Protein Kinase C {delta} Activates Topoisomerase II{alpha} To Induce Apoptotic Cell Death in Response to DNA Damage

DNA topoisomerase II is an essential nuclear enzyme that modulatesDNA processes by altering the topological state of double-strandedDNA. This enzyme is required for chromosome condensation andsegregation; however, the regulatory mechanism of its activationis largely unknown. Here we demonstrate that topoisomerase II ${alpha}$ is activated in response to genotoxic stress. Concomitant withthe activation, the expression of topoisomerase II ${alpha}$ is increasedfollowing DNA damage. The results also demonstrate that theproapoptotic kinase protein kinase C ${delta}$ (PKC ${delta}$ ) interacts with topoisomeraseII ${alpha}$ . This association is in an S-phase-specific manner and isrequired for stabilization and catalytic activation of topoisomeraseII ${alpha}$ in response to DNA damage. Conversely, inhibition of PKC ${delta}$ activity attenuates DNA damage-induced activation of topoisomeraseII ${alpha}$ . Finally, aberrant activation of topoisomerase II ${alpha}$ by PKC ${delta}$ is associated with induction of apoptosis upon exposure to genotoxicagents. These findings indicate that PKC ${delta}$ regulates topoisomeraseII ${alpha}$ and thereby cell fate in the genotoxic stress response.

INTRODUCTION

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Introduction

DNA topoisomerase II is a nuclear enzyme that regulates DNAtopology via transient double-strand breaks in the DNA helix(5, 38, 39). Topoisomerase II is involved in cell proliferationand has been implicated in indispensable cellular processes,such as replication, transcription, recombination, and chromosomalcondensation and segregation (38). This enzyme also has an essentialfunction as a structural component of mitotic chromosome andinterphase nuclear scaffolds (9, 14). Given the fact that expressionof topoisomerase II in proliferating cells is higher than thatin quiescent cells, this enzyme is a clinically useful targetto elicit cytotoxic effects in proliferating tumor cells. Indeed,a variety of anticancer agents target topoisomerase II (20)and interfere with its catalytic activity by trapping the enzymein a form that is covalently bound to DNA. These stable enzyme-associatedDNA complexes induce DNA damage and cell death. In additionto catalytically inactivating topoisomerase II, previous studieshave shown that aberrant expression of topoisomerase II is associatedwith the induction of apoptosis (28, 37). While forced expressionof topoisomerase II ${alpha}$ in cells triggered apoptotic cell death,nuclear localization of the enzyme was required for efficientapoptotic induction. By contrast, another report demonstratedthat depletion of topoisomerase II ${alpha}$ conferred induction of apoptosis(1). Taken together, these results suggest that appropriateregulation of topoisomerase II ${alpha}$ expression is essential for cellviability and proliferation. In this regard, deregulated expressionof topoisomerase II ${alpha}$ is associated with the commitment of apoptoticcell death; however, the mechanism remains unclear.

The protein kinase C (PKC) family of serine/threonine kinasesis subdivided into (i) conventional PKCs (PKC ${alpha}$ [PKC ${alpha}$ ], PKCß,and PKC ${gamma}$ ) that are calcium dependent and activated by diacylglycerol(DAG), (ii) novel PKCs (PKC ${delta}$ , PKC ${varepsilon}$ , PKC ${theta}$ , and PKCµ) thatare calcium independent and activated by DAG, and (iii) atypicalPKCs (PKC ${zeta}$ and PKC ${lambda}$ ) that are calcium independent and not activatedby DAG (32). The ubiquitously expressed novel PKC, PKC ${delta}$ , is tyrosinephosphorylated and activated by c-Abl and Lyn in the responseto DNA damage (45, 48). PKC ${delta}$ interacts with the nuclear DNA-dependentprotein kinase catalytic subunit (DNA-PKcs) (2). Phosphorylationof DNA-PKcs by PKC ${delta}$ inhibits the function of DNA-PKcs to formcomplexes with DNA and to phosphorylate downstream targets (2).In addition, cells deficient in DNA-PK are resistant to apoptosisinduced by overexpression of the PKC ${delta}$ catalytic domain. Otherstudies have demonstrated that the nuclear complex of c-Abland Lyn includes the protein tyrosine phosphatase SHPTP1 (Srchomology 2 domain [SH2]-containing protein tyrosine phosphatase1) (21, 42) and that PKC ${delta}$ phosphorylates and inactivates SHPTP1in response to DNA damage (44). In cells that respond to genotoxicstress with apoptosis, PKC ${delta}$ is cleaved by caspase-3 into a constitutivelyactive catalytic fragment (PKC ${delta}$ CF) (10, 11). The finding thatPKC ${delta}$ CF induced nuclear condensation and DNA fragmentation indicatesthat cleavage of PKC ${delta}$ contributes to the apoptotic response (15).In this context, a recent study has demonstrated that PKC ${delta}$ translocatedto the nucleus and regulated Rad9 by phosphorylation in theapoptotic response to DNA damage (46). Furthermore, anotherstudy showed that cells derived from PKC ${delta}$ -null transgenic micewere defective in mitochondrion-dependent apoptosis inducedby various agents such as UV irradiation and hydrogen peroxide(27). These findings collectively support an essential rolefor PKC ${delta}$ in the induction of apoptosis in the genotoxic stressresponse.

The present study demonstrates that PKC ${delta}$ interacts with topoisomeraseII ${alpha}$ . This interaction is required for stabilization and activationof topoisomerase II ${alpha}$ following DNA damage. The results also demonstratethat genotoxic stress-induced topoisomerase II ${alpha}$ activation confersthe induction of PKC ${delta}$ -mediated apoptosis.

MATERIALS AND METHODS

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Materials and Methods

Cell culture. Human MOLT-4, U-937, and HL-60 leukemia cells were culturedin RPMI 1640 medium supplemented with 10% heat-inactivated fetalbovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin,and 2 mM L-glutamine. Human U2-OS osteosarcoma cells, 293T embryonalkidney cells, mouse embryo fibroblasts (MEFs), and pkc ${delta}$ ^–/–MEFs (29) were grown in Dulbecco's modified Eagle's medium containing10% heat-inactivated fetal bovine serum and antibiotics. Cellswere treated with 10 µM 1-ß-D-arabinofuranosylcytosine(ara-C; Sigma-Aldrich), 500 µM cisplatin (CDDP; Sigma-Aldrich),5 µM rottlerin (Sigma-Aldrich), 100 nM bistratene A (Sigma-Aldrich),500 ng/ml nocodazole (Sigma-Aldrich), 5 µg/ml aphidicolin(WAKO), or 10 µM ICRF-193 (Zenyaku).

Plasmids. PKC ${delta}$ expression plasmids were described previously (44, 45).Plasmids for the expression of topoisomerase II ${alpha}$ glutathioneS-transferase (GST) fusion proteins were constructed by PCRusing topoisomerase II ${alpha}$ cDNA fused with green fluorescent protein(GFP) (GFP-TopoII ${alpha}$ ) (30). To construct the GFP-topoisomeraseII ${alpha}$ mutant in which the C-terminal domain was deleted (GFP-TopoII ${alpha}$ ${Delta}$ C),GFP-TopoII ${alpha}$ was digested with restriction enzymes PstI and SmaI.After the PstI site was blunt ended, the DNA was subjected toself-ligation, resulting in the truncated form of GFP-TopoII ${alpha}$ encoding amino acid residues 1 to 1145.

Cell transfections. Cell transfections were performed as described previously (43,47). The total DNA concentration was kept constant by includingan empty vector.

