proteins. (A) Subcellular distribution of PP2A/C, Ku 70, or Ku 86 in H1299 cells was examined by immunofluorescent staining. (B) H1299 cells were treated with increased concentrations of CPT for 60 minutes. A coIP experiment was performed using the isolated nuclear fraction and PP2A/C or Ku 86 antibody, respectively. PP2A/C, Ku 70 or Ku 86 was analyzed by Western blot. Rabbit preimmune serum (Pre) was used as a control.

Depletion of PP2A/C by RNA Interference Results in Phosphorylation of Ku and DNA-PKcs, Suppression of DSB Repair, and Increased Chromosomal Aberrations

sensitizes H1299 cells to the DNA-damaging agent CPT. These data support and extend the findings of another group [11].

To test a role of PP2A in DSB repair, a gene silencing approach was used. Transfection of PP2A/C siRNA significantly reduces the expression level of endogenous PP2A/C by more than 95% in H1299 cells (Figure 5A). This effect of siRNA on PP2A/C expression is highly specific because the control siRNA has no effect. Functionally, depletion of PP2A/C by RNA interference (RNAi) results in increased phosphorylation of Ku 70, Ku 86, and DNA-PKcs, decreased Ku/DNA-PKcs activity, and dissociation of the Ku/DNA-PKcs complex, which suppress the repair of CPT-induced DSBs as determined by either PFGE or analysis of γ-H2AX by immunostaining or Western blot (Figures 5, W5, and W6A). These findings provide strong evidence that physiologically expressed PP2A can promote DSB repair by a mechanism involving dephosphorylation of the NHEJ factors. Importantly, depletion of PP2A/C by RNAi suppresses DNA end-joining and enhances cytogenetic abnormalities including chromosomal and chromatid breaks (Figure W6, B and C), suggesting that PP2A may be essential for maintenance of genetic stability. To test the effect of PP2A on survival after exposure of cells to CPT, H1299 cells were transfected with PP2A siRNA and control siRNA. After 24 hour, cells were treated with increasing concentrations of CPT (i.e., 0-500 nM) for another 24 hours. The apoptotic cells and viable cells were detected using an ApoAlert Annexin-V Kit. Results reveal that depletion of PP2A by RNAi significantly increases apoptosis after CPT treatment (Figure 5E), indicating that depletion of PP2A not only suppresses DNA repair but also

Ku 70/86 Heterodimer Is Required for PP2A Promotion of DSB Repair Ku 70 and Ku 86 usually exist in cells as functional heterodimers that are required for the repair of DSBs [5]. Interestingly, no detectable levels of Ku 86 as well as Ku 70 can be observed in Ku 86−/− MEF cells compared with wild type MEF cells (Figure 6A), suggesting that the presence of Ku 86 for heterodimerization with Ku 70 is also essential for a stable expression of Ku 70. This is consistent with others’ observations that expression of Ku 86 is required to stabilize the Ku 70 protein [34,35]. To test the effect of PP2A on DSB repair in Ku-deficient cells [36], wild type and Ku 86−/− MEF cells overexpressing PP2A/C or vector-only control were treated with CPT (5 μM) for 1 hour, then washed and incubated in normal culture medium for various times up to 24 hours. Time course experiments show that overexpression of HA-tagged PP2A/C significantly accelerates DSB repair in wild type MEF cells but has no such effect in Ku 86−/− MEF cells (Figures 6 and W7). This supports the notion that the Ku-mediated NHEJ pathway is required for PP2A promotion of DSB repair. Discussion The process of the NHEJ pathway is initiated by the association of DNA ends with the Ku 70/86 heterodimer, a protein with a ring-shaped structure that displays an extraordinary affinity for open DNA ends.

