Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells Ikuko Kitazumi, Yoko Maseki, Yoshiko Nomura, Akiko Shimanuki, Yumi Sugita and Masayoshi Tsukahara Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd, Hagiwara, Takasaki, Gunma, Japan Introduction Apoptosis is a crucial cellular mechanism that is involved in inflammation, cell differentiation, and cell proliferation. Apoptotic cells are characterized by dis- tinctive morphological and biochemical changes, including plasma membrane blebbing, nuclear conden- sation, DNA fragmentation, and phosphatidylserine (PS) exposure. These changes are largely mediated by the activation of caspases, a family of cysteinyl aspartate-specific proteases whose target proteins are particularly important indicators of the apoptotic sig- nal pathway. The activation of other proteases also plays a key role in apoptotic cell death. They consti- tute alternative signal pathways that frequently overlap the caspase-dependent pathway. Among them, caspase-3 is involved in nuclear changes by cleaving substrates such as poly(ADP-ribose) polymerase (PARP), an Keywords apoptosis; caspase-3; caspase inhibitor; DNA fragmentation; okadaic acid Correspondence M. Tsukahara, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd, 100-1 Hagiwara, Takasaki, Gunma 370-0013, Japan Fax: +81 27 353 7400 Tel: +81 27 353 7382 E-mail: masayoshi.tsukahara@ kyowa-kirin.co.jp Database The sequences for CHO-K1 caspase-3 cDNA reported in this article have been submitted to the GenBank database under the accession number FJ940732 (Received 10 June 2009, revised 23 October 2009, accepted 11 November 2009) doi:10.1111/j.1742-4658.2009.07493.x DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine⁄ threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase- 3 ) ⁄ ) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycar- bonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3 ) ⁄ ) cells, sug- gesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA frag- mentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmenta- tion. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmenta- tion were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3. Abbreviations AAD, aminoactinomycin; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; CHO, Chinese hamster ovary; DIG, digoxigenin; NaCl ⁄ P i (–), calcium and magnesium-free phosphate buffered saline; OA, okadaic acid; PARP, poly(ADP-ribose) polymerase; PP, protein phosphatase;p PS, phosphatidylserine; TLCK, N-a-tosyl- L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone. 404 FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS inhibitor of caspase-activated DNase ⁄ DNA fragmenta- tion factor 45 (ICAD ⁄ DFF45) [1–3]. Cells have multiple cleavage mechanisms, as shown by cleavage induction of these substrates in caspase-3-deficient cells [4,5]. Various methods are used for blocking caspase activa- tion. One example is the addition of caspase inhibitors, which are frequently used to define caspase-independent events. However, it is unclear whether caspase inhibitors actually prevent caspase activation or whether they effectively block caspase-dependent apoptotic events. Caspase inhibition is ineffective [6], although some reports suggest that the caspase-3 ⁄ 7 inhibitor is effective at blocking DNA fragmentation [7–9]. Furthermore, caspase inhibitors have been found to inhibit more than just the targeted caspase. This uncertainty over the requirement of caspase-3 for DNA fragmentation led us to target it for gene knockout in the pres- ent study. We determined the genome sequence of Chinese hamster ovary (CHO)-K1 cells, and produced caspase-3-deficient CHO-K1 (caspase-3 ) ⁄ ) ) cells to examine the differences between treatment with inhibi- tors and gene knockout on DNA fragmentation, and to investigate whether caspase-3 is required for DNA fragmentation. We used okadaic acid (OA), a potent inhibitor of the serine ⁄ threonine protein phosphatase (PP) type 1 (PP1) and type 2 (PP2A), to induce caspase-3 activa- tion and apoptotic cellular reactions, including