Protein identification by mass spectrometry analysis. 293T cells were transfected with Flag vector or PKC ${delta}$ tagged witha Flag epitope (Flag-PKC ${delta}$ ). At 48 h posttransfection, cells werelysed with 0.1% NP-40 lysis buffer (0.1% NP-40, 50 mM Tris-Cl,pH 7.6, 150 mM NaCl, 2 µg/ml aprotinin, 1 mM dithiothreitol[DTT], 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF],10 µg/ml leupeptin, 1 µM pepstatin, and 1 mM Na₃VO₄).Lysates were centrifuged at 14,000 x g for 15 min, and the supernatantswere subjected to immunoprecipitation with anti-Flag agarose(Sigma-Aldrich). Flag-PKC ${delta}$ -associated complexes were then separatedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and visualized by silver staining. Protein bandswere cut out from gels, digested with trypsin, and analyzedby matrix-assisted laser desorption ionization-time of flightmass spectrometry as described previously (12).

Immunoprecipitation. For coimmunoprecipitation of PKC ${delta}$ and topoisomeraseII ${alpha}$ , nuclear lysates from MOLT-4 cells were prepared as describedpreviously (7). Precleared lysates (1 mg) were incubated withanti-topoisomerase II ${alpha}$ (Alexis Biochemicals) or anti-PKC ${delta}$ (SantaCruz Biotechnology) antibodies for 2 h at 4°C followed by1 h of incubation with protein A-Sepharose beads (Amersham Biosciences).The immune complexes were washed three times with 0.1% NP-40lysis buffer and then eluted by boiling for 5 min in 50 mM Tris-Cl,pH 6.8, containing 2% SDS, 6% 2-mercaptoethanol, 0.01% bromophenolblue, and 10% glycerol. The eluted samples were subjected toimmunoblot analysis.

Immunoblot analysis. Cells were lysed on ice for 30 min with 1% NP-40 lysis buffer(1% NP-40, 50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 2 µg/mlaprotinin, 1 mM DTT, 10 mM NaF, 1 mM PMSF, 10 µg/ml leupeptin,1 µM pepstatin, and 1 mM Na₃VO₄). Lysates were centrifugedat 14,000 x g for 15 min, and the supernatants were analyzedby immunoblotting. Cell lysates or immunoprecipitates were separatedby SDS-PAGE and transferred to nitrocellulose filters. The filterswere then incubated with anti-Flag, anti-GST (Nacalai Tesque),anti-GFP (Nacalai Tesque), anti-topoisomerase II ${alpha}$ (MBL or Topogen),anti-topoisomerase II ${alpha}$ and ß (MBL), anti-PKC ${delta}$ , anti-phospho-PKC ${delta}$ (Thr505) (Cell Signaling Technology) or antitubulin (Sigma-Aldrich).The antigen-antibody complexes were visualized by chemiluminescence(Perkin-Elmer).

In vitro binding assays. Cell lysates were incubated with purified proteins fused toGST in lysis buffer for 2 h at 4°C. The adsorbates wereresolved by SDS-PAGE and analyzed by immunoblotting with anti-topoisomeraseII ${alpha}$ or anti-PKC ${delta}$ .

Preparation of nuclear extracts. To prepare nuclear extracts for topoisomerase II catalytic activityassays, 3 x 10⁷ to 5 x 10⁷ cells were washed first with phosphate-bufferedsaline and then with 1 ml of buffer A (10 mM HEPES, pH 7.6,15 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 10 µg/mlleupeptin) and resuspended in buffer A containing 0.2% NP-40.After centrifugation, cells were resuspended in 1 ml of bufferA containing 0.25 M sucrose. Subsequently, samples were collectedby centrifugation and resuspended in buffer D (50 mM HEPES,pH 7.6, 400 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5mM PMSF, and 10 µg/ml leupeptin). After the samples weremixed and gently rocked for 30 min at 4°C, they were centrifugedfor 15 min at 1,400 rpm. Supernatants were used as nuclear extracts.

Topoisomerase II ${alpha}$ catalytic activity assays. Topoisomerase II activity was assayed by the decatenation ofkinetoplast DNA (KDNA) or relaxation of pBluescript. The decatenationassays were performed by incubating 0.2 µg KDNA (NipponGene) with nuclear extracts or recombinant topoisomerase II ${alpha}$ (Topogen) in assay buffer A (50 mM Tris-HCl, pH 8.0, 120 mMKCl, 10 mM MgCl₂, 0.5 mM ATP, 0.5 mM DTT, and 30 µg/mlbovine serum albumin). After incubation for 20 min at 37°C,the reactions were stopped by the addition of stop buffer (5%Sarkosyl, 0.0025% bromophenol blue, and 25% glycerol). The reactionproducts were resolved on a 1% agarose gel containing 0.5 µg/mlethidium bromide. The relaxation assays were performed by incubating0.2 µg pBluescript with nuclear extracts or recombinanttopoisomerase II ${alpha}$ in assay buffer B (30 mM Tris-HCl, pH 7.6,60 mM KCl, 8 mM MgCl₂, 3 mM ATP, 15 mM 2-mercaptoethanol, and30 µg/ml bovine serum albumin). After incubation for 15min at 37°C, the reactions were quenched by the additionof 0.1 volume of 10% SDS. The reaction products were resolvedon a 1% native agarose gel.

siRNA transfections. Small interfering RNA duplexes (siRNAs) targeting PKC ${delta}$ were synthesizedand purified by Invitrogen (Stealth RNAi). Stealth RNAi sequenceswere 5'-AUUAGCACAAUCUGGAUGACGCGCC-3' for PKC ${delta}$ siRNA1, 5'-AAACUCAUGGUUCUUGAUGUAGUGG-3'for PKC ${delta}$ siRNA2, 5'-AAAGAAGGUGGCGAUAAACUCAUGG-3' for PKC ${delta}$ siRNA3,and 5'-AACUCCGGUCUUCUUCUCGAAACCC-3' for PKC ${delta}$ siRNA4. siRNAs targetingtopoisomerase II ${alpha}$ were synthesized and purified by QIAGEN. ThesiRNA sequences were 5'-AAGACUGUCUGUUGAAAGATT-3' for topoisomeraseII ${alpha}$ siRNA1 and 5'-CAUAUUUUGCUCCGCCCAGTT-3' for topoisomerase II ${alpha}$ siRNA2.Scrambled siRNA was purchased from QIAGEN and used as a negativecontrol. Transfection of siRNAs was performed using Lipofectamine2000 (Invitrogen).

In vitro kinase assays. In vitro kinase assays were performed as described previously(44).

RT-PCR analysis for topoisomerase II ${alpha}$ gene expression. Total cellular RNA was extracted using the RNeasy kit (QIAGEN).First-strand cDNA synthesis and the following PCR were performedwith 500 ng of total RNA using SuperScript one-step reversetranscriptase PCR (RT-PCR) system (Invitrogen) according tothe manufacturer's protocol. For topoisomerase II ${alpha}$ gene expression,the nucleotide sequence of 5'-GCCCTCCTGCTACACATTTC-3' was usedas the sense primer, and 5'-AACACTTGGGCTTTACTTCACTT-3' was usedas the antisense primer. For ß-actin gene expression,the nucleotide sequence of 5'-CAGGGCGTGATGGTGGGCA-3' was usedas the sense primer, and 5'-CAAACATCATCTGGGTCATCTTCTC-3' wasused as the antisense primer. The reaction products were resolvedon a 2% agarose gel.

Cell cycle analysis. DNA content was assessed by staining ethanol-fixed cells withpropidium iodide and monitoring by using a FACScan (Becton Dickinson).Cell cycle phases were determined by using the CellQuest program(Becton Dickinson).

Assessment of apoptosis. Apoptotic cells were detected by terminal deoxynucleotidyltransferase-mediateddUTP-biotin nick end labeling (TUNEL) assays using the DeadEndcolorimetric TUNEL system (Promega).

