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DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis
Experimental Cell Research 342 (2016) 135–144
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Research Article
DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis Katsuya Iuchi n, Tatsuo Yagura Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 28 December 2015 Received in revised form 8 March 2016 Accepted 9 March 2016 Available online 11 March 2016
Ku protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku protein during apoptosis remains unclear. Here, we show characterization of Ku protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the nontreated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion, Ku is intact and retains DNA binding activity in early apoptotic cells. & 2016 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Ku protein DNA-PK
1. Introduction Ku was originally identified as a major autoantigen in the sera of patients with autoimmune diseases [1]. Ku protein is a heterodimer composed of 70 and 80 kDa subunits and binds to ends of double-stranded DNA [2,3]. The binding of Ku to ends of doublestranded DNA is largely sequence-independent. Double-stranded DNA (ds DNA) of 14–18 bp in length is sufficient for binding with a single molecule of Ku protein [4,5]. In the presence of DNA ends, Ku can interact with DNA-PKcs, which phosphorylate serine/ threonine [4,6,7]. The interaction between Ku and DNA-PKcs has been implicated in DNA repair (non-homologous end-joining, NHEJ) [8–15], V(D)J recombination [16], and apoptosis [17]. In the first step of NHEJ, Ku binds to DNA double-strand breaks (DSBs) induced by ionizing radiation and DNA damaging agents. DNA-PK (Ku and DNA-PKcs) are widely-known as DNA damage sensors, which recruit other DNA repair proteins. Binding of DNA-PK to dsDNA breaks also stimulates transcriptional activation of p53 [18] and/or NF-κB [19]. n Correspondence to: Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-machi, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan. E-mail address: [email protected] (K. Iuchi).
http://dx.doi.org/10.1016/j.yexcr.2016.03.011 0014-4827/& 2016 Elsevier Inc. All rights reserved.
Ku plays a key role in inhibiting apoptosis through not only the DSB repair pathway but also the other pathways. Several studies have identified associations between cell death and the loss of nuclear Ku70 [20] and/or cytosolic accumulation of Ku70 [21]. Ku70-deficient cells are more sensitive than Ku70 þ / cells to X-ray irradiation [22]. Ku generally prevents cell death via initiating the DNA repair process in response to DNA damage, but severe DNA damage triggers apoptosis. The relative balance between DNA repair and cell death responses determines the ultimate fate of the cell. X-ray induces binding of Ku to nuclear clusterin/XIP8, and the binding is relative to dramatically reduced cell growth, colony-forming ability, and increased cell death [23]. In addition to the roles in DSB repair, Ku has a cytoprotective function in the cytosol [17]. Ku70 blocks the translocation of Bax, pro-apoptotic Bcl-2 family protein, to mitochondria. Ku protein expression and DNA-binding activity are regulated by proteolytic degradation and post-transcriptional modification. During caspase- or granzyme B-mediated apoptosis, the repair of DSBs is blocked by caspase-dependent cleavage of DNA-PKcs [24]. Ku80 is cleaved by caspase-3 in the cell nucleus during apoptosis [25]. Granzyme A is a protease, and it activates caspase-independent cell death with morphological features of apoptosis by directly degrading Ku70 after Arg301 and thus disrupting Ku complex binding to DNA [26]. Ku level decreased during
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β-carotene-induced apoptosis through caspase activation and ROS production in the gastric cancer cell line AGS [27]. In addition, the steady-state level and localization of Ku protein are regulated by heterodimerization. Ku80 is required for the stabilization of Ku70 in the cytoplasm, and Ku70 is required for the nuclear localization of Ku80 [28]. Ku70 is indirectly stabilized by increased cellular SUMOylation [29]. DSB induced ubiquitylation of Ku80 and removal of Ku80 from DNA [30]. These reports indicate that Ku level and DNA-binding activity are down-regulated during apoptosis. Various stimuli activate both DNA repair and cell death signals at the same time. Thus, it seems that Ku is involved in the dual regulation of DSB repair and apoptosis. Although extensive DSBs trigger apoptosis, the biological role of Ku at the start of apoptosis remains unclear. This study aims to investigate the role of Ku70 and Ku80 in apoptotic cell death induced by DSBs. In order to study the role of Ku protein during early apoptosis, we used etoposide-treated HL-60 cells. Etoposide is a widely used and effective anticancer agent that inhibits the essential enzyme DNA topoisomerase II, resulting in DSBs, and triggers apoptosis in a variety of malignant cells. Here, we report evidence that Ku is intact and retains DNA binding activity in cells that undergo apoptotic cell death induced by DSBs and binds to apoptotic DNA fragments.
2. Materials and methods 2.1. Materials and chemicals Anti-DNA-PKcs rabbit IgG antibody (sc-9051) was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Goat anti-rabbit IgG conjugated-AP antibody (4050-04) was obtained from Southern Biotechnology Associates (Birmingham, AL). Goat anti-human IgA, IgG, and IgM conjugated-AP antibody (59284) was obtained from CAPPEL (West Chester, PA, USA, CA). Anti-Ku antiserum was kindly provided by Dr. Tsuneyo Mimori. All other chemicals were obtained from Sigma-Aldrich or Wako and were of the highest quality commercially available. 2.2. Cells culture HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 50 units/mL streptomycin at 37 °C with 5% CO2. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum and kanamycin at 37 °C with 5% CO2. 2.3. Apoptosis induction HL-60 cells were treated with 10 mM etoposide or 1 mM staurosporine for various durations. To induce macrophagic terminal differentiation followed by apoptosis, HL-60 cells were treated with 80 nM TPA (12-O-tetradecanoylphorbol 13-acetate) for various durations. 2.4. Determination of differentiation or apoptotic cells treated with TPA To determine apoptosis, cells were fixed with 1% glutaraldehyde and stained with Hoechst 33258. Cells were examined under a fluorescence light microscope when the nucleus exhibited typical apoptotic features such as chromatin condensation around the periphery and/or fragmentation. Macrophage-differentiation was measured by counting the adherent cells using a BeckmanCoulter Counter.