DNA fragmentation. Inhibition of PP2A (low OA concen- tration) reduces apoptosis by decreasing mitochon- drial cytochrome c release, whereas inhibition of PP1 (high OA concentration) induces apoptosis through p53-dependent cell death pathways [10–12]. It is unclear whether OA-induced caspase-3 activation is necessary for apoptosis; one study demonstrated that caspase-3 is important in the process of OA-induced apoptosis [13], whereas another showed that OA induces apoptosis in the human breast carcinoma cell line MCF-7, which lacks expression of caspase-3 [14]. Furthermore, a caspase inhibitor was reported to block OA-induced caspase-3 activity but not apopto- sis [15]. Here, OA induced DNA fragmentation in both CHO-K1 caspase-3 ) ⁄ ) cells and CHO-K1 cells treated with the caspase-3 ⁄ 7 inhibitor benzyloxycarbonyl-Asp- Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD- fmk), indicating that caspase-3 is not essential for DNA fragmentation. However, z-DEVD-fmk blocked OA-induced DNA fragmentation in caspase-3 ) ⁄ ) cells, but not in CHO-K1 cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation indepen- dently from DEVDase activity, and that DEVDase or DEVDase-like activity is involved in DNA fragmenta- tion in caspase-3 ) ⁄ ) cells. Furthermore, the wide- ranging serine protease inhibitor 4-(2-aminoethyl)- benzenesulfonyl fluoride hydrochloride (AEBSF) blocked caspase-3 activation and DNA fragmentation, suggesting that serine proteases are involved in DNA fragmentation, probably upstream of caspase-3 activation. Results Sequencing of CHO-K1 full-length caspase-3 cDNA and genomic structure Full-length CHO-K1 cell caspase-3 cDNA was isolated using 5¢-RACE and 3¢-RACE, and used to determine the genomic sequence of caspase-3. We confirmed that the cDNA sequence is coincident with the genomic sequence. Our caspase-3 cDNA sequence differed from the GenBank database sequence (accession no. AY479976; see Fig. S1). A caspase-3 gene-targeting vector was constructed according to our sequence, and used to knock out exons 4–6 of the CHO-K1 cell cas- pase-3 gene (Fig. 1A). Exon 6 contains the cleavage site that is required for its activation [16]. Gene knock- out was confirmed by Southern blot analysis, which revealed additional diagnostic bands from the targeted alleles, as expected (Fig. 1B). Western blotting with a caspase-3 antibody did not detect pro-caspase-3 expression after gene knockout (Fig. 1C). The caspase-3/7 inhibitor z-DEVD-fmk causes differential effects on OA-induced DNA fragmentation in CHO-K1 and caspase-3 ) / ) cells To establish whether caspase-3 is required for OA-induced DNA fragmentation in CHO-K1 cells, we investigated the differences in OA response between CHO-K1 and caspase-3 ) ⁄ ) cells. As shown in Fig. 2A,B, both cell types similarly showed PS expo- sure and DNA fragmentation, which are characteristic of apoptosis. OA induced DNA fragmentation both with and without caspase-3, indicating that caspase-3 is not essential for the induction of DNA fragmenta- tion. Next, CHO-K1 cells were stimulated with OA in the presence of z-DEVD-fmk. DNA fragmentation still occurred (Fig. 2A), as well as PS exposure and cleav- age of the caspase-3 active form, although caspase-3- specific DEVDase activity was completely blocked (Fig. 2B,C). These results indicate that z-DEVD-fmk is unable to prevent apoptotic cell reactions, including DNA fragmentation, supporting the above finding that caspase-3 is not essential for OA-induced DNA frag- mentation in CHO-K1 cells. I. Kitazumi et al. Involvement of caspase-3 in DNA fragmentation FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS 405 Although caspase-3 gene knockout or inhibition of caspase-3 activity is ineffective in preventing OA-induced DNA fragmentation in CHO-K1 cells, z-DEVD-fmk was successful at inhibiting OA-induced DNA fragmentation in caspase-3 ) ⁄ ) cells (Fig. 2A). The difference between CHO-K1 cells and caspase-3 ) ⁄ ) cells is the presence of caspase-3, and it is hypothesized that caspase-3 activity, and not DEVDase activity of cas- pase-3, is