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RNAi knockdown of caspase-activated DNase inhibits rotenone-induced DNA fragmentation in HeLa cells
Neurochemistry International 50 (2007) 601–606 www.elsevier.com/locate/neuint
RNAi knockdown of caspase-activated DNase inhibits rotenone-induced DNA fragmentation in HeLa cells Tadamiki Tsuruta, Kentaro Oh-hashi, Yoshihito Ueno, Yukio Kitade, Kazutoshi Kiuchi, Yoko Hirata * Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Received 8 July 2006; received in revised form 21 November 2006; accepted 5 December 2006 Available online 18 January 2007
Abstract Rotenone, an inhibitor of mitochondrial complex I, induces apoptosis in a variety of cells. However, little is known about endogenous endonucleases involved in rotenone-induced DNA fragmentation, a biochemical hallmark of apoptosis. We used a chemically modified siRNA which is thought to be more effective than a non-modified siRNA to study whether caspase-activated DNase (CAD) contributes to this phenomenon. Western blot analysis showed that CAD protein decreased to 8% of control levels in human cervical carcinoma HeLa cells after a 48 h transfection of siRNA. Consistent with the reduction of the protein level, the siRNA was found to inhibit rotenone-induced DNA fragmentation. These results suggest that CAD is the endogenous endonuclease that mediates internucleosomal DNA degradation in rotenone-induced apoptosis. # 2006 Elsevier Ltd. All rights reserved. Keywords: CAD; ICAD; Rotenone; siRNA
1. Introduction Apoptosis is a programmed, spontaneous cell death process that eliminates injured or aberrant cells, and removes useless cells during mammalian development. Several morphological and biochemical hallmarks are observed in apoptotic cells, including cellular shrinkage, nuclear fragmentation, caspase cascade activation and internucleosomal DNA fragmentation (Earnshaw et al., 1999; Martelli et al., 2001; Nagata, 2005). It is well known that dysregulation of apoptosis can contribute to the pathogenesis of many disorders including neurodegeneration, autoimmune diseases and cancer (Fischer and Schulze-Osthoff, 2005; Green and Evan, 2002; Nicholson, 2000; Reed, 2002; Thompson, 1995). One frequent hallmark of apoptotic cell death is the degradation of DNA into nucleosomal units. It has been suggested that several DNases, including DNase I, DNase
Abbreviations: CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, short interfering RNA * Corresponding author. Tel.: +81 58 293 2609; fax: +81 58 230 1893. E-mail address: [email protected] (Y. Hirata). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.12.002
II, DNase g, cyclophilin, caspase-activated DNase (CAD; also known as DNA fragmentation factor, or DFF40), and endonuclease G, are involved in apoptotic DNA degradation (Enari et al., 1998; Li et al., 2001; Liu et al., 1997; Montague et al., 1994; Rauch et al., 1997; Tanuma and Shiokawa, 1994; van Loo et al., 2001). Among these, CAD is the candidate most likely to be responsible for DNA fragmentation in many cells since cells that lack CAD or ICAD, inhibitor of CAD which is also required for functional folding of CAD do not undergo DNA fragmentation in vitro in response to various apoptotic stimuli (McIlroy et al., 2000; Nagase et al., 2003). Rotenone impairs oxidative phosphorylation by inhibiting complex I of the mitochondrial respiratory chain that results in cellular ATP depletion and oxidative stress (Chinopoulos et al., 2000). Recent studies have shown that rotenone induces cell death in a variety of cultured cells (Hartley et al., 1994; Wolvetang et al., 1994) and in primary dopaminergic culture (Radad et al., 2006). Rotenone also induces neuropathological and behavioral symptoms similar to Parkinson’s disease in rat (Betarbet et al., 2000; Hoglinger et al., 2003; Sherer et al., 2003). Parkinson’s disease is progressive and is one of the most common neurological disorders. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta
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resulting in a decrease in dopamine levels in the striatum where the nerve terminals from these neurons are found. Numerous studies have suggested that apoptosis plays a role in the cell death in Parkinson’s disease (Heidenreich, 2003; Hirsch et al., 1999). Although little is known about endogenous DNases involved in rotenone-induced apoptosis, it has been reported that rotenone activates caspase-3 (Lee et al., 2005; Reinecke et al., 2006), suggesting the involvement of CAD in rotenoneinduced DNA fragmentation. In this study, we used an RNA interference technique with short interfering RNAs (siRNAs) to knockdown CAD and investigate whether CAD contributes to rotenone-induced DNA fragmentation. The siRNA used in this study is a chemically modified form that has thymidine dimers with a carbamate linkage on the 30 terminus in each strand (Ueno et al., 2005). It is suggested that these modifications increase the stability of the siRNA against nucleolytic degradation by nucleases, and therefore it may suppress the CAD expression more effectively than the siRNA having natural phosphodiester linked thymidine dimer.
