Oncogene (2014), 1–7 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc
ORIGINAL ARTICLE
DNA-PK/Chk2 induces centrosome amplification during prolonged replication stress C-Y Wang1,2, EY-H Huang1,3, S-c Huang1 and B-c Chung1 The antineoplastic drug hydroxyurea (HU), when used at subtoxic doses, induces prolonged replication stress and centrosome amplification. This causes genomic instability and increases the malignancy of the recurring tumor. The mechanism of centrosome amplification induced by prolonged replication stress, however, is still unclear. Here, we examined the involvement of ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) and found that HU-induced centrosome amplification was inhibited by the depletion of DNA-PKcs, but not ATM and ATR. Inactivation of ATM/ATR in U2OS cells instead caused aneuploidy and cell death. We found DNA-PKcs depletion also abrogated ATM phosphorylation, indicating that ATM activation during prolonged replication stress depends on DNA-PK. Depletion of DNA-PK abrogated checkpoint kinase (Chk)2 activation and partially reduced Chk1 activation. Chk2 depletion blocked HU-induced centrosome amplification, indicating a function of Chk2 in centrosome amplification. We further found that Chk2 was phosphorylated at Thr68 on the mother centriole at late G2 and mitosis when unstressed and on all amplified centrioles induced by HU. In summary, we have elucidated that DNA-PK/Chk2 signaling induces centrosome amplification upon long-term HU treatment, therefore increasing our insight into tumor recurrence after initial chemotherapy. Oncogene advance online publication, 24 March 2014; doi:10.1038/onc.2014.74
INTRODUCTION Hydroxyurea (HU) and 5-fluorouracil are common anticancer drugs that cause replication stress and activate DNA damage response.1 These drugs are often highly toxic, killing cancer cells but also generating many side effects. Yet, when subtoxic doses of the drug are administered, some cancer cells can still survive although they are arrested at the S phase; and their centrosomes are often amplified.2,3 Centrosome amplification induced by DNA damage results in the elimination of cells with damaged genomes.4 However, when coupled with a deficiency in DNA repair machinery, cells harboring amplified centrosomes are more susceptible to malignancy.5 The amplification of centrosomes upon DNA damage requires the activation of checkpoint proteins. Members of the phosphatidylinositol 3-kinase-like kinase superfamily, including ATM (ataxia telangiectasia, mutated), ATR (ataxia telangiectasia, mutated and Rad3-related) and DNA-PK (DNAdependent protein kinase), initiate damage signals in response to DNA damages.6 ATM is activated by DNA double-strand breaks to repair DNA damage via homologous recombination and to stop cell cycle progression.7 Upon DNA double-strand breakage, ATM is activated by phosphorylation at Ser1981.8 ATM then phosphorylates checkpoint kinase 2 (Chk2) at Thr68,9 which is critical for cell cycle control.10 ATM activation by high levels of DNA damage, together with Rad51 deficiency, induces centrosome amplification.11 The second kinase induced by DNA double-strand breaks is DNA-PK, which is activated in response to DNA damage to facilitate DNA repair and to halt the cell cycle.12 DNA-PK is composed of the DNA-binding components Ku70, Ku80 and the catalytic subunit DNA-PKcs.13 Ku heterodimer binds to the broken 1
ends of DNA and recruits DNA-PKcs to form an active complex that initiates DNA damage repair by non-homologous end-joining. DNA-PK also activates Akt signaling to arrest cell cycle progression and to maintain cell survival.12 The third kinase that activates the damage response is ATR, which is induced by DNA single-strand breaks.14 Once recruited to the DNA, ATR activates its substrate checkpoint kinase 1 (Chk1) and initiates the downstream cascade to limit cell cycle progression. The activation of ATR-Chk1 signaling also results in Thr160 phosphorylation of cyclin-dependent kinase 2 (CDK2) followed by centrosome amplification.15 Inactivation of Chk1 abrogates centrosome amplification induced by DNA alkylation damage.16 In this report we have investigated the signaling pathways induced by HU treatment. We found that DNA-PK/Chk was activated upon HU treatment and promoted centrosome amplification. We also found that activated Chk2 resided on the mother centriole at late G2 and mitosis. Our studies elucidate the mechanism of centrosome amplification in response to prolonged replication stress via the DNA-PK/Chk signaling pathway.
RESULTS Activation of DNA-PK induces centrosome amplification during prolonged replication stress The signaling pathways that respond to prolonged replication stress were investigated. HU treatment of U2OS cells for 48 h induced all three checkpoint proteins, DNA-PK, ATM and ATR (Figure 1a); additionally, phosphorylated H2AX appeared as speckles (Figure 1b). These are all hallmarks of severe DNA
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan and 2Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Correspondence: Professor B-c Chung, Institute of Molecular Biology, Academia Sinica, 128 Academia Road Section 2, Taipei 115, Taiwan. E-mail: [email protected] 3 Current address: Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA Received 26 November 2012; revised 21 December 2013; accepted 1 January 2014
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
2
Figure 1. HU induces activation of DNA-PK at the damaged DNA loci and induces centrosome amplification depending on DNA-PK. (a) DNAPK, ATM and ATR are activated by HU treatment. Extracts of U2OS treated with or without HU were analyzed by immunoblot with antibodies against Thr2609-phosphorylated DNA-PKcs (p-PKcs), Ser428-phosphorylated ATR (p-ATR), Ser1981-phosphorylated ATM (p-ATM) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (b) Phosphorylated DNA-PK colocalizes with γH2AX foci. HU or etoposide (ETO)-treated Y1 cells were co-stained with DNA dye (DAPI) and antibodies against phospho-Ser139 of H2AX (γH2AX) and Thr2609-phosphorylated DNAPKcs (p-PKcs). (c and d) Vanillin reverses centrosome overduplication caused by HU treatment. Quantification of a population of U2OS (c) and H1299 (d) cells with multiple centrosomes upon HU treatment in the presence or absence of DNA-PK inhibitor vanillin. (e) Inactivation of DNAPK does not disturb the cell cycle profile. Quantification of different cell cycle stages in U2OS cells with or without HU in the presence or absence of vanillin. (f) HU treatment induces the formation of multiple centrioles. Immunofluorescence staining of centrioles with acetylated tubulin (Ace-tub, green) and of centrosomes with γ-tubulin (γ-tub, red) in Y1 cells. Scale bars are 5 μm. (g) Vanillin reverses centriole overduplication caused by HU treatment. Quantification of the population of Y1 cells with multiple centrioles upon HU treatment in the presence or absence of DNA-PK inhibitor vanillin. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001.
