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Shinya Kobayashi^1,2,, Shinichi Kajino^1,2,, Naoko Takahashi¹, Satoshi Kanazawa¹, Kenichi Imai¹, Yurina Hibi¹, Hirotaka Ohara², Makoto Itoh² and Takashi Okamoto^1,*

¹ Department of Molecular and Cellular Biology, and ² Department of Internal Medicine and Bioregulation, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan

	Abstract

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The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor B (NF-B). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans-membrane potential (m) and activation of caspase-9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.

	Introduction

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Apoptosis is a well-defined biochemical pathway and is essential for the maintenance of cellular homeostasis in metazoans. Accumulating evidences indicate that the normal apoptotic pathway is affected in the pathological processes such as cancer and autoimmunity (Fisher et al. 1995; Green & Reed 1998; Jackson & Puck 1999; Daniel & Korsmeyer 2004). The induction of apoptosis occurs through two distinct pathways, the one elicited by death receptors in the plasma membrane (‘extrinsic pathway’) and the other directly involving mitochondria (‘intrinsic pathway’). Whereas the former primarily involves activation of caspase-8, the latter apoptosis pathway is associated with the release of cytochrome C from mitochondria and activation of caspase-9 (for a review see Judith et al. 2004).

The p53 binding protein 2 (53BP2) has been initially identified as an interacting protein to p53 (Iwabuchi et al. 1994) and implicated in the biological action of p53. It was also shown that the 53BP2 binding site in the p53 core domain is evolutionarily conserved and is frequently mutated in human cancer (Iwabuchi et al. 1994; Gorina & Pavletich 1996). The subsequent studies have revealed that it interacts with Bcl-2 (Naumovski & Cleary 1996) and p65 subunit of nuclear factor B (NF-B) (Yang et al. 1999). Interestingly, 53BP2 has been shown to induce apoptosis (Yang et al. 1999), which was confirmed by others (Lopez et al. 2000; Ao et al. 2001; Samuels-Lev et al. 2001; Bergamaschi et al. 2004). However, the mechanism by which 53BP2 induces apoptosis has not been clarified.

53BP2 protein is encoded by a single copy gene TP53BP2 located in the long arm of chromosome 1 at q42.1 (Yang et al. 1997). We have recently found that it encodes two distinct mRNA species, either with or without exon 3, by alternative splicing (Takahashi et al. 2004) (Fig. 1A). These splicing variants encode two 53BP2 proteins containing 1005 and 1128 amino acids (aa) with the longer isoform containing additional 123 amino acids in the N-terminus where no known functional motif or distinct intracellular localization signal is found. Although Samuels-Lev et al. (2001) renamed the longer 53BP2 isoform as ASPP2 (apoptosis stimulating protein of p53 2), we have proposed to call these proteins as 53BP2S (short) and 53BP2L (long) based on the genome organization of TP53BP2 transcripts (Takahashi et al. 2004). 53BP2 proteins contain several structural and functional motifs including Gln-rich -helical region, Pro-rich regions, ankyrin repeats, and Src-homology 3 domain.

View larger version (36K): [in this window] [in a new window]   Figure 1  Induction of apoptosis by 53BP2 proteins. (A) Diagrammatic representation of 53BP2S and 53BP2L/ASPP2 proteins. Locations of Gln-rich region, putative ‘-helical region,’ Pro-rich region, ankyrin repeats, and SH3 domain are indicated. Two splicing variants, 53BP2S and 53BP2L/ASPP2, containing 1005 amino acids and 1128 amino acids residues, respectively, are encoded by the same gene TP53BP2 (Takahashi et al. 2004). 53BP2L contains additional 123 amino acid N-terminal region containing no apparent functional/structural motifs. (B) The localization of endogenous 53BP2 proteins in MIA PaCa-2 cells. Subcellular localization of endogenous 53BP2 was examined by immunostaining with anti-53BP2 mouse monoclonal antibody. The dim staining of 53BP2 proteins was repeatedly observed, which is presumably due to the low protein stability as previously indicated (Yang et al. 1999; Lopez et al. 2000). (C) Cleavage of PARP by 53BP2 proteins. MIA PaCa-2 cells and HeLa cells were transfected with pcDNA3.1–53BP2 (‘53BP2S’) or pCEP4-ASPP2 (‘53BP2L’) plasmids and the cell lysates were immunoblotted with anti-PARP antibody. The intact form of PARP (116 kDa) and its cleavage form (89 kDa) were detected by an anti-PARP rabbit polyclonal antibody (indicated by arrows). Note that no significant difference of the amounts of the cleaved form of PARP was found in cells expressing 53BP2S and 53BP2L. Cont, cells transfected with a control expression vector pcDNA3.1. (D) Induction of apoptosis by over-expression of 53BP2 proteins. MIA PaCa-2 cells were transfected with pcDNA3.1–53BP2 or pCEP4-ASPP2 and cells undergoing apoptosis were detected by flow cytometry. Live and dead cells were discriminated on the basis of their forward and side light-scattering properties. In order to evaluate cells undergoing apoptosis, cells were stained by both annexin V-PE and 7-AAD and those cells expressing 7-AAD were excluded from the measurement. The transfection efficiency was estimated to be approximately 65% by the GFP expression from the co-transfected pEGFP plasmid. The experiments were repeated more than three times with the same results.