chloroform (1:1), followed by ethanol precipitation, essentially as described earlier (Hockenbery et al., 1990). The DNA dissolved in TE buffer was incubated with RNase A (50 mg/ml) at 37 8C for 1 h. Approximately half of the recovered soluble DNA per condition was separated by electrophoresis in 1.2% agarose gels and visualized with an UV transilluminator. Images were taken with a Gel Print 2000i/VGA (RMLuton, Inc., Jackson, MI, USA).
2.5. Western blot analysis Cells were lysed in sodium dodecyl sulfate (SDS)–Laemmli sample buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol) and sonicated for 20 s. The protein concentration was measured by the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) using g-immunoglobulin as a standard. Total cell lysates containing 10 or 15 mg protein were resolved on 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Biosciences Corp., Piscataway, NJ, USA). Membranes were blocked in blocking buffer (phosphatebuffered saline with 0.05% Tween 20 (PBS-T) containing 5% nonfat dry milk) for 1.5 h at room temperature, and probed with the appropriate antibodies at the dilutions recommended by the manufacturer overnight at 4 8C, and subsequently incubated with anti-rabbit Ig F(ab0 )2-HRP (Amersham Biosciences Corp.) for 90 min at room temperature. All antibodies were diluted in blocking buffer. The membranes were visualized using the enhanced chemiluminescence (ECL) system (Amersham Biosciences Corp.) or the SuperSignal West Dura Extended Duration Substrate (Pierce Chemical, Rockford, IL, USA). Bands were quantified with the Bio Image Intelligent Quantifier (RMLuton, Inc.).
2. Materials and methods 2.6. RT-PCR analysis 2.1. Materials Anti-CAD antibody was obtained from ProSci Incorporated (Poway, CA, USA); anti-ICAD antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-cleaved-caspase-3 antibody was from Cell Signaling Technology (Beverly, MA, USA). Anti-hamster CAD monoclonal antibody was kindly supplied by Shigekazu Nagata (Nagase et al., 2003). z-VAD-fmk was obtained from Peptide Institute, Inc. (Osaka, Japan).
2.2. Cell culture Human cervical carcinoma HeLa cells (RIKEN BioResource Center, Tsukuba, Japan) were grown in MEM (Sigma–Aldrich, St. Louis, MO, USA, M4655) supplemented with 10% bovine serum (BS) (Invitrogen Corporation, Carlsbad, CA, USA) at 37 8C in an atmosphere of 5% CO2. After the original medium was replaced with fresh medium, cells were treated with the indicated concentrations of rotenone for 20 h.
2.3. Transfection of HeLa cells with siRNA The CAD siRNA sequence (GAGAAGTGGACTGGGAGTA) was designed to correspond to nucleotides 845–863 of the human CAD (AB013918). Chemically modified siRNA was synthesized as previously described (Ueno et al., 2005). For siRNA-directed silencing, a purified CAD siRNA was transfected with Lipofectamine2000 (Invitrogen Corporation) in HeLa cells plated in six-well plates. siRNA was diluted in 250 ml OPTI-MEM and mixed with 10 ml Lipofectamine2000 diluted in 250 ml OPTI-MEM and incubated for 20 min at room temperature to form the siRNA–lipid complexes. The solution was added to HeLa cells plated in six-well plates containing 2 ml OPTI-MEM and the transfection was carried out for 6 h. After 6 h, the solution was removed and replaced with complete growth medium. At 24 or 48 h post-transfection, cells were harvested and analyzed. To transfect cells in 10-cm dishes, double the amounts of Lipofectamine2000, siRNA and medium.