damage. Phosphorylated DNA-PKcs also colocalized with phosphorylated H2AX foci, indicating HU treatment induced a DNA damage response and activated DNA-PK. We have previously shown that activation of DNA-PK due to the depletion of NR5A1 induces centrosome amplification in adrenocortical Y1 cells,17 and here further tested whether DNA-PK also induced centrosome amplification during replication stress. DNAPK inhibitor vanillin itself had no effect, but it suppressed the formation of multiple centrosomes caused by HU in U2OS Oncogene (2014), 1 – 7
(Figure 1c) and H1299 (Figure 1d) cells. Vanillin (1 mM) alone or combined with HU, however, affected neither cell viability (Supplementary Figures S1A and B) nor apoptosis (Supplementary Figures S1C and D) and had no effect on cell cycle profiles (Figure 1e; Supplementary Figure S1E). Thus, DNA-PK participated in HU-induced centrosome overduplication without affecting the cell cycle or cell viability. The effect of HU on centrosome amplification was further examined by the staining of centrosomes with γ-tubulin and of © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
3 centrioles stained by nocodazole-resistant acetylated tubulin (Acetub) followed by counting centriole numbers. Normal Y1 cells contained one centrosome with two centrioles at G1 and duplicated centrosomes with four centrioles at the S/G2 phase (Figure 1f). HU treatment induced multiple γ-tubulin spots (>2) and multiple centrioles (>4 Ace-tub) (Figure 1f), which were reversed by vanillin (Figure 1g). Thus, inhibition of DNA-PK activation prevented centriole amplification during prolonged HU treatment. In addition to DNA-PK, the roles of ATM/ATR were also examined by treating cells with the common ATM/ATR inhibitor, caffeine. Caffeine alone had no effect on centrosome numbers, but it aggravated the multiple centrosome phenotype induced by HU (Figure 2a). When examined in more detail, these cells with multiple centrosomes were divided into two groups: cells with a single nucleus (Figure 2b, mono-Nucl; >2Cent, upper panel) and cells with di-nuclei (Figure 2b, di-Nucl; >2Cent, lower panel). HU alone induced multiple centrosomes with a single intact nucleus (Figure 2c), which was not affected by the treatment with caffeine, but was inhibited by vanillin. This indicates that DNA-PK, but not ATM/ATR, participates in centrosome amplification caused by replication stress. Cells with multiple centrosomes/di-nuclei were induced when caffeine was present together with HU, whereas vanillin could not repress this (Figure 2d). These data indicates that inactivation of ATM/ATR results in aneuploidy with multiple centrosomes, and this phenotype is independent of DNA-PK during prolonged replication stress. To further confirm the role of DNA-PK in centrosome amplification, DNA-PKcs in U2OS cells was depleted by small interfering RNA (siRNA). This caused the absence of DNA-PKcs phosphorylation in the presence of HU, but had no effect on the levels of Ku70 and Ku80 (Figure 3a). Depletion of DNA-PKcs inhibited centrosome amplification caused by HU in U2OS cells (Figure 3b). This data combined with the above inhibitor data demonstrate that DNA-PK is involved in centrosome amplification. We also tested the effect of Ku70 and Ku80 by depleting them individually with siRNA. Immunoblot assay showed that efficient depletion of Ku70 led to the depletion of Ku80, and vice versa (Figure 3c). Depletion of Ku70 and Ku80 inhibited HU-induced centrosome amplification (Figure 3d), indicating that all DNA-PK subunits, including Ku70 and Ku80, are involved in centrosome amplification caused by HU. Besides the stress condition, the role of DNA-PK in centriole numbers without stress was also examined. After DNA-PKcs depletion (Figure 3e), the populations of cells with either unduplicated (centriole number = 2) or duplicated (centriole number = 3–4) centrioles were not changed compared with the control group (Figure 3f). Thus, DNA-PK does not participate in centriole duplication when unstressed; only during prolonged replication stress is it activated to induce centrosome overduplication. Activation of Chk2 induces centrosome amplification upon HU treatment As HU induced the activation of ATM, ATR and DNA-PK, their downstream effectors were examined. Upon HU treatment, Akt phosphorylation was not changed, but phosphorylation of Chk1 and Chk2 were increased (Figure 4a). Upon DNA-PKcs depletion, Thr68 phosphorylation of Chk2 (p-Chk2) was abrogated and the phosphorylation of Chk1 was partially reduced (Figure 4a). Both ATM and ATR were activated by HU, and depletion of DNA-PKcs inhibited phosphorylation of ATM, but not ATR (Figure 4a). These data indicate that DNA-PK activation during prolonged replication stress leads to phosphorylation of ATM, Chk2 and to a lower extent Chk1. We tested the effect of ATM by examining the activation of its substrates Chk1/2 during prolonged replication stress. Depletion © 2014 Macmillan Publishers Limited
Figure 2. Caffeine induces aberration of multiple nuclei, whereas vanillin abrogates centrosome amplification during prolonged replication stress. (a) Caffeine induces centrosome aberration in the presence of HU. Quantification of U2OS cells with multiple centrosomes in the presence or absence of HU and/or caffeine. (b) Immunofluorescence examination of centrosomes (stained with anti-γ-tubulin antibody) in U2OS cells treated with HU and caffeine. Insets are lower magnifications showing nuclei stained by DAPI and centrosomes. Two types of cells were detected: those with a single nucleus and multiple centrosomes (mono-Nucl; >2Cent; upper panel) or with di-nuclei with multiple centrosomes (di-Nucl; >2Cent; lower panel). Scale bars are 5 μm. (c and d) Caffeine induces aberration of multiple nuclei, whereas vanillin abrogates centrosome amplification during prolonged replication stress. Quantification of U2OS cells containing (c) single nucleus with multiple centrosomes (mono-Nucl; >2Cent), or (d) di-nuclei with multiple centrosomes (di-Nucl; >2Cent). These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001.
of ATM had no effect on the phosphorylation of Chk1, Chk2 and ATR, but it slightly reduced DNA-PKcs phosphorylation (Figure 4b). This data indicates that upon HU treatment Chk2 is not activated by ATM but by DNA-PK. The role of ATR was also checked. Depletion of ATR did not affect phosphorylation of DNA-PKcs and Chk2, but it greatly reduced the activation of Chk1 and perhaps slightly of ATM (Figure 4c). These data show that Chk1 phosphorylation depends on both DNA-PK and ATR, and Chk2 phosphorylation depends on DNA-PK upon prolonged replication stress induced by HU. The effects of Chk1 and Chk2 on centrosome amplification were further examined by treating U2OS cells with UCN-01 that inhibits mostly Chk1 but also Chk2. We also inhibited Chk2 specifically with Chk2 inhibitor II (Chk2i). UCN-01 but not Chk2i treatment induced considerable cell death (Supplementary Figure S2A), consistent with the earlier report that inactivation of Chk1 induces Oncogene (2014), 1 – 7
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
4
apoptosis.18 Examining the remaining live cells, we found that UCN-01 abrogated the multiple centrosomal phenotype induced by HU (Figure 5a). The role of Chk2 was examined by siRNA
depletion. Depletion of Chk2 by siRNA did not abrogate DNA-PK, ATM, ATR or Chk1 activation (Figure 5b), but lowered the multiple centrosome phenotype caused by HU treatment (Figure 5c). The Chk2-specific inhibitor, Chk2i, also blocked centriole (Figure 5d) and centrosome (Supplementary Figure S2B) amplification caused by HU. Chk2i also blocked centrosome amplification in Y1 cells (Supplementary Figure S2C). These data showed that Chk2 contributed to centrosome amplification induced by HU.
Activated Chk2 resides on the mother centriole The subcellular localization of p-Chk2 was examined by immunofluorescence staining. In the presence of HU, both nuclear and centriolar p-Chk2 levels were increased (Figure 6a), and p-Chk2 was present on over-duplicated centrioles. In the absence of HU, p-Chk2 started to increase at the centrosome at the late G2 phase and peaked at mitosis in U2OS (Figure 6b) and HeLa cells (Supplementary Figure S3A). Centrioles were examined by staining with Ace-tub after microtubule depolymerization in the cold, and were found co-stained with p-Chk2 (Figure 6c). This result implies that p-Chk2 is targeted to the centriole independent of microtubule transport. We adjusted the image exposure time in order to obtain higher immunofluorescence signals for closer examination and found that only one of the two centrioles was decorated with p-Chk2 (Figure 6c). Co-staining with Centrin also confirmed the asymmetric localization of p-Chk2 in one of the centrioles during mitosis (Supplementary Figure S3B). To identify which centriole was decorated by p-Chk2, we examined the mother centriole by staining with known mother centriolar protein Odf2.19 At the G1 phase, two centrioles were stained with Ace-tub but only one was decorated with Odf2
Figure 3. DNA-PKcs and Ku participate in centrosome amplification induced by HU. (a) Efficient depletion of DNA-PKcs by siRNA transfection. Extracts of U2OS cells after transfection with siRNA against scrambled control (siCTL) or DNA-PKcs (siPKcs) in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs), Thr2609-phosphorylated DNAPKcs (p-PKcs), Ku70, Ku80 and Hsc70. (b) Centrosome amplification in HU-treated U2OS depends on DNA-PKcs. Quantification of the population of U2OS cells containing multiple centrosomes with or without HU in the DNA-PKcs (siPKcs) siRNA-transfected cells. (c) Efficient depletion of Ku70 and Ku80 by siRNA. Extracts of U2OS cells after transfection with Ku70 (siKu70) or Ku80 (siKu80) siRNA in the presence or absence of HU were analyzed by immunoblot with antibodies against Ku70, Ku80 or actin. (d) Depletion of Ku70 or Ku80 inhibit centrosome amplification caused by HU treatment. Quantification of population of U2OS cells with multiple centrosomes after transfection with siRNA against Ku70 (siKu70) or Ku80 (siKu80) with or without HU. (e and f) Depletion of DNA-PKcs does not affect centriole duplication. (e) Extracts of U2OS cells after transfection with siRNA against DNA-PKcs (PKcs) in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs) or Hsc70. (f) Quantification of a population of U2OS cells containing multiple centrosomes with or without HU in the scrambled control (siCTL) or DNA-PKcs (siPKcs) siRNAtransfected cells. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001. Oncogene (2014), 1 – 7
Figure 4. Depletion of DNA-PKcs prevents HU-induced Chk2 activation. Extracts of U2OS cells after transfection with siRNA against scrambled control (CTL) and (a) DNA-PKcs (PKcs), (b) ATM or (c) ATR in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs), Thr2609phosphorylated DNA-PKcs (p-PKcs), Ser473-phosphorylated Akt (p-Akt), Ser345-phosphorylated Chk1 (p-Chk1), Chk1, Ser428phosphorylated ATR (p-ATR), Thr68-phosphorylated Chk2 (p-Chk2), Chk2, Ku70, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α-tubulin (α-tub), ATM and Ser1981-phosphorylated ATM (p-ATM). © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
5
Figure 5. Involvement of Chk1/2 in centrosome amplification induced by HU. (a) Quantification of a population of U2OS cells containing multiple centrosomes with or without HU in UCN-01 (Chk1 inhibitor)-treated cells. (b) Efficient depletion of Chk2 by siRNA transfection. Following transfection with siRNA against scrambled control (siCTL) or Chk2 (siChk2), extracts of U2OS cells were analyzed by immunoblot with antibodies against Thr68phosphorylated Chk2 (p-Chk2), Chk2, Ser345-phosphorylated Chk1 (p-Chk1), Thr2609-phosphorylated DNA-PKcs (p-PKcs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Ser1981phosphorylated ATM (p-ATM) and Ser428-phosphorylated ATR (p-ATR). (c and d) Centrosome amplification in U2OS cells during prolonged replication stress depends on Chk2 activity. Quantification of a population of U2OS cells containing multiple (c) centrosomes or (d) centrioles with or without HU in (c) Chk2 siRNA (siChk2)-transfected or (d) Chk2 inhibitor (Chk2i)-treated cells. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. *P ⩽ 0.05, **P ⩽ 0.01, ***P ⩽ 0.001.