2.5. Electrophoretic analysis of apoptotic DNA degradation Approximately 5 105 cells were harvested, washed in PBS, and centrifuged. The cell pellets were lysed in 200 μL of lysis buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5 mg/mL RNase A, 0.5 mg/mL proteinase K), and incubated at 37 °C for 30 min. The lysates were resuspended in 300 mL of NaI solution (6 M NaI, 10 mM Tris–HCl pH 8.0, 13 mM EDTA, 0.5% sodium Nlaulyl sarcosine, 30 mg/mL glycogen), and incubated at 60 °C for 15 min. DNA was precipitated by 1 volume of 100% isopropanol, and then washed with 50% isopropanol. The DNA pellets were airdried and re-dissolved in Tris-EDTA buffer. DNA was electrophoresed on a 2.5% agarose gel and visualized with ethidium bromide, and the DNA pattern was subsequently examined by ultraviolet transillumination. 2.6. Flow cytometric analysis Measurements of DNA content and cell cycle analysis were performed as described previously [31]. HL-60 cells or HeLa treated with etoposide were washed with PBS and fixed with ice-cold 70% ethanol, and stored at 20 °C until use. To examine cell cycle status, the stored cells were pelleted, washed with PBS, resuspended in PBS containing 0.5 mg/mL of RNase A, and incubated at 37 °C for 20 min. The cells were then pelleted, resuspended in PBS containing 50 mg/mL of propidium iodide, and incubated at 4 °C for 10 min in the dark. Finally, the stained cells were analyzed with a Becton-Dickinson FACSCalibur flow cytometer. A minimum of 10,000 cells/sample were analyzed. Data were collected and analyzed using CellQuest software. 2.7. Whole cell extract preparation The preparation of protein lysates was performed as described [32]. Approximately 5 105 cells were harvested at different times after etoposide treatment. Cells were resuspended in extraction buffer (50 mM NaF, 20 mM HEPES-KOH pH 7.8, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, proteinase inhibitors (antipain, leupeptin, pepstain A, chymostain)) and then frozen in liquid nitrogen and thawed in tap water three times. After centrifugation at 8,000 g for 30 min at 4 °C, clear supernatant was transferred to a fresh tube and stored at 80 °C. 2.8. Western blot Western blot analysis was performed as described previously [33,34]. HL-60 whole cell extracts were electrophoresed on SDSpolyacrylamide gels. Ku was analyzed on 10% SDS-polyacrylamide gels, and DNA-PKcs on 8% SDS-polyacrylamide gels. The proteins were transferred to a PVDF membrane for 1 h at 20 mA, and then 16 h at 40 mA, at 4 °C. The blot was rinsed in rinsing buffer (10 mM Tris–HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100), and placed in blocking buffer (5% non-fat milk, in rinsing buffer) for 1 h. Next, the blot was incubated with primary antibody at 1:1000 dilution in blocking buffer for 1 h to overnight for anti-Ku antiserum (OM) and anti-DNA-PKcs antibody. Following incubation with the primary antibody, the blot was rinsed twice with rinsing buffer, followed by one 15-min and three 5-min washes in rinsing buffer on a rocking platform. Anti-human or anti-rabbit IgG-AP secondary antibody was used at 1:5000 dilution in rinsing buffer. Incubation with secondary antibodies was performed at room temperature for 1 h. On following the same washing steps as described above, the complexes were detected with BCIP/NBT.
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Fig. 1. Effect of etoposide on the DNA probe-binding activity and protein level of Ku. (A) RI-EMSA: Two mg of protein extract obtained from untreated HL-60 cells was incubated with 32P-labeled dsDNA probe (15-bp or 28-bp) in the presence of 1 mg of closed circular DNA. (B) RI-EMSA: Two mg of protein extract obtained from untreated HL60 cells was incubated with 32P-labeled dsDNA probe (15-bp) and anti-human Ku polyclonal antiserum in the presence of 1 mg of closed circular DNA. (C) RI-EMSA: Two micrograms of protein extract obtained from cells treated with etoposide for the duration indicated was incubated with 32P-labeled, 15-bp dsDNA probe in the presence of 1 mg of closed circular DNA. (D) Western blot: 20 mg of protein extract obtained from either control or treated cells was run on a 10% SDS polyacrylamide gel under reducing conditions. Ku70 and Ku80 were detected using human polyclonal antiserum. Twenty micrograms of protein extract was run on a 8% SDS polyacrylamide gel under reducing conditions, and DNA-PKcs was detected using a rabbit polyclonal antibody.
2.9. Electrophoretic mobility shift assay (EMSA) RI-EMSA was performed using a modification of the RI-EMSA method used by Muller et al. [32]. Sequences of the oligonucleotides used for making double-stranded probes were 5′-cacaccgcatacgtc-3′, 3′-gtgtggcgtatgcag-5′, 5′-cacaccgcatacgtcaaagcaaccatag-3′, and 3′gtgtggcgtatgcagtttcgttggtatc-5′. For this analysis, 10 pmol of the 15 or 28 oligomer, T4 DNA polynucleotide kinase, and 3 mL of [γ-32P]ATP (3000 Ci/mmol) were incubated for 30 min at 37 °C. Then, 10 pmol of the complementary oligonucleotide was added; the mixture was incubated at 85 °C for 5 min, and subsequently cooled at room temperature. The DNA probe was purified with CENTRI-SEP COLUMNS (Princeton separation). Radiolabeled DNA, 1 mg of circular plasmid and 2 mg of whole cell extract were incubated with 1 binding buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 10% glycerol, 50 mM KCl) for 15 min at 27 °C. For experiments with mixed extracts from control and apoptotic cells, the extracts were co-incubated in 1 binding buffer for 5 min on ice. Radiolabeled DNA and circular plasmid were then added, and the reaction mixture was incubated for an additional 10 min prior to gel electrophoresis. The samples were electrophoresed on a 6% polyacrylamide gel in TBE at 4 °C for
1 h at 150 V. The gel was dried on Whatman paper and exposed to a phosphor imager plate for desired durations. Bands were visualized by phosphor imaging using the Bio-Image analyzer BAS 2500 (Fujifilm). Radioactivity in each band was quantified with an imaging analyzer. WB-EMSA: Unlabeled ds DNA (15-bp or 160-bp), circular plasmid, and 10 mg of whole cell extract were incubated with 1 binding buffer for 15 min at 27 °C. The samples were electrophoresed on a 6% polyacrylamide gel in TBE at 4 °C, for 1.5 h at 150 V. The proteins were transferred to a PVDF membrane. The membrane was analyzed as described in the western blot section above. 2.10. Immunofluorescence of Ku HL-60 cells were treated with etoposide for 4 h. The cells were fixed for 5 min by 70% methanol. After washing, the cells were blocked with 5% BSA in TBS for 30 min. The cells were incubated with 1:100 anti-Ku70 antibody (A9: sc-5309, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at RT followed by incubation with 1:100 FITC-conjugated anti-rabbit antibody for 1 h
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Fig. 2. Inhibitory effects of extract from etoposide-treated cells on Ku-DNA probe binding activity. (A) Radiolabeled ds DNA (15-bp) was incubated with extracts in binding buffer at 27 °C for 15 min (lanes 1 and 2). After mixing the extracts prepared from untreated cells and etoposide-treated cells, radiolabeled ds DNA was added and the mixture was incubated in binding buffer at 27 °C for 15 min (lane 3). Etoposide-treated cell extract was heat-denatured at 90 °C for 3 min and mixed with untreated cell extracts, and subsequently analyzed by RI-EMSA (lane 4). (B) Apoptotic cells with sub-G1 DNA content were analyzed by flow cytometry. The percentage of cells in the subG1 population is shown in each panel. (C) DNA fragmentation after treatment of HL-60 cells with etoposide. DNA was extracted from HL-60 cells treated with or without 10 mM etoposide for 4 h and subjected to agarose gel electrophoresis.
at 25 °C. To stain the DNA, the cells were incubated with Hoechst33825 for a few minutes, and then observed under a fluorescent microscope. 2.11. Measurement of Ku in apoptotic cells using flow cytometry HL-60 cells were treated with etoposide for 4 h, and then fixed for 5 min by 70% methanol. After washing, the cells were blocked with 5% BSA in TBS for 30 min. The cells were incubated with 1:100 anti-Ku70 antibody (A9: sc-5309, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at RT followed by incubation with 1:100 FITC-conjugated anti-rabbit antibody for 1 h at 25 °C. To measure the DNA content, the cells were washed with TBS, resuspended in TBS containing 0.5 mg/mL of RNase A, and incubated at 37 °C for 20 min. The cells were incubated with 50 mg/mL
propidium iodide in sodium citrate buffer for 10 min, and subsequently analyzed with a Becton-Dickinson FACSCalibur flow cytometer. 2.12. Rough purification of Ku Whole cell extract (100 mL) was diluted (1:10) with column buffer (50 mM Tris–HCl pH 7.6, 1 mM EDTA, 5% glycerol, 0.05% Tween 20, 0.5 mM PMSF) and cleared by passing through a 0.22μm filter. The filtrate was loaded on a Resource Q column (Amersham Biosciences, 1 mL) and equilibrated in column buffer containing 0.05 M KCl. The column was eluted with 5 mL of 0.05– 0.8 M linear gradient of KCl. The fractions were frozen in small aliquots and stored at 80 °C.