2.4. Analysis of DNA fragmentation The cells (approximately 107 cells) were resuspended in lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 0.5% Triton X-100), and kept on ice for 10 min. After centrifugation at 14,000 g for 10 min, the soluble DNA was isolated and extracted with Tris/EDTA (TE)-saturated phenol and phenol/
Total RNA was isolated with TRIzol reagent (Invitrogen Corporation) from HeLa cells plated in six-well plates. Single-strand cDNA was synthesized from 0.5 mg total RNA in a mixture containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2. Oligo(dT)12–18 primer (0.5 mg), 0.5 mM dNTP, 5 mM DTT, RNaseOUT (40 units) and SuperScript III reverse transcriptase (200 units) (Invitrogen Corporation). The mixture was incubated at 42 8C for 60 min, and subsequently heated at 70 8C for 15 min. PCR amplification of human CAD cDNA was carried out for 32 cycles with rTaq DNA polymerase (Takara Bio Inc., Otsu, Shiga, Japan). The primer sequences were as follows: forward, 50 GGCTATGTGAGCGACATCAGGCG-30 (AB013918, 238–260) and reverse, 50 -CTCTCACAGCTGTATCTCAG-30 (AB013918, 466–485). These primers resulted in a 248 bp product. To ensure comparable amplification efficiencies in all RNA samples, 28S ribosomal RNA RT-PCR was also performed for 19 cycles using the following primers: forward, 50 -GTTCACCCACTAATAGGGAACGTG-30 (M11167, 4459–4482) and reverse, 50 -GATTCTGACTTAGAGGCGTTCAGT-30 (M11167, 4647–4670), which gave a 212-bp product. PCR products were separated by 1.2% agarose gel electrophoresis and visualized under UV light. Gel photographs were taken using a Gel Print 200i/VGA, and analyzed by the Bio Image Intelligent Quantifier (RMLuton). Levels of CAD mRNA were calculated in arbitrary units as the proportion of CAD PCR product intensity to 28S ribosomal RNA PCR product intensity from the same RNA sample and expressed as percentage of control.
3. Result 3.1. Rotenone-induced DNA fragmentation is dependent on the activation of caspase-3 in HeLa cells The treatment of HeLa cells with various concentrations of rotenone resulted in DNA fragmentation and caspase-3 cleavage (Fig. 1A). In accordance with the caspase-3 activation, rotenone also increased cleaved ICAD that is considered to be a substrate of caspase-3 (Fig. 1B). Furthermore, rotenoneinduced DNA fragmentation was prevented by the synthetic peptide z-VAD-fmk, a pan-caspase inhibitor as shown in Fig. 1C. These results show that rotenone causes caspasedependent cell death in HeLa cells.
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Fig. 1. Rotenone-induced apoptosis in HeLa cells. (A) Rotenone-induced DNA fragmentation. HeLa cells were incubated with the indicated concentrations of rotenone for 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. M, HindIII digests of l-DNA. (B) Western blot analysis of caspase-3 and ICAD cleavage. Total cell lysates were separated by SDSPAGE and transferred to nitrocellulose membranes as described in Section 2. (C) z-VAD-fmk prevented rotenone-induced DNA fragmentation. HeLa cells were preincubated with indicated concentrations of z-VAD-fmk for 1 h and rotenone (0.5 mM) was added. The cells were cultured for another 20 h.
3.2. Knockdown of CAD mRNA and protein by siRNA To knockdown CAD mRNA and protein, we used a chemically modified siRNA (Ueno et al., 2005). It has been reported that the modified siRNAs are more potent than nonmodified siRNAs due to the increased stability against nucleolytic degradation. RT-PCR analysis showed that CAD mRNA decreased to 40% of control at 10 nM and 20% at 50 nM, respectively (Fig. 2A and B) at 24 h post-transfection. Similarly, Western blotting showed that CAD protein decreased to 16% at 10 nM and 8% at 50 nM, respectively (Fig. 3A and B), at 48 h post-transfection. Alpha-tubulin protein level served as a loading control. Similar results were obtained using an anti-hamster CAD monoclonal antibody (data not shown). These results indicate that the CAD siRNA used in this study efficiently suppresses CAD protein. 3.3. CAD contributes to rotenone-induced DNA fragmentation in HeLa cells To investigate whether CAD contributes to rotenoneinduced DNA fragmentation in HeLa cells, we examined the
effect of CAD knockdown on rotenone-induced DNA fragmentation. As shown in Fig. 4A, silencing CAD expression with 10 or 50 nM CAD siRNA prevented rotenone-induced DNA fragmentation. ICAD and caspase-3 were cleaved in siRNA treated cells just as in non-treated cells, suggesting that apoptotic signaling is not affected by siRNA (Fig. 4B). These results indicate that CAD involves in rotenone-induced DNA fragmentation in HeLa cells. Finally, we compared the effect of chemical modification of CAD siRNA on rotenone-induced DNA fragmentation. At 1 nM, both an unmodified siRNA (a) and a chemically modified siRNA (b) did not show remarkable inhibitory effect (Fig. 5). In contrast, at 50 nM, a chemically modified siRNA clearly showed a strong inhibitory effect compared with an unmodified siRNA. 4. Discussion Rotenone has been investigated extensively as an etiological model for Parkinson’s disease (Betarbet et al., 2000; Sherer et al., 2003) in which apoptosis has been suggested to play a role in the cell death (Heidenreich, 2003; Hirsch et al., 1999), but little is known about endogenous DNases involved in
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Fig. 2. Effect of siRNA on CAD mRNA expression. RT-PCR analysis of CAD mRNA expression. Total RNA was isolated at 24 h post-transfection of siRNA into HeLa cells. (A) Represents the typical agarose gel electrophoresis data. (B) Semi-quantitative analysis of the intensity of bands corresponding to CAD was carried out as described in Section 2. Data are mean S.D. (n = 4). **p < 0.01.