(Figure 6d). Following centriole duplication, two pairs of centrioles were present but only one of the duplicated centrioles stained with Odf2 before and after centrosome separation, confirming that Odf2 is a marker for the mother centriole. These Odf2 spots colocalized with p-Chk2 at the late G2 phase, indicating that p-Chk2 resides in the mother centriole (Figure 6c). DISCUSSION In this report we have examined the long-term effect of the anticancer drug HU on centrosome homeostasis, and show that the DNA-PK/Chk2 signaling was activated leading to centrosome overduplication. Depletion of DNA-PK or Chk2 alleviates centrosome amplification induced by HU. We also show that Chk2 is activated in the mother centriole at G2/M during normal cell cycles. © 2014 Macmillan Publishers Limited
Actions of ATM, ATR and DNA-PK during prolonged replication stress DNA damages induced by replication stress can elicit different responses along the time course of the stress. Upon rapid replication fork stalling (2 h, 5 mM HU), ATM activation depends on ATR.9 During prolonged replication stress (48 h, 2 mM HU) here we show that ATM is phosphorylated by DNA-PK, as depletion of DNA-PK, but not ATR, inhibits ATM phosphorylation. The transition from ATR to DNA-PK as the sensor of the DNA damage depends on the time. The initial stage of replication fork stalling is the exposure of single-stranded DNA, which activates ATR followed by ATM to initiate the DNA damage response. When the replication fork stalls persistently, both strands of DNA would break and ATM is activated by DNA-PK instead of ATR. These kinases work independently, but they also regulate each other in complex networks depending on the type of damage. In this study, we show that centrosome amplification was induced by DNA-PK but not by ATM or ATR during prolonged replication stress, although all three kinases were activated. This is consistent with our previous study that centrosomal DNA-PK activation induces centrosome overduplication in steroidogenic cells.17 Here, we show that inactivation of ATM and ATR by caffeine induced aneuploidy upon prolonged replication stress, thus distinguishing the functions of ATM/ATR from DNA-PK during long-term replication stress. In response to this prolonged replication stress DNA-PK controlled centrosome homeostasis, whereas ATM and ATR regulated chromosome stability. Although DNA-PK and ATM/ATR appeared to exert different roles in response to prolonged replication stress, their regulations were interdependent. Here, we show that DNA-PK could phosphorylate ATM in response to long-term replication stress, and vice versa to a smaller extent. Thus these proteins can cooperate with and activate each other for their own separate functions. DNA-PK/Chk2 signaling induces centrosome amplification during prolonged replication stress We show here that activation of DNA-PK/Chk2 during prolonged replication stress induced centrosome amplification. As amplified centrosomes become aberrant mitotic apparatus during mitosis, DNA-PK/Chk2 appeared to trigger aberrant mitosis when the S phase is prolonged. Yet DNA-PK/Chk2 signaling is also required for the maintenance of mitotic chromosomes during mitosis.20–22 Activation of the same signaling at different cell cycle stages thus results in different cell fates (protection versus destruction). During normal cell cycles, activation of mitotic DNA-PK/Chk2 helps maintain genomic integrity.22 However, during prolonged replication stress, genomic DNA gets damaged; and as a protective mechanism centrosomes are amplified to eliminate cells with severely damaged genomes. We therefore propose that DNA-PK/ Chk2 signaling both actively and passively prevent tumorigenesis. During normal cell growth, it actively maintains the integrity of the mitotic genome. During stress, DNA-PK/Chk2 induces centrosome amplification and results in aberrant mitosis followed by mitotic catastrophe to prevent tumorigenesis passively. The mechanism by which Chk2 induces centrosome amplification is still unclear. Irradiation-induced centrosome amplification requires CDK2.15 Upon irradiation, ATR/Chk1 signaling activates CDK2 by phosphorylating Thr160 directly. Chk1 and Chk2 share common substrates, such as p53 and CDC25. Thus, during replication stress, Chk2 signaling may also activate CDK2 by direct phosphorylation at Thr160, but this hypothesis needs to be tested. Chk2 is activated only on the mother centriole Chk2 localizes to the centrosome independent of DNA damage.23 Here, we show that p-Chk2 was located only on the mother Oncogene (2014), 1 – 7
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
6
Figure 6. Asymmetric localization of phosphorylated Chk2 on the mother centriole from late G2 and mitosis. Double staining of U2OS cells with antibodies against phospho-Thr68 of Chk2 (p-Chk2) and (b and d) acetylated tubulin (Ace-tub), (c) γ-tubulin (γ-tub) or (e) Odf2. DNA was stained with DAPI. Scale bars are 5 μm. Insets are magnification of centrioles. (a) Phosphorylated Chk2 accumulates in the nucleus and centrioles of U2OS cells in the presence of HU. (b and c) Asymmetric localization of p-Chk2 on the centriole at late G2 and mitosis. (d) Asymmetric distribution of Odf2 before and after centriole duplication. (e) Phosphorylated Chk2 resides on the mother centriole at the late G2 phase.
centriole. The function of p-Chk2 on the centrosome has been little studied. Chk2 may coordinate centrosome functions with cell cycle progression23 and contribute to cytokinesis as it is also localized to the mid-body.24 Here, we find p-Chk2 resides on the mother centriole. It has been shown that disruption of proteins in the mother centriole may result in centriole splitting, appendage disorganization, poor microtubule nucleation or disruption of primary cilium formation.25,26 It will be interesting to identify the function of p-Chk2 in the mother centriole.
MATERIALS AND METHODS Cell culture and drug treatment Mouse adrenocortical Y1 cells were grown in Dulbecco’s modified Eagle medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO2. Human cervical tumor cells HeLa, human embryonic kidney 293 T, mouse myoblasts C2C12 and human osteosarcoma cells U2OS were grown in DMEM and human lung cancer cells H1299 were grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO2. These cells were regularly examined by Oncogene (2014), 1 – 7
4′,6-diamino-2-phenylindole staining and immunofluorescence detection for the absence of mycoplasma contamination. For drug treatment, cells were incubated with or without 2 mM HU in the presence or absence of 1 mM vanillin, 2 mM caffeine, 200 nM UCN-01 or 10 μM Chk2i II for 24 h or 48 h before analysis.
RNAi Targeted genes in U2OS cells were depleted using annealed siRNA with the following target sequences: siDNA-PKcs: 5′-gggcgcuaaucguacugaa [dt] [dt]-3′27 siKu70: 5′-gaugcccuuuacugaaaaa [dt] [dt]-3′28 siKu80: 5′-cagagaagauucuucauggg [dt] [dt]-3′29 siATM: 5′-aacauacuacucaaagacauu [dt] [dt ]-3′30 siATR: 5′-aaccuccgugauguugcuuga [dt] [dt]-3′30 siChk2: 5′-aagaaccugaggaccaagaac [dt] [dt]-3′31 Scrambled siRNA with the target sequence: 5′-gaucauacgugcgaucaga [dt] [dt]-3′ was purchased from Sigma (Sigma, St Louis, MO, USA). For siRNA transfection, 10 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was mixed first with 500 μl Opti-MEM medium (Life Technologies, Grand Island, NY, USA) for 5 min, then with 200 nM siRNA in 500 μl OptiMEM medium and incubated at room temperature for 20 min before the mixture was layered onto cells in 1 ml DMEM medium. Cells were harvested 48 h after transfection. © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
7 Antibodies The following antibodies were obtained commercially: anti-γ-tubulin, antiα-tubulin and anti-acetylated-tubulin (Sigma), anti-Chk2, anti-Chk2 phospho-Thr68, anti-Akt, anti-Akt phospho-Ser473, anti-ATR and anti-ATR phospho-Ser428 (Cell Signaling, Beverly, MA, USA), anti-centrin 20H5 (Millipore, Billerica, MA, USA), polyclonal anti-centrin, anti-Chk1 and antiChk1 phospho-Ser345 (Abcam, Cambridge, UK), anti-Ku70, anti-Ku80 and anti-ATM (Genetex, Trvine, CA, USA), anti-ATM phospho-Ser1981 (Epitomics, Burlingame, CA, USA) anti-actin, anti-GAPDH, anti-DNA-PKcs and anti-DNA-PKcs phospho-Thr2609 (Santa Cruz Biotech, Santa Cruz, CA, USA).