RESULTS

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Results

Identification of topoisomerase II ${alpha}$ as a novel PKC ${delta}$ -interacting protein. To identify cellular proteins that interact with PKC ${delta}$ , 293T cellswere transiently transfected with PKC ${delta}$ tagged with a Flag epitope.Anti-Flag immunoprecipitates were resolved by SDS-PAGE, andthe coimmunoprecipitating proteins were analyzed by mass spectrometry.The results revealed that topoisomerase II ${alpha}$ was one of the PKC ${delta}$ -interactingproteins (Fig. 1A). To assess whether endogenous PKC ${delta}$ associateswith endogenous topoisomerase II ${alpha}$ in cells, anti-PKC ${delta}$ immunoprecipitatesfrom MOLT-4 cell lysates were analyzed by immunoblotting withanti-topoisomerase II ${alpha}$ . PKC ${delta}$ and topoisomerase II ${alpha}$ formed complexesin cells (Fig. 1B). In reciprocal experiments, immunoblot analysisof anti-topoisomerase II ${alpha}$ immunoprecipitates with anti-PKC ${delta}$ confirmedthe association of PKC ${delta}$ and topoisomerase II ${alpha}$ (Fig. 1B). Similarresults were obtained from U-937 and HL-60 cells (data not shown).To determine whether PKC ${delta}$ associates with topoisomerase IIß,lysates from MOLT-4 cells were subjected to immunoprecipitationwith anti-PKC ${delta}$ followed by immunoblotting with an anti-topoisomeraseII antibody that recognizes both topoisomerase II ${alpha}$ (170 kDa)and IIß (180 kDa). The results demonstrated that PKC ${delta}$ interacts preferably with topoisomerase II ${alpha}$ and little, if any,with topoisomerase IIß (Fig. 1C). To further definethe association of PKC ${delta}$ and topoisomerase II ${alpha}$ , cell lysates wereincubated with purified GST, GST-PKC ${delta}$ regulatory domain (RD)or GST-PKC ${delta}$ catalytic fragment (CF) (Fig. 2A). Analysis of adsorbateswith anti-topoisomerase II ${alpha}$ showed the binding of topoisomeraseII ${alpha}$ to GST-PKC ${delta}$ CF, but not to GST or GST-PKC ${delta}$ RD (Fig. 2B). To mapthe PKC ${delta}$ -interacting domain on topoisomerase II ${alpha}$ , cell lysateswere incubated with GST fusion proteins containing topoisomeraseII ${alpha}$ (amino acid residues 1 to 403, 388 to 864, 852 to 1233, or1021 to 1531) (Fig. 2C). The results demonstrated that topoisomeraseII ${alpha}$ (amino acid residues 1021 to 1531) contains the determinantsresponsible for binding to PKC ${delta}$ (Fig. 2D). These findings indicatethat PKC ${delta}$ CF interacts with the C-terminal region of topoisomeraseII ${alpha}$ . To further confirm this in cells, we constructed a GFP-topoisomeraseII ${alpha}$ mutant in which the C-terminal domain was deleted (GFP-TopoII ${alpha}$ ${Delta}$ C).GFP-TopoII ${alpha}$ or GFP-TopoII ${alpha}$ ${Delta}$ C was transfected into 293T cells. Thefinding that PKC ${delta}$ interacts with full-length topoisomerase II ${alpha}$ ,but not with topoisomerase II ${alpha}$ with the C-terminal domain deleted,indicates that the C-terminal region of topoisomerase II ${alpha}$ isrequired for binding to PKC ${delta}$ in cells (Fig. 2E). To determinewhether this interaction is direct, purified GST or GST-PKC ${delta}$ was incubated with recombinant topoisomerase II ${alpha}$ . The findingthat GST-PKC ${delta}$ , and not GST, bound to recombinant topoisomeraseII ${alpha}$ provides support that binding is direct (Fig. 2F). To assessfurther whether PKC ${delta}$ phosphorylates topoisomerase II ${alpha}$ , purifiedGST-PKC ${delta}$ was incubated with recombinant topoisomerase II ${alpha}$ . Analysisof the products by SDS-PAGE and autoradiography showed thattopoisomerase II ${alpha}$ is a substrate for PKC ${delta}$ (Fig. 2G). These datademonstrate that PKC ${delta}$ directly binds to and phosphorylates topoisomeraseII ${alpha}$ .

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FIG. 1. Association of PKC ${delta}$ with topoisomerase II ${alpha}$ (TopoII ${alpha}$ ). (A) Cell lysates from 293T cells transfected with Flag vector or Flag-PKC ${delta}$ were immunoprecipitated (IP) with anti-Flag. Immunoprecipi- tates were resolved by SDS-PAGE and analyzed by silver staining. The polypeptides identified by mass spectrometric analyses are indicated to the right of the blot (IgH, immunoglobulin heavy chain). (B) Nuclear lysates from MOLT-4 cells were subjected to immunoprecipitation (IP) with preimmune rabbit serum (PIRS), anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ), or anti-PKC ${delta}$ . Cell lysates and immunoprecipitates were analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ or anti-PKC ${delta}$ . The finding that the binding stoichiometry of PKC ${delta}$ with topoisomerase II ${alpha}$ was relatively high is mainly due to using nuclear lysates for immunoprecipitation. (C) Nuclear lysates from MOLT-4 cells were subjected to immunoprecipitation with PIRS or anti-PKC ${delta}$ . Cell lysates and immunoprecipitates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ and IIß (anti-TopoII) or anti-PKC ${delta}$ .

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FIG. 2. PKC ${delta}$ directly interacts with and phosphorylates topoisomerase II ${alpha}$ . (A) Schematic representation of PKC ${delta}$ . RD, regulatory domain; CF, catalytic fragment. (B) MOLT-4 cell lysates were incubated with GST, GST-PKC ${delta}$ RD, or GST-PKC ${delta}$ CF bound to glutathione beads. The adsorbates were analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or anti-GST. GST-PKC ${delta}$ fr., GST-PKC ${delta}$ fragment. (C) Schematic representation of topoisomerase II ${alpha}$ . C-ter. domain, C-terminal domain. (D) MOLT-4 cell lysates were incubated with GST or GST-topoisomerase II ${alpha}$ fragments (GST-TopoII ${alpha}$ fr.) bound to glutathione beads. The adsorbates were analyzed by immunoblotting (IB) with anti-PKC ${delta}$ or anti-GST. Each one of the bands specific for GST-topoisomerase II ${alpha}$ fragments is indicated by an asterisk. (E) 293T cells were transfected with GFP-TopoII ${alpha}$ or a truncated form of GFP-TopoII ${alpha}$ that encodes amino acid residues 1 to 1145 (GFP-TopoII ${alpha}$ ${Delta}$ C). Lysates were subjected to immunoprecipitation (IP) with anti-PKC ${delta}$ or PIRS followed by immunoblot (IB) analysis with anti-GFP or anti-PKC ${delta}$ . IgH, immunoglobulin heavy chain. (F) Recombinant topoisomerase II ${alpha}$ was incubated with glutathione beads containing GST or GST-PKC ${delta}$ . The adsorbates were subjected to immunoblot (IB) analysis with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or anti-GST. (G) GST-PKC ${delta}$ was incubated with or without recombinant topoisomerase II ${alpha}$ and [ ${gamma}$ -³²P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography or by Coomassie brilliant blue R-250 (CBB) staining.