Fig. 3. Effect of siRNA on CAD protein expression. Western analysis of CAD expression. (A) Total cell lysates were separated by SDS-PAGE and Western blotting was carried out as described in Section 2. This panel represents a typical blot. (B) Semi-quantitative analysis of the intensity of bands corresponding to CAD was performed as described in Section 2. Data are mean S.D. (n = 4). ** p < 0.01.
Fig. 4. (A) Rotenone-induced DNA fragmentation was suppressed by CAD siRNA. siRNA were transfected by Lipofectamine2000 into HeLa cells plated in 10-cm dishes for 6 h. At 28-h post-transfection, rotenone was added and incubated for another 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. (B) Caspase-3 and ICAD were cleaved in siRNA treated cells. Total cell lysates were separated by SDSPAGE and Western blotting was carried out as described in Section 2.
Fig. 5. A modified siRNA (b) suppressed rotenone-induced DNA fragmentation more efficiently than an unmodified siRNA (a). siRNA were transfected by Lipofectamine2000 into HeLa cells plated in 10-cm dishes for 6 h. At 28-h posttransfection, rotenone was added, and the incubation was continued for another 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. HindIII digests of l-DNA are shown in the left lane.
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rotenone-induced DNA fragmentation, a biochemical hallmark of apoptosis. Rotenone induces apoptosis in a caspase-3-dependent manner in many cell lines including rat pheochromocytoma PC12 cells (Hartley et al., 1994), human promyelocytic leukemia HL60 cells (Li et al., 2003; Matsunaga et al., 1996), human neuroblastoma SH-SY5Y cells (King et al., 2001) and SK-N-MC cells (Sherer et al., 2002). On the other hand, it induces caspase-9/3-dependent cell death in differentiated human neural stem cells and caspase-9/3-independent cell death in undifferentiated human neural stem cells (Li et al., 2005). They suggested that the extent and rapidity of ATP depletion determines whether cells undergo apoptotic or necrotic cell death in a given rotenone model. In this paper, we showed that rotenone-induced DNA fragmentation is dependent on caspase-3 activation, suggesting the involvement of CAD. To examine the role of CAD in this event, we used siRNA to knockdown CAD expression. Introducing siRNA caused decreases in both CAD mRNA and protein with concomitant prevention of rotenone-induced DNA fragmentation. Rotenone-induced DNA fragmentation was almost completely blocked by siRNA at 50 nM. Since RNA interference provides efficient and highly specific gene silencing, it is unlikely that CAD siRNA acts on other DNases. In addition, rotenone-induced DNA fragmentation was completely blocked by the pretreatment of z-VAD-fmk, a pan-caspase inhibitor. These results suggest that CAD is the sole enzyme that degrades DNA into nucleosomal units in response to rotenone treatment in HeLa cells. Another study shows the existence of nucleases other than CAD that are involved in etoposide- and long term-culture-induced DNA fragmentation in a caspase-3dependent manner in HeLa cells (Torriglia et al., 1999). They suggested the existence of distinct endonucleases activated by different stimuli in the same cell line. As previously reported, siRNAs chemically modified with thymidine dimers consisting of a carbamate linkage on 30 terminus in each strand which are designed for a human RNase L are much more effective compared with siRNAs that have the natural phosphodiester linkage (Ueno et al., 2005). In the present study, we used a siRNA with the same modification. A chemically modified siRNA results in more remarkable inhibition of rotenone-induced DNA fragmentation. These results support the idea that the modification of the 30 overhang regions might increase the stability of the siRNA and enhance its silencing activity. Taken together, our results indicate that rotenone induces caspase-dependent apoptosis in HeLa cells and that CAD is the only endonuclease involved in rotenone-induced DNA fragmentation. Chemical modification of siRNA might augment the ability of the siRNA to inhibit protein expression. Acknowledgments We thank Dr. Shigekazu Nagata (Osaka University) for an anti-hamster CAD monoclonal antibody. This work was supported in part by the Grants-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science.