Immunofluorescence microscopy Cells were grown on glass coverslips at 37 °C before fixation with ice-cold methanol at − 20 °C for 6 min. To visualize centriolar Ace-tub staining, microtubules were depolymerized on ice for 1 h followed by fixation with − 20 °C methanol for 6 min. After blocking with 5% bovine serum albumin for 1 h, cells were incubated with antibodies for 24 h at 4 °C. After extensive washing with phosphate-buffered saline (PBS), cells were incubated with fluorescein isothiocyanate-conjugated and Cy3-conjugated secondary antibodies (Invitrogen) for 1 h in the dark. The nuclei were stained with 4′,6-diamino-2phenylindole (0.1 μg/ml) simultaneously. After extensive washing, the coverslips were mounted in 50% glycerol on glass slides. Fluorescent cells were examined with an AxioImager Z1 fluorescence microscope or an LSM 510 confocal microscope (both from Zeiss, Oberkochen, Germany). The number of centrosomes and centrioles from >100 cells were counted under the microscope in three independent experiments and shown as mean ± s.d. Student’s t-test was performed to analyze the difference between groups as indicated.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay Cells were washed with PBS followed by adding 1 ml MTT solution (2 mg/ ml in PBS) in each well. After incubation for 3 h at 37 oC, 2 ml dimethyl sulfoxide was added and cells were incubated in the dark for an additional 30 min. Absorbance was measured at the wavelength of 570 nm.
FACS analysis For fluorescence-activated cell sorting (FACS) analysis, cells were collected by trypsinization and resuspended with PBS. Following centrifugation at 1000 r.p.m. for 5 min, cells were resuspended with 1 mM EDTA in PBS (PBSE). After centrifugation, the pellet was resuspended with 0.5 ml PBS-E and followed by fixation with ice-cold 70% ethanol overnight at 4 °C. Fixed cells were washed with PBS-E and stained with propidium iodide (SouthernBiotech, Birmingham, AL, USA) at room temperature for 1 h. DNA content of propidium iodide-stained cells was measured by FACScalibur (Becton-Dickinson, San Diego, CA, USA) and further analyzed by Kaluza software (Beckman Coulter, Brea, CA, USA).
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We would like to thank Ya-Min Lin for technical assistance in FACS analysis. This study was supported by grants from Academia Sinica, NHRI-EX102-10210SI and NSC1022923-B-001-003-MY3 to B-cC and NSC102-2320-B-006–051 to C-YW.
REFERENCES 1 Stehman FB, Bundy BN, Kucera PR, Deppe G, Reddy S, O'Connor DM. Hydroxyurea 5fluorouracil infusion, and cisplatin adjunct to radiation therapy in cervical carcinoma: a phase I-II trial of the Gynecologic Oncology Group. Gynecol Oncol 1997; 66: 262–267. 2 Prosser SL, Straatman KR, Fry AM. Molecular dissection of the centrosome overduplication pathway in S-phase-arrested cells. Mol Cell Biol 2009; 29: 1760–1773. 3 Khodjakov A, Rieder CL, Sluder G, Cassels G, Sibon O, Wang CL. De novo formation of centrosomes in vertebrate cells arrested during S phase. J Cell Biol 2002; 158: 1171–1181. 4 Sato N, Mizumoto K, Nakamura M, Ueno H, Minamishima YA, Farber JL et al. A possible role for centrosome overduplication in radiation-induced cell death. Oncogene 2000; 19: 5281–5290.
5 Bennett RA, Izumi H, Fukasawa K. Induction of centrosome amplification and chromosome instability in p53-null cells by transient exposure to subtoxic levels of S-phase-targeting anticancer drugs. Oncogene 2004; 23: 6823–6829. 6 Durocher D, Jackson SP. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 2001; 13: 225–231. 7 Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003; 3: 155–168. 8 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003; 421: 499–506. 9 Stiff T, Walker SA, Cerosaletti K, Goodarzi AA, Petermann E, Concannon P et al. ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling. EMBO J 2006; 25: 5775–5782. 10 Smith J, Tho LM, Xu N, Gillespie DA. The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res 2010; 108: 73–112. 11 Dodson H, Bourke E, Jeffers LJ, Vagnarelli P, Sonoda E, Takeda S et al. Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM. EMBO J 2004; 23: 3864–3873. 12 Bozulic L, Surucu B, Hynx D, Hemmings BA. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 2008; 30: 203–213. 13 Collis SJ, DeWeese TL, Jeggo PA, Parker AR. The life and death of DNA-PK. Oncogene 2005; 24: 949–961. 14 Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300: 1542–1548. 15 Bourke E, Brown JA, Takeda S, Hochegger H, Morrison CG. DNA damage induces Chk1-dependent threonine-160 phosphorylation and activation of Cdk2. Oncogene 2010; 29: 616–624. 16 Robinson HM, Black EJ, Brown R, Gillespie DA. DNA mismatch repair and Chk1dependent centrosome amplification in response to DNA alkylation damage. Cell Cycle 2007; 6: 982–992. 17 Wang CY, Kao YH, Lai PY, Chen WY, Chung BC. Steroidogenic factor-1 (NR5A1) maintains centrosome homeostasis in steroidogenic cells by restricting centrosomal DNA-PK activation. Mol Cell Biol 2013; 33: 476–484. 18 Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000; 14: 1448–1459. 19 Nakagawa Y, Yamane Y, Okanoue T, Tsukita S, Tsukita S. Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. Mol Biol Cell 2001; 12: 1687–1697. 20 Lee KJ, Lin YF, Chou HY, Yajima H, Fattah KR, Lee SC et al. Involvement of DNAdependent protein kinase in normal cell cycle progression through mitosis. J Biol Chem 2011; 286: 12796–12802. 21 Stolz A, Ertych N, Kienitz A, Vogel C, Schneider V, Fritz B et al. The CHK2-BRCA1 tumour suppressor pathway ensures chromosomal stability in human somatic cells. Nat Cell Biol 2010; 12: 492–499. 22 Shang ZF, Huang B, Xu QZ, Zhang SM, Fan R, Liu XD et al. Inactivation of DNAdependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2 Phosphorylation in response to DNA damage. Cancer Res 2010; 70: 3657–3666. 23 Golan A, Pick E, Tsvetkov L, Nadler Y, Kluger H, Stern DF. Centrosomal Chk2 in DNA damage responses and cell cycle progression. Cell Cycle 2010; 9: 2647–2656. 24 Tsvetkov L, Xu X, Li J, Stern DF. Polo-like kinase 1 and Chk2 interact and colocalize to centrosomes and the midbody. J Biol Chem 2003; 278: 8468–8475. 25 Joo K, Kim CG, Lee MS, Moon HY, Lee SH, Kim MJ et al. CCDC41 is required for ciliary vesicle docking to the mother centriole. Proc Natl Acad Sci USA 2013; 110: 5987–5992. 26 Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M et al. Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol 2007; 179: 321–330. 27 An J, Yang DY, Xu QZ, Zhang SM, Huo YY, Shang ZF et al. DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein. Mol Cancer 2008; 7: 32. 28 Ayene IS, Ford LP, Koch CJ. Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation. Mol Cancer Ther 2005; 4: 529–536. 29 Rampakakis E, Di Paola D, Zannis-Hadjopoulos M. Ku is involved in cell growth DNA replication and G1-S transition. J Cell Sci 2008; 121: 590–600. 30 Andreassen PR, D'Andrea AD, Taniguchi T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev 2004; 18: 1958–1963. 31 Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, Nigg EA et al. Centrosome-associated Chk1 prevents premature activation of cyclin-BCdk1 kinase. Nat Cell Biol 2004; 6: 884–891.
Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc) © 2014 Macmillan Publishers Limited
Oncogene (2014), 1 – 7
ORIGINAL ARTICLE
DNA-PK/Chk2 induces centrosome amplification during prolonged replication stress C-Y Wang1,2, EY-H Huang1,3, S-c Huang1 and B-c Chung1 The antineoplastic drug hydroxyurea (HU), when used at subtoxic doses, induces prolonged replication stress and centrosome amplification. This causes genomic instability and increases the malignancy of the recurring tumor. The mechanism of centrosome amplification induced by prolonged replication stress, however, is still unclear. Here, we examined the involvement of ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) and found that HU-induced centrosome amplification was inhibited by the depletion of DNA-PKcs, but not ATM and ATR. Inactivation of ATM/ATR in U2OS cells instead caused aneuploidy and cell death. We found DNA-PKcs depletion also abrogated ATM phosphorylation, indicating that ATM activation during prolonged replication stress depends on DNA-PK. Depletion of DNA-PK abrogated checkpoint kinase (Chk)2 activation and partially reduced Chk1 activation. Chk2 depletion blocked HU-induced centrosome amplification, indicating a function of Chk2 in centrosome amplification. We further found that Chk2 was phosphorylated at Thr68 on the mother centriole at late G2 and mitosis when unstressed and on all amplified centrioles induced by HU. In summary, we have elucidated that DNA-PK/Chk2 signaling induces centrosome amplification upon long-term HU treatment, therefore increasing our insight into tumor recurrence after initial chemotherapy. Oncogene advance online publication, 24 March 2014; doi:10.1038/onc.2014.74
INTRODUCTION Hydroxyurea (HU) and 5-fluorouracil are common anticancer drugs that cause replication stress and activate DNA damage response.1 These drugs are often highly toxic, killing cancer cells but also generating many side effects. Yet, when subtoxic doses of the drug are administered, some cancer cells can still survive although they are arrested at the S phase; and their centrosomes are often amplified.2,3 Centrosome amplification induced by DNA damage results in the elimination of cells with damaged genomes.4 However, when coupled with a deficiency in DNA repair machinery, cells harboring amplified centrosomes are more susceptible to malignancy.5 The amplification of centrosomes upon DNA damage requires the activation of checkpoint proteins. Members of the phosphatidylinositol 3-kinase-like kinase superfamily, including ATM (ataxia telangiectasia, mutated), ATR (ataxia telangiectasia, mutated and Rad3-related) and DNA-PK (DNAdependent protein kinase), initiate damage signals in response to DNA damages.6 ATM is activated by DNA double-strand breaks to repair DNA damage via homologous recombination and to stop cell cycle progression.7 Upon DNA double-strand breakage, ATM is activated by phosphorylation at Ser1981.8 ATM then phosphorylates checkpoint kinase 2 (Chk2) at Thr68,9 which is critical for cell cycle control.10 ATM activation by high levels of DNA damage, together with Rad51 deficiency, induces centrosome amplification.11 The second kinase induced by DNA double-strand breaks is DNA-PK, which is activated in response to DNA damage to facilitate DNA repair and to halt the cell cycle.12 DNA-PK is composed of the DNA-binding components Ku70, Ku80 and the catalytic subunit DNA-PKcs.13 Ku heterodimer binds to the broken 1
ends of DNA and recruits DNA-PKcs to form an active complex that initiates DNA damage repair by non-homologous end-joining. DNA-PK also activates Akt signaling to arrest cell cycle progression and to maintain cell survival.12 The third kinase that activates the damage response is ATR, which is induced by DNA single-strand breaks.14 Once recruited to the DNA, ATR activates its substrate checkpoint kinase 1 (Chk1) and initiates the downstream cascade to limit cell cycle progression. The activation of ATR-Chk1 signaling also results in Thr160 phosphorylation of cyclin-dependent kinase 2 (CDK2) followed by centrosome amplification.15 Inactivation of Chk1 abrogates centrosome amplification induced by DNA alkylation damage.16 In this report we have investigated the signaling pathways induced by HU treatment. We found that DNA-PK/Chk was activated upon HU treatment and promoted centrosome amplification. We also found that activated Chk2 resided on the mother centriole at late G2 and mitosis. Our studies elucidate the mechanism of centrosome amplification in response to prolonged replication stress via the DNA-PK/Chk signaling pathway.
RESULTS Activation of DNA-PK induces centrosome amplification during prolonged replication stress The signaling pathways that respond to prolonged replication stress were investigated. HU treatment of U2OS cells for 48 h induced all three checkpoint proteins, DNA-PK, ATM and ATR (Figure 1a); additionally, phosphorylated H2AX appeared as speckles (Figure 1b). These are all hallmarks of severe DNA
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan and 2Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Correspondence: Professor B-c Chung, Institute of Molecular Biology, Academia Sinica, 128 Academia Road Section 2, Taipei 115, Taiwan. E-mail: [email protected] 3 Current address: Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA Received 26 November 2012; revised 21 December 2013; accepted 1 January 2014
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
2
Figure 1. HU induces activation of DNA-PK at the damaged DNA loci and induces centrosome amplification depending on DNA-PK. (a) DNAPK, ATM and ATR are activated by HU treatment. Extracts of U2OS treated with or without HU were analyzed by immunoblot with antibodies against Thr2609-phosphorylated DNA-PKcs (p-PKcs), Ser428-phosphorylated ATR (p-ATR), Ser1981-phosphorylated ATM (p-ATM) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (b) Phosphorylated DNA-PK colocalizes with γH2AX foci. HU or etoposide (ETO)-treated Y1 cells were co-stained with DNA dye (DAPI) and antibodies against phospho-Ser139 of H2AX (γH2AX) and Thr2609-phosphorylated DNAPKcs (p-PKcs). (c and d) Vanillin reverses centrosome overduplication caused by HU treatment. Quantification of a population of U2OS (c) and H1299 (d) cells with multiple centrosomes upon HU treatment in the presence or absence of DNA-PK inhibitor vanillin. (e) Inactivation of DNAPK does not disturb the cell cycle profile. Quantification of different cell cycle stages in U2OS cells with or without HU in the presence or absence of vanillin. (f) HU treatment induces the formation of multiple centrioles. Immunofluorescence staining of centrioles with acetylated tubulin (Ace-tub, green) and of centrosomes with γ-tubulin (γ-tub, red) in Y1 cells. Scale bars are 5 μm. (g) Vanillin reverses centriole overduplication caused by HU treatment. Quantification of the population of Y1 cells with multiple centrioles upon HU treatment in the presence or absence of DNA-PK inhibitor vanillin. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001.
