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B and Bcl-2 family proteins
1 Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
2 Laboratory of Molecular Genetics, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Abstract |
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B (NF-B). It was demonstrated that over-expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF-B activation elicited by a physiological NF-B inducer, interleukin-1ß (IL-1ß), and anti-apoptotic Bcl-2 family proteins on the 53BP2S-mediated apoptosis. We found that both NF-B activation and Bcl-2 family proteins could prevent the 53BP2S-mediated depression of mitochondrial transmembrane potential, activation of caspase-9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti-apoptotic Bcl-2 family proteins. The regulatory network of the 53BP2S-mediated apoptosis cascade including its interacting proteins is discussed. |
Introduction |
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B (NF-B) (Yang et al. 1999), suggesting its role in carcinogenesis. We recently found that although the 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), two isoform proteins, 53BP2S and 53BP2L, formerly named 53BP2 (Yang et al. 1999) or Bcl-2 binding protein (Bbp) (Naumovski & Cleary 1996) and ASPP2 (Samuels-Lev et al. 2001), respectively, are generated by alternative splicing (Takahashi et al. 2004). We and others reported the proapoptotic action of 53BP2S/Bbp by demonstrating the annexin V staining, nuclear fragmentation, and induction of cell death (Yang et al. 1999; Lopez et al. 2000; Ao et al. 2001; Samuels-Lev et al. 2001; Bergamaschi et al. 2004). In addition, we have recently found that 53BP2S/Bbp is translocated to the mitochondria and induces cell death through the mitochondrial death pathway (Kobayashi et al. 2005). 53BP2 proteins interact with p53, p65 and Bcl-2 through the C-terminal ankyrin repeats and SH3 domain (Iwabuchi et al. 1994; Naumovski & Cleary 1996; Yang et al. 1999). Previous studies indicated 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), suggesting that 53BP2 proteins may participate in the biological actions of p53. In fact, we found that the levels of 53BP2 mRNA expression in various human cancer cell lines was correlated with the sensitivity to DNA damaging agents irrespectively of the p53 status (Mori et al. 2000), indicating the biological relevance in vivo.
In this study, we have further explored the effects of NF-B and Bcl-2 family proteins on the proapoptotic action of 53BP2S/Bbp. We found that both NF-B and Bcl-2 family proteins could prevent the 53BP2S-mediated apoptosis. The biological roles of 53BP2 proteins and these interacting proteins in the regulation of apoptosis, and their possible roles in carcinogenesis are discussed.
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Results |
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To assess the proapoptotic effect of 53BP2S/Bbp, we transfected 53BP2S gene in MIA PaCa-2 cells and the induction of apoptosis was evaluated by typical nuclear morphology and trypan blue dye exclusion assay. In Fig. 1A, when MIA PaCa-2 cells were transfected with pEGFP-53BP2 (Yang et al. 1999), expressing 53BP2S/Bbp, the typical apoptotic morphology, such as nuclear condensation and fragmentation, was observed in these transiently transfected cells. Approximately 32% of cells exhibited apoptosis when 53BP2S was transduced, which was significantly higher than the control (6.2%; P < 0.01) (Fig. 1B). To further confirm the effect of 53BP2S/Bbp, 293/53BP2 cells, a stable cell line in which 53BP2S/Bbp expression is under the stringent control of ponasteron A (pon A), were treated with pon A to induce 53BP2S/Bbp expression. After 24 h of postinduction, similar apoptotic nuclear changes were observed in 293/53BP2 cells whereas the control 293/LZ cells did not show such changes (Fig. 1C). In Fig. 1D, the number of surviving cells in 293/53BP2 and 293/LZ cultures were counted by trypan blue dye exclusion assay, with or without pon A, over time following the induction. The survival rate of 293/53BP2 cells decreased upon 53BP2S/Bbp expression and reached 54%, whereas that of the untreated cells (without pon A) was only 3%, and was similar to the background level of control cells (293/LZ).