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RNAi knockdown of caspase-activated DNase inhibits rotenone-induced DNA fragmentation in HeLa cells Tadamiki Tsuruta, Kentaro Oh-hashi, Yoshihito Ueno, Yukio Kitade, Kazutoshi Kiuchi, Yoko Hirata * Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Received 8 July 2006; received in revised form 21 November 2006; accepted 5 December 2006 Available online 18 January 2007
Abstract Rotenone, an inhibitor of mitochondrial complex I, induces apoptosis in a variety of cells. However, little is known about endogenous endonucleases involved in rotenone-induced DNA fragmentation, a biochemical hallmark of apoptosis. We used a chemically modified siRNA which is thought to be more effective than a non-modified siRNA to study whether caspase-activated DNase (CAD) contributes to this phenomenon. Western blot analysis showed that CAD protein decreased to 8% of control levels in human cervical carcinoma HeLa cells after a 48 h transfection of siRNA. Consistent with the reduction of the protein level, the siRNA was found to inhibit rotenone-induced DNA fragmentation. These results suggest that CAD is the endogenous endonuclease that mediates internucleosomal DNA degradation in rotenone-induced apoptosis. # 2006 Elsevier Ltd. All rights reserved. Keywords: CAD; ICAD; Rotenone; siRNA
1. Introduction Apoptosis is a programmed, spontaneous cell death process that eliminates injured or aberrant cells, and removes useless cells during mammalian development. Several morphological and biochemical hallmarks are observed in apoptotic cells, including cellular shrinkage, nuclear fragmentation, caspase cascade activation and internucleosomal DNA fragmentation (Earnshaw et al., 1999; Martelli et al., 2001; Nagata, 2005). It is well known that dysregulation of apoptosis can contribute to the pathogenesis of many disorders including neurodegeneration, autoimmune diseases and cancer (Fischer and Schulze-Osthoff, 2005; Green and Evan, 2002; Nicholson, 2000; Reed, 2002; Thompson, 1995). One frequent hallmark of apoptotic cell death is the degradation of DNA into nucleosomal units. It has been suggested that several DNases, including DNase I, DNase
Abbreviations: CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, short interfering RNA * Corresponding author. Tel.: +81 58 293 2609; fax: +81 58 230 1893. E-mail address: [email protected] (Y. Hirata). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.12.002
II, DNase g, cyclophilin, caspase-activated DNase (CAD; also known as DNA fragmentation factor, or DFF40), and endonuclease G, are involved in apoptotic DNA degradation (Enari et al., 1998; Li et al., 2001; Liu et al., 1997; Montague et al., 1994; Rauch et al., 1997; Tanuma and Shiokawa, 1994; van Loo et al., 2001). Among these, CAD is the candidate most likely to be responsible for DNA fragmentation in many cells since cells that lack CAD or ICAD, inhibitor of CAD which is also required for functional folding of CAD do not undergo DNA fragmentation in vitro in response to various apoptotic stimuli (McIlroy et al., 2000; Nagase et al., 2003). Rotenone impairs oxidative phosphorylation by inhibiting complex I of the mitochondrial respiratory chain that results in cellular ATP depletion and oxidative stress (Chinopoulos et al., 2000). Recent studies have shown that rotenone induces cell death in a variety of cultured cells (Hartley et al., 1994; Wolvetang et al., 1994) and in primary dopaminergic culture (Radad et al., 2006). Rotenone also induces neuropathological and behavioral symptoms similar to Parkinson’s disease in rat (Betarbet et al., 2000; Hoglinger et al., 2003; Sherer et al., 2003). Parkinson’s disease is progressive and is one of the most common neurological disorders. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta
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resulting in a decrease in dopamine levels in the striatum where the nerve terminals from these neurons are found. Numerous studies have suggested that apoptosis plays a role in the cell death in Parkinson’s disease (Heidenreich, 2003; Hirsch et al., 1999). Although little is known about endogenous DNases involved in rotenone-induced apoptosis, it has been reported that rotenone activates caspase-3 (Lee et al., 2005; Reinecke et al., 2006), suggesting the involvement of CAD in rotenoneinduced DNA fragmentation. In this study, we used an RNA interference technique with short interfering RNAs (siRNAs) to knockdown CAD and investigate whether CAD contributes to rotenone-induced DNA fragmentation. The siRNA used in this study is a chemically modified form that has thymidine dimers with a carbamate linkage on the 30 terminus in each strand (Ueno et al., 2005). It is suggested that these modifications increase the stability of the siRNA against nucleolytic degradation by nucleases, and therefore it may suppress the CAD expression more effectively than the siRNA having natural phosphodiester linked thymidine dimer.
chloroform (1:1), followed by ethanol precipitation, essentially as described earlier (Hockenbery et al., 1990). The DNA dissolved in TE buffer was incubated with RNase A (50 mg/ml) at 37 8C for 1 h. Approximately half of the recovered soluble DNA per condition was separated by electrophoresis in 1.2% agarose gels and visualized with an UV transilluminator. Images were taken with a Gel Print 2000i/VGA (RMLuton, Inc., Jackson, MI, USA).