PKC ${delta}$ induces the catalytic activity of topoisomerase II ${alpha}$ . To examine whether PKC ${delta}$ is involved in topoisomerase II ${alpha}$ activity,purified topoisomerase II ${alpha}$ was incubated in the presence or absenceof recombinant kinase-active PKC ${delta}$ . We assayed catalytic activityof topoisomerase II ${alpha}$ by the decatenation of kinetoplast DNA.Decatenation activity was substantially enhanced in the presenceof PKC ${delta}$ (Fig. 3A). To determine whether stimulatory effects ofPKC ${delta}$ are confined to decatenation activity, we performed DNArelaxation assays. Purified topoisomerase II ${alpha}$ was incubated withsupercoiled plasmids in the presence or absence of PKC ${delta}$ . In concertwith the decatenation assays, coincubation of PKC ${delta}$ increasedrelaxation activity of topoisomerase II ${alpha}$ (Fig. 3B). To definePKC ${delta}$ -mediated topoisomerase II ${alpha}$ activation in vitro more quantitatively,serially diluted PKC ${delta}$ recombinant proteins were incubated with0.25 U of topoisomerase II ${alpha}$ . Decatenation assays revealed thatat least 20 ng of active PKC ${delta}$ was necessary for full activationof topoisomerase II ${alpha}$ (Fig. 3C). To determine whether kinase activityis required for topoisomerase II ${alpha}$ activation, purified kinase-activeor -inactive PKC ${delta}$ CF proteins were incubated with purified topoisomeraseII ${alpha}$ . The kinase-active, but not kinase-inactive, PKC ${delta}$ proteinwas capable of topoisomerase II ${alpha}$ activation, resulting in thedecatenation of KDNA (Fig. 3D). These findings indicate thatPKC ${delta}$ induces catalytic activity of topoisomerase II ${alpha}$ in vitro.To assess involvement of PKC ${delta}$ in topoisomerase II ${alpha}$ activationin vivo, MOLT-4 cells were treated with or without the PKC ${delta}$ inhibitorrottlerin (18). Decatenation assays using nuclear lysates revealedthat rottlerin treatment was associated with inhibition of topoisomeraseII ${alpha}$ activity (Fig. 4A). Similar findings were obtained for U-937and HL-60 cells (data not shown). To determine whether kinaseactivity of PKC ${delta}$ is required for the activation of topoisomeraseII ${alpha}$ , 293T cells were transfected with Flag vector, wild-type(wt) Flag-PKC ${delta}$ CF or the Flag-PKC ${delta}$ CF(K-R) mutant, which is catalyticallyinactive. Analysis of decatenation assays demonstrated thatexpression of the PKC ${delta}$ CF, but not the vector or the PKC ${delta}$ CF(K-R)mutant, induced topoisomerase II ${alpha}$ activity (Fig. 4B). Similarresults were obtained with DNA relaxation assays (data not shown).To further establish the essential role for PKC ${delta}$ in topoisomeraseII ${alpha}$ regulation, pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were analyzedby the decatenation and DNA relaxation assays. The finding thattopoisomerase II ${alpha}$ activity was diminished in pkc ${delta}$ ^–/–MEFs provided further support for a pivotal role of PKC ${delta}$ in topoisomeraseII ${alpha}$ activation (Fig. 4C and D). To exclude the possibility thatrottlerin is directly involved in the inhibition of topoisomeraseII ${alpha}$ activity, pkc ${delta}$ ^–/– MEFs were left untreated ortreated with rottlerin for 1 h. Decatenation assays using nuclearlysates demonstrated that rottlerin treatment had no effecton topoisomerase II ${alpha}$ activity in pkc ${delta}$ ^–/– MEFs (Fig.4E).

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FIG. 3. Topoisomerase II ${alpha}$ is activated by PKC ${delta}$ in vitro. (A) The indicated amount of purified topoisomerase II ${alpha}$ (TopoII ${alpha}$ ) was incubated with (+) or without (–) kinase-active recombinant PKC ${delta}$ (50 ng). Decatenation assays using reaction mixtures containing kinetoplast DNA were performed, and the reaction products were analyzed on a 1% agarose gel. Cat. KDNA, catenated KDNA; Decat. KDNA, decatenated KDNA. (B) Purified topoisomerase II ${alpha}$ was incubated with or without recombinant PKC ${delta}$ . Relaxation assays using reaction mixtures containing pBluescript were performed, and the reaction products were analyzed on a 1% agarose gel. (C) Purified topoisomerase II ${alpha}$ (0.25 U) was incubated with or without (–) the indicated amount of kinase-active recombinant PKC ${delta}$ . Decatenation assays using reaction mixtures containing KDNA were performed, and the reaction products were analyzed on a 1% agarose gel. (D) Purified topoisomerase II ${alpha}$ (0.25 U) was incubated with recombinant GST, GST-PKC ${delta}$ CF, or GST-PKC ${delta}$ CF(K-R). Decatenation assays using reaction mixtures containing KDNA were performed, and the reaction products were analyzed on a 1% agarose gel.

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FIG. 4. Topoisomerase II ${alpha}$ is activated by PKC ${delta}$ in cells. (A) MOLT-4 cells were incubated in the presence (+) or absence (–) of rottlerin. Decatenation assays using nuclear lysates (Nuc. Lysates) were performed, and the reaction products were resolved on a 1% agarose gel. Cat. KDNA, catenated KDNA; Decat. KDNA, decatenated KDNA. (B) 293T cells were transfected with Flag vector, Flag-PKC ${delta}$ CF, or Flag-PKC ${delta}$ CF(K-R). Nuclear lysates were analyzed by decatenation assays (top blot). Cell lysates were subjected to immunoblot (IB) analysis with anti-Flag or antitubulin. (C and D) Nuclear lysates from pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were analyzed by the decatenation (C) and DNA relaxation (D) assays. (E) pkc ${delta}$ ^–/– MEFs were left untreated or treated with rottlerin for 1 h. Nuclear lysates were analyzed by the decatenation assays. DMSO, dimethyl sulfoxide.