damage. Phosphorylated DNA-PKcs also colocalized with phosphorylated H2AX foci, indicating HU treatment induced a DNA damage response and activated DNA-PK. We have previously shown that activation of DNA-PK due to the depletion of NR5A1 induces centrosome amplification in adrenocortical Y1 cells,17 and here further tested whether DNA-PK also induced centrosome amplification during replication stress. DNAPK inhibitor vanillin itself had no effect, but it suppressed the formation of multiple centrosomes caused by HU in U2OS Oncogene (2014), 1 – 7
(Figure 1c) and H1299 (Figure 1d) cells. Vanillin (1 mM) alone or combined with HU, however, affected neither cell viability (Supplementary Figures S1A and B) nor apoptosis (Supplementary Figures S1C and D) and had no effect on cell cycle profiles (Figure 1e; Supplementary Figure S1E). Thus, DNA-PK participated in HU-induced centrosome overduplication without affecting the cell cycle or cell viability. The effect of HU on centrosome amplification was further examined by the staining of centrosomes with γ-tubulin and of © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
3 centrioles stained by nocodazole-resistant acetylated tubulin (Acetub) followed by counting centriole numbers. Normal Y1 cells contained one centrosome with two centrioles at G1 and duplicated centrosomes with four centrioles at the S/G2 phase (Figure 1f). HU treatment induced multiple γ-tubulin spots (>2) and multiple centrioles (>4 Ace-tub) (Figure 1f), which were reversed by vanillin (Figure 1g). Thus, inhibition of DNA-PK activation prevented centriole amplification during prolonged HU treatment. In addition to DNA-PK, the roles of ATM/ATR were also examined by treating cells with the common ATM/ATR inhibitor, caffeine. Caffeine alone had no effect on centrosome numbers, but it aggravated the multiple centrosome phenotype induced by HU (Figure 2a). When examined in more detail, these cells with multiple centrosomes were divided into two groups: cells with a single nucleus (Figure 2b, mono-Nucl; >2Cent, upper panel) and cells with di-nuclei (Figure 2b, di-Nucl; >2Cent, lower panel). HU alone induced multiple centrosomes with a single intact nucleus (Figure 2c), which was not affected by the treatment with caffeine, but was inhibited by vanillin. This indicates that DNA-PK, but not ATM/ATR, participates in centrosome amplification caused by replication stress. Cells with multiple centrosomes/di-nuclei were induced when caffeine was present together with HU, whereas vanillin could not repress this (Figure 2d). These data indicates that inactivation of ATM/ATR results in aneuploidy with multiple centrosomes, and this phenotype is independent of DNA-PK during prolonged replication stress. To further confirm the role of DNA-PK in centrosome amplification, DNA-PKcs in U2OS cells was depleted by small interfering RNA (siRNA). This caused the absence of DNA-PKcs phosphorylation in the presence of HU, but had no effect on the levels of Ku70 and Ku80 (Figure 3a). Depletion of DNA-PKcs inhibited centrosome amplification caused by HU in U2OS cells (Figure 3b). This data combined with the above inhibitor data demonstrate that DNA-PK is involved in centrosome amplification. We also tested the effect of Ku70 and Ku80 by depleting them individually with siRNA. Immunoblot assay showed that efficient depletion of Ku70 led to the depletion of Ku80, and vice versa (Figure 3c). Depletion of Ku70 and Ku80 inhibited HU-induced centrosome amplification (Figure 3d), indicating that all DNA-PK subunits, including Ku70 and Ku80, are involved in centrosome amplification caused by HU. Besides the stress condition, the role of DNA-PK in centriole numbers without stress was also examined. After DNA-PKcs depletion (Figure 3e), the populations of cells with either unduplicated (centriole number = 2) or duplicated (centriole number = 3–4) centrioles were not changed compared with the control group (Figure 3f). Thus, DNA-PK does not participate in centriole duplication when unstressed; only during prolonged replication stress is it activated to induce centrosome overduplication. Activation of Chk2 induces centrosome amplification upon HU treatment As HU induced the activation of ATM, ATR and DNA-PK, their downstream effectors were examined. Upon HU treatment, Akt phosphorylation was not changed, but phosphorylation of Chk1 and Chk2 were increased (Figure 4a). Upon DNA-PKcs depletion, Thr68 phosphorylation of Chk2 (p-Chk2) was abrogated and the phosphorylation of Chk1 was partially reduced (Figure 4a). Both ATM and ATR were activated by HU, and depletion of DNA-PKcs inhibited phosphorylation of ATM, but not ATR (Figure 4a). These data indicate that DNA-PK activation during prolonged replication stress leads to phosphorylation of ATM, Chk2 and to a lower extent Chk1. We tested the effect of ATM by examining the activation of its substrates Chk1/2 during prolonged replication stress. Depletion © 2014 Macmillan Publishers Limited
Figure 2. Caffeine induces aberration of multiple nuclei, whereas vanillin abrogates centrosome amplification during prolonged replication stress. (a) Caffeine induces centrosome aberration in the presence of HU. Quantification of U2OS cells with multiple centrosomes in the presence or absence of HU and/or caffeine. (b) Immunofluorescence examination of centrosomes (stained with anti-γ-tubulin antibody) in U2OS cells treated with HU and caffeine. Insets are lower magnifications showing nuclei stained by DAPI and centrosomes. Two types of cells were detected: those with a single nucleus and multiple centrosomes (mono-Nucl; >2Cent; upper panel) or with di-nuclei with multiple centrosomes (di-Nucl; >2Cent; lower panel). Scale bars are 5 μm. (c and d) Caffeine induces aberration of multiple nuclei, whereas vanillin abrogates centrosome amplification during prolonged replication stress. Quantification of U2OS cells containing (c) single nucleus with multiple centrosomes (mono-Nucl; >2Cent), or (d) di-nuclei with multiple centrosomes (di-Nucl; >2Cent). These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001.
of ATM had no effect on the phosphorylation of Chk1, Chk2 and ATR, but it slightly reduced DNA-PKcs phosphorylation (Figure 4b). This data indicates that upon HU treatment Chk2 is not activated by ATM but by DNA-PK. The role of ATR was also checked. Depletion of ATR did not affect phosphorylation of DNA-PKcs and Chk2, but it greatly reduced the activation of Chk1 and perhaps slightly of ATM (Figure 4c). These data show that Chk1 phosphorylation depends on both DNA-PK and ATR, and Chk2 phosphorylation depends on DNA-PK upon prolonged replication stress induced by HU. The effects of Chk1 and Chk2 on centrosome amplification were further examined by treating U2OS cells with UCN-01 that inhibits mostly Chk1 but also Chk2. We also inhibited Chk2 specifically with Chk2 inhibitor II (Chk2i). UCN-01 but not Chk2i treatment induced considerable cell death (Supplementary Figure S2A), consistent with the earlier report that inactivation of Chk1 induces Oncogene (2014), 1 – 7
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
4
apoptosis.18 Examining the remaining live cells, we found that UCN-01 abrogated the multiple centrosomal phenotype induced by HU (Figure 5a). The role of Chk2 was examined by siRNA
depletion. Depletion of Chk2 by siRNA did not abrogate DNA-PK, ATM, ATR or Chk1 activation (Figure 5b), but lowered the multiple centrosome phenotype caused by HU treatment (Figure 5c). The Chk2-specific inhibitor, Chk2i, also blocked centriole (Figure 5d) and centrosome (Supplementary Figure S2B) amplification caused by HU. Chk2i also blocked centrosome amplification in Y1 cells (Supplementary Figure S2C). These data showed that Chk2 contributed to centrosome amplification induced by HU.
Activated Chk2 resides on the mother centriole The subcellular localization of p-Chk2 was examined by immunofluorescence staining. In the presence of HU, both nuclear and centriolar p-Chk2 levels were increased (Figure 6a), and p-Chk2 was present on over-duplicated centrioles. In the absence of HU, p-Chk2 started to increase at the centrosome at the late G2 phase and peaked at mitosis in U2OS (Figure 6b) and HeLa cells (Supplementary Figure S3A). Centrioles were examined by staining with Ace-tub after microtubule depolymerization in the cold, and were found co-stained with p-Chk2 (Figure 6c). This result implies that p-Chk2 is targeted to the centriole independent of microtubule transport. We adjusted the image exposure time in order to obtain higher immunofluorescence signals for closer examination and found that only one of the two centrioles was decorated with p-Chk2 (Figure 6c). Co-staining with Centrin also confirmed the asymmetric localization of p-Chk2 in one of the centrioles during mitosis (Supplementary Figure S3B). To identify which centriole was decorated by p-Chk2, we examined the mother centriole by staining with known mother centriolar protein Odf2.19 At the G1 phase, two centrioles were stained with Ace-tub but only one was decorated with Odf2
Figure 3. DNA-PKcs and Ku participate in centrosome amplification induced by HU. (a) Efficient depletion of DNA-PKcs by siRNA transfection. Extracts of U2OS cells after transfection with siRNA against scrambled control (siCTL) or DNA-PKcs (siPKcs) in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs), Thr2609-phosphorylated DNAPKcs (p-PKcs), Ku70, Ku80 and Hsc70. (b) Centrosome amplification in HU-treated U2OS depends on DNA-PKcs. Quantification of the population of U2OS cells containing multiple centrosomes with or without HU in the DNA-PKcs (siPKcs) siRNA-transfected cells. (c) Efficient depletion of Ku70 and Ku80 by siRNA. Extracts of U2OS cells after transfection with Ku70 (siKu70) or Ku80 (siKu80) siRNA in the presence or absence of HU were analyzed by immunoblot with antibodies against Ku70, Ku80 or actin. (d) Depletion of Ku70 or Ku80 inhibit centrosome amplification caused by HU treatment. Quantification of population of U2OS cells with multiple centrosomes after transfection with siRNA against Ku70 (siKu70) or Ku80 (siKu80) with or without HU. (e and f) Depletion of DNA-PKcs does not affect centriole duplication. (e) Extracts of U2OS cells after transfection with siRNA against DNA-PKcs (PKcs) in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs) or Hsc70. (f) Quantification of a population of U2OS cells containing multiple centrosomes with or without HU in the scrambled control (siCTL) or DNA-PKcs (siPKcs) siRNAtransfected cells. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. ***P ⩽ 0.001. Oncogene (2014), 1 – 7
Figure 4. Depletion of DNA-PKcs prevents HU-induced Chk2 activation. Extracts of U2OS cells after transfection with siRNA against scrambled control (CTL) and (a) DNA-PKcs (PKcs), (b) ATM or (c) ATR in the presence or absence of HU were analyzed by immunoblot with antibodies against DNA-PKcs (PKcs), Thr2609phosphorylated DNA-PKcs (p-PKcs), Ser473-phosphorylated Akt (p-Akt), Ser345-phosphorylated Chk1 (p-Chk1), Chk1, Ser428phosphorylated ATR (p-ATR), Thr68-phosphorylated Chk2 (p-Chk2), Chk2, Ku70, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α-tubulin (α-tub), ATM and Ser1981-phosphorylated ATM (p-ATM). © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
5
Figure 5. Involvement of Chk1/2 in centrosome amplification induced by HU. (a) Quantification of a population of U2OS cells containing multiple centrosomes with or without HU in UCN-01 (Chk1 inhibitor)-treated cells. (b) Efficient depletion of Chk2 by siRNA transfection. Following transfection with siRNA against scrambled control (siCTL) or Chk2 (siChk2), extracts of U2OS cells were analyzed by immunoblot with antibodies against Thr68phosphorylated Chk2 (p-Chk2), Chk2, Ser345-phosphorylated Chk1 (p-Chk1), Thr2609-phosphorylated DNA-PKcs (p-PKcs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Ser1981phosphorylated ATM (p-ATM) and Ser428-phosphorylated ATR (p-ATR). (c and d) Centrosome amplification in U2OS cells during prolonged replication stress depends on Chk2 activity. Quantification of a population of U2OS cells containing multiple (c) centrosomes or (d) centrioles with or without HU in (c) Chk2 siRNA (siChk2)-transfected or (d) Chk2 inhibitor (Chk2i)-treated cells. These results are mean ± s.d. from three independent experiments; >300 cells were counted in each individual group. *P ⩽ 0.05, **P ⩽ 0.01, ***P ⩽ 0.001.