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Fig. 3. Effect of DNase I on the inhibitory effect of the apoptotic cell extract on Ku-DNA binding. Radiolabeled ds DNA (15-bp) was incubated with extracts in binding buffer at 27 °C for 15 min (lanes 1, 2). The mixture of extracts from untreated and etoposide-treated cells was incubated for 5 min on ice; then radiolabeled ds DNA was added, and the mixture was incubated in binding buffer at 27 °C for 15 min. Etoposide-treated extract was treated with DNase I with 2.5 mM MgCl2 at 37 °C for 30 min, and subjected to inactivation of DNase I by adding 2.5 mM EDTA and by heat-treatment at 65 °C for 10 min (lanes 3–7).
3. Results 3.1. Ku-DNA probe binding activity is decreased in apoptotic cells Ku protein plays an important role in DNA damage repair and prevention of cell death. Apoptosis causes a decrease in cellular DNA repair proteins. We examined whether Ku-DNA probe binding activity and Ku protein level were altered in the apoptotic cells. Whole cell extracts of the untreated and etoposide-treated cells were analyzed by RI-EMSA and western blot. In RI-EMSA, we mainly used a radiolabeled 15-bp DNA probe which only bound Ku70/80 heterodimer but not DNA-PKcs (Fig. 1A, lane 1), because Ku-DNA complex and other complexes were formed when using a 28-bp DNA probe (Fig. 1A, lane 2). The 15-bp DNA probe/Ku band was supershifted by anti-Ku antiserum (Fig. 1B). Ku-DNA probe binding activity dramatically decreased after 2 h of etoposide treatment, although the activity increased after 1 h of treatment (Fig. 1C). We investigated whether apoptosis led to a loss in the amount of DNA-PKcs and Ku in the cell. Ku protein was intact in the apoptotic cells, and etoposide-treatment did not result in loss of Ku protein. In contrast, DNA-PKcs appeared to be degraded after the induction of apoptosis with etoposide (Fig. 1D). These results demonstrated that Ku protein level was not regulated by proteases in the apoptotic cells. Nevertheless, Ku in apoptotic cells hardly bound to additional DNA probes. 3.2. Apoptotic cell extracts inhibit Ku-DNA probe binding activity To test the hypothesis that apoptotic cell extract has an inhibitory effect on Ku-DNA probe binding activity, we performed RI-EMSA with a mixture of extracts from apoptotic and untreated cells. Apoptotic cell extract inhibited Ku-DNA probe binding activity in untreated cell extract when examined by the RI-EMSA method (Fig. 2A, lane 3). Furthermore, heat-treated extract from apoptotic cells retained the inhibitory effect on Ku-DNA probe binding in untreated cell extract (Fig. 2A, lane 4). Our results suggest that a heat-resistant inhibitor is present in the apoptotic cells. DNA-gel electrophoresis showed that the HL-60 cells treated
with etoposide displayed DNA fragmentation in multiples of 180– 200 bp (Fig. 2C). Flow cytometric analysis of DNA content also showed that the percentage of cells with a sub-G1 DNA content increased after 2 h of etoposide treatment (Fig. 2B). These results indicate that nucleosome or/and endogenous DNA fragments is a candidate as the inhibitor of Ku-DNA probe binding activity in the apoptotic cells. 3.3. The inhibitory effect of apoptotic cell extract is decreased by DNase I treatment To further investigate the inhibitory effect of apoptotic cell extract on Ku-DNA probe binding activity, apoptotic cell extract was treated with DNase I. DNase I-treated extract from apoptotic cells could not inhibit Ku-DNA probe binding activity in untreated cells (Fig. 3). This result suggests that free DNA fragments or nucleosomes were present in the whole cell extract of apoptotic cells. 3.4. Ku-DNA probe binding activity is decreased during apoptosis but not differentiation HL-60 cells were induced macrophage-like differentiation and subsequently apoptosis by TPA-treatment (Sup. Fig. S2A). We investigated whether Ku in these cells bound to the DNA probe. KuDNA probe binding activity dramatically decreased after 3 days of TPA treatment (Sup. Fig. S2B), although the protein level is not changed by TPA-treatment (Sup. Fig. S2C). HL-60 cells treated with TPA displayed DNA fragmentation (Sup. Fig. S2D). Moreover apoptotic cell extract inhibited Ku-DNA probe binding activity in untreated cell extract. DNase I-treated extract from apoptotic cells could not inhibit Ku-DNA probe binding activity in untreated cells (Sup. Fig. S2E). These results suggest that Ku-DNA probe binding activity is decreased during apoptosis but not differentiation. 3.5. EMSA in conjunction with western blot (WB-EMSA) successfully detected the Ku/DNA complex Generally, Ku-DNA binding activity is analyzed by EMSA with a radiolabeled DNA probe. In the RI-EMSA assay, only Ku bound to
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Fig. 4. Analysis of Ku by WB-EMSA (Combination of EMSA with 15-bp non-labeled ds DNA and western blot). (A) Extracts from etoposide-treated HL-60 cells were separated by 6% native PAGE, and then analyzed by western blot with polyclonal antiserum raised against Ku (lane 1). Extracts from untreated HL-60 cells were incubated with 160-bp DNA, followed by western blot (lane 2). Extract from untreated or etoposide treated HL-60 cells was pre-incubated with 15-bp DNA, followed by western blot (lanes 3–6). (B) Extracts from untreated HL-60 cells were analyzed (lane 1). Extract from untreated or etoposide-treated HL-60 cells was pre-incubated with 15-bp DNA, followed by western blot (lanes 2, 3). After mixing the extracts prepared from untreated cells and etoposide treated cells, non-labeled ds DNA was added, and the mixture was incubated in binding buffer at 27 °C for 15 min (lane 4). (C) Comparison of EMSA combined with western blot (WB-EMSA) to conventional EMSA with 15-bp 32P-labeled DNA probe (RIEMSA). The diagram of RI-EMSA is a part of Fig. 1C, and the figure of WB-EMSA is a part of Fig. 4B.
the DNA probe can be detected, and Ku bound to endogenous DNA fragments of chromatin cannot be detected. To detect complexes between Ku and endogenous DNA, we performed EMSA in conjunction with western blot (WB-EMSA). WB-EMSA is a method which combines EMSA and western blot. This method allows distinction between the complex of additional 15-bp DNA/Ku and complexes of endogenous DNA/Ku (Fig. 4A and B). Activity of Ku
binding to 15-bp DNA was dramatically decreased after 2 h and 4 h of etoposide treatment (Fig. 4A, lanes 5 and 6). WB-EMSA of the apoptotic extract resembled that of the mixture of 160-bp DNA fragment and untreated cell extract (Fig. 4A, lanes 1 and 2). In addition, apoptotic cell extract inhibited Ku/15-bp DNA binding in untreated cell extract in WB-EMSA (Fig. 4B, lane 4). As shown in Fig. 4C, 15-bp DNA/Ku complex was detected by WB-EMSA as well
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Fig. 5. Purification of Ku on Resource Q column. (A) Whole cell extract from untreated or etoposide-treated HL-60 cells was loaded onto a 1 mL Resource Q column, and protein was subsequently eluted by a linear KCl gradient (50–800 mM). (B) The presence of Ku in the elutes was determined by immunoblotting after SDS-PAGE. (C) Analysis of Ku purified with Resource Q column with WB-EMSA. (D) The elutes were analyzed with RI-EMSA.