2.5. Western blot analysis Cells were lysed in sodium dodecyl sulfate (SDS)–Laemmli sample buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol) and sonicated for 20 s. The protein concentration was measured by the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) using g-immunoglobulin as a standard. Total cell lysates containing 10 or 15 mg protein were resolved on 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Biosciences Corp., Piscataway, NJ, USA). Membranes were blocked in blocking buffer (phosphatebuffered saline with 0.05% Tween 20 (PBS-T) containing 5% nonfat dry milk) for 1.5 h at room temperature, and probed with the appropriate antibodies at the dilutions recommended by the manufacturer overnight at 4 8C, and subsequently incubated with anti-rabbit Ig F(ab0 )2-HRP (Amersham Biosciences Corp.) for 90 min at room temperature. All antibodies were diluted in blocking buffer. The membranes were visualized using the enhanced chemiluminescence (ECL) system (Amersham Biosciences Corp.) or the SuperSignal West Dura Extended Duration Substrate (Pierce Chemical, Rockford, IL, USA). Bands were quantified with the Bio Image Intelligent Quantifier (RMLuton, Inc.).
2. Materials and methods 2.6. RT-PCR analysis 2.1. Materials Anti-CAD antibody was obtained from ProSci Incorporated (Poway, CA, USA); anti-ICAD antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-cleaved-caspase-3 antibody was from Cell Signaling Technology (Beverly, MA, USA). Anti-hamster CAD monoclonal antibody was kindly supplied by Shigekazu Nagata (Nagase et al., 2003). z-VAD-fmk was obtained from Peptide Institute, Inc. (Osaka, Japan).
2.2. Cell culture Human cervical carcinoma HeLa cells (RIKEN BioResource Center, Tsukuba, Japan) were grown in MEM (Sigma–Aldrich, St. Louis, MO, USA, M4655) supplemented with 10% bovine serum (BS) (Invitrogen Corporation, Carlsbad, CA, USA) at 37 8C in an atmosphere of 5% CO2. After the original medium was replaced with fresh medium, cells were treated with the indicated concentrations of rotenone for 20 h.
2.3. Transfection of HeLa cells with siRNA The CAD siRNA sequence (GAGAAGTGGACTGGGAGTA) was designed to correspond to nucleotides 845–863 of the human CAD (AB013918). Chemically modified siRNA was synthesized as previously described (Ueno et al., 2005). For siRNA-directed silencing, a purified CAD siRNA was transfected with Lipofectamine2000 (Invitrogen Corporation) in HeLa cells plated in six-well plates. siRNA was diluted in 250 ml OPTI-MEM and mixed with 10 ml Lipofectamine2000 diluted in 250 ml OPTI-MEM and incubated for 20 min at room temperature to form the siRNA–lipid complexes. The solution was added to HeLa cells plated in six-well plates containing 2 ml OPTI-MEM and the transfection was carried out for 6 h. After 6 h, the solution was removed and replaced with complete growth medium. At 24 or 48 h post-transfection, cells were harvested and analyzed. To transfect cells in 10-cm dishes, double the amounts of Lipofectamine2000, siRNA and medium.