	Results

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Induction of apoptosis by 53BP2 proteins

Figure 1A illustrates the organization of 53BP2 isoforms as previously reported (Takahashi et al. 2004). As shown in Fig. 1B, the endogenous 53BP2 proteins were localized predominantly in the cytoplasm, confirming the previous reports wherein 53BP2 was over-expressed (Iwabuchi et al. 1998; Yang et al. 1999). To compare the effect of 53BP2S and 53BP2L, these proteins were transduced in MIA PaCa-2 and HeLa cells. After 48 h of transfection, the cleaved form of poly ADP ribose polymerase (PARP; 89 kDa product), a hallmark of apoptosis, was detected (Fig. 1C). When 53BP2S and 53BP2L were over-expressed in MIA PaCa-2 cells, approximately 16% and 27% of cells were found undergoing early apoptotic process (annexin V (+), 7-AAD (–)), respectively, whereas the percentage of apoptotic cells in the control was only 1.8% (Fig. 1D). The extents of apoptosis were similar to our previous observations using various DNA damaging agents (Mori et al. 2000).

Induction of apoptosis in a stable transfectant (293/53BP2)

We then examined the action of 53BP2 using the 293/53BP2 cells, in which expression of 53BP2S is under stringent control by ponasteron A (pon A). In Fig. 2A, both 293/53BP2 and its control 293/LZ were treated with pon A. The 53BP2S protein became detectable after 12 h of induction by pon A (5 µM) in a time-dependent manner and induced apoptosis as early as 24 h after pon A treatment. As shown in Fig. 2B, after 72 h of 53BP2S expression, a significant number (26%) of cells underwent apoptosis as revealed by positive staining for annexin V, whereas only the background level (6.5%) was stained in control cells. Cells at early apoptotic process (annexin (+), 7-AAD (–)) were found 14% and 3% in 293/53BP2 cells and control cells, respectively (Fig. 2B). No cleavage of PARP or a significant annexin V staining was detected with the control 293/LZ cells (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 2  Induction of apoptosis in 293/53BP2 cell line. (A) Time course of induction of 53BP2S and cleavage of PARP in 293/53BP2 cells. Cells were treated with pon A (5 µM) using an ecdysone-inducible expression system for the indicated periods (h), and the cell lysate (10 µg protein) was examined for the expression of 53BP2S and PARP. The intact full-length PARP (116 kDa) was cleaved into 89 kDa during the apoptotic process. Longer exposure of chemiluminescence for protein detection revealed the endogenous 53BP2L protein in these cells. ß-tubulin was used as an internal control. (B) Flow cytometric detection of apoptotic cells. 293/53BP2 cells were stimulated with pon A (5 µM) and cultured for the indicated periods (h). The percentages of cells at apoptosis (annexin V (+)) and cells at early apoptosis (annexin V positive and 7-AAD (–)) were counted and indicated separately.