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The DNA-Ku complex then functions as a scaffold to assemble the other key NHEJ proteins, including DNA-PKcs, at the DNA termini [37]. After the introduction of a DSB, the DNA-PKcs enzyme is quickly recruited to the DNA-Ku scaffold. Because both Ku and DNA-PKcs are phosphoproteins, phosphorylation of these major NHEJ factors may play an essential role in regulating DNA-PK activity as well as DSB repair. Intriguingly, DNA-dependent phosphorylation of purified DNA-PK proteins (Ku 70, Ku 86, and DNA-PKcs) occurs at serine and threonine residues, which correlates with loss of kinase activity and decreased interaction between phosphorylated DNA-PKcs and DNA-bound Ku [7]. DNA-PKcs can be phosphorylated at multiple sites [37], and phosphorylation at different site(s) distinctly regulates its kinase activity. For example, phosphorylation of Thr 2609 can enhance DNA-PK activity, whereas phosphorylation at Ser 2056 or Thr 3950 is associated with loss of DNA-PK kinase activity [7,37,38]. Ku 70/86 can also be phosphorylated by DNA-PK. However, phosphorylation of Ku 70/86 is not required for the NHEJmediated DSB repair [10,39]. In contrast, the catalytic subunit of PP2A is able to directly dephosphorylate Ku 70, Ku 86, and DNA-PKcs in vitro, which results in the restoration of DNA-PK protein kinase activity [6], suggesting that PP2A-mediated dephosphorylation of these NHEJ factors may positively regulate DSB repair. PP2A has recently been reported to facilitate DSB repair by dephosphorylating γ-H2AX [11]. Because the Ku and DNA-PKcs–mediated NHEJ pathway is required for DSB repair [37], PP2A-mediated promotion of DSB repair may occur, at least in part, in a mechanism

Neoplasia Vol. 11, No. 10, 2009 involving the NHEJ pathway. It is known that PP2A-mediated dephosphorylation of Ku and/or DNA-PKcs enhances DNA-PK activity in vitro [6], suggesting that PP2A may maintain DNA-PK in a dephosphorylated active state. Here, we provide strong evidence that PP2A actually functions as a physiological phosphatase for both Ku and DNA-PKcs to dephosphorylate and activate these NHEJ factors, which leads to accelerated DSB repair in vivo (Figures 2, 4, W3, and W4). In contrast, specific disruption of PP2A activity, either by expression of small T antigen or by depletion of PP2A/C by RNAi, results in increased phosphorylation and decreased activity of Ku and DNA-PKcs with suppression of DNA end-joining and DSB repair (Figures 1, 4, and 5). PP2A colocalizes and interacts with Ku 70/86 and directly dephosphorylates Ku 70/86 and DNA-PKcs, indicating its potential direct role as a Ku/DNA-PKcs phosphatase (Figures 3 and 4). Confirmation of PP2A as a physiological Ku/DNA-PKcs phosphatase was obtained in vivo from results of transfection studies that demonstrate that overexpression of PP2A/C results in the dephosphorylation of Ku and DNA-PKcs (Figure 4B) in association with enhanced Ku and DNA-PK activities and accelerated DSB repair (Figures 2 and W3), suggesting that the dephosphorylated form of DNAPK functions as an active form in the NHEJ-mediated DSB repair pathway. These findings support the notion that PP2A promotion of DSB repair may occur, at least in part, by directly dephosphorylating and activating the major NHEJ factors (Ku and DNA-PKcs). DSBs are toxic, genomic DNA lesions that, if unrepaired or repaired incorrectly, can lead to mutations, chromosomal breaks, or

Figure 4. PP2A-induced dephosphorylation of Ku and DNA-PKcs facilitates their association. (A) Phosphorylated Ku 70, Ku 86, or DNAPKcs was immunoprecipitated and incubated with increasing concentrations of purified PP2A at 30°C for 10 minutes as described in the Materials and Methods section. Phosphorylation of Ku 70, Ku 86, and DNA-PKcs was determined by autoradiography. (B) H1299 cells overexpressing HA-PP2A/C or vector-only control cells were metabolically labeled with 32P-orthophosphoric acid for 90 minutes. Ku 70, Ku 86, or DNA-PKcs was immunoprecipitated, and phosphorylation was determined by autoradiography. (C) H1299 cells expressing HA-PP2A/C or vector-only control were disrupted in EBC lysis buffer. CoIP was performed using Ku 86 antibody. The Ku-associated DNA-PKcs, Ku 70, and Ku 86 were then analyzed by Western blot. Rabbit preimmune serum (Pre) was used as a control.