Association of PKC ${delta}$ with topoisomerase II ${alpha}$ is required for topoisomerase II ${alpha}$ stabilization and activation. Previous studies have demonstrated that expression of topoisomeraseII ${alpha}$ is regulated in a cell cycle-dependent manner with levelsthat are low at G₁, gradually increase from S to G₂, and peakat M phase (19, 23, 35, 40). By contrast, topoisomerase IIßexpression is relatively constant throughout the cell cycle(19, 40). Furthermore, topoisomerase II ${alpha}$ is markedly degradedat the transition from M into G₁ phase. Several studies havesuggested that this degradation is associated with the ubiquitin-proteasomepathway (22, 31, 33). To examine PKC ${delta}$ -mediated topoisomeraseII ${alpha}$ activation in mitosis, MOLT-4 cells were synchronized inM phase by a microtubule inhibitor, nocodazole, in the presenceor absence of rottlerin. Analysis of decatenation assays demonstratedthat inhibition of PKC ${delta}$ attenuated topoisomerase II ${alpha}$ activityin M phase (Fig. 5A). Similar results were obtained in U-937cells (Fig. 5B). To verify and extend these findings, MOLT-4cells or U-937 cells were synchronized at M phase in the presenceor absence of PKC ${delta}$ activator bistratene A (17). The finding thattreatment with bistratene A increased the catalytic activityof topoisomerase II ${alpha}$ further supports the involvement of PKC ${delta}$ in topoisomerase II ${alpha}$ activation in mitosis (Fig. 5C and datanot shown). To define whether topoisomerase II ${alpha}$ regulation byPKC ${delta}$ is cell cycle dependent, MOLT-4 cells synchronized at Mphase were released by the removal of nocodazole from the cellculture medium. Consistent with previous studies, the activityof topoisomerase II ${alpha}$ gradually decreased after cells exited Mphase and entered into G₁ phase (Fig. 5D). Importantly, pretreatmentof cells with rottlerin substantially attenuated topoisomeraseII activity (Fig. 5D). Similar findings were obtained in U-937cells (data not shown). To extend these analyses to S phase,MOLT-4 cells were synchronized in S phase by aphidicolin andthen released by its removal. In concert with previous findings,topoisomerase II ${alpha}$ activation was induced from S to G₂/M phase,reduced at G₁ phase, and restored in S phase (Fig. 5E). Moreover,cell cycle-dependent topoisomerase II ${alpha}$ activation was in partabrogated by the pretreatment with rottlerin throughout thecell cycle (Fig. 5E). Similar results were obtained in U-937cells (data not shown). To determine whether PKC ${delta}$ activates topoisomeraseII ${alpha}$ in a distinct cell cycle phase(s), cells were synchronizedin M or S phase and then treated with or without rottlerin for1 or 2 h. Decatenation assays demonstrated that treatment withrottlerin in M phase had no significant effect on topoisomeraseII ${alpha}$ activity (Fig. 5F). By contrast, there was substantial attenuationof topoisomerase II ${alpha}$ activity in response to rottlerin in S phase(Fig. 5F). To further establish the involvement of PKC ${delta}$ on topoisomeraseII ${alpha}$ activation in S phase, pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFswere synchronized in S phase by aphidicolin. Analysis of decatenationassays demonstrated that topoisomerase II ${alpha}$ activity is substantiallyabrogated in pkc ${delta}$ ^–/– MEFs (Fig. 5G). These findingsindicate that PKC ${delta}$ modulates topoisomerase II ${alpha}$ from S to G₂/Mphases. In concert with these results, association of PKC ${delta}$ withtopoisomerase II ${alpha}$ was confined to S phase (Fig. 6A, blot a).Moreover, pretreatment of cells with rottlerin was associatedwith down-regulation of this interaction and topoisomerase II ${alpha}$ expression (Fig. 6A, blots a and c). The finding that PKC ${delta}$ wasactivated from late G₁ to S phase further supports the S-phase-specificrole for PKC ${delta}$ in topoisomerase II ${alpha}$ regulation (Fig. 6A, blot d).Taken together, these results indicate that PKC ${delta}$ is requiredfor topoisomerase II ${alpha}$ stabilization and activation during S phase.To confirm that activity of PKC ${delta}$ is required for interactionwith topoisomerase II ${alpha}$ , MOLT-4 cells were left untreated or treatedwith rottlerin. The PKC ${delta}$ -topoisomerase II ${alpha}$ complex formation wasabrogated by treatment with rottlerin (Fig. 6B). In addition,treatment with rottlerin for longer periods (4 and 10 h) wasassociated with substantial attenuation of topoisomerase II ${alpha}$ expression (Fig. 6B). These results support a model in whichPKC ${delta}$ activation triggers stabilization and interaction with topoisomeraseII ${alpha}$ . Previous studies have shown that PKC ${delta}$ localizes to both thecytoplasm and the nucleus, and cytoplasmic PKC ${delta}$ translocatesto the nucleus upon exposure to various genotoxic agents (2,6, 46, 48). In this regard, S-phase-specific interaction ofPKC ${delta}$ with topoisomerase II ${alpha}$ may be partially due to transientnuclear targeting of PKC ${delta}$ in S phase. To examine this possibility,MOLT-4 cells were treated with or without aphidicolin. Subcellularfractionation assays demonstrated that the expression ratioof PKC ${delta}$ in the nucleus and cytoplasm in asynchronous cells wascomparable with that of S-phase-enriched cells (Fig. 6C). Todefine whether PKC ${delta}$ modulates the mRNA levels of topoisomeraseII ${alpha}$ , asynchronized and synchronized (S or G₂/M) MOLT-4 cellswere treated with or without rottlerin. RT-PCR assays revealedthat the mRNA levels of topoisomerase II ${alpha}$ remained unchangedregardless of PKC ${delta}$ activity in asynchronous, S-phase, and G₂/M-phasecells (Fig. 7A). To further determine whether topoisomeraseII ${alpha}$ stabilization by PKC ${delta}$ is mainly due to posttranslational modification,293T cells were stably transfected with GFP-tagged topoisomeraseII ${alpha}$ (Fig. 7B). 293T/GFP-topoisomerase II ${alpha}$ cells were treated withaphidicolin to arrest cells in S phase in the presence or absenceof rottlerin and then released by its removal. As shown withendogenous protein (Fig. 6A, blot c), inhibition of PKC ${delta}$ by rottlerindown-regulated ectopic expression of topoisomerase II ${alpha}$ in bothS and G₂/M phases (Fig. 7C). These results demonstrate thatPKC ${delta}$ modulates topoisomerase II ${alpha}$ at a posttranslational level,and not at a transcriptional level.

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FIG. 5. PKC ${delta}$ is involved in cell cycle-dependent activation of topoisomerase II ${alpha}$ . (A and B) MOLT-4 (A) and U-937 (B) cells were treated with nocodazole (+) in the presence (+) or absence (–) of rottlerin. Topoisomerase II ${alpha}$ activity was analyzed by decatenation assays (top blot). Lysates were analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or anti-PKC ${delta}$ . Nuc. Lysates, nuclear lysates; Cat. KDNA, catenated KDNA; Decat. KDNA, decatenated KDNA. (C) MOLT-4 cells were treated with nocodazole in the presence or absence of bistratene A or rottlerin. Nuclear lysates were analyzed by decatenation assays (top blot). Lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or antitubulin (bottom blot). (D) MOLT-4 cells were treated with nocodazole for 16 h in the presence or absence of rottlerin and then released by nocodazole removal and harvested at the indicated times. Nuclear (Nuc.) lysates were prepared, and topoisomerase II ${alpha}$ activity was analyzed by decatenation assays (top blot). Lysates were analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ or antitubulin (bottom blot). The cell cycle was monitored by using a FACscan and represented as the percentage of population in each cell cycle phase in the graph at the bottom of panel D. (E) MOLT-4 cells were treated with aphidicolin for 16 h in the presence or absence of rottlerin and then released by aphidicolin removal and harvested at the indicated times. Nuclear lysates were prepared, and topoisomerase II ${alpha}$ activity was analyzed by decatenation assays (top blot). Lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ or antitubulin (bottom blot). The cell cycle was monitored by using a FACscan and represented as the percentage of population in each cell cycle phase in the graph. (F) MOLT-4 cells were treated with nocodazole or aphidicolin for 16 h and then with rottlerin for the indicated times. Topoisomerase II ${alpha}$ activity was analyzed by decatenation assays (top blot). Lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ or antitubulin (bottom blot). The cell cycle was monitored by using a FACscan and represented as the percentage of population in each cell cycle phase in the graph. (G) pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were treated with aphidicolin for 16 h. Nuclear lysates were analyzed by the decatenation assays (top blot). Lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ , anti-PKC ${delta}$ , or antitubulin.

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FIG. 6. S-phase-specific interaction of PKC ${delta}$ with topoisomerase II ${alpha}$ . (A) MOLT-4 cells were synchronized in G₂/M phase by treatment with nocodazole in the presence or absence of rottlerin and then released into the cell cycle by its removal. Cells were harvested at the indicated times, and lysates were analyzed by immunoprecipitation (IP) with anti-PKC ${delta}$ followed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) (a) or anti-PKC ${delta}$ (b). Cell lysates were also analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ (c), anti-phospho-Thr505 PKC ${delta}$ (d), anti-PKC ${delta}$ (e), or antitubulin (f). The cell cycle was monitored by using a FACscan. DMSO, dimethyl sulfoxide; IgH, immunoglobulin heavy chain; P-Thr505 PKC ${delta}$ , phospho-Thr505 PKC ${delta}$ . (B) MOLT-4 cells were left untreated or treated with rottlerin for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-PKC ${delta}$ followed by immunoblot analysis with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or anti-PKC ${delta}$ . Lysates were also analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ , anti-PKC ${delta}$ , or antitubulin. (C) MOLT-4 cells were left untreated or treated with aphidicolin to synchronize cells in S phase. Lysates from nuclear (N) and cytoplasmic (C) fractions were subjected to immunoblot (IB) analysis with anti-PKC ${delta}$ , anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ), or anti-I ${kappa}$ B ${alpha}$ .

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FIG. 7. PKC ${delta}$ regulates a posttranslational modification of topoisomerase II ${alpha}$ . (A) MOLT-4 cells were left untreated or treated with aphidicolin or nocodazole for 16 h in the presence (+) or absence (–) of rottlerin. Total RNA was subjected to RT-PCR analysis using primer sets for topoisomerase II ${alpha}$ (TopoII ${alpha}$ ) or ß-actin. (B) 293T cells were stably transfected with GFP vector (293T/GFP) or GFP-topoisomerase II ${alpha}$ (293T/GFP-TopoII ${alpha}$ ). Cell lysates were analyzed by immunoblotting (IB) with anti-GFP or antitubulin. (C) 293T/GFP-TopoII ${alpha}$ cells were treated with aphidicolin or nocodazole for 16 h in the presence or absence of rottlerin. After the cells were washed with phosphate-buffered saline twice, cells were mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and analyzed with a Nikon Eclipse TE2000-U microscope. The cell cycle was determined by using a FACscan. DMSO, dimethyl sulfoxide.