In Fig. 3A, intracellular localization of 53BP2S was examined by transfection of pEGFP53BP2 expressing 53BP2S in fusion with green fluorescence protein (GFP). A punctate vesicular pattern was noted, localized predominantly in the cytoplasm of the transfected cells. To confirm the localization of 53BP2S, we co-transfected pDsRed2-Mito, expressing red fluorescent protein targeted to mitochondria. As demonstrated in Fig. 3A and 53BP2S was shown to be partly localized in the mitochondria in addition to the cytoplasm. In most cells only portions of mitochondria were costained with 53BP2S, suggesting that small amounts of 53BP2S molecules could be sufficient to induce apoptosis. In Fig. 3B, subcellular fractionation was performed and the presence of 53BP2S was examined. Protein expression was induced by pon A for 48 h and each subcellular fraction was subjected to Western blotting with anti-53BP2 antibody. Although majority of the 53BP2S protein was detected in the cytosolic fraction, it was also detected in the mitochondrial fraction (Fig. 3B).

View larger version (22K): [in this window] [in a new window]   Figure 3  Intracellular localization of 53BP2S. (A) Co-localization of 53BPS and mitochondria marker. 293 cells were transiently transfected with pEGFP-53BP2 and mitochondria-targeting plasmid pDsRed2-Mit and examined under confocal microscope. The GFP fluorescence, DsRed fluorescence and merged images of cells are shown. Note that only portions of mitochondria (visualized by DsRed) were costained with GFP (53BP2S). (B) Subcellular fractionation. Upon induction of 53BP2 by pon A (5 µM, 48 h) in 293/53BP2 cells, whole cell extract (WCE) was prepared. The cytoplasmic (Cy), mitochondrial (Mit) and nuclear (Nuc) fractions were separated as described in Experimental procedures. Each protein fraction was separated by 10% SDS-PAGE, and probed with antibodies to 53BP2S, PCNA (nuclear marker), mitochondrial heat shock protein (Mit hsp70) and LDH (cytoplasmic marker). The same cell equivalents were loaded on each lane. Contamination of the cytoplasmic fraction into the mitochondria fraction was considered negligible because of the absence of LDH. The identical results were obtained repeatedly.

Depression of m by 53BP2S expression

These findings suggested the involvement of the ‘intrinsic’ death pathway. We thus examined the change in m following 53BP2S expression (Fig. 4). Several cationic, lipophilic, fluorescent dyes such as CMXRos and rhodamine 123, can readily detect changes in m as they are selectively sequestered by respiring mitochondria by virtue of their negative charges on the inner membrane and are washed out when m is lost. As shown in Fig. 4A, the extent of CMXRos staining in pEGFP53BP2-transfected cells (visualized by the expression of GFP) was diminished, indicating a decrease in m. In contrast, no such changes were observed in the control cells transfected with pEGFP. In Fig. 4B, the temporal change of m in 293/53BP2 cells is shown. When 53BP2S was expressed, progressive reduction of m detected by the rhodamine123 fluorescence intensity was observed over time, concomitantly with the appearance of apoptotic cells (compare with Fig. 2B). No such change was observed in control 293/LZ cells.

View larger version (23K): [in this window] [in a new window]   Figure 4  Alteration of the mitochondria transmembrane potential (m) by 53BP2S. (A) Reduction of m by 53BP2S. After 48 h of transfection with pEGFP (a) or pEGFP53BP2 (b–d) plasmids, MIA PaCa-2 cells were stained with CMXRos. Typical cells are shown. In cells expressing 53BP2S, progressive reduction of m was observed in association with nuclear fragmentation (from b–d). The same exposure time was used in each picture. (B) Temporal change of m following 53BP2S induction. 293/53BP2 and 293/LZ cells were treated with pon A for indicated periods (h), stained with rhodamine 123, and flow cytometric analysis was performed. Distribution of fluorescence intensity of cells with sham treatment (only the solvent ethanol was added) is shown in gray shadow. ‘Control,’ uninduced cells.

Finally, to examine the upstream caspase cascade involved in the 53BP2S-mediated apoptosis, 293/53BP2 cell lysates were prepared after 24 and 48 h of pon A treatment. The presence of activated (cleaved) forms of caspase-8 and caspase-9 were examined in these cells. Figure 5A shows that caspase-9, but not caspase-8, was activated following the induction of 53BP2S. To confirm these observations, the effects of specific inhibitors for caspase-3, -8, and -9 were examined. As shown in Fig. 5B, the effects of peptide inhibitors among all types of known caspases (VAD), caspase-3 (DEVD), caspase-8 (IETD) and caspase-9 (LEHD) were shown. Although VAD, DEVD and LEHD effectively blocked the PARP cleavage induced by 53BP2S, only a minimal effect was observed with IETD. These findings indicate that 53BP2S induces apoptosis through the mitochondrial (‘intrinsic’) death pathway.