involved in the mechanism by which OA induces DNA fragmentation in CHO-K1 cells. In the case of caspase-3 ) ⁄ ) cells, DEVDase or DEVDase-like activity may play an important role in inducing DNA fragmentation. OA induces caspase-dependent and caspase-independent DNA fragmentation in the presence of caspase-3 We next examined whether the activation of other caspases is required for DNA fragmentation involving caspase-3. In the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk), OA-induced DNA fragmentation was reduced but not completely inhibited in CHO-K1 cells (Fig. 2A). Therefore, OA induced DNA fragmentation in both a caspase activation-independent and caspase activation-dependent manner. By contrast, z-VAD-fmk completely blocked OA-induced DNA fragmentation in caspase-3 ) ⁄ ) cells, indicating that only caspase Probe Cleavage cite Pro -caspase-3 n.s. A ScaI pCasp3-Hygro pCasp3-Zeo Wild-type allele Hygro-target allele Zeo-target allele C Wild type (5 kb) Zeo Hygro Zeo-targeted Hygro-targeted (3.1 kb) NS (4.5 kb) CHO-K1 B Caspase-3 –/– CHO-K1 Caspase-3 –/– Hygro Zeo Fig. 1. Establishment of caspase-3 ) ⁄ ) cells. (A) Targeted disruption of the caspase-3 gene. Targeting vector constructs, the wild-type allele and targeted mutant alleles are shown. The open box indi- cates hygromycin (Hygro)-resistance or zeocin (Zeo)-resistance gene; the shaded box in the wild-type allele indicates targeted ex- ons. The arrow indicates the caspase-3 cleavage site in the target exon. (B) Strategy to differentiate homologous recombinants from the wild-type allele by Southern blot analysis, using a 3¢ external probe (black box). Hybridization of ScaI-digested CHO-K1 cell geno- mic DNA to the probe gives a 5 kb DNA band. Targeted integration is revealed by the appearance of an additional 3.1 kb diagnostic band from the target allele with the pCasp3–Hygro vector and a 4.5 kb band from the targeted allele with the pCasp3–Zeo vector. (C) Lack of pro-caspase-3 expression in untreated cells, detected by western blot analysis using antibody against caspase-3. A B C CHO-K1 Caspase-3 –/– OA z-DEVD-fmk z-VAD-fmk DNA fragmentation – – – + – – + + – + – + + + + – – – + – – + + – + – + + + + CHO-K1 OA z-DEVD-fmk z-VAD-fmk Cleaved caspase-3 /inhibitor Cleaved cas p ase-3 Pro-caspase-3 – – – + – – + + – + – + + + + CHO-K1 Caspase-3 –/– PS (+) cells 20 - 40 - 60 - 80 - 0 - 100 - Caspase3 activity 150 - 100 - 50 - 0 - OA z-DEVD-fmk z-VAD-fmk – – – + – – + + – + – + + + + – – – + – – + + – + – + + + + Fig. 2. Effects of caspase-3 gene knockout and caspase inhibitors in apoptotic cell reactions. CHO-K1 and caspase-3 ) ⁄ ) cells were preincubated with 50 l M caspase-3 ⁄ 7 inhibitor z-DEVD-fmk and pan-caspase inhibitor z-VAD-fmk for 1 h, and then stimulated with 300 n M OA for 24 h. (A) DNA fragmentation was analyzed on 2% agarose gels. (B) Caspase-3-specific DEVDase activity and percentages of PS-exposing cells. Upper panel: DEVDase enzyme activities were measured as pmol pNA liberated ⁄ h ⁄ lg total protein. Lower panel: percentage of PS-exposing cells (annexin V + ⁄ 7-AAD ) and annexin V + ⁄ 7-AAD + ) was determined by double staining with annexin–phycoerythrin to detect PS-exposing cells, and 7-AAD to detect dead cells. Values are expressed as means ± standard deviations from four separate experiments. (C) No inhibitory effect of caspase inhibitors in caspase-3 processing. Cell extracts were subjected to western blot analysis using anti- body against caspase-3. Involvement of caspase-3 in DNA fragmentation I. Kitazumi et al. 406 FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS activation-dependent DNA fragmentation occurred in the case of caspase-3 deficiency. In addition, z-VAD- fmk blocked caspase-3-specific DEVDase activity but did not inhibit caspase-3 cleavage and PS exposure like z-DEVD-fmk (Fig. 2B,C). There was no change when z-DEVD-fmk and z-VAD-fmk pretreatments were applied together, suggesting that caspase-3, with the exception of DEVDase activity, contributes to both OA-induced caspase activity-independent and caspase