as RI-EMSA (Fig. 4C). These results indicate that Ku binds to the apoptotic DNA fragments and/or nucleosomes in the apoptotic cells. 3.6. Ku protein in apoptotic cell extract purified by Resource Q column binds to additional 15-bp DNA Ku protein in apoptotic cells was purified by Resource Q column and subsequently analyzed by western blot (Fig. 5A, B). The majority of Ku in both control and apoptotic cell extract was present in fractions #13 and #14. Purified Ku from control and apoptotic cell extracts displayed similar bands in WB-EMSA (Fig. 5C, lanes 1 and 2). These results indicate that the charge of Ku
protein in apoptotic and untreated cells is unchanged. Next, we investigated whether purified Ku was capable of binding 15-bp DNA. Purified Ku from apoptotic cell extract bound 15-bp DNA in WB-EMSA (Fig. 5C, lane 6), whereas Ku in pre-purified apoptotic extract did not (Fig. 5C, lane 4). Moreover, we investigated whether Ku protein in the apoptotic cell extract purified by Resource Q column bound DNA probe. As shown in Fig. 5D, Ku protein in the apoptotic cell extract purified by Resource Q column could bind radiolabeled DNA probe, while pre-purified Ku protein from apoptotic cells did not bind radiolabeled DNA probe. This suggests that Ku is intact and retains DNA binding activity in cells that undergo apoptotic induced by DSBs.
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Fig. 6. Localization and expression of Ku70 in apoptotic cells. (A) Localization of Ku70 in apoptotic cells with condensed chromatin. HL-60 cells were treated with etoposide for 4 h. Ku70 was stained with anti-Ku70 antibodies and FITC-conjugated anti-mouse antibody. (B) Expression of Ku70 in apoptotic cells. HL-60 cells were treated with etoposide for 4 h. Ku70 protein was stained as above, and apoptotic cells containing fragmented DNA were detected by staining with propidium iodide.
3.7. Localization and expression of Ku protein in apoptotic cells As shown in the figures above, Ku protein in apoptotic cell extract binds apoptotic DNA fragments and/or nucleosomes. We investigated whether Ku localized to the condensed chromatin in the apoptotic nuclei. As shown in Fig. 6A, Ku70 was localized to the condensed chromatin in HL-60 cells treated with etoposide (Fig. 6A). To assess the expression of Ku70 in apoptotic cells, HL-60 cells were immunostained with anti-Ku70 antibodies and the cellular DNA content was measured by flow cytometry. Ku70 expression was detected in apoptotic HL-60 cells in sub-G1 region (Fig. 6B, UL). These results indicate that Ku binds to fragmented DNA and/or nucleosomes during early apoptosis.
4. Discussion This study analyzed the protein expression of Ku and its DNA binding activity during early apoptosis. In the present study, Ku was neither cleaved nor degraded during early apoptosis induced by etoposide treatment. These results are consistent with those from a previous report [35]. Moreover, Ku DNA binding activity in the apoptotic cells is retained. Some previous studies have shown that Ku protein levels [36,37] and DNA binding activity [38,39] were reduced in the cells undergoing apoptosis. The discrepancy between results may be attributed to the cell death inducers, culture cells, or method used to measure Ku-DNA binding activity. The expression of Ku protein was decreased during staurosporineinduced apoptosis (Sup. Fig. S4). The present study is the first report showing that Ku can bind endogenous DNA fragments produced during apoptosis by using the two EMSA methods (RI-EMSA
and WB-EMSA). In most previous studies, Ku DNA binding activity was assayed with only RI-EMSA, and the influence of DNA fragments present in apoptotic cells was overlooked. However we could not completely exclude the possibility that Ku is digested by some proteases or post-translated. Ku proteins that are not binding to DNA may be modified or damaged as reported by other groups. The present study shows that apoptotic cell extracts inhibited Ku-DNA binding activity in non-treated cell extract in RI-EMSA, but not in WB-EMSA. Moreover, Ku in purified apoptotic cell extracts was able to bind exogenous 15-bp DNA, while Ku in unpurified extracts could not bind. These results suggest that Ku binds to cleaved chromosomal DNA in apoptotic cells. Chromatin DNA contains DNase I- and MNase-hypersensitive sites. Early in apoptosis, there is a rapid degradation of nuclease-hypersensitive euchromatin that contains hyperacetylated histones [40]. Ku protein can bind unfolded nucleosomes that lack histone H1 [41]. Therefore, Ku may bind free DNA fragments from chromatin DNA digested by caspase-activated DNase CAD (also known as DFF40) during early apoptosis. In this study, DNA/multiple Ku complex could be digested by DNase I, resulting in a DNA/single Ku complex (Sup. Fig. S1). This result suggested that Ku protein did not interfere with the digestion of apoptotic DNA fragment by DNase I. Ku in the extract from etoposide-treated HeLa can bind additional 15-bp DNA (Sup. Fig. S3A). Treatment with etoposide for 36 h could not increase the sub-G1 population in HeLa cells (Sup. Fig. S3C). These results suggest that the additional DNA binding activity of Ku is decreased during apoptosis with DNA fragmentation but not cell cycle arrest and topoisomerase II inactivation by etoposide. DNA-PK can be activated by nucleosomes through Ku binding to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes [42]. UV light exposure and etoposide treatment resulted in an increase of all histones in cell lysates compared to activated lymphoblasts. Furthermore, this increase of histone amount in cell lysates was accompanied by a considerable increase of early apoptotic cells, but no significant changes in late apoptotic cells or necrotic cells were observed [43]. In vitro studies showed that histone H1 and high mobility group (HMGB) enhance DFF40 function [44,45]. These data suggest that Ku may be related to chromatin component proteins and be involved in regulating after apoptosis system such as genomic DNA digestion and phagocytosis. In the present study, Ku is intact during early apoptosis, despite degradation of DNA repair proteins such as DNA-PKcs, according to our western blot analysis. Previous reports have also shown that these DNA repair proteins (DNA-PKcs and ATM) are inactivated by cleavage during apoptosis [35,46,47]. Cells undergoing apoptosis exhibit specific morphological changes, which include membrane blebbing, cytoplasmic and chromatin condensation, nuclear breakdown, and assembly of membrane-enclosed vesicles termed apoptotic bodies that are eventually subjected to phagocytosis. Normally, apoptotic cells are removed rapidly by phagocytic cells before apoptotic blebs can be released [43]. Hence, Ku may bind to fragmented chromatin DNA during early apoptosis and finally become cleaved upon phagocytosis. Binding of Ku to chromatin DNA fragments may prevent the release of Ku into the extracellular space. HMGB1, a chromatin protein, is capable of binding to chromatin in apoptotic cells. But HMGB1 in necrotic cells is released into the extracellular space, leading to promotion of inflammation [48]. Ku is a target of autoimmune conditions including systemic lupus erythematosus and Graves’ disease. Failure of apoptosis can lead to autoimmune diseases. Therefore, binding of Ku to apoptotic DNA fragments may be essential for preventing it from causing autoimmune diseases. In conclusion, our data demonstrated that Ku is intact and
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retains DNA binding activity in early apoptotic cells. In addition, Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. These findings may allow for a further understanding of the mechanism of interaction of Ku with DNA during apoptosis and the cause of autoimmune diseases.
Acknowledgement We thank Prof. Fumiaki Yamao and Dr. Toyoaki Natsume for scientific advice and technical support on RI-EMSA. We thank Prof. Shinsuke Fujiwara and Prof. Yoshinori Toyoshima for many useful discussions. We thank Ms. Megumi Mizuno for experimental support.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2016.03.011.