PKC ${delta}$ stabilizes topoisomerase II ${alpha}$ expression in response to DNA damage. To examine the status of topoisomerase II ${alpha}$ expression levelsin response to DNA damage, MOLT-4 cells were treated with 1-ß-D-arabinofuranocylcytosine.ara-C is incorporated into elongating DNA strands and causesarrest of DNA replication by functioning as a relative chainterminator and induces DNA double-strand breaks (26). Immunoblotanalysis of cell lysates with anti-topoisomerase II ${alpha}$ demonstratedthat ara-C treatment induced a transient increase of topoisomeraseII ${alpha}$ expression (Fig. 8A). Similar findings were obtained withU-937 and HL-60 cells (Fig. 8C and data not shown). Moreover,as shown for ara-C, cisplatin treatment was also associatedwith up-regulation of topoisomerase II ${alpha}$ expression (Fig. 8B).These results indicate that certain types of genotoxic stressinduce the expression of topoisomerase II ${alpha}$ . To assess the involvementof PKC ${delta}$ in topoisomerase II ${alpha}$ expression, MOLT-4 cells were pretreatedwith rottlerin followed by ara-C treatment. Rottlerin pretreatmentwas associated with attenuation of ara-C-induced topoisomeraseII ${alpha}$ expression (Fig. 8A). Similar results were obtained withCDDP treatment (Fig. 8B). These findings indicate that PKC ${delta}$ isinvolved in the up-regulation of topoisomerase II ${alpha}$ expressionin response to DNA damage. To assess whether the PKC ${delta}$ -mediatedincrease of topoisomerase II ${alpha}$ expression following genotoxicstress is caused by a transcriptional modification, MOLT-4 cellswere pretreated with or without rottlerin followed by treatmentwith ara-C. RT-PCR assays revealed that the mRNA levels of topoisomeraseII ${alpha}$ remained unchanged regardless of PKC ${delta}$ activity, in controland ara-C treated cells (Fig. 8D). To examine the involvementof PKC ${delta}$ in posttranslational modulation of topoisomerase II ${alpha}$ uponexposure to genotoxic agents, 293T/topoisomerase II ${alpha}$ cells weretreated with ara-C in the presence or absence of rottlerin.ara-C enhanced both endogenous and exogenous expression of topoisomeraseII ${alpha}$ (Fig. 8E). Moreover, inactivation of PKC ${delta}$ by rottlerin inhibitedup-regulation of topoisomerase II ${alpha}$ expression (Fig. 8E). Theseresults demonstrate that PKC ${delta}$ induces topoisomerase II ${alpha}$ expressionfollowing ara-C treatment by a posttranslational, and not atranscriptional, regulation. To further define whether kinaseactivity of PKC ${delta}$ is required for topoisomerase II ${alpha}$ expression,293T cells were transfected with the Flag vector, wt Flag-PKC ${delta}$ CF,or Flag-PKC ${delta}$ CF(K-R) mutant. The finding that ectopic expressionof the wt Flag-PKC ${delta}$ CF, but not the Flag vector or the Flag-PKC ${delta}$ CF(K-R)mutant, up-regulated topoisomerase II ${alpha}$ expression supports therole for PKC ${delta}$ in kinase activity-dependent induction of topoisomeraseII ${alpha}$ expression (Fig. 9A). To determine whether DNA damage inducesthe expression of topoisomerase II ${alpha}$ by a PKC ${delta}$ -dependent mechanism,PKC ${delta}$ was knocked down by transfection of U2-OS cells with siRNAsthat target PKC ${delta}$ (Fig. 9B). As shown in various cell types, treatmentof U2-OS cells with ara-C also induced topoisomerase II ${alpha}$ expression(Fig. 9C). Importantly, knocking down PKC ${delta}$ inhibited the up-regulationof topoisomerase II ${alpha}$ elicited by ara-C (Fig. 9C). To furtherdefine the direct role for PKC ${delta}$ in DNA damage-induced topoisomeraseII ${alpha}$ expression, pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were treatedwith ara-C. Immunoblot analysis with anti-topoisomerase II ${alpha}$ demonstratedthat ara-C induced an increase of topoisomerase II ${alpha}$ expressionin pkc ${delta}$ ^+/+ MEFs (Fig. 9D). By contrast, there was no detectableinduction of topoisomerase II ${alpha}$ in ara-C-treated pkc ${delta}$ ^–/–MEFs (Fig. 9D). These findings indicate that topoisomerase II ${alpha}$ expression is up-regulated by a PKC ${delta}$ -dependent mechanism in theresponse to genotoxic stress.

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FIG. 8. Inhibition of PKC ${delta}$ by rottlerin abrogates DNA damage-induced expression of topoisomerase II ${alpha}$ . (A and B) MOLT-4 cells were pretreated with or without rottlerin for 1 h followed by treatment with ara-C (A) or cisplatin (CDDP) (B) for the indicated periods. Cell lysates were subjected to immunoblot (IB) analysis with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or anti-PKC ${delta}$ . DMSO, dimethyl sulfoxide. (C) U-937 cells were treated and analyzed as described above for panel A. (D) MOLT-4 cells were left untreated or treated with ara-C for 4 h in the presence or absence of rottlerin. Total RNA was subjected to RT-PCR analysis using primer sets for topoisomerase II ${alpha}$ (TopoII ${alpha}$ ) or ß-actin. (E) 293T/GFP-TopoII ${alpha}$ cells were left untreated or treated with rottlerin for 1 h followed by treatment with ara-C for 4 h. Cell lysates were subjected to immunoblot analysis with anti-topoisomerase II ${alpha}$ or anti-PKC ${delta}$ .

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FIG. 9. PKC ${delta}$ -dependent induction of topoisomerase II ${alpha}$ expression in response to DNA damage. (A) 293T cells were transfected with Flag vector, Flag-PKC ${delta}$ CF, or Flag-PKC ${delta}$ CF(K-R). Cell lysates were analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ), anti-Flag, or antitubulin. (B) U2-OS cells were left untransfected (Control) or transfected with the indicated siRNAs. Cell lysates were subjected to immunoblot (IB) analysis with anti-PKC ${delta}$ or antitubulin. (C) U2-OS cells transfected with scrambled siRNA or PKC ${delta}$ siRNA2 were left untreated or treated with ara-C for 4 h. Cell lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ , anti-PKC ${delta}$ , or antitubulin. (D) pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were left untreated or treated with ara-C for 4 h. Cell lysates were analyzed by immunoblotting with anti-topoisomerase II ${alpha}$ , anti-PKC ${delta}$ , or antitubulin.

PKC ${delta}$ activates topoisomerase II ${alpha}$ for the DNA damage response. Previous studies have shown that PKC ${delta}$ is activated in responseto DNA damage (11). In this context and given the finding thatPKC ${delta}$ induces topoisomerase II ${alpha}$ expression following genotoxicstress, it is conceivable that PKC ${delta}$ induces enzymatic activityof topoisomerase II ${alpha}$ in response to DNA damage. To address thisissue, MOLT-4 cells were treated with ara-C in the presenceor absence of rottlerin. Decatenation assays showed that topoisomeraseII ${alpha}$ was activated by ara-C treatment (Fig. 10A). Importantly,pretreatment with rottlerin substantially attenuated ara-C-inducedtopoisomerase II ${alpha}$ activation (Fig. 10A). To verify these findings,we performed DNA relaxation assays. In concert with the decatenationassays, inhibition of PKC ${delta}$ activation by rottlerin attenuatedara-C-induced topoisomerase II ${alpha}$ activity (Fig. 10B). Similarstudies performed on U-937 and HL-60 cells confirmed these results(Fig. 10C and data not shown). Moreover, as found for ara-C,comparable results were obtained in the treatment of cells withCDDP (Fig. 10D and data not shown). To further assess whetherDNA damage induces topoisomerase II ${alpha}$ activity by a PKC ${delta}$ -dependentmechanism, PKC ${delta}$ was knocked down by transfection of U2-OS cellswith siRNAs. Treatment with ara-C induced topoisomerase II ${alpha}$ activity(Fig. 10E). Importantly, knocking down PKC ${delta}$ inhibited the up-regulationof topoisomerase II ${alpha}$ activity elicited by ara-C (Fig. 10E).To confirm the involvement of PKC ${delta}$ on topoisomerase II ${alpha}$ activationupon exposure to genotoxic agents, pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/–MEFs were left untreated or treated with ara-C. Analysis ofdecatenation assays demonstrated that topoisomerase II ${alpha}$ activitywas substantially enhanced in pkc ${delta}$ ^+/+, but not pkc ${delta}$ ^–/–,MEFs (Fig. 10F). Taken together, these findings indicate thattopoisomerase II ${alpha}$ is activated in response to certain genotoxicagents in a PKC ${delta}$ -dependent mechanism.