activity-dependent DNA fragmentation. Serine proteases are involved in OA-induced DNA fragmentation upstream of caspase-3 It was previously reported that the serine protease inhibitors N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and N-a-tosyl-l-lysine chloromethyl ketone (TLCK) did not block caspase-3 cleavage, although they substantially inhibited DEVDase activity [17]. On the other hand, it was also reported that the general serine protease inhibitor AEBSF indirectly inhibits cas- pase-3 cleavage and DEVDase activity [18]. To inhibit caspase-3 activity in an alternative way, CHO-K1 cells were pretreated with TPCK, TLCK, and AEBSF. Like the caspase inhibitors, TPCK and TLCK were unable to inhibit DNA fragmentation and cleavage of caspase-3, but caspase-3-specific DEVDase activity was reduced (Fig. 3A–C). AEBSF blocked caspase-3-spe- cific DEVDase activity and cleavage of caspase-3 (Fig. 3B,C), and also inhibited OA-induced DNA frag- mentation (Fig. 3A), suggesting that AEBSF-sensitive, but TPCK-insensitive or TLCK-insensitive serine pro- teases, are involved in OA-induced DNA fragmenta- tion upstream of caspase-3 cleavage. In the case of caspase-3 ) ⁄ ) cells, OA-induced DNA fragmentation was also blocked by AEBSF but not by TPCK or TLCK (Fig. 3A), indicating that AEBSF-sensitive but TPCK-insensitive or TLCK-insensitive serine proteases mediate OA-induced DNA fragmentation indepen- dently of caspase-3. Additionally, AEBSF inhibited OA-induced PS exposure in both CHO-K1 cells and caspase-3 ) ⁄ ) cells (Fig. 3B), suggesting that AEBSF- sensitive, but TPCK-insensitive or TLCK-insensitive, serine proteases mediate apoptotic reactions, inducing DNA fragmentation, upstream of caspase-3 cleavage or caspase-3-independently. Caspase inhibitors decrease PARP cleavage only in the absence of caspase-3 The nuclear repair enzyme PARP is activated in response to DNA damage. PARP degradation is used as an indicator of caspase-3 activity because it is one of the main caspase-3 substrates involved in genomic processes [19]. Knockout of caspase-3 and treatment with z-DEVD-fmk had no effect on OA-induced PARP degradation (Fig. 4), so caspase-3 is not essen- tial for PARP degradation. Treatment with z-VAD- fmk had little effect on PARP degradation in CHO-K1 cells; by contrast, z-DEVD-fmk slightly inhibited and z-VAD-fmk partially inhibited OA-induced PARP deg- radation in caspase-3 ) ⁄ ) cells (Fig. 4). These results indicate that caspase-3 is involved in caspase-indepen- dent intranuclear reactions and that OA-induced PARP degradation is highly dependent on caspase activation in the absence of caspase-3. In addition, TPCK and TLCK did not inhibit PARP degradation, but it was completely blocked by AEBSF in both cell types, supporting the idea that AEBSF-sensitive, but A B C OA TPCK TLCK AEBSF CHO-K1 Cleaved caspase-3 Pro-caspase-3 – – – – + – – – + + – – + – + – + – – + CHO-K1 Caspase-3 –/– OA TPCK TLCK AEBSF – – – – DNA fragmentation + – – – + + – – + – + – + – – + – – – – + – – – + + – – + – + – + – – + CHO-K1 Caspase-3 –/– OA TPCK TLCK AEBSF – – – – + – – – + + – – + – + – + – – + – – – – + – – – + + – – + – + – + – – + - - - - - - - - - - 150 100 50 0 Caspase3 activity 20 40 60 80 0 100 PS (+) cells Fig. 3. Effect of serine protease inhibition on OA-induced apoptotic cell reactions. CHO-K1 and caspase-3 ) ⁄ ) cells were stimulated with 300 n M OA for 24 h in the presence of 10 lM TPCK, 100 lM TLCK, or 400 l M AEBSF. Inhibitors were added 1 h before OA addition. (A) DNA fragmentation. (B) Caspase-3-specific DEVDase activity (upper panel) and percentages of PS-exposing cells (lower panel). (C) Cleavage of caspase-3. I. Kitazumi et al. Involvement of caspase-3 in DNA fragmentation FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS 407 TPCK-insensitive or TLCK-insensitive, serine prote- ases mediate apoptotic reactions upstream of caspase-3 cleavage or caspase-3-independently. Discussion Capase-3 is a key mediator of apoptosis, and most apoptotic pathways lead to its activation, resulting in the cleavage of a wide range