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Research Article
DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis Katsuya Iuchi n, Tatsuo Yagura Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 28 December 2015 Received in revised form 8 March 2016 Accepted 9 March 2016 Available online 11 March 2016
Ku protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku protein during apoptosis remains unclear. Here, we show characterization of Ku protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the nontreated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion, Ku is intact and retains DNA binding activity in early apoptotic cells. & 2016 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Ku protein DNA-PK
1. Introduction Ku was originally identified as a major autoantigen in the sera of patients with autoimmune diseases [1]. Ku protein is a heterodimer composed of 70 and 80 kDa subunits and binds to ends of double-stranded DNA [2,3]. The binding of Ku to ends of doublestranded DNA is largely sequence-independent. Double-stranded DNA (ds DNA) of 14–18 bp in length is sufficient for binding with a single molecule of Ku protein [4,5]. In the presence of DNA ends, Ku can interact with DNA-PKcs, which phosphorylate serine/ threonine [4,6,7]. The interaction between Ku and DNA-PKcs has been implicated in DNA repair (non-homologous end-joining, NHEJ) [8–15], V(D)J recombination [16], and apoptosis [17]. In the first step of NHEJ, Ku binds to DNA double-strand breaks (DSBs) induced by ionizing radiation and DNA damaging agents. DNA-PK (Ku and DNA-PKcs) are widely-known as DNA damage sensors, which recruit other DNA repair proteins. Binding of DNA-PK to dsDNA breaks also stimulates transcriptional activation of p53 [18] and/or NF-κB [19]. n Correspondence to: Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-machi, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan. E-mail address: [email protected] (K. Iuchi).
http://dx.doi.org/10.1016/j.yexcr.2016.03.011 0014-4827/& 2016 Elsevier Inc. All rights reserved.
Ku plays a key role in inhibiting apoptosis through not only the DSB repair pathway but also the other pathways. Several studies have identified associations between cell death and the loss of nuclear Ku70 [20] and/or cytosolic accumulation of Ku70 [21]. Ku70-deficient cells are more sensitive than Ku70 þ / cells to X-ray irradiation [22]. Ku generally prevents cell death via initiating the DNA repair process in response to DNA damage, but severe DNA damage triggers apoptosis. The relative balance between DNA repair and cell death responses determines the ultimate fate of the cell. X-ray induces binding of Ku to nuclear clusterin/XIP8, and the binding is relative to dramatically reduced cell growth, colony-forming ability, and increased cell death [23]. In addition to the roles in DSB repair, Ku has a cytoprotective function in the cytosol [17]. Ku70 blocks the translocation of Bax, pro-apoptotic Bcl-2 family protein, to mitochondria. Ku protein expression and DNA-binding activity are regulated by proteolytic degradation and post-transcriptional modification. During caspase- or granzyme B-mediated apoptosis, the repair of DSBs is blocked by caspase-dependent cleavage of DNA-PKcs [24]. Ku80 is cleaved by caspase-3 in the cell nucleus during apoptosis [25]. Granzyme A is a protease, and it activates caspase-independent cell death with morphological features of apoptosis by directly degrading Ku70 after Arg301 and thus disrupting Ku complex binding to DNA [26]. Ku level decreased during
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β-carotene-induced apoptosis through caspase activation and ROS production in the gastric cancer cell line AGS [27]. In addition, the steady-state level and localization of Ku protein are regulated by heterodimerization. Ku80 is required for the stabilization of Ku70 in the cytoplasm, and Ku70 is required for the nuclear localization of Ku80 [28]. Ku70 is indirectly stabilized by increased cellular SUMOylation [29]. DSB induced ubiquitylation of Ku80 and removal of Ku80 from DNA [30]. These reports indicate that Ku level and DNA-binding activity are down-regulated during apoptosis. Various stimuli activate both DNA repair and cell death signals at the same time. Thus, it seems that Ku is involved in the dual regulation of DSB repair and apoptosis. Although extensive DSBs trigger apoptosis, the biological role of Ku at the start of apoptosis remains unclear. This study aims to investigate the role of Ku70 and Ku80 in apoptotic cell death induced by DSBs. In order to study the role of Ku protein during early apoptosis, we used etoposide-treated HL-60 cells. Etoposide is a widely used and effective anticancer agent that inhibits the essential enzyme DNA topoisomerase II, resulting in DSBs, and triggers apoptosis in a variety of malignant cells. Here, we report evidence that Ku is intact and retains DNA binding activity in cells that undergo apoptotic cell death induced by DSBs and binds to apoptotic DNA fragments.
2. Materials and methods 2.1. Materials and chemicals Anti-DNA-PKcs rabbit IgG antibody (sc-9051) was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Goat anti-rabbit IgG conjugated-AP antibody (4050-04) was obtained from Southern Biotechnology Associates (Birmingham, AL). Goat anti-human IgA, IgG, and IgM conjugated-AP antibody (59284) was obtained from CAPPEL (West Chester, PA, USA, CA). Anti-Ku antiserum was kindly provided by Dr. Tsuneyo Mimori. All other chemicals were obtained from Sigma-Aldrich or Wako and were of the highest quality commercially available. 2.2. Cells culture HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 50 units/mL streptomycin at 37 °C with 5% CO2. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum and kanamycin at 37 °C with 5% CO2. 2.3. Apoptosis induction HL-60 cells were treated with 10 mM etoposide or 1 mM staurosporine for various durations. To induce macrophagic terminal differentiation followed by apoptosis, HL-60 cells were treated with 80 nM TPA (12-O-tetradecanoylphorbol 13-acetate) for various durations. 2.4. Determination of differentiation or apoptotic cells treated with TPA To determine apoptosis, cells were fixed with 1% glutaraldehyde and stained with Hoechst 33258. Cells were examined under a fluorescence light microscope when the nucleus exhibited typical apoptotic features such as chromatin condensation around the periphery and/or fragmentation. Macrophage-differentiation was measured by counting the adherent cells using a BeckmanCoulter Counter.