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Figure 5. Depletion of PP2A/C by RNAi results in enhanced phosphorylation of Ku and DNA-PKcs in association with inhibition of the Ku/ DNA-PKcs association and activity, which suppresses DSB repair and DNA end-joining leading to increased genetic instability. (A) H1299 cells were transfected with PP2A/C siRNA or control siRNA. Expression of PP2A/C was analyzed by Western blot. (B) H1299 cells were transfected with PP2A/C siRNA or control siRNA. After 48 hours, cells were metabolically labeled with 32P-orthophosphoric acid for 90 minutes. Phosphorylation of Ku 70, Ku 86, and DNA-PKcs was determined by autoradiography. (C) H1299 cells expressing PP2A/C siRNA or control siRNA were treated with CPT (5 μM) for 1 hour. Cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by PFGE. (D) Cytogenetic abnormalities were analyzed by T-FISH in H1299 cells expressing PP2A/C siRNA or control siRNA as described in the legend of Figure 1. (E) H1299 cells were transfected with PP2A/C siRNA or control siRNA. After 24 hours, cells were treated with increasing concentrations of CPT (i.e., 0-500 nM) for another 24 hours. Cell viability was determined by analyzing annexin-V binding on FACS. Data represent the mean ± SD of three separate determinations.

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Figure 6. Expression of Ku 70 and Ku 86 is essential for PP2A promotion of DSB repair. (A) HA-tagged PP2A/C was transfected into wild type or Ku 86−/− MEF cell lines. Expression of Ku 70, Ku 86, or HA-PP2A/C was analyzed by Western blot using Ku 70, Ku 86, or HA antibody, respectively. (B and C) Wild type or Ku 86−/− MEF cells overexpressing HA-PP2A/C or vector-only control were treated with CPT (5 μM) for 1 hour. Then, cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by analysis of γ-H2AX foci.

translocations with malignant transformation [28,40]. Inhibition of PP2A activity by expression of small T or depletion of PP2A/C by RNAi results in decreased DSB repair and increased genetic instability (i.e., chromosomal and chromatid breaks; Figures 1, 5, W2, W5, and W6), indicating that PP2A plays an important role in maintaining genetic stability. Our findings help explain why PP2A can function as a potent tumor suppressor [13]. Recruitment of DNA-PKcs by the Ku complex to the broken DNA ends and formation of functional Ku/DNA-PKcs complexes are essential for repairing DSBs [2]. Intriguingly, overexpression of PP2A/C reduces phosphorylation of Ku and DNA-PKcs in association with increased Ku/DNA-PKcs association (Figure 4). In contrast, inhibition of PP2A by expression of small T or by depletion of PP2A/C by RNAi results in increased phosphorylation of Ku and DNA-PKcs and decreased formation of the Ku/DNA-PKcs complex (Figures 5B, W4, and W5A), indicating that phosphorylation may cause dissociation of the Ku/DNA-PKcs complex and that dephosphorylation may facilitate Ku to interact with DNA-PKcs. These findings uncover a novel signal mechanism by which PP2A enhances Ku and DNA-PK activities with acceleration of DSB repair.