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B activation
To examine the effect of NF-B activation on the 53BP2S-induced cell death, IL-1ß was added after the production of 53BP2S protein by ponA treatment for 24 h in 293/53BP2 cells. As shown in Fig. 2A, the 53BP2S-mediated cell death was prevented. There was a time lag of approximately 48 h between the addition of IL-1ß and the appearance of inhibition of cell death. In the experiment demonstrated in Fig. 2A, 53(B)P2S protein was detectable after 12 h ponA treatment (data not shown). The NF-B activation by IL-1ß was monitored by the induction of its DNA binding activity as demonstrated by the electrophoretic mobility shift assay (EMSA) (Fig. 2B). Whereas the cell survival rate of the 53BP2S-expressing cells was 42% after 5 days of pon A treatment, that of the cells treated with IL-1ß (24 h after the pon A treatment) exhibited an 84% survival rate, which was very close to the level of control 293/LZ cells not expressing 53BP2S/Bbp (Fig. 2A). The IL-1ß treatment had no effect on the level of 53BP2S/Bbp expression per se in 293/53BP2 cells (data not shown). In addition, we detected expression of Bcl-2 and Bcl-XL proteins and their levels were not further up-regulated by the IL-1ß treatment (data not shown).
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We then examined the effect of Bcl-2 family proteins on the 53BP2S-mediated apoptosis. Bcl-2, Bcl-XL, and 
BH4 mutant of Bcl-XL lacking the crucial BH4 domain, were expressed together with 53BP2S/Bbp in MIA PaCa-2 cells and the number of apoptotic cells were counted. As shown in Fig. 4, both Bcl-2 and Bcl-XL expression inhibited 53BP2S-induced cell death in a dose-dependent manner, with greater inhibitory effect of Bcl-XL. No such effect was observed with BH4 mutant.
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m), we examined m of the transfected cells by staining with a fluorescence dye CMX-ROS (Fig. 5). Whereas 53BP2S/Bbp induced the reduction of m, observed by the reduction of mitochondrial staining with CMX-ROS, expression of Bcl-2 or Bcl-XL, but not BH4 mutant, prevented its reduction. These results confirmed the anti-apoptotic effect of anti-apoptotic Bcl-2 proteins shown in Fig. 4.
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Discussion |
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B and Bcl-2 family proteins on the 53BP2S-mediated apoptosis. The partial clone of 53BP2 was initially identified as one of the interacting proteins of p53 (Iwabuchi et al. 1994). Since NF-B p65 subunit and Bcl-2 are known to inhibit apoptosis (DeLuca et al. 1998; Haddad 2004) and have previously been reported to interact with 53BP2S/Bbp (Naumovski & Cleary 1996; Yang et al. 1999), our findings indicate that anti-apoptotic actions of these proteins appear to be through blocking the pro-apoptotic actions of 53BP2S/Bbp at least in a part. Thus, 53BP2S-mediated apoptosis is regulated by p53, NF-B and Bcl-2/Bcl-XL (Fig. 6).
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It is not clear whether p53 is required for the proapoptotic action of 53BP2 proteins. Lopez et al. (2000) observed that the DNA damage induced the 53BP2S/Bbp expression and protein stabilization leading to apoptosis in the context of wild-type p53, although p53 suppressed 53BP2S/Bbp expression in undamaged cells. We and others have previously reported very low 53BP2 protein levels in spite of the highly abundant mRNA and suggested a possibility that the 53BP2 level might be regulated at the post-translational level (Naumovski & Cleary 1996; Yang et al. 1999; Lopez et al. 2000). Thus, the p53 interaction may stabilize 53BP2 proteins and enhance its apoptotic action. In addition, it was reported that p53-mediated transactivation was augmented by 53BP2S/Bbp (Iwabuchi et al. 1998) and 53BP2L/ASPP2 (Samuels-Lev et al. 2001). Samuels-Lev et al. (2001) proposed a model in which 53BP2L/ASPP2 interacts with p53 in the nucleus and specifically enhances gene expression of p53 responsive proapoptotic genes such as Bax. However, although MIA PaCa-2 cells contain p53 mutation at R248 (Yoshikawa et al. 1999), known to be involved in the interaction with 53BP2 at least in a crystal structure of the p53–53BP2 protein complex (Gorina & Pavletich 1996), we found that 53BP2S/Bbp could induce apoptosis in MIA PaCa-2 cells much more efficiently than in 293 cells, expressing wild-type p53 (Fig. 1). Thus, it is yet to be investigated whether 53BP2-mediated apoptosis requires the interaction with p53 and whether 53BP2S/Bbp and 53BP2L/ASPP2 have distinct actions in cells. It is also possible that proapoptotic actions of 53BP2 proteins are exerted at multiple levels.