2.5. Electrophoretic analysis of apoptotic DNA degradation Approximately 5 105 cells were harvested, washed in PBS, and centrifuged. The cell pellets were lysed in 200 μL of lysis buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5 mg/mL RNase A, 0.5 mg/mL proteinase K), and incubated at 37 °C for 30 min. The lysates were resuspended in 300 mL of NaI solution (6 M NaI, 10 mM Tris–HCl pH 8.0, 13 mM EDTA, 0.5% sodium Nlaulyl sarcosine, 30 mg/mL glycogen), and incubated at 60 °C for 15 min. DNA was precipitated by 1 volume of 100% isopropanol, and then washed with 50% isopropanol. The DNA pellets were airdried and re-dissolved in Tris-EDTA buffer. DNA was electrophoresed on a 2.5% agarose gel and visualized with ethidium bromide, and the DNA pattern was subsequently examined by ultraviolet transillumination. 2.6. Flow cytometric analysis Measurements of DNA content and cell cycle analysis were performed as described previously [31]. HL-60 cells or HeLa treated with etoposide were washed with PBS and fixed with ice-cold 70% ethanol, and stored at 20 °C until use. To examine cell cycle status, the stored cells were pelleted, washed with PBS, resuspended in PBS containing 0.5 mg/mL of RNase A, and incubated at 37 °C for 20 min. The cells were then pelleted, resuspended in PBS containing 50 mg/mL of propidium iodide, and incubated at 4 °C for 10 min in the dark. Finally, the stained cells were analyzed with a Becton-Dickinson FACSCalibur flow cytometer. A minimum of 10,000 cells/sample were analyzed. Data were collected and analyzed using CellQuest software. 2.7. Whole cell extract preparation The preparation of protein lysates was performed as described [32]. Approximately 5 105 cells were harvested at different times after etoposide treatment. Cells were resuspended in extraction buffer (50 mM NaF, 20 mM HEPES-KOH pH 7.8, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, proteinase inhibitors (antipain, leupeptin, pepstain A, chymostain)) and then frozen in liquid nitrogen and thawed in tap water three times. After centrifugation at 8,000 g for 30 min at 4 °C, clear supernatant was transferred to a fresh tube and stored at 80 °C. 2.8. Western blot Western blot analysis was performed as described previously [33,34]. HL-60 whole cell extracts were electrophoresed on SDSpolyacrylamide gels. Ku was analyzed on 10% SDS-polyacrylamide gels, and DNA-PKcs on 8% SDS-polyacrylamide gels. The proteins were transferred to a PVDF membrane for 1 h at 20 mA, and then 16 h at 40 mA, at 4 °C. The blot was rinsed in rinsing buffer (10 mM Tris–HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100), and placed in blocking buffer (5% non-fat milk, in rinsing buffer) for 1 h. Next, the blot was incubated with primary antibody at 1:1000 dilution in blocking buffer for 1 h to overnight for anti-Ku antiserum (OM) and anti-DNA-PKcs antibody. Following incubation with the primary antibody, the blot was rinsed twice with rinsing buffer, followed by one 15-min and three 5-min washes in rinsing buffer on a rocking platform. Anti-human or anti-rabbit IgG-AP secondary antibody was used at 1:5000 dilution in rinsing buffer. Incubation with secondary antibodies was performed at room temperature for 1 h. On following the same washing steps as described above, the complexes were detected with BCIP/NBT.
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Fig. 1. Effect of etoposide on the DNA probe-binding activity and protein level of Ku. (A) RI-EMSA: Two mg of protein extract obtained from untreated HL-60 cells was incubated with 32P-labeled dsDNA probe (15-bp or 28-bp) in the presence of 1 mg of closed circular DNA. (B) RI-EMSA: Two mg of protein extract obtained from untreated HL60 cells was incubated with 32P-labeled dsDNA probe (15-bp) and anti-human Ku polyclonal antiserum in the presence of 1 mg of closed circular DNA. (C) RI-EMSA: Two micrograms of protein extract obtained from cells treated with etoposide for the duration indicated was incubated with 32P-labeled, 15-bp dsDNA probe in the presence of 1 mg of closed circular DNA. (D) Western blot: 20 mg of protein extract obtained from either control or treated cells was run on a 10% SDS polyacrylamide gel under reducing conditions. Ku70 and Ku80 were detected using human polyclonal antiserum. Twenty micrograms of protein extract was run on a 8% SDS polyacrylamide gel under reducing conditions, and DNA-PKcs was detected using a rabbit polyclonal antibody.
2.9. Electrophoretic mobility shift assay (EMSA) RI-EMSA was performed using a modification of the RI-EMSA method used by Muller et al. [32]. Sequences of the oligonucleotides used for making double-stranded probes were 5′-cacaccgcatacgtc-3′, 3′-gtgtggcgtatgcag-5′, 5′-cacaccgcatacgtcaaagcaaccatag-3′, and 3′gtgtggcgtatgcagtttcgttggtatc-5′. For this analysis, 10 pmol of the 15 or 28 oligomer, T4 DNA polynucleotide kinase, and 3 mL of [γ-32P]ATP (3000 Ci/mmol) were incubated for 30 min at 37 °C. Then, 10 pmol of the complementary oligonucleotide was added; the mixture was incubated at 85 °C for 5 min, and subsequently cooled at room temperature. The DNA probe was purified with CENTRI-SEP COLUMNS (Princeton separation). Radiolabeled DNA, 1 mg of circular plasmid and 2 mg of whole cell extract were incubated with 1 binding buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 10% glycerol, 50 mM KCl) for 15 min at 27 °C. For experiments with mixed extracts from control and apoptotic cells, the extracts were co-incubated in 1 binding buffer for 5 min on ice. Radiolabeled DNA and circular plasmid were then added, and the reaction mixture was incubated for an additional 10 min prior to gel electrophoresis. The samples were electrophoresed on a 6% polyacrylamide gel in TBE at 4 °C for
1 h at 150 V. The gel was dried on Whatman paper and exposed to a phosphor imager plate for desired durations. Bands were visualized by phosphor imaging using the Bio-Image analyzer BAS 2500 (Fujifilm). Radioactivity in each band was quantified with an imaging analyzer. WB-EMSA: Unlabeled ds DNA (15-bp or 160-bp), circular plasmid, and 10 mg of whole cell extract were incubated with 1 binding buffer for 15 min at 27 °C. The samples were electrophoresed on a 6% polyacrylamide gel in TBE at 4 °C, for 1.5 h at 150 V. The proteins were transferred to a PVDF membrane. The membrane was analyzed as described in the western blot section above. 2.10. Immunofluorescence of Ku HL-60 cells were treated with etoposide for 4 h. The cells were fixed for 5 min by 70% methanol. After washing, the cells were blocked with 5% BSA in TBS for 30 min. The cells were incubated with 1:100 anti-Ku70 antibody (A9: sc-5309, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at RT followed by incubation with 1:100 FITC-conjugated anti-rabbit antibody for 1 h
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Fig. 2. Inhibitory effects of extract from etoposide-treated cells on Ku-DNA probe binding activity. (A) Radiolabeled ds DNA (15-bp) was incubated with extracts in binding buffer at 27 °C for 15 min (lanes 1 and 2). After mixing the extracts prepared from untreated cells and etoposide-treated cells, radiolabeled ds DNA was added and the mixture was incubated in binding buffer at 27 °C for 15 min (lane 3). Etoposide-treated cell extract was heat-denatured at 90 °C for 3 min and mixed with untreated cell extracts, and subsequently analyzed by RI-EMSA (lane 4). (B) Apoptotic cells with sub-G1 DNA content were analyzed by flow cytometry. The percentage of cells in the subG1 population is shown in each panel. (C) DNA fragmentation after treatment of HL-60 cells with etoposide. DNA was extracted from HL-60 cells treated with or without 10 mM etoposide for 4 h and subjected to agarose gel electrophoresis.
at 25 °C. To stain the DNA, the cells were incubated with Hoechst33825 for a few minutes, and then observed under a fluorescent microscope. 2.11. Measurement of Ku in apoptotic cells using flow cytometry HL-60 cells were treated with etoposide for 4 h, and then fixed for 5 min by 70% methanol. After washing, the cells were blocked with 5% BSA in TBS for 30 min. The cells were incubated with 1:100 anti-Ku70 antibody (A9: sc-5309, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at RT followed by incubation with 1:100 FITC-conjugated anti-rabbit antibody for 1 h at 25 °C. To measure the DNA content, the cells were washed with TBS, resuspended in TBS containing 0.5 mg/mL of RNase A, and incubated at 37 °C for 20 min. The cells were incubated with 50 mg/mL
propidium iodide in sodium citrate buffer for 10 min, and subsequently analyzed with a Becton-Dickinson FACSCalibur flow cytometer. 2.12. Rough purification of Ku Whole cell extract (100 mL) was diluted (1:10) with column buffer (50 mM Tris–HCl pH 7.6, 1 mM EDTA, 5% glycerol, 0.05% Tween 20, 0.5 mM PMSF) and cleared by passing through a 0.22μm filter. The filtrate was loaded on a Resource Q column (Amersham Biosciences, 1 mL) and equilibrated in column buffer containing 0.05 M KCl. The column was eluted with 5 mL of 0.05– 0.8 M linear gradient of KCl. The fractions were frozen in small aliquots and stored at 80 °C.