View larger version (49K): [in this window] [in a new window]   Figure 5  Involvement of caspase-9 in apoptosis induced by 53BP2S. (A) Activation of caspase-9 by 53BP2S. 293/53BP2 cells were treated by pon A (5 µM) for the indicated periods (h). Each cell lysate (10 µg protein) was examined for the activated (‘cleaved’) form of caspase-9 by Western blotting with anti-caspase-9 (cleaved form) or anti-caspase-8 (cleaved form) antibodies (upper panel). ß-tubulin was used as an internal control. In the lower panel, 293/53BP2 cells were either stimulated with the agonistic anti-Fas antibody (CH-11) or treated with pon A and the activation of caspase-8 or caspase-9 was similarly examined. (B) Inhibition of the PARP cleavage by caspase inhibitors. 293/53BP2 cells were cultured with or without caspase inhibitors for 12 h and treated with pon A (5 µM) for 48 h. The cell lysate was prepared and examined for the PARP cleavage by Western blotting. The same amounts of each cell lysate (10 µg protein) were analyzed. Cont, DMSO alone; VAD, common inhibitor for the caspase-family (Z-VAD-FMK); DEVD, caspase-3-specific inhibitor (Z-DEVD-FMK); IETD, caspase-8-specific inhibitor (Z-IETD-FMK); LEHD, caspase-9-specific inhibitor (Z-IETD-FMK).

	Discussion

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Regarding the possible involvement of 53BP2 in the cellular response to DNA damage, we have previously reported the positive correlation between the level of 53BP2 mRNA expression and the sensitivity to DNA damaging agents in various human cancer cell lines although no mutation of 53BP2 gene was detected (Mori et al. 2000). In addition, Ao et al. (2001) found that 53BP2S expression augmented the cellular apoptotic response to the DNA damage. Lopez et al. (2000) observed that the DNA damage induced the 53BP2 expression and protein stabilization leading to apoptosis. Bergamaschi et al. (2004) recently reported similar observations with ASPP2 (53BP2L). Therefore, it is likely that the activated p53 may augment the 53BP2-mediated cell death. In support of this action of p53, Marchenko et al. (2000) demonstrated the mitochondrial translocation of p53 upon irradiation and induction of apoptosis through the intrinsic death pathway. Mihara et al. (2003) further explored the mitochondrial involvement of p53 and found that p53 formed a complex with Bcl-2 and BclX_L followed by permeabilization of the outer mitochondrial membrane.

Intriguingly, Iwabuchi et al. (1998) and Samuels-Lev et al. (2001) found that p53-mediated transactivation was augmented by 53BP2S and 53BP2L (ASPP2), respectively. Samuels-Lev et al. (2001) proposed a model that 53BP2L interacts with p53 in the nucleus and specifically enhances gene expression of p53 responsive proapoptotic genes such as Bax. Although the 3D structure model of p53 and 53BP2 complex (Gorina & Pavletich 1996) does not support their hypothesis because when 53BP2 binds to p53, it involves the L3 loop of p53 (required for its DNA binding) and the H1 helix (required for p53 dimerization), thus precluding the p53 binding to DNA, there may be multiple mechanisms by which 53BP2 induces apoptosis.

In addition to the possible involvement of p53 and 53BP2 in apoptosis, 53BP2 abnormality is implicated in autoimmunity such as systemic lupus erythematosus (SLE) since one of the genetic loci of the familial incidence of SLE was shown to be located to 1q42.1 (Tsao et al. 1997), to which TP53BP2 is located (Yang et al. 1997). This is coincided with the fact that abnormalities of various apoptosis-associated factors were reported in SLE and its animal models (Fisher et al. 1995; Sneller et al. 1997; Jackson & Puck 1999). Further genetic studies are needed to find a link between 53BP2 and autoimmunity.