Neoplasia Vol. 11, No. 10, 2009 PP2A-induced dephosphorylation of Ku and DNA-PKcs enhances their activities with accelerated DSB repair, indicating that the NHEJ factors may act as the functional PP2A downstream substrates. Because PP2A can accelerate the repair of DSBs in wild type MEF cells but failed to promote DSB repair in the Ku-deficient MEF cells (Figures 6 and W7), this supports the notion that the Ku 70/86 heterodimer is required for the effect of PP2A on DSB repair. In addition to PP2A, PP5 and PP6 have been previously reported to dephosphorylate DNA-PKcs and regulate DNA-PKcs activity [41,42]. PP5 is the phosphatase that is responsible for removing the phosphate group from T2609 and S2056 of DNA-PKcs after irradiation damage. Dephosphorylation of T2609 caused by PP5 has been suggested to block end processing and lead to radiation sensitivity [41]. By contrast, dephosphorylation of DNA-PKcs by PP6 offers a mechanism for the activation of DNA-PK protein kinase as part of the cellular damage response [42]. Importantly, PP6-induced dephosphorylation of DNA-PKcs facilitates DNA repair [42]. Compared with our findings, it seems that the functional consequence of PP6-mediated dephosphorylation is similar with PP2A. PP6-mediated dephosphorylation of DNA-PKcs may not occur at T2609 or S2056 because phosphorylation levels of T2609 and S2056 were not affected by depletion of either PP6R1 or PP6c [42]. Therefore, further work is required to identify the site(s) of DNA-PKcs dephosphorylation induced by PP6 and PP2A. In summary, our findings have identified PP2A as a novel physiological phosphatase of Ku and DNA-PKcs, which can activate Ku and DNA-PK activities by dephosphorylation leading to the promotion of DSB repair. Disruption of PP2A activity by small T or RNAi downregulates Ku and DNA-PKcs activities, which results in decreased DSB repair and increased genetic instability. PP2A-mediated dephosphorylation of Ku and DNA-PKcs facilitates a functional interaction between Ku and DNA-PKcs. Intriguingly, the Ku-mediated NHEJ pathway is required for PP2A to promote DSB repair. Thus, PP2A acceleration of DSB repair through the NHEJ pathway may contribute to the maintenance of genetic stability and/or prevention of tumorigenesis. Acknowledgments The authors thank David J. Chen (University of Texas Southwestern Medical Center, Dallas, TX) for providing wild type and Ku 86−/− MEF cells. References [1] Riha K, Heacock M, and Shippen D (2006). The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology. Annu Rev Genet 40, 237–277. [2] Hefferin M and Tomkinson A (2005). Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair (Amst.) 4, 639–648. [3] Khanna K and Jackson S (2001). DNA double-strand breaks: signal, repair and the cancer connection. Nat Genet 27, 247–254. [4] Van Gent D, Hoeijmakers J, and Kanaar R (2001). Chromosomal stability and DNA double-strand break connection. Nat Rev Genet 2, 196–206. [5] Burma S, Chen B, and Chen D (2006). Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst.) 5, 1042–1048. [6] Douglas P, Moorhead G, Ye R, and Lees-Miller S (2001). Protein phosphatases regulates DNA-dependent protein kinase activity. J Biol Chem 276, 18992–18998. [7] Chan D and Lees-Miller S (1996). The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J Biol Chem 271, 8936–8941. [8] Cui X, Yu Y, Gupta S, Cho Y, Lees-Miller S, and Meek K (2005). Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing

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Supplemental Methods

Measurement of Ku 70/86 Activity Ku 70/Ku 86 DNA binding activity was analyzed using a Ku 70/ 86 DNA Repair Kit (Active Motif). Briefly, cells were washed and resuspended in hypotonic buffer. The nuclear extract was isolated for Ku activity analysis. Five micrograms of nuclear protein was loaded into the oligonucleotide-coated 96-well plate and incubated for 60 minutes at room temperature. Ku 70 or Ku 86 antibody was added and incubated for another 60 minutes. After washing, HRP-conjugated secondary antibody was added and incubated for 60 minutes. Then, developing solution and stop solution were added. Optical density was read on a spectrophotometer at 450 nm. Each experiment was repeated three times, and data represent the mean ± SD of three separate determinations.