2.4. Analysis of DNA fragmentation The cells (approximately 107 cells) were resuspended in lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 0.5% Triton X-100), and kept on ice for 10 min. After centrifugation at 14,000 g for 10 min, the soluble DNA was isolated and extracted with Tris/EDTA (TE)-saturated phenol and phenol/
Total RNA was isolated with TRIzol reagent (Invitrogen Corporation) from HeLa cells plated in six-well plates. Single-strand cDNA was synthesized from 0.5 mg total RNA in a mixture containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2. Oligo(dT)12–18 primer (0.5 mg), 0.5 mM dNTP, 5 mM DTT, RNaseOUT (40 units) and SuperScript III reverse transcriptase (200 units) (Invitrogen Corporation). The mixture was incubated at 42 8C for 60 min, and subsequently heated at 70 8C for 15 min. PCR amplification of human CAD cDNA was carried out for 32 cycles with rTaq DNA polymerase (Takara Bio Inc., Otsu, Shiga, Japan). The primer sequences were as follows: forward, 50 GGCTATGTGAGCGACATCAGGCG-30 (AB013918, 238–260) and reverse, 50 -CTCTCACAGCTGTATCTCAG-30 (AB013918, 466–485). These primers resulted in a 248 bp product. To ensure comparable amplification efficiencies in all RNA samples, 28S ribosomal RNA RT-PCR was also performed for 19 cycles using the following primers: forward, 50 -GTTCACCCACTAATAGGGAACGTG-30 (M11167, 4459–4482) and reverse, 50 -GATTCTGACTTAGAGGCGTTCAGT-30 (M11167, 4647–4670), which gave a 212-bp product. PCR products were separated by 1.2% agarose gel electrophoresis and visualized under UV light. Gel photographs were taken using a Gel Print 200i/VGA, and analyzed by the Bio Image Intelligent Quantifier (RMLuton). Levels of CAD mRNA were calculated in arbitrary units as the proportion of CAD PCR product intensity to 28S ribosomal RNA PCR product intensity from the same RNA sample and expressed as percentage of control.
3. Result 3.1. Rotenone-induced DNA fragmentation is dependent on the activation of caspase-3 in HeLa cells The treatment of HeLa cells with various concentrations of rotenone resulted in DNA fragmentation and caspase-3 cleavage (Fig. 1A). In accordance with the caspase-3 activation, rotenone also increased cleaved ICAD that is considered to be a substrate of caspase-3 (Fig. 1B). Furthermore, rotenoneinduced DNA fragmentation was prevented by the synthetic peptide z-VAD-fmk, a pan-caspase inhibitor as shown in Fig. 1C. These results show that rotenone causes caspasedependent cell death in HeLa cells.
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Fig. 1. Rotenone-induced apoptosis in HeLa cells. (A) Rotenone-induced DNA fragmentation. HeLa cells were incubated with the indicated concentrations of rotenone for 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. M, HindIII digests of l-DNA. (B) Western blot analysis of caspase-3 and ICAD cleavage. Total cell lysates were separated by SDSPAGE and transferred to nitrocellulose membranes as described in Section 2. (C) z-VAD-fmk prevented rotenone-induced DNA fragmentation. HeLa cells were preincubated with indicated concentrations of z-VAD-fmk for 1 h and rotenone (0.5 mM) was added. The cells were cultured for another 20 h.
3.2. Knockdown of CAD mRNA and protein by siRNA To knockdown CAD mRNA and protein, we used a chemically modified siRNA (Ueno et al., 2005). It has been reported that the modified siRNAs are more potent than nonmodified siRNAs due to the increased stability against nucleolytic degradation. RT-PCR analysis showed that CAD mRNA decreased to 40% of control at 10 nM and 20% at 50 nM, respectively (Fig. 2A and B) at 24 h post-transfection. Similarly, Western blotting showed that CAD protein decreased to 16% at 10 nM and 8% at 50 nM, respectively (Fig. 3A and B), at 48 h post-transfection. Alpha-tubulin protein level served as a loading control. Similar results were obtained using an anti-hamster CAD monoclonal antibody (data not shown). These results indicate that the CAD siRNA used in this study efficiently suppresses CAD protein. 3.3. CAD contributes to rotenone-induced DNA fragmentation in HeLa cells To investigate whether CAD contributes to rotenoneinduced DNA fragmentation in HeLa cells, we examined the
effect of CAD knockdown on rotenone-induced DNA fragmentation. As shown in Fig. 4A, silencing CAD expression with 10 or 50 nM CAD siRNA prevented rotenone-induced DNA fragmentation. ICAD and caspase-3 were cleaved in siRNA treated cells just as in non-treated cells, suggesting that apoptotic signaling is not affected by siRNA (Fig. 4B). These results indicate that CAD involves in rotenone-induced DNA fragmentation in HeLa cells. Finally, we compared the effect of chemical modification of CAD siRNA on rotenone-induced DNA fragmentation. At 1 nM, both an unmodified siRNA (a) and a chemically modified siRNA (b) did not show remarkable inhibitory effect (Fig. 5). In contrast, at 50 nM, a chemically modified siRNA clearly showed a strong inhibitory effect compared with an unmodified siRNA. 4. Discussion Rotenone has been investigated extensively as an etiological model for Parkinson’s disease (Betarbet et al., 2000; Sherer et al., 2003) in which apoptosis has been suggested to play a role in the cell death (Heidenreich, 2003; Hirsch et al., 1999), but little is known about endogenous DNases involved in
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Fig. 2. Effect of siRNA on CAD mRNA expression. RT-PCR analysis of CAD mRNA expression. Total RNA was isolated at 24 h post-transfection of siRNA into HeLa cells. (A) Represents the typical agarose gel electrophoresis data. (B) Semi-quantitative analysis of the intensity of bands corresponding to CAD was carried out as described in Section 2. Data are mean S.D. (n = 4). **p < 0.01.