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Fig. 3. Effect of DNase I on the inhibitory effect of the apoptotic cell extract on Ku-DNA binding. Radiolabeled ds DNA (15-bp) was incubated with extracts in binding buffer at 27 °C for 15 min (lanes 1, 2). The mixture of extracts from untreated and etoposide-treated cells was incubated for 5 min on ice; then radiolabeled ds DNA was added, and the mixture was incubated in binding buffer at 27 °C for 15 min. Etoposide-treated extract was treated with DNase I with 2.5 mM MgCl2 at 37 °C for 30 min, and subjected to inactivation of DNase I by adding 2.5 mM EDTA and by heat-treatment at 65 °C for 10 min (lanes 3–7).
3. Results 3.1. Ku-DNA probe binding activity is decreased in apoptotic cells Ku protein plays an important role in DNA damage repair and prevention of cell death. Apoptosis causes a decrease in cellular DNA repair proteins. We examined whether Ku-DNA probe binding activity and Ku protein level were altered in the apoptotic cells. Whole cell extracts of the untreated and etoposide-treated cells were analyzed by RI-EMSA and western blot. In RI-EMSA, we mainly used a radiolabeled 15-bp DNA probe which only bound Ku70/80 heterodimer but not DNA-PKcs (Fig. 1A, lane 1), because Ku-DNA complex and other complexes were formed when using a 28-bp DNA probe (Fig. 1A, lane 2). The 15-bp DNA probe/Ku band was supershifted by anti-Ku antiserum (Fig. 1B). Ku-DNA probe binding activity dramatically decreased after 2 h of etoposide treatment, although the activity increased after 1 h of treatment (Fig. 1C). We investigated whether apoptosis led to a loss in the amount of DNA-PKcs and Ku in the cell. Ku protein was intact in the apoptotic cells, and etoposide-treatment did not result in loss of Ku protein. In contrast, DNA-PKcs appeared to be degraded after the induction of apoptosis with etoposide (Fig. 1D). These results demonstrated that Ku protein level was not regulated by proteases in the apoptotic cells. Nevertheless, Ku in apoptotic cells hardly bound to additional DNA probes. 3.2. Apoptotic cell extracts inhibit Ku-DNA probe binding activity To test the hypothesis that apoptotic cell extract has an inhibitory effect on Ku-DNA probe binding activity, we performed RI-EMSA with a mixture of extracts from apoptotic and untreated cells. Apoptotic cell extract inhibited Ku-DNA probe binding activity in untreated cell extract when examined by the RI-EMSA method (Fig. 2A, lane 3). Furthermore, heat-treated extract from apoptotic cells retained the inhibitory effect on Ku-DNA probe binding in untreated cell extract (Fig. 2A, lane 4). Our results suggest that a heat-resistant inhibitor is present in the apoptotic cells. DNA-gel electrophoresis showed that the HL-60 cells treated
with etoposide displayed DNA fragmentation in multiples of 180– 200 bp (Fig. 2C). Flow cytometric analysis of DNA content also showed that the percentage of cells with a sub-G1 DNA content increased after 2 h of etoposide treatment (Fig. 2B). These results indicate that nucleosome or/and endogenous DNA fragments is a candidate as the inhibitor of Ku-DNA probe binding activity in the apoptotic cells. 3.3. The inhibitory effect of apoptotic cell extract is decreased by DNase I treatment To further investigate the inhibitory effect of apoptotic cell extract on Ku-DNA probe binding activity, apoptotic cell extract was treated with DNase I. DNase I-treated extract from apoptotic cells could not inhibit Ku-DNA probe binding activity in untreated cells (Fig. 3). This result suggests that free DNA fragments or nucleosomes were present in the whole cell extract of apoptotic cells. 3.4. Ku-DNA probe binding activity is decreased during apoptosis but not differentiation HL-60 cells were induced macrophage-like differentiation and subsequently apoptosis by TPA-treatment (Sup. Fig. S2A). We investigated whether Ku in these cells bound to the DNA probe. KuDNA probe binding activity dramatically decreased after 3 days of TPA treatment (Sup. Fig. S2B), although the protein level is not changed by TPA-treatment (Sup. Fig. S2C). HL-60 cells treated with TPA displayed DNA fragmentation (Sup. Fig. S2D). Moreover apoptotic cell extract inhibited Ku-DNA probe binding activity in untreated cell extract. DNase I-treated extract from apoptotic cells could not inhibit Ku-DNA probe binding activity in untreated cells (Sup. Fig. S2E). These results suggest that Ku-DNA probe binding activity is decreased during apoptosis but not differentiation. 3.5. EMSA in conjunction with western blot (WB-EMSA) successfully detected the Ku/DNA complex Generally, Ku-DNA binding activity is analyzed by EMSA with a radiolabeled DNA probe. In the RI-EMSA assay, only Ku bound to
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Fig. 4. Analysis of Ku by WB-EMSA (Combination of EMSA with 15-bp non-labeled ds DNA and western blot). (A) Extracts from etoposide-treated HL-60 cells were separated by 6% native PAGE, and then analyzed by western blot with polyclonal antiserum raised against Ku (lane 1). Extracts from untreated HL-60 cells were incubated with 160-bp DNA, followed by western blot (lane 2). Extract from untreated or etoposide treated HL-60 cells was pre-incubated with 15-bp DNA, followed by western blot (lanes 3–6). (B) Extracts from untreated HL-60 cells were analyzed (lane 1). Extract from untreated or etoposide-treated HL-60 cells was pre-incubated with 15-bp DNA, followed by western blot (lanes 2, 3). After mixing the extracts prepared from untreated cells and etoposide treated cells, non-labeled ds DNA was added, and the mixture was incubated in binding buffer at 27 °C for 15 min (lane 4). (C) Comparison of EMSA combined with western blot (WB-EMSA) to conventional EMSA with 15-bp 32P-labeled DNA probe (RIEMSA). The diagram of RI-EMSA is a part of Fig. 1C, and the figure of WB-EMSA is a part of Fig. 4B.
the DNA probe can be detected, and Ku bound to endogenous DNA fragments of chromatin cannot be detected. To detect complexes between Ku and endogenous DNA, we performed EMSA in conjunction with western blot (WB-EMSA). WB-EMSA is a method which combines EMSA and western blot. This method allows distinction between the complex of additional 15-bp DNA/Ku and complexes of endogenous DNA/Ku (Fig. 4A and B). Activity of Ku
binding to 15-bp DNA was dramatically decreased after 2 h and 4 h of etoposide treatment (Fig. 4A, lanes 5 and 6). WB-EMSA of the apoptotic extract resembled that of the mixture of 160-bp DNA fragment and untreated cell extract (Fig. 4A, lanes 1 and 2). In addition, apoptotic cell extract inhibited Ku/15-bp DNA binding in untreated cell extract in WB-EMSA (Fig. 4B, lane 4). As shown in Fig. 4C, 15-bp DNA/Ku complex was detected by WB-EMSA as well
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Fig. 5. Purification of Ku on Resource Q column. (A) Whole cell extract from untreated or etoposide-treated HL-60 cells was loaded onto a 1 mL Resource Q column, and protein was subsequently eluted by a linear KCl gradient (50–800 mM). (B) The presence of Ku in the elutes was determined by immunoblotting after SDS-PAGE. (C) Analysis of Ku purified with Resource Q column with WB-EMSA. (D) The elutes were analyzed with RI-EMSA.