We confirmed the inhibitory effect of NF-B activation on the proapoptotic action of 53BP2S/Bbp. We demonstrate that anti-apoptotic action of NF-B was evident even after the initiation of 53BP2S-mediated cell death (Fig. 2). The anti-apoptotic nature of NF-B has been well established (DeLuca et al. 1998; Chen et al. 2001; Karin & Lin 2002). There are at least two mechanisms by which NF-B inhibits apoptosis: (i) by induction of gene expression of anti-apoptotic proteins such as c-IAP2 (Chu et al. 1997), IEX-1 L (Wu et al. 1998) and even Bcl-XL (Chen et al. 2000) and (ii) through blocking the action of proapoptotic factors by direct or indirect interaction (Yang et al. 1999). However, these two distinct mechanisms are not mutually exclusive for neither one mechanism alone can fully explain the strong anti-apoptotic action of NF-B. In suppor of the latter mechanism, we observed that NF-B could inhibit the TNF-
-mediated apoptosis without de novo protein synthesis at least in some cell lines (Kajino et al. 2000). Although the intracellular location where NF-B interacts with 53BP2S/Bbp is not known, it may occur in the vicinity of mitochondria since NF-B and IB are found in the mitochondrial intermembrane space and TNF- can liberate the NF-B within mitochondria (Bottero et al. 2001; Cogswell et al. 2003). Thus, 53BP2S/Bbp could serve as one of the molecular targets for the anti-apoptotic actions of NF-B. However, since the delay of the anti-apoptotic effect of IL-1ß on 53BP2S-mediated apoptosis was observed, it is possible that both direct and indirect effects of NF-B actions are involved in 53BP2S-induced apoptosis.
These observations have indicated the role of NF-B in various pathologies and implicated NF-B as a common therapeutic target (Karin & Lin 2002). For example, Arlt et al. (2002) reported that the blockade of NF-B activation cascade by its inhibitors MG132 and sulfasalazine greatly augmented the effect of doxorubicin or etoposide in inducing the cell death of human pancreatic cancer cell lines. Use of these compounds and derivatives is expected to augment the therapeutic efficacy of conventional cancer therapy.
These findings support a possibility that 53BP2 proteins are involved in various biological processes such as carcinogenesis and the cellular response to DNA damage. Mori et al. (2000) reported that the level of 53BP2 mRNA expression in various human cancer cell lines was correlated with the sensitivity to DNA damaging agents although no mutation of 53BP2 gene was detected. In addition, Ao et al. (2001) observed that 53BP2S/Bbp expression augmented the cellular apoptotic response to the DNA damage. Thus, selective action of 53BP2 with p53, Bcl-2 and NF-B p65 subunit may determine the susceptibility of cells to trigger the apoptotic pathway in response to the DNA damage (Fig. 6).
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Experimental procedures |
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Human recombinant cytokine IL-1ß (Boehringer Mannheim, Mannheim, Germany), Hoechst-33258 (Molecular Probes, Eugene, OR, USA), CMX-ROS (Molecular Probes), Ponasterone A (pon A) (Invitrogen, La Jolla, CA, USA) were commercially obtained. SuperFect transfection reagents was purchased from QIAGEN (Qiagen Inc., Valencia, CA, USA). Mouse monoclonal antibodies to human 53BP2 proteins (BD Transduction Laboratories, San Diego, CA, USA), and rabbit polyclonal antibody to human p65 (Santa Cruz Biotech, Santa Cruz, CA, USA), goat polyclonal antibody to human p50 (Santa Cruz Biotech) 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/Bbp expression plasmids, pcDNA3.1–53BP2 and pEGFP-53BP2, expressing 53BP2S/Bbp protein (1005 amino acids) either alone or in fusion with green fluorescence protein (GFP), was reported previously (Yang et al. 1999). Human bcl-2, bcl-xL, and bclxL mutant (BH4) cDNAs were subcloned into the pUC-CAGGS expression vector as previously described (Shimizu et al. 1996, 2000).
Cell lines and cultures
The 53BP2S/Bbp 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 fetal 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. 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 fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
Microscopic examination
In order to examine the apoptotic cell morphology, 293/53BP2 cells were cultured on 2-well Laboratory-Tek tissue culture chamber slides (Nunc, Inc., Naperville, IL, USA) and stimulated by pon A. The cells were fixed with 4.0% paraformaldehyde in PBS for 15 min at room temperature, rinsed twice in PBS and stained with Hoechst-33258. The apoptotic cells were identified by their shrunken morphology and the condensed and fragmented nuclear morphology.
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 immumoreactive proteins were visualized by ECL (Asamitsu et al. 2003).
Determination of mitochondrial m in cultured cells
To visualize the cells with depressed m, cells growing on Laboratory-TekII chambered cover glass were stained with