(Figure 6d). Following centriole duplication, two pairs of centrioles were present but only one of the duplicated centrioles stained with Odf2 before and after centrosome separation, confirming that Odf2 is a marker for the mother centriole. These Odf2 spots colocalized with p-Chk2 at the late G2 phase, indicating that p-Chk2 resides in the mother centriole (Figure 6c). DISCUSSION In this report we have examined the long-term effect of the anticancer drug HU on centrosome homeostasis, and show that the DNA-PK/Chk2 signaling was activated leading to centrosome overduplication. Depletion of DNA-PK or Chk2 alleviates centrosome amplification induced by HU. We also show that Chk2 is activated in the mother centriole at G2/M during normal cell cycles. © 2014 Macmillan Publishers Limited
Actions of ATM, ATR and DNA-PK during prolonged replication stress DNA damages induced by replication stress can elicit different responses along the time course of the stress. Upon rapid replication fork stalling (2 h, 5 mM HU), ATM activation depends on ATR.9 During prolonged replication stress (48 h, 2 mM HU) here we show that ATM is phosphorylated by DNA-PK, as depletion of DNA-PK, but not ATR, inhibits ATM phosphorylation. The transition from ATR to DNA-PK as the sensor of the DNA damage depends on the time. The initial stage of replication fork stalling is the exposure of single-stranded DNA, which activates ATR followed by ATM to initiate the DNA damage response. When the replication fork stalls persistently, both strands of DNA would break and ATM is activated by DNA-PK instead of ATR. These kinases work independently, but they also regulate each other in complex networks depending on the type of damage. In this study, we show that centrosome amplification was induced by DNA-PK but not by ATM or ATR during prolonged replication stress, although all three kinases were activated. This is consistent with our previous study that centrosomal DNA-PK activation induces centrosome overduplication in steroidogenic cells.17 Here, we show that inactivation of ATM and ATR by caffeine induced aneuploidy upon prolonged replication stress, thus distinguishing the functions of ATM/ATR from DNA-PK during long-term replication stress. In response to this prolonged replication stress DNA-PK controlled centrosome homeostasis, whereas ATM and ATR regulated chromosome stability. Although DNA-PK and ATM/ATR appeared to exert different roles in response to prolonged replication stress, their regulations were interdependent. Here, we show that DNA-PK could phosphorylate ATM in response to long-term replication stress, and vice versa to a smaller extent. Thus these proteins can cooperate with and activate each other for their own separate functions. DNA-PK/Chk2 signaling induces centrosome amplification during prolonged replication stress We show here that activation of DNA-PK/Chk2 during prolonged replication stress induced centrosome amplification. As amplified centrosomes become aberrant mitotic apparatus during mitosis, DNA-PK/Chk2 appeared to trigger aberrant mitosis when the S phase is prolonged. Yet DNA-PK/Chk2 signaling is also required for the maintenance of mitotic chromosomes during mitosis.20–22 Activation of the same signaling at different cell cycle stages thus results in different cell fates (protection versus destruction). During normal cell cycles, activation of mitotic DNA-PK/Chk2 helps maintain genomic integrity.22 However, during prolonged replication stress, genomic DNA gets damaged; and as a protective mechanism centrosomes are amplified to eliminate cells with severely damaged genomes. We therefore propose that DNA-PK/ Chk2 signaling both actively and passively prevent tumorigenesis. During normal cell growth, it actively maintains the integrity of the mitotic genome. During stress, DNA-PK/Chk2 induces centrosome amplification and results in aberrant mitosis followed by mitotic catastrophe to prevent tumorigenesis passively. The mechanism by which Chk2 induces centrosome amplification is still unclear. Irradiation-induced centrosome amplification requires CDK2.15 Upon irradiation, ATR/Chk1 signaling activates CDK2 by phosphorylating Thr160 directly. Chk1 and Chk2 share common substrates, such as p53 and CDC25. Thus, during replication stress, Chk2 signaling may also activate CDK2 by direct phosphorylation at Thr160, but this hypothesis needs to be tested. Chk2 is activated only on the mother centriole Chk2 localizes to the centrosome independent of DNA damage.23 Here, we show that p-Chk2 was located only on the mother Oncogene (2014), 1 – 7
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
6
Figure 6. Asymmetric localization of phosphorylated Chk2 on the mother centriole from late G2 and mitosis. Double staining of U2OS cells with antibodies against phospho-Thr68 of Chk2 (p-Chk2) and (b and d) acetylated tubulin (Ace-tub), (c) γ-tubulin (γ-tub) or (e) Odf2. DNA was stained with DAPI. Scale bars are 5 μm. Insets are magnification of centrioles. (a) Phosphorylated Chk2 accumulates in the nucleus and centrioles of U2OS cells in the presence of HU. (b and c) Asymmetric localization of p-Chk2 on the centriole at late G2 and mitosis. (d) Asymmetric distribution of Odf2 before and after centriole duplication. (e) Phosphorylated Chk2 resides on the mother centriole at the late G2 phase.
centriole. The function of p-Chk2 on the centrosome has been little studied. Chk2 may coordinate centrosome functions with cell cycle progression23 and contribute to cytokinesis as it is also localized to the mid-body.24 Here, we find p-Chk2 resides on the mother centriole. It has been shown that disruption of proteins in the mother centriole may result in centriole splitting, appendage disorganization, poor microtubule nucleation or disruption of primary cilium formation.25,26 It will be interesting to identify the function of p-Chk2 in the mother centriole.
MATERIALS AND METHODS Cell culture and drug treatment Mouse adrenocortical Y1 cells were grown in Dulbecco’s modified Eagle medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO2. Human cervical tumor cells HeLa, human embryonic kidney 293 T, mouse myoblasts C2C12 and human osteosarcoma cells U2OS were grown in DMEM and human lung cancer cells H1299 were grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO2. These cells were regularly examined by Oncogene (2014), 1 – 7
4′,6-diamino-2-phenylindole staining and immunofluorescence detection for the absence of mycoplasma contamination. For drug treatment, cells were incubated with or without 2 mM HU in the presence or absence of 1 mM vanillin, 2 mM caffeine, 200 nM UCN-01 or 10 μM Chk2i II for 24 h or 48 h before analysis.
RNAi Targeted genes in U2OS cells were depleted using annealed siRNA with the following target sequences: siDNA-PKcs: 5′-gggcgcuaaucguacugaa [dt] [dt]-3′27 siKu70: 5′-gaugcccuuuacugaaaaa [dt] [dt]-3′28 siKu80: 5′-cagagaagauucuucauggg [dt] [dt]-3′29 siATM: 5′-aacauacuacucaaagacauu [dt] [dt ]-3′30 siATR: 5′-aaccuccgugauguugcuuga [dt] [dt]-3′30 siChk2: 5′-aagaaccugaggaccaagaac [dt] [dt]-3′31 Scrambled siRNA with the target sequence: 5′-gaucauacgugcgaucaga [dt] [dt]-3′ was purchased from Sigma (Sigma, St Louis, MO, USA). For siRNA transfection, 10 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was mixed first with 500 μl Opti-MEM medium (Life Technologies, Grand Island, NY, USA) for 5 min, then with 200 nM siRNA in 500 μl OptiMEM medium and incubated at room temperature for 20 min before the mixture was layered onto cells in 1 ml DMEM medium. Cells were harvested 48 h after transfection. © 2014 Macmillan Publishers Limited
DNA-PK/Chk2 induces centrosome amplification C-Y Wang et al
7 Antibodies The following antibodies were obtained commercially: anti-γ-tubulin, antiα-tubulin and anti-acetylated-tubulin (Sigma), anti-Chk2, anti-Chk2 phospho-Thr68, anti-Akt, anti-Akt phospho-Ser473, anti-ATR and anti-ATR phospho-Ser428 (Cell Signaling, Beverly, MA, USA), anti-centrin 20H5 (Millipore, Billerica, MA, USA), polyclonal anti-centrin, anti-Chk1 and antiChk1 phospho-Ser345 (Abcam, Cambridge, UK), anti-Ku70, anti-Ku80 and anti-ATM (Genetex, Trvine, CA, USA), anti-ATM phospho-Ser1981 (Epitomics, Burlingame, CA, USA) anti-actin, anti-GAPDH, anti-DNA-PKcs and anti-DNA-PKcs phospho-Thr2609 (Santa Cruz Biotech, Santa Cruz, CA, USA).