DNA-PK Activity Assay The SignaTECT DNA-PK Assay Kit was used to measure DNAPK activity (Promega). The following reaction mixtures, in the presence or absence of activator, were prepared: control buffer, 2.5 μl of DNA-PK activation buffer, 5.0 μl of DNA-PK in 5 × reaction buffer, 2.5 μl of DNA-PK biotinylated peptide substrate, 0.2 μl of BSA (10 mg/ml), and 5 μl of γ 32P-ATP. The mixture was preincubated at 30°C for 4 minutes. The whole-cell extract sample was diluted in the enzyme buffer, and the reaction was initiated by adding the appropriate amount of enzyme to the sample and incubated at 30°C for 5 minutes. The reaction was stopped by addition of the termination buffer. The sample was spotted onto the SAM2R Biotin Capture membrane (Promega, Madison, WI). The membrane was then washed and dried under a heat lamp for 10 minutes. DNA-PK enzyme activity was determined by

scintillation counting. Each experiment was repeated three times, and data represent the mean ± SD of three separate determinations.

In Vivo DNA End-joining Assay The pGL3 plasmid (Promega), in which expression of the luciferase gene is controlled by the CMV promoter, was used to evaluate correct nonhomologous end-joining activity that precisely rejoins broken DNA ends in vivo as described [3]. The pGL3 plasmid was completely linearized by the restriction endonuclease NarI, which cleaves within the luciferase-coding region as confirmed by agarose gel electrophoresis. The linearized DNA was purified and then dissolved in sterilized water. A 20:1 mixture of the linearized pGL3 plasmid and pTK Renilla control luciferase reporter vector (an internal control) were transfected into H1299 cells expressing small T or PP2A siRNA. After 48 hours, luciferase activity was measured using a dual-luciferase assay system following the manufacture’s instruction (Promega). Because the pGL3 reporter plasmid was digested to completion with NarI within the luciferase-coding region, only precise DNA end-joining can restore the luciferase activity. Each experiment was repeated three times, and data represent the mean ± SD of three separate determinations. Supplemental References [1] Balajee A and Geard C (2004). Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks. Exp Cell Res 300, 320–334. [2] Chowdhury D, Keogh M, Ishii H, Peterson C, Buratowski S, and Lieberman J (2005). γ-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell 20, 801–809. [3] Shin K, Kang M, Dicterow E, Kameta A, Baluda M, and Park N (2004). Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity. Clin Cancer Res 10, 2551–2560.

Figure W1. Expression of Ku 70, Ku 86, DNA-PKcs, and PP2A in human lung cancer cells. Expression levels of endogenous Ku 70, Ku 86, DNA-PKcs, and PP2A/C in human lung cancer cells were analyzed by Western blot using Ku 70, Ku 86, DNA-PKcs, or PP2A/C antibody, respectively. Tubulin was used as a loading control.

Figure W2. Disruption of PP2A activity by expression of small T antigen downregulates Ku and DNA-PK activity in association with suppression of DNA end-joining and DSB repair. (A) Ku DNA binding activity or DNA-PK activity was measured in H1299 cells expressing small T or vector-only control using a Ku 70/86 DNA Repair Kit or a SignaTECT DNA-PK Assay Kit, respectively. Data represent the mean ± SD of three separate determinations. (B) H1299 cells expressing small T or vector-only control were treated with CPT (5 μM) for 1 hour. Then, cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by analysis of γ-H2AX by immunostaining or Western blot. The number of γ-H2AX foci per cell was determined on a cell to cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [1]. The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [2]. (C) H1299 cells expressing small T or vector-only control were treated as previously mentioned. γ-H2AX was analyzed Western blot using a γ-H2AX antibody. (D) DNA end-joining activity was measured in H1299 cells expressing small T or vector-only control as described in the Materials and Methods section. Data represent the mean ± SD of three separate determinations.