of cytoplasmic and nuclear proteins. However, it is widely reported that inactivation or an absence of caspase-3 does not inhi- bit apoptosis [20,21]. DNA fragmentation, one of the hallmarks of apoptotic cells, is also evident in caspase- 3-deficient cells [22]. By contrast, it has been shown that caspase-3-deficient cells fail to exhibit DNA frag- mentation [23,24]. It does not necessarily appear that the requirement for caspase-3 for DNA fragmentation may depend on the combination of cell type and level of stimulation. In this study, we found that OA induced DNA frag- mentation in both caspase-3 ) ⁄ ) cells and z-DEVD- treated CHO-K1 cells (Fig. 2A), indicating that caspase-3 is not essential for OA-induced DNA frag- mentation in CHO-K1 cells. It is unlikely that the discrepancy between previously reported caspase-3- deficient cells and our own caspase-3 ) ⁄ ) cells in the induction of DNA fragmentation is caused by different mechanisms of caspase-3 deletion or different stimuli. For instance, the caspase-3-deficient cell line MCF-7 is frequently used to investigate the role of caspase-3 in apoptosis. MCF-7 caspase-3 deficiency is due to exon 3 skipping during splicing of the caspase-3 pre-mRNA [23], but although our caspase-3 ) ⁄ ) cells lacked exons 4–6, both cell types underwent apoptosis in response to OA. We also confirmed that the protein kinase C inhibitor staurosporine, which has been shown to induce caspase activation and DNA fragmentation [6], induced DNA fragmentation in caspase-3 ) ⁄ ) cells (data not shown). However, even if gene knockout and treatment with inhibitors do not affect a reaction, the factor being blocked may nevertheless still be involved in the reaction. For example, this study showed that z- DEVD-fmk blocked DNA fragmentation in caspase-3- deficient cells but not in caspase-3-expressing cells (Fig. 2A). Although caspase-3 is therefore not essential for DNA fragmentation, it still maintains its function to induce DNA fragmentation in the absence of DEVDase activity. Apoptosis is induced not only by the caspase-depen- dent process, but also by other processes that do not require caspase activation. As shown in Fig. 2A,C, OA-induced DNA fragmentation was not completely blocked by z-VAD-fmk in CHO-K1 cells. In the pres- ence of caspase-3, therefore, OA can induce DNA fragmentation without caspase activation. Addition- ally, z-VAD-fmk partially blocked DNA fragmenta- tion, as compared with only slight inhibition by z-DEVD-fmk. Although OA can induce caspase-inde- pendent DNA fragmentation, it is highly dependent on caspase activation. By contrast, OA-induced DNA fragmentation in caspase-3-deficient cells was depen- dent on caspase activity. In support of these findings, it was previously shown that DNA fragmentation occurs through at least two redundant parallel path- ways: caspase-dependent and caspase-independent [22,25]. In the case of CHO-K1 cells, OA appears to induce DNA fragmentation by at least three different pathways: caspase-3 and other caspase activation- dependent pathways not involving DEVDase activity; caspase-3-dependent and other caspase activation-inde- pendent pathways not involving DEVDase activity; and other caspase activation-dependent pathways involving DEVDase activity. It therefore appears that the induction pathway of apoptosis varies according to the type of caspase involved. Caspase-3 is initially synthesized as a zymogen. During apoptosis, pro-caspase-3 is cleaved sequentially to generate the active p17 and p12 subunits that form the active heterotetramer [26]. Here, treatment of CHO-K1 cells with z-DEVD-fmk and z-VAD-fmk caused cleavage of caspase-3 despite inhibition of cas- pase-3 DEVDase activity (Fig. 2B,C). It is unlikely that the failure to inhibit caspase-3 cleavage in CHO- K1 cells was due to insufficient amounts of caspase inhibitors, because OA-induced caspase-3 cleavage was not inhibited when CHO-K1 cells were treated with high concentrations of caspase inhibitors (data not shown). The caspase inhibitors used in this study irre- versibly bind to