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FIG. 10. PKC ${delta}$ -dependent activation of topoisomerase II ${alpha}$ in response to DNA damage. (A and B) MOLT-4 cells were pretreated with or without rottlerin for 1 h followed by treatment with ara-C. Nuclear lysates (Nuc. Lysates) were analyzed by decatenation (A) and DNA relaxation (B) assays. DMSO, dimethyl sulfoxide; Cat. KDNA, catenated KDNA; Decat. KDNA, decatenated KDNA. (C) U-937 cells were treated as described above for panel A, and nuclear lysates were analyzed by the decatenation assays. (D) MOLT-4 cells were left untreated or treated with rottlerin for 1 h followed by the treatment with CDDP for the indicated times. Topoisomerase II ${alpha}$ activity was analyzed by decatenation assays. (E) U2-OS cells transfected with scrambled siRNA or PKC ${delta}$ siRNA2 were left untreated or treated with ara-C for 4 h. Nuclear lysates were analyzed by the decatenation assays. (F) pkc ${delta}$ ^+/+ and pkc ${delta}$ ^–/– MEFs were left untreated or treated with ara-C for 4 h. Nuclear lysates were analyzed by the decatenation assays.

Activation of topoisomerase II ${alpha}$ is involved in PKC ${delta}$ -mediated apoptosis in response to DNA damage. Previous studies have shown that DNA damage elicited by ara-Cefficiently induces apoptosis (26). Moreover, inhibition ofPKC ${delta}$ by (i) expression of dominant-negative PKC ${delta}$ , (ii) rottlerintreatment, or (iii) knocking down PKC ${delta}$ , attenuated ara-C-inducedapoptosis (15, 46). These findings indicate that activationof PKC ${delta}$ following genotoxic stress is associated with apoptosisexecution; however, this mechanism is largely unknown. Importantly,the present study demonstrates that ara-C treatment inducesexpression and activation of topoisomerase II ${alpha}$ by a PKC ${delta}$ -dependentmechanism. In this context, to examine the possibility thatactivation of topoisomerase II ${alpha}$ is involved in PKC ${delta}$ -mediated apoptosisfollowing DNA damage, cells were treated with ara-C in the presenceor absence of a non-DNA-damaging catalytic inhibitor of topoisomeraseII ${alpha}$ , ICRF-193 (36). Treatment of cells with ara-C was associatedwith apoptosis induction (Fig. 11A). In contrast, pretreatmentwith ICRF-193 substantially attenuated ara-C-induced apoptosis(Fig. 11A). These results suggest that ara-C-induced apoptosisis, at least in part, a topoisomerase II ${alpha}$ -dependent mechanism.To further define the role for topoisomerase II ${alpha}$ in ara-C-inducedapoptosis, topoisomerase II ${alpha}$ was knocked down in U2-OS cellsby transfection with topoisomerase II ${alpha}$ siRNAs (Fig. 11B). Knockingdown topoisomerase II ${alpha}$ attenuated the induction of apoptosiselicited by ara-C treatment (Fig. 11C). Moreover, as previouslyreported (46), pretreatment with rottlerin conferred a protectiveeffect on ara-C-mediated apoptosis (Fig. 11C). By contrast,inhibition of PKC ${delta}$ activity by rottlerin had little, if any,effect on attenuation of apoptosis when knocking down topoisomeraseII ${alpha}$ (Fig. 11C). These findings collectively support the involvementof topoisomerase II ${alpha}$ as a positive regulator of apoptosis byPKC ${delta}$ -mediated activation in response to DNA damage.

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FIG. 11. Topoisomerase II ${alpha}$ is required for PKC ${delta}$ -induced apoptotic cell death in response to genotoxic stress. (A) MOLT-4 cells were treated with ara-C for the indicated times in the presence (open bar) or absence (closed bar) of ICRF-193. The percentages of apoptotic cells were determined by TUNEL assays. The results are represented as means ± standard deviations (error bars) obtained from four fields of 100 to 300 cells (each field) and three independent experiments. (B) U2-OS cells were transfected with the indicated siRNAs for 48 h and then left untreated (–) or treated (+) with ara-C for 4 h. Cell lysates were subjected to immunoblot (IB) analysis with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or antitubulin. Nuclear lysates were analyzed by decatenation assays (bottom blot). The cells had been transfected with scrambled siRNA (lanes S), topoisomerase II ${alpha}$ siRNA1 (lanes 1), or topoisomerase II ${alpha}$ siRNA2 (lanes 2). Nuc. Lysates, nuclear lysates; Cat. KDNA, catenated KDNA; Decat. KDNA, decatenated KDNA. (C) U2-OS cells transfected with the indicated siRNAs were left untreated (–) or treated (+) with rottlerin for 1 h followed by treatment with ara-C (+) for 24 h. The percentages of apoptotic cells were determined by TUNEL assays. The results are represented as means ± standard deviations (error bars) obtained from four fields of 100 to 300 cells, each performed over three independent experiments. Cell lysates were also analyzed by immunoblotting (IB) with anti-topoisomerase II ${alpha}$ (anti-TopoII ${alpha}$ ) or antitubulin.

DISCUSSION

Top

Discussion

Activation of PKC ${delta}$ in the DNA damage response. Involvement of PKC ${delta}$ in the DNA damage response is supported bythe findings that both arrest of DNA replication and inductionof DNA lesions are associated with PKC ${delta}$ activation (44-46, 48).The available evidence indicates that full-length PKC ${delta}$ is activatedas an early event within 1 h of exposure to genotoxic agents.Activation of PKC ${delta}$ by tyrosine phosphorylation upon exposureto genotoxic agents is mediated in part by the c-Abl kinase(48). Another study demonstrates that Lyn also phosphorylatesPKC ${delta}$ and that Lyn-mediated tyrosine phosphorylation of PKC ${delta}$ contributesto PKC ${delta}$ activation (45). PKC ${delta}$ is also activated as a later eventin the genotoxic stress response by caspase-3-mediated proteolyticcleavage (10, 11, 25). The cleaved C-terminal 40-kDa fragmentcontains the ATP-binding and kinase domains and is constitutivelyactive (10, 11, 15). The finding that tyrosine phosphorylationof PKC ${delta}$ is required for activation of caspase-3 and thereby PKC ${delta}$ cleavage supports a link between both mechanisms of PKC ${delta}$ activation(3). Whereas expression of PKC ${delta}$ CF induces apoptotic cell death(15), the precise events responsible for this response are unknownbut may involve an interaction between PKC ${delta}$ CF and DNA-PKcs orRad9 (2, 46). In addition, the present studies show for thefirst time that PKC ${delta}$ CF interacts with topoisomerase II ${alpha}$ . Thisinteraction is necessary for PKC ${delta}$ -mediated induction of apoptosisin response to certain types of genotoxic agents. Importantly,cleavage of PKC ${delta}$ into the constitutively active catalytic fragmentis irreversible and thus may function in prolonged stimulationof multiple proapoptotic pathways.