Fig. 3. Effect of siRNA on CAD protein expression. Western analysis of CAD expression. (A) Total cell lysates were separated by SDS-PAGE and Western blotting was carried out as described in Section 2. This panel represents a typical blot. (B) Semi-quantitative analysis of the intensity of bands corresponding to CAD was performed as described in Section 2. Data are mean S.D. (n = 4). ** p < 0.01.
Fig. 4. (A) Rotenone-induced DNA fragmentation was suppressed by CAD siRNA. siRNA were transfected by Lipofectamine2000 into HeLa cells plated in 10-cm dishes for 6 h. At 28-h post-transfection, rotenone was added and incubated for another 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. (B) Caspase-3 and ICAD were cleaved in siRNA treated cells. Total cell lysates were separated by SDSPAGE and Western blotting was carried out as described in Section 2.
Fig. 5. A modified siRNA (b) suppressed rotenone-induced DNA fragmentation more efficiently than an unmodified siRNA (a). siRNA were transfected by Lipofectamine2000 into HeLa cells plated in 10-cm dishes for 6 h. At 28-h posttransfection, rotenone was added, and the incubation was continued for another 20 h. The soluble DNA was isolated and analyzed by agarose gel electrophoresis. DNA ladders were visualized under a UV light after ethidium bromide staining as described in Section 2. HindIII digests of l-DNA are shown in the left lane.
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rotenone-induced DNA fragmentation, a biochemical hallmark of apoptosis. Rotenone induces apoptosis in a caspase-3-dependent manner in many cell lines including rat pheochromocytoma PC12 cells (Hartley et al., 1994), human promyelocytic leukemia HL60 cells (Li et al., 2003; Matsunaga et al., 1996), human neuroblastoma SH-SY5Y cells (King et al., 2001) and SK-N-MC cells (Sherer et al., 2002). On the other hand, it induces caspase-9/3-dependent cell death in differentiated human neural stem cells and caspase-9/3-independent cell death in undifferentiated human neural stem cells (Li et al., 2005). They suggested that the extent and rapidity of ATP depletion determines whether cells undergo apoptotic or necrotic cell death in a given rotenone model. In this paper, we showed that rotenone-induced DNA fragmentation is dependent on caspase-3 activation, suggesting the involvement of CAD. To examine the role of CAD in this event, we used siRNA to knockdown CAD expression. Introducing siRNA caused decreases in both CAD mRNA and protein with concomitant prevention of rotenone-induced DNA fragmentation. Rotenone-induced DNA fragmentation was almost completely blocked by siRNA at 50 nM. Since RNA interference provides efficient and highly specific gene silencing, it is unlikely that CAD siRNA acts on other DNases. In addition, rotenone-induced DNA fragmentation was completely blocked by the pretreatment of z-VAD-fmk, a pan-caspase inhibitor. These results suggest that CAD is the sole enzyme that degrades DNA into nucleosomal units in response to rotenone treatment in HeLa cells. Another study shows the existence of nucleases other than CAD that are involved in etoposide- and long term-culture-induced DNA fragmentation in a caspase-3dependent manner in HeLa cells (Torriglia et al., 1999). They suggested the existence of distinct endonucleases activated by different stimuli in the same cell line. As previously reported, siRNAs chemically modified with thymidine dimers consisting of a carbamate linkage on 30 terminus in each strand which are designed for a human RNase L are much more effective compared with siRNAs that have the natural phosphodiester linkage (Ueno et al., 2005). In the present study, we used a siRNA with the same modification. A chemically modified siRNA results in more remarkable inhibition of rotenone-induced DNA fragmentation. These results support the idea that the modification of the 30 overhang regions might increase the stability of the siRNA and enhance its silencing activity. Taken together, our results indicate that rotenone induces caspase-dependent apoptosis in HeLa cells and that CAD is the only endonuclease involved in rotenone-induced DNA fragmentation. Chemical modification of siRNA might augment the ability of the siRNA to inhibit protein expression. Acknowledgments We thank Dr. Shigekazu Nagata (Osaka University) for an anti-hamster CAD monoclonal antibody. This work was supported in part by the Grants-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science.
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