Our observations together with those of others suggest that 53BP2 is involved in apoptosis at multiple steps and is implicated in various pathological processes. Since 53BP2 has been shown to interact with a number of proteins responsible for the regulation of apoptosis such as p53, Bcl-2 and NF-B p65 subunit, selective interaction of 53BP2 with these proteins may determine the susceptibility of cells to trigger the apoptotic pathway.

	Experimental procedures

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Reagents and antibodies

FuGENE 6 and SuperFect transfection reagents were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA) and QIAGEN (Qiagen Inc., Valencia, CA, USA), respectively. PE-conjugated annexin V and 7-AAD (Becton Dickenson, Mountain View, CA, USA), ponasterone A (pon A) (Invitrogen, La Jolla, CA, USA) were commercially obtained. The caspase inhibitors (caspase-3 inhibitor, Z-DEVD-FMK; caspase-8 inhibitor, Z-IETD-FMK; caspase-9 inhibitor, Z-LEHD-FMK; caspase-family inhibitor, Z-VAD-FMK) were purchased from MBL. Mouse monoclonal antibodies to human 53BP2 (BD Transduction Laboratories, San Diego, CA, USA), ß-tubulin (Sigma Chemical Co., St. Louis, MO, USA), human lactate dehydrogenase (LDH) (MBL, Nagoya, Japan) and human Fas (Sigma), and mouse polyclonal antibody to human mitochondrial heat shock protein 70 (Affinity Bioreagents, Golden, CO, USA) were purchased from individual suppliers. The rabbit polyclonal antibody to human 53BP2 was a generous gift from L. Naumovski (Stanford University, CA, USA). Mouse monoclonal antibodies to caspase-8 (cleaved form) and caspase-9 (cleaved form) and rabbit polyclonal antibody to PARP were purchased from Cell Signaling Technology (Beverly, MA, USA).

Plasmids

Construction of the 53BP2S expression plasmids, pcDNA3.1–53BP2 and pEGFP-53BP2, expressing 53BP2S protein (1005 amino acids) either alone or in fusion with green fluorescence protein (GFP), was reported previously (Yang et al. 1999). pCEP4-ASPP2, expressing 53BP2L (1128 amino acids), was a gift from L. Naumovski. pDsRed2-Mito, expressing a fusion protein of the Discosoma sp. red fluorescent protein linked to the mitochondrial targeting sequence from subunit VIII of human cytochrome oxidase, was purchased from BD Bioscience Clontech (Palo Alto, CA, USA).

Cell lines and cultures

The 53BP2S inducible cell line 293/53BP2 and its control cell line 293/LZ were kindly provided by Charles D. Lopez, Stanford University, CA, USA and previously described (Lopez et al. 2000). These cells were grown at 37 °C in 5% CO₂ in Dulbecco's modified Eagle medium (DMEM) with 10% (v/v) heat-inactivated foetal calf serum, 290 µg/mL of L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 600 µg/mL G418 and 500 µg/mL Zeocin. The parental 293 and HeLa cells were grown at 37 °C in DMEM with 10% (v/v) heat-inactivated foetal calf serum (IBL, Maebashi, Japan), 1 mM glutamate, 100 U/mL penicillin, and 100 µg/mL streptomycin. A human pancreatic cancer cell line MIA PaCa-2 was grown in Eagle minimal essential medium supplemented with nonessential amino acids, 10% (v/v) heat-inactivated foetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Immunostaining

Semi-confluent MIA PaCa-2 cells on Laboratory-Tek tissue culture chamber slides were fixed with 4.5% paraformaldehyde in PBS for 15 min at room temperature, rinsed twice with PBS, and incubated with PBS containing 0.5% Triton X-100 for 20 min at room temperature. They were subsequently incubated with the primary anti-53BP2 mouse antibody (B92320 [GenBank] , Transduction Laboratories, Lexington, KY, USA) for 1 h at 37 °C, rinsed three times with PBS containing 0.05% Triton X-100, and incubated with the secondary antibody, rhodamine-conjugated goat anti-mouse IgG (Calbiochem-Novabiochem, La Jolla, CA, USA), for 1 h at 37 °C. The slides were rinsed with PBS three times and mounted with buffered glycerol for fluorescent microscopic examination. Primary and secondary antibodies were diluted at 1 : 100 and 1 : 200, respectively, in PBS containing 3% bovine serum albumin.