Immunofluorescence microscopy Cells were grown on glass coverslips at 37 °C before fixation with ice-cold methanol at − 20 °C for 6 min. To visualize centriolar Ace-tub staining, microtubules were depolymerized on ice for 1 h followed by fixation with − 20 °C methanol for 6 min. After blocking with 5% bovine serum albumin for 1 h, cells were incubated with antibodies for 24 h at 4 °C. After extensive washing with phosphate-buffered saline (PBS), cells were incubated with fluorescein isothiocyanate-conjugated and Cy3-conjugated secondary antibodies (Invitrogen) for 1 h in the dark. The nuclei were stained with 4′,6-diamino-2phenylindole (0.1 μg/ml) simultaneously. After extensive washing, the coverslips were mounted in 50% glycerol on glass slides. Fluorescent cells were examined with an AxioImager Z1 fluorescence microscope or an LSM 510 confocal microscope (both from Zeiss, Oberkochen, Germany). The number of centrosomes and centrioles from >100 cells were counted under the microscope in three independent experiments and shown as mean ± s.d. Student’s t-test was performed to analyze the difference between groups as indicated.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay Cells were washed with PBS followed by adding 1 ml MTT solution (2 mg/ ml in PBS) in each well. After incubation for 3 h at 37 oC, 2 ml dimethyl sulfoxide was added and cells were incubated in the dark for an additional 30 min. Absorbance was measured at the wavelength of 570 nm.
FACS analysis For fluorescence-activated cell sorting (FACS) analysis, cells were collected by trypsinization and resuspended with PBS. Following centrifugation at 1000 r.p.m. for 5 min, cells were resuspended with 1 mM EDTA in PBS (PBSE). After centrifugation, the pellet was resuspended with 0.5 ml PBS-E and followed by fixation with ice-cold 70% ethanol overnight at 4 °C. Fixed cells were washed with PBS-E and stained with propidium iodide (SouthernBiotech, Birmingham, AL, USA) at room temperature for 1 h. DNA content of propidium iodide-stained cells was measured by FACScalibur (Becton-Dickinson, San Diego, CA, USA) and further analyzed by Kaluza software (Beckman Coulter, Brea, CA, USA).
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We would like to thank Ya-Min Lin for technical assistance in FACS analysis. This study was supported by grants from Academia Sinica, NHRI-EX102-10210SI and NSC1022923-B-001-003-MY3 to B-cC and NSC102-2320-B-006–051 to C-YW.
REFERENCES 1 Stehman FB, Bundy BN, Kucera PR, Deppe G, Reddy S, O'Connor DM. Hydroxyurea 5fluorouracil infusion, and cisplatin adjunct to radiation therapy in cervical carcinoma: a phase I-II trial of the Gynecologic Oncology Group. Gynecol Oncol 1997; 66: 262–267. 2 Prosser SL, Straatman KR, Fry AM. Molecular dissection of the centrosome overduplication pathway in S-phase-arrested cells. Mol Cell Biol 2009; 29: 1760–1773. 3 Khodjakov A, Rieder CL, Sluder G, Cassels G, Sibon O, Wang CL. De novo formation of centrosomes in vertebrate cells arrested during S phase. J Cell Biol 2002; 158: 1171–1181. 4 Sato N, Mizumoto K, Nakamura M, Ueno H, Minamishima YA, Farber JL et al. A possible role for centrosome overduplication in radiation-induced cell death. Oncogene 2000; 19: 5281–5290.
5 Bennett RA, Izumi H, Fukasawa K. Induction of centrosome amplification and chromosome instability in p53-null cells by transient exposure to subtoxic levels of S-phase-targeting anticancer drugs. Oncogene 2004; 23: 6823–6829. 6 Durocher D, Jackson SP. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 2001; 13: 225–231. 7 Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003; 3: 155–168. 8 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003; 421: 499–506. 9 Stiff T, Walker SA, Cerosaletti K, Goodarzi AA, Petermann E, Concannon P et al. ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling. EMBO J 2006; 25: 5775–5782. 10 Smith J, Tho LM, Xu N, Gillespie DA. The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res 2010; 108: 73–112. 11 Dodson H, Bourke E, Jeffers LJ, Vagnarelli P, Sonoda E, Takeda S et al. Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM. EMBO J 2004; 23: 3864–3873. 12 Bozulic L, Surucu B, Hynx D, Hemmings BA. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 2008; 30: 203–213. 13 Collis SJ, DeWeese TL, Jeggo PA, Parker AR. The life and death of DNA-PK. Oncogene 2005; 24: 949–961. 14 Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300: 1542–1548. 15 Bourke E, Brown JA, Takeda S, Hochegger H, Morrison CG. DNA damage induces Chk1-dependent threonine-160 phosphorylation and activation of Cdk2. Oncogene 2010; 29: 616–624. 16 Robinson HM, Black EJ, Brown R, Gillespie DA. DNA mismatch repair and Chk1dependent centrosome amplification in response to DNA alkylation damage. Cell Cycle 2007; 6: 982–992. 17 Wang CY, Kao YH, Lai PY, Chen WY, Chung BC. Steroidogenic factor-1 (NR5A1) maintains centrosome homeostasis in steroidogenic cells by restricting centrosomal DNA-PK activation. Mol Cell Biol 2013; 33: 476–484. 18 Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000; 14: 1448–1459. 19 Nakagawa Y, Yamane Y, Okanoue T, Tsukita S, Tsukita S. Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. Mol Biol Cell 2001; 12: 1687–1697. 20 Lee KJ, Lin YF, Chou HY, Yajima H, Fattah KR, Lee SC et al. Involvement of DNAdependent protein kinase in normal cell cycle progression through mitosis. J Biol Chem 2011; 286: 12796–12802. 21 Stolz A, Ertych N, Kienitz A, Vogel C, Schneider V, Fritz B et al. The CHK2-BRCA1 tumour suppressor pathway ensures chromosomal stability in human somatic cells. Nat Cell Biol 2010; 12: 492–499. 22 Shang ZF, Huang B, Xu QZ, Zhang SM, Fan R, Liu XD et al. Inactivation of DNAdependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2 Phosphorylation in response to DNA damage. Cancer Res 2010; 70: 3657–3666. 23 Golan A, Pick E, Tsvetkov L, Nadler Y, Kluger H, Stern DF. Centrosomal Chk2 in DNA damage responses and cell cycle progression. Cell Cycle 2010; 9: 2647–2656. 24 Tsvetkov L, Xu X, Li J, Stern DF. Polo-like kinase 1 and Chk2 interact and colocalize to centrosomes and the midbody. J Biol Chem 2003; 278: 8468–8475. 25 Joo K, Kim CG, Lee MS, Moon HY, Lee SH, Kim MJ et al. CCDC41 is required for ciliary vesicle docking to the mother centriole. Proc Natl Acad Sci USA 2013; 110: 5987–5992. 26 Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M et al. Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol 2007; 179: 321–330. 27 An J, Yang DY, Xu QZ, Zhang SM, Huo YY, Shang ZF et al. DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein. Mol Cancer 2008; 7: 32. 28 Ayene IS, Ford LP, Koch CJ. Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation. Mol Cancer Ther 2005; 4: 529–536. 29 Rampakakis E, Di Paola D, Zannis-Hadjopoulos M. Ku is involved in cell growth DNA replication and G1-S transition. J Cell Sci 2008; 121: 590–600. 30 Andreassen PR, D'Andrea AD, Taniguchi T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev 2004; 18: 1958–1963. 31 Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, Nigg EA et al. Centrosome-associated Chk1 prevents premature activation of cyclin-BCdk1 kinase. Nat Cell Biol 2004; 6: 884–891.
Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc) © 2014 Macmillan Publishers Limited
Oncogene (2014), 1 – 7