activated caspases. Following inhibitor treatment, we detected caspase-3 fragments with a slightly higher molecular mass than fragments that had not been treated (Fig. 2C). This molecular mass kDa CHO-K1 Caspase-3 –/– OA z-DEVD-fmk z-VAD-fmk TPCK TLCK AEBSF – – – – – – + – – – – – + + – – – – + – + – – – + + + – – – + – – + – – + – – – + – + – – – – + – – – – – – + – – – – – + + – – – – + – + – – – + + + – – – + – – + – – + – – – + – + – – – – + 150 - 100 - PA RP Cleaved PARP Fig. 4. Differential effect of caspase inhibitors and serine protease inhibitors on PARP degradation between CHO-K1 and caspase-3 ) ⁄ ) cells. CHO-K1 and caspase-3 ) ⁄ ) cells were preincubated with each inhibitor for 1 h, and then stimulated with 300 n M OA for 24 h. Cell extracts were subjected to western blot analysis using antibody against PARP. Involvement of caspase-3 in DNA fragmentation I. Kitazumi et al. 408 FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS change may be caused by binding of caspase inhibi- tors. It was previously shown that processing of cas- pase-3 still occurred in the presence of z-VAD-fmk, but that fragments were inactive because they bound caspase inhibitors [18]. Although caspase inhibitors bind to activated caspase-3 and inhibit DEVDase activity, proteolytic activity of caspase-3, including substrate cleavage and DNA fragmentation, still occurs in CHO-K1 cells. Activities inhibited by caspase inhibitors (e.g. DEVDase activity) do not appear to be involved in inducing apoptotic responses. It was previously reported that serine proteases were involved in the cascade to affect the cleavage step of caspase-3 and PARP degradation [27]. As an example, the proapoptotic mitochondrial serine protease Omi ⁄ HtrA2 is released into the cytosol during apopto- sis, and enhances caspase activation by inactivating inhibitor of apoptosis proteins. The serine protease activity of Omi ⁄ HtrA2 is also able to induce direct degradation of inhibitor of apoptosis proteins and cas- pase-independent cell death [28,29]. In this study, TPCK and TLCK could not inhibit caspase-3 cleav- age, and thus subsequently induce PARP degradation and DNA fragmentation. On the other hand, AEBSF completely blocked caspase-3 cleavage, PARP degrada- tion, and DNA fragmentation. It is possible that TPCK-insensitive or TLCK-insensitive but AEBSF- sensitive serine proteases are involved in PARP degra- dation and DNA fragmentation via caspase-3 process- ing. However, AEBSF also blocked PARP degradation and DNA fragmentation in the absence of caspase-3, indicating that serine proteases are involved in nuclear changes via caspase-3-independent pathways. As DEVDase activity plays an important role in OA-induced DNA fragmentation in caspase-3-deficient cells, what induces DNA fragmentation in caspase-3- deficient CHO-K1 cells remains to be clarified. Cas- pase-7 also demonstrates DEVDase activity, and it has been reported that caspase-3 and caspase-7 have some overlapping substrate specificities, because they share a common DXXD motif, although they are functionally distinct. In the absence of caspase-3, caspase-7 is a potential substitute and also cleaves PARP but at a different level of activity [30]. It has also been shown that caspase-7 is as efficient as caspase-3 for inducing DNA fragmentation in caspase-3 knockout mice [31], and has a much higher affinity for PARP than cas- pase-3 in vitro [19]. We confirmed that OA also induced caspase-7 cleavage in CHO-K1 cells and cas- pase-3 ) ⁄ ) cells, but caspase-7 cleavage still occurred even though DNA fragmentation was inhibited in caspase inhibitor-treated caspase-3 ) ⁄ ) cells (Fig. S2). If caspase-7 is involved in DNA fragmentation, it requires DEVDase activity. However, it was previously reported that OA cleaves caspase-2 but not caspase-7 in MCF-7 cells [14], and that factors other than cas- pase-7 are involved in OA-induced DNA fragmenta- tion. Although caspase-7 is the most likely substitute factor for caspase-3, it is uncertain whether it plays an important role in DEVDase-dependent DNA fragmentation. Different