Figure W3. Overexpression of PP2A upregulates Ku and DNA-PK activities leading to accelerated DSB repair. (A) Ku DNA binding activity or DNA-PK activity was measured in H1299 cells overexpressing HA-PP2A/C or vector-only control using a Ku 70/86 DNA Repair Kit or a SignaTECT DNA-PK Assay Kit, respectively. Data represent the mean ± SD of three separate determinations. (B and C) H1299 cells overexpressing HA-PP2A/C or vector-only control were treated with CPT (5 μM) for 1 hour. Cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by analysis of γ-H2AX by immunostaining. The number of γ-H2AX foci per cell was determined on a cell to cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [1]. The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [2].

Figure W4. Expression of small T results in increased phosphorylation of Ku 70, Ku 86 and DNA-PKcs, which leads to dissociation of Ku/DNA-PKcs complex. (A) H1299 cells overexpressing small T or vector-only control cells were metabolically labeled with 32Porthophosphoric acid for 90 minutes. Ku 70, Ku 86, or DNA-PKcs was immunoprecipitated and phosphorylation was determined by autoradiography. (B) H1299 cells expressing small T or vector-only control cells were disrupted in EBC lysis buffer. CoIP was performed using Ku 86 antibody. The Ku-associated DNA-PKcs, Ku 70, and Ku 86 were then analyzed by Western blot. Rabbit preimmune serum (Pre) was used as a control.

Figure W5. Depletion of PP2A/C by RNAi results in disruption of the Ku/DNA-PKcs complex and suppression of Ku and DNA-PK activities leading to decreased DSB repair. (A) H1299 cells expressing PP2A/C siRNA or control siRNA were disrupted in EBC lysis buffer. CoIP was performed using a Ku 86 antibody. The Ku-associated DNA-PKcs, Ku 70, and Ku 86 were then analyzed by Western blot. Rabbit preimmune serum (Pre) was used as a control. (B) Ku DNA binding activity or DNA-PK activity was measured in H1299 cells expressing PP2A siRNA or control siRNA using a Ku 70/86 DNA Repair Kit or a SignaTECT DNA-PK Assay Kit, respectively. Data represent the mean ± SD of three separate determinations. (C and D) H1299 cells expressing PP2A/C siRNA or control siRNA were treated with CPT (5 μM) for 1 hour. Cells were washed three times and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by analysis of γ-H2AX by immunostaining. The number of γ-H2AX foci per cell was determined on a cell to cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [1]. (E) The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [2].

Figure W6. Specific knockdown of PP2A/C suppresses DSB repair and DNA end-joining leading to genetic instability. (A) H1299 cells expressing PP2A/C siRNA or control siRNA were treated with CPT (5 μM) for 1 hour. Cells were washed three times and incubated in normal culture medium for various times up to 24 hours. γ-H2AX was determined by Western blot using a γ-H2AX antibody. (B) DNA end-joining activity was measured in H1299 cells expressing PP2A/C siRNA or control siRNA as described in the Materials and Methods section. Data represent the mean ± SD of three separate determinations. (C) Cytogenetic abnormalities were analyzed by T-FISH in H1299 cells expressing PP2A/C siRNA or control siRNA. DAPI-stained chromosomes are blue. Red dots come from telomere signals. Color-coded arrowheads indicate a normal chromosome and different kinds of cytogenetic abnormalities (white indicates normal chromosome with four telomere signals; green, chromosomal break with two telomere signals; yellow, chromatid break with three telomere signals).

Figure W7. Ku 70 and Ku 86 are required for PP2A promotion of DSB repair. (A and B) Wild type or Ku 86−/− MEF cells overexpressing HA-PP2A/C or vector-only control were treated with CPT (5 μM) for 1 hour. Then, cells were washed three times, and incubated in normal culture medium for various times up to 24 hours. DSBs were determined by analysis of γ-H2AX foci. The number of γ-H2AX foci per cell was determined on a cell to cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [1]. The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [2].