as RI-EMSA (Fig. 4C). These results indicate that Ku binds to the apoptotic DNA fragments and/or nucleosomes in the apoptotic cells. 3.6. Ku protein in apoptotic cell extract purified by Resource Q column binds to additional 15-bp DNA Ku protein in apoptotic cells was purified by Resource Q column and subsequently analyzed by western blot (Fig. 5A, B). The majority of Ku in both control and apoptotic cell extract was present in fractions #13 and #14. Purified Ku from control and apoptotic cell extracts displayed similar bands in WB-EMSA (Fig. 5C, lanes 1 and 2). These results indicate that the charge of Ku
protein in apoptotic and untreated cells is unchanged. Next, we investigated whether purified Ku was capable of binding 15-bp DNA. Purified Ku from apoptotic cell extract bound 15-bp DNA in WB-EMSA (Fig. 5C, lane 6), whereas Ku in pre-purified apoptotic extract did not (Fig. 5C, lane 4). Moreover, we investigated whether Ku protein in the apoptotic cell extract purified by Resource Q column bound DNA probe. As shown in Fig. 5D, Ku protein in the apoptotic cell extract purified by Resource Q column could bind radiolabeled DNA probe, while pre-purified Ku protein from apoptotic cells did not bind radiolabeled DNA probe. This suggests that Ku is intact and retains DNA binding activity in cells that undergo apoptotic induced by DSBs.
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Fig. 6. Localization and expression of Ku70 in apoptotic cells. (A) Localization of Ku70 in apoptotic cells with condensed chromatin. HL-60 cells were treated with etoposide for 4 h. Ku70 was stained with anti-Ku70 antibodies and FITC-conjugated anti-mouse antibody. (B) Expression of Ku70 in apoptotic cells. HL-60 cells were treated with etoposide for 4 h. Ku70 protein was stained as above, and apoptotic cells containing fragmented DNA were detected by staining with propidium iodide.
3.7. Localization and expression of Ku protein in apoptotic cells As shown in the figures above, Ku protein in apoptotic cell extract binds apoptotic DNA fragments and/or nucleosomes. We investigated whether Ku localized to the condensed chromatin in the apoptotic nuclei. As shown in Fig. 6A, Ku70 was localized to the condensed chromatin in HL-60 cells treated with etoposide (Fig. 6A). To assess the expression of Ku70 in apoptotic cells, HL-60 cells were immunostained with anti-Ku70 antibodies and the cellular DNA content was measured by flow cytometry. Ku70 expression was detected in apoptotic HL-60 cells in sub-G1 region (Fig. 6B, UL). These results indicate that Ku binds to fragmented DNA and/or nucleosomes during early apoptosis.
4. Discussion This study analyzed the protein expression of Ku and its DNA binding activity during early apoptosis. In the present study, Ku was neither cleaved nor degraded during early apoptosis induced by etoposide treatment. These results are consistent with those from a previous report [35]. Moreover, Ku DNA binding activity in the apoptotic cells is retained. Some previous studies have shown that Ku protein levels [36,37] and DNA binding activity [38,39] were reduced in the cells undergoing apoptosis. The discrepancy between results may be attributed to the cell death inducers, culture cells, or method used to measure Ku-DNA binding activity. The expression of Ku protein was decreased during staurosporineinduced apoptosis (Sup. Fig. S4). The present study is the first report showing that Ku can bind endogenous DNA fragments produced during apoptosis by using the two EMSA methods (RI-EMSA
and WB-EMSA). In most previous studies, Ku DNA binding activity was assayed with only RI-EMSA, and the influence of DNA fragments present in apoptotic cells was overlooked. However we could not completely exclude the possibility that Ku is digested by some proteases or post-translated. Ku proteins that are not binding to DNA may be modified or damaged as reported by other groups. The present study shows that apoptotic cell extracts inhibited Ku-DNA binding activity in non-treated cell extract in RI-EMSA, but not in WB-EMSA. Moreover, Ku in purified apoptotic cell extracts was able to bind exogenous 15-bp DNA, while Ku in unpurified extracts could not bind. These results suggest that Ku binds to cleaved chromosomal DNA in apoptotic cells. Chromatin DNA contains DNase I- and MNase-hypersensitive sites. Early in apoptosis, there is a rapid degradation of nuclease-hypersensitive euchromatin that contains hyperacetylated histones [40]. Ku protein can bind unfolded nucleosomes that lack histone H1 [41]. Therefore, Ku may bind free DNA fragments from chromatin DNA digested by caspase-activated DNase CAD (also known as DFF40) during early apoptosis. In this study, DNA/multiple Ku complex could be digested by DNase I, resulting in a DNA/single Ku complex (Sup. Fig. S1). This result suggested that Ku protein did not interfere with the digestion of apoptotic DNA fragment by DNase I. Ku in the extract from etoposide-treated HeLa can bind additional 15-bp DNA (Sup. Fig. S3A). Treatment with etoposide for 36 h could not increase the sub-G1 population in HeLa cells (Sup. Fig. S3C). These results suggest that the additional DNA binding activity of Ku is decreased during apoptosis with DNA fragmentation but not cell cycle arrest and topoisomerase II inactivation by etoposide. DNA-PK can be activated by nucleosomes through Ku binding to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes [42]. UV light exposure and etoposide treatment resulted in an increase of all histones in cell lysates compared to activated lymphoblasts. Furthermore, this increase of histone amount in cell lysates was accompanied by a considerable increase of early apoptotic cells, but no significant changes in late apoptotic cells or necrotic cells were observed [43]. In vitro studies showed that histone H1 and high mobility group (HMGB) enhance DFF40 function [44,45]. These data suggest that Ku may be related to chromatin component proteins and be involved in regulating after apoptosis system such as genomic DNA digestion and phagocytosis. In the present study, Ku is intact during early apoptosis, despite degradation of DNA repair proteins such as DNA-PKcs, according to our western blot analysis. Previous reports have also shown that these DNA repair proteins (DNA-PKcs and ATM) are inactivated by cleavage during apoptosis [35,46,47]. Cells undergoing apoptosis exhibit specific morphological changes, which include membrane blebbing, cytoplasmic and chromatin condensation, nuclear breakdown, and assembly of membrane-enclosed vesicles termed apoptotic bodies that are eventually subjected to phagocytosis. Normally, apoptotic cells are removed rapidly by phagocytic cells before apoptotic blebs can be released [43]. Hence, Ku may bind to fragmented chromatin DNA during early apoptosis and finally become cleaved upon phagocytosis. Binding of Ku to chromatin DNA fragments may prevent the release of Ku into the extracellular space. HMGB1, a chromatin protein, is capable of binding to chromatin in apoptotic cells. But HMGB1 in necrotic cells is released into the extracellular space, leading to promotion of inflammation [48]. Ku is a target of autoimmune conditions including systemic lupus erythematosus and Graves’ disease. Failure of apoptosis can lead to autoimmune diseases. Therefore, binding of Ku to apoptotic DNA fragments may be essential for preventing it from causing autoimmune diseases. In conclusion, our data demonstrated that Ku is intact and
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retains DNA binding activity in early apoptotic cells. In addition, Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. These findings may allow for a further understanding of the mechanism of interaction of Ku with DNA during apoptosis and the cause of autoimmune diseases.
Acknowledgement We thank Prof. Fumiaki Yamao and Dr. Toyoaki Natsume for scientific advice and technical support on RI-EMSA. We thank Prof. Shinsuke Fujiwara and Prof. Yoshinori Toyoshima for many useful discussions. We thank Ms. Megumi Mizuno for experimental support.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2016.03.011.
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