levels of involvement in the apoptotic pathway may be partly due to the cellular localization of caspases. Caspase-3 and caspase-7 have different subcellular distributions, and are functionally distinct. Caspase-7 translocates from the cytosol to the micro- somes during apoptosis, but does not translocate to the nucleus. By contrast, pro-caspase-3 is localized in the cytoplasm and, when activated, caspase-3 trans- locates to the nucleus after inducing apoptosis [32]. The nuclear translocation of active caspase-3 requires the cleavage of caspase-3 and substrate recognition, but does not require substrate degradation [16]. In this study, induction of DNA fragmentation is correlated with induction of caspase-3 cleavage. An inability to block caspase-3 cleavage may cause the nuclear trans- location of active caspase-3 and DNA fragmentation in CHO-K1 cells. It appears that caspase-3 can trans- locate to the nucleus and degrade intranuclear proteins regardless of whether inhibitors are bound. Following AEBSF treatment, active caspase-3 cannot be formed, and therefore caspase-3 cannot translocate to the nucleus and induce DNA fragmentation. In conclusion, we have shown that caspase-3 is involved in OA-induced DNA fragmentation, even though gene knockout of caspase-3 and treatment with caspase inhibitors are ineffective in inhibiting DNA fragmentation. It appears that activation of caspase-3 without DEVDase activity is required for reacting sub- strates. Additionally, OA induces caspase-dependent and caspase-independent DNA fragmentation, but cas- pase-independent DNA fragmentation only occurs in the presence of caspase-3. Caspase-3 is an important contributor to apoptosis, but alternative pathways exist that provide different mechanisms of apoptotic reactions to those involving caspase-3. Treatment with caspase inhibitors may therefore be insufficient for examination of the involvement of caspases. Experimental procedures Reagents and antibodies The apoptosis-inducing drug OA (sodium salt) was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). The caspase-3 ⁄ 7 inhibitor z-DEVD-fmk I. Kitazumi et al. Involvement of caspase-3 in DNA fragmentation FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS 409 and the pan-caspase inhibitor z-VAD-fmk were purchased from Medical & Biological Laboratories (Aichi, Japan). The serine protease inhibitors TPCK and TLCK were purchased from Sigma-Aldrich (St Louis, MO, USA), and the wide-ranging serine protease inhibitor AEBSF was obtained from Roche Diagnostics (Basel, Switzerland). Human caspase-3 (CPP32) Ab-4 rabbit polyclonal antibody was purchased from Lab Vision (Fremont, CA, USA), and human mouse monoclonal antibody against PARP (A6.4.12) was obtained from Abcam (Cambridge, UK). Cell lines The CHO-K1 cell line was obtained from the ATCC. Cell lines were cultured in MEM-a (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO 2 . Cloning of CHO-K1 caspase-3 cDNA and genome structure Single-stranded caspase-3 cDNA was synthesized from total RNA extracted from CHO-K1 cells using Isogen (Nippon Gene, Tokyo, Japan). Double-stranded cDNA was synthe- sized, and its sequence was determined by 5¢-RACE and 3¢-RACE. RACE primers (5¢-GGAGAACACTGAAAACT CAGTGGATTC-3¢ and 5¢-TGGATGAACCAGGAGCCA TCC-3¢) were designed according to GenBank database coding sequence (CDs) of CHO-K1 caspase-3 cDNA (accession no. AY479976) and Syrian hamster mRNA (accession no. U27463). The caspase-3 genome sequence was determined from our cDNA (Genbank accession no. FJ940732; see Fig. S1). Caspase-3 gene knockout Targeting vectors containing a hygromycin resistance or zeocin resistance gene driven by the cytomegalovirus pro- moter were constructed to include exons 4–6 of the cas- pase-3 gene. Genomic DNA corresponding to 8.3 kb of the 5¢-homologous arm and 2.2 kb of the 3¢-homologous arm was subcloned into the 5¢-sites and 3¢-sites of the hygromy- cin resistance (pCasp3–Hygro) and zeocin resistance (pCasp3–Zeo) genes, respectively (Fig. 1A). Cells were transfected with targeting vectors