PKC ${delta}$ interacts with topoisomerase II ${alpha}$ . Previous studies have shown that PKC ${delta}$ localizes to both the nucleusand cytoplasm (2, 6, 46, 48). While nuclear PKC ${delta}$ associates constitutivelywith DNA-PKcs and Rad9 (2, 46), the nuclear targets of PKC ${delta}$ areotherwise largely unknown. The present study demonstrates thatnuclear PKC ${delta}$ also associates with topoisomerase II ${alpha}$ . Our resultsshow that the catalytic fragment of PKC ${delta}$ directly binds to theC-terminal region of topoisomerase II ${alpha}$ . Notably, the findingthat this interaction is confined to the S phase suggests arole for PKC ${delta}$ in the gradual increase of topoisomerase II ${alpha}$ expressionand activity during S phase. Moreover, the results demonstratethat, upon DNA damage in S phase by ara-C, activated PKC ${delta}$ inducesaberrant expression and activation of topoisomerase II ${alpha}$ . In thiscontext, PKC ${delta}$ -mediated apoptosis by ara-C is, at least in part,required for topoisomerase II ${alpha}$ activation. These findings collectivelysupport a model in which modulation of topoisomerase II ${alpha}$ by PKC ${delta}$ during S phase is essential for determination of cell fate.

PKC ${delta}$ induces topoisomerase II ${alpha}$ expression and activation. Activity of topoisomerase II ${alpha}$ is tightly regulated by posttranslationalmodifications, such as phosphorylation and ubiquitination. Thetopoisomerase II enzyme has been shown to be phosphorylatedon multiple serine and threonine residues, the majority of whichare located in the C-terminal region. Several lines of studyhave been conducted to examine whether topoisomerase II ${alpha}$ is phosphorylatedin a cell cycle-dependent manner. The previous findings haveindicated that this enzyme is hyperphosphorylated from S phaseto G₂/M phase. In concert with phosphorylation, expression andactivation of topoisomerase II ${alpha}$ are induced and reach their maximallevels in M phase. Whereas there are a number of proteins involvedin topoisomerase II ${alpha}$ regulation, our present study revealed PKC ${delta}$ as a newly identified modulator of topoisomerase II ${alpha}$ expressionand activation. Indeed, the present study demonstrates thatPKC ${delta}$ regulates the expression level of topoisomerase II ${alpha}$ . Forexample, inhibition of PKC ${delta}$ was associated with down-regulationof topoisomerase II ${alpha}$ expression (Fig. 5A to G, 6A and B, and7C). Induction of DNA damage-elicited topoisomerase II ${alpha}$ expressionwas also diminished by inhibition of PKC ${delta}$ activity (Fig. 8A to C).Importantly, this regulation, at least in part, depends on thekinase activity of PKC ${delta}$ (Fig. 9A). Taken together, these resultssuggest the possibility that PKC ${delta}$ regulates topoisomerase II ${alpha}$ expression, resulting in the modulation of its activity. Thepresent results also demonstrate that PKC ${delta}$ is associated withinduction of topoisomerase II ${alpha}$ stabilization and activation inS phase. The mechanism by which PKC ${delta}$ achieves this is, at present,unclear. A potential explanation is that degradation of topoisomeraseII ${alpha}$ is inhibited by PKC ${delta}$ -mediated modification, while PKC ${delta}$ phosphorylation of topoisomerase II ${alpha}$ could induce its activation.Importantly, the finding that PKC ${delta}$ is activated and interactswith topoisomerase II ${alpha}$ during S phase further supports the S-phase-specificrole for PKC ${delta}$ in topoisomerase II ${alpha}$ modulation. In this context,other reports demonstrated that PKC ${delta}$ is activated from late G₁to S phase (24, 34). Activation of PKC ${delta}$ stimulated G₁ phase cellcycle progression. Furthermore, activated PKC ${delta}$ in S phase triggeredcaspase-dependent apoptotic cell death (34). Meanwhile, suppressionof PKC ${delta}$ activity was sufficient to inhibit DNA synthesis (24).Thus, taken together with the present findings, it is conceivablethat PKC ${delta}$ functions in cell cycle progression by modulating topoisomeraseII ${alpha}$ in S phase. Obviously, further studies will be needed todefine the precise role for PKC ${delta}$ in cell cycle progression.

Our results also demonstrate that PKC ${delta}$ promotes topoisomeraseII ${alpha}$ expression and subsequent activation in response to certaintypes of genotoxic stress, such as ara-C treatment. Since ara-Cis incorporated into DNA and causes DNA strand breaks, ara-Ctreatment arrests cells in S phase (13, 26). In this regard,the finding that activation of PKC ${delta}$ following ara-C treatmentcaused more pronounced expression and activation of topoisomeraseII ${alpha}$ further supports the model in which regulation of topoisomeraseII ${alpha}$ by PKC ${delta}$ is S phase specific.

Topoisomerase II ${alpha}$ is of functional importance in regulation of the PKC ${delta}$ -dependent apoptotic response to DNA damage. Whereas PKC ${delta}$ is involved in the apoptotic response to DNA damage,the mechanism by which PKC ${delta}$ induces DNA damage-elicited activationof the intrinsic apoptotic pathway is largely unknown. Certaininsights have been derived from our previous finding that PKC ${delta}$ regulates the interaction of human Rad9 (hRad9) with Bcl-2 and,consequently, the hRad9-mediated apoptotic response to DNA damage(46). Moreover, the results indicated that PKC ${delta}$ translocatesto the nucleus and thereby regulates hRad9 (46). Recently, wedemonstrated that PKC ${delta}$ regulates p53 by Ser46 phosphorylation(41). In that study, we show that p53-dependent apoptosis elicitedby etoposide is attenuated by pretreatment of cells with rottlerin,indicating a pivotal role for PKC ${delta}$ in induction of p53-mediatedapoptosis. Thus, DNA damage-induced nuclear targeting of PKC ${delta}$ could contribute to the induction of the intrinsic apoptoticpathway. In the present study, we further demonstrate that topoisomeraseII ${alpha}$ functions as a novel nuclear effector of PKC ${delta}$ -mediated apoptosis.We found that inappropriate expression of topoisomerase II ${alpha}$ byPKC ${delta}$ is associated with genotoxic stress-elicited apoptosis.Moreover, the finding that PKC ${delta}$ -mediated deregulation of topoisomeraseII ${alpha}$ was S phase specific suggested the disruption of cell cyclecheckpoints, resulting in the execution of apoptosis. In thisregard, another study demonstrated that topoisomerase II ${alpha}$ -mediatedcell death was triggered after progression through the G₁-Sphase transition but before the G₂-M phase transition (28).Given the evidence for the involvement of topoisomerase II ${alpha}$ inthe G₂ checkpoint that regulates the entry into mitosis (8),aberrant activation of topoisomerase II ${alpha}$ during S phase couldcause failure to arrest before mitosis. Unscheduled entry intomitosis could then trigger the intrinsic apoptotic pathway,which is characterized as "mitotic catastrophe" (4). Additionalstudies will be required to prove this scenario. However, asshown in the previous studies, the tight control of topoisomeraseII ${alpha}$ is fundamental to the appropriate operation of the cell cycle(16, 19, 40). Thus, the findings in the present work supportthe model in which deregulation of topoisomerase II ${alpha}$ activityby PKC ${delta}$ sensitizes the cells to genotoxic stress-induced catastrophiccell death.

ACKNOWLEDGMENTS

We thank W. T. Beck for providing GFP-TopoII ${alpha}$ plasmid and T.Kasama for assisting in the mass spectrometry analysis.

This work was supported by grants from the Ministry of Education,Science and Culture of Japan (to K.Y., K.I.N., and Y.M.), theNakajima Foundation (to K.Y.), Takeda Science Foundation (toK.Y.), Public Trust Haraguchi Memorial Cancer Research Fund(to K.Y.), Kanae Foundation for Life & Socio-medical Science(to K.Y.) and Kowa Life Science Foundation (to K.Y.).

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81 3 5803 5826. Fax: 81 3 5803 0242. E-mail: yos.mgen{at}mri.tmd.ac.jp.

REFERENCES

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