Cell fractionation

In order to examine the cellular localization of 53BP2 in the 293/53BP2 cells, cells were pretreated with pon A (5 µM, 48 h) and subjected to fractionation using commercial kits (Nuclear/Cytosol Fractionation Kit and Mitochondria/Cytosol Fractionation Kit, BioVision, Mountain View, CA, USA). The heavy membrane precipitate containing mitochondria was extensively washed in order to avoid the contamination of cytoplasmic proteins. The identification of 53BP2 and validation of cell fractionation were performed by Western blotting with antibodies to 53BP2, LDH (cytoplasmic marker) and mitochondrial heat shock protein 70 (mitochondria marker).

Flow cytometric analysis of apoptosis

In order to assess apoptosis, flow cytometric analysis was performed using FACScan (Becton Dickinson). MIA PaCa-2 cells were transiently transfected with pcDNA3.1–53BP2 expressing 53BP2S or pCEP4-ASPP2 expressing 53BP2L using SuperFect according to the manufacturer's recommendations. Cells at a concentration of approximately 1 x 10⁶ cells/mL were washed twice with cold PBS and resuspended in annexin V binding buffer (10 mM HEPES-NaOH (pH 7.4), 140 mM NaCl and 2.5 mM CaCl₂). In some experiments, cells were double-stained with annexin V and 7-Amino-actinomycin D (7-AAD). 293/53BP2 cells were induced to express 53BP2 by incubation with 5 µM pon A and apoptotic cells were similarly counted.

Microscopic examination

In order to examine the cellular localization of 53BP2S, 293 cells were cultured on 2-well Laboratory-Tek tissue culture chamber slides and transfected with 0.4 µg of pEGFP-53BP2 expressing 53BP2S together with 0.1 µg of the mitochondria targeting plasmid, pDsRed2-Mito (BD Bioscience Clontech). The transfected cells were fixed with 4.0% paraformaldehyde in PBS for 15 min at room temperature, and observed under the confocal microscope (RADIANCE2000; Bio-Rad, Hercules, CA, USA). Each fluorophore was recorded separately using narrow-band filters centered at 522 nm for GFP fluorescence and 605 nm for DsRed2 fluorescence.

Evaluation of apoptosis by Western blotting

Apoptosis was also assessed by the cleavage of PARP, and caspases-8 and -9 by Western blotting using relevant antibodies described above. Briefly, whole cell extracts were lyzed in 200 µL of ice-cold lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 2 mM dithiothreitol, 0.25% Nonidet P-40, 1 mM phenylmethyl-sulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin and 1 µg/mL pepstatin (A). The lysate was cleared by centrifugation and the protein concentration of the whole cell extract was measured using Bio-Rad DC protein assay kit (Bio-Rad). Equal amounts of cell lysates (10 µg protein) were resolved by 10% SDS-PAGE and transferred on nitrocellulose membrane followed by incubating with individual antibodies. The immunoreactive proteins were visualized by ECL.

Determination of mitochondrial m in cultured cells

To evaluate m, cells were treated with 10 µg/mL Rh123 for 15 min at 37 °C. After incubation, cells were washed with PBS(+) three times, resuspended in PBS(+), and fluorescence was scored immediately by flow cytometer. To visualize the cells with depressed m, cells growing on Laboratory-TekII chambered cover glass were stained with 40 nM CMXRos in PBS(+) for 15 min, washed with PBS(+) three times and observed under the confocal microscope (Bio-Rad MRC600UVF). The acquisitions of the mitochondrial images were provided by 585LP emission filter with same setting (Iris: 2.0, Gain: 1.4).

	Acknowledgements

 
We thank Dr Louie Naumovski (Stanford University) for his generous gifts of 293/53BP2 cells, polyclonal antibody to 53BP2, and a plasmid expressing 53BP2L. This work was supported in part by grants-in-aid from the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japanese Human Sciences Foundation.

	Footnotes

 
Communicated by: Masayuki M. Yamamoto

These two authors contributed equally to this work.

^* Correspondence: E-mail: tokamoto{at}med.nagoya-cu.ac.jp

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Received: 8 October 2004
Accepted: 12 December 2004

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