using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, and selected with hygromycin or zeocin. Genomic southern blot analysis Genomic DNA isolated from CHO-K1 and caspase-3 ) ⁄ ) cells was digested with ScaI, electrophoresed, and transferred to Hybond-N + membranes (GE Healthcare, Chalfont St Giles, UK). Membranes were hybridized in digoxigenin (DIG) Easy Hyb (Roche Diagnostics) with a DIG-labeled probe prepared using the PCR DIG Probe Synthesis Kit (Roche Diagnostics). PCR labeling was carried out with the primer pair 5¢-CAGTACAGCT ACCTCAAGTGCAACA TC-3¢ and 5¢-GGTGACAGTC CTTTCTGAAGCTGTG-3¢, according to the manufac- turer’s instructions. Signals were visualized with the DIG system (Roche Diagnostics). Detection of DNA fragmentation Treated cells were washed with cold NaCl ⁄ P i (–) (calcium- and magnesium-free phosphate buffered saline) and fixed in ice-cold 70% ethanol at )20 °C. Ethanol was removed, and cells were resuspended in PC buffer (192 lm Na 2 HPO 4 ,4lm citric acid). After incubation for 20 min at room tempera- ture, cell suspensions were centrifuged at 17 400 g and 4 °C for 20 min. Supernatants were collected and incubated with DNase-free RNase A (100 mgÆmL )1 ; Qiagen, Hilden, Ger- many) for 1 h at 37 °C, and then incubated with proteinase K (Qiagen) for 30 min at 50 °C. DNA was analyzed on 2% agarose gels and visualized by ethidium bromide staining. Measurement of PS exposure The level of PS exposure was measured by the extent of annexin V–phycoerythrin binding, using the Guava Nexin Reagent and Guava EasyCyte Plus System (Millipore, Bill- erica, MA, USA). After treatment, cells were harvested, washed with cold NaCl ⁄ P i (–), and then mixed with Guava Nexin Reagent according to the manufacturer’s instruc- tions. Stained cells were acquired on a Guava EasyCyte Plus System. The two-dye strategy allows for identification of four cell populations: nonapoptotic cells, annexin V ) ⁄ 7- aminoactinomycin (AAD) ) ; early apoptotic cells, annexin V + ⁄ 7-AAD ) ; late-stage apoptotic and dead cells, annexin V + ⁄ 7-AAD + ; and necrotic cells and mostly nuclear debris, annexin V ) ⁄ 7-AAD + . Western blot analysis After treatment, cells were washed with cold NaCl ⁄ P i (–) and lysed in lysis buffer [30 mm Tris ⁄ HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 1 mm dithiothreitol, 10% glycerol]. Equal amounts of total protein were subjected to SDS ⁄ PAGE and transferred to nitrocellulose membranes. The membranes were blocked with blocking buffer [18 mm Tris, 450 mm NaCl, 0.09% Tween-20, 0.4% Block Ace (DS Pharma Biomedical, Osaka, Japan)] for 1 h at room tem- perature, and incubated overnight at 4 °C with primary antibodies in blocking buffer. They were then washed in 20 mm Tris, 500 mm NaCl, and 0.1% Tween-20, incubated for 5 h at room temperature with secondary antibodies in blocking buffer, washed in 20 mm Tris, 500 mm NaCl, and Involvement of caspase-3 in DNA fragmentation I. Kitazumi et al. 410 FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS 0.1% Tween-20, and rinsed in NaCl ⁄ Tris (20 mm Tris, 500 mm NaCl). Proteins were detected using SuperSignal West Dura Extended Duration Substrate [Pierce (part of Thermo Fisher Scientific), Waltham, MA, USA]. 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J Neurosci 24, 9977–9984. 32 Chandler JM, Cohen GM & MacFarlane M (1998) Dif- ferent subcellular distribution of caspase-3 and caspase- 7 following Fas-induced apoptosis in mouse liver. J Biol Chem 273, 10815–10818. Supporting information The following supplementary material is available: Fig. S1. CHO-K1 cell caspase-3 cDNA sequence. Fig. S2. Okadaic acid (OA)-induced caspase-7 cleavage. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Involvement of caspase-3 in DNA fragmentation I. Kitazumi et al. 412 FEBS Journal 277 (2010) 404–412 ª 2009 The Authors Journal compilation ª 2009 FEBS . Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells Ikuko. blocked OA-induced DNA fragmentation in caspase-3 ) ⁄ ) cells, but not in CHO-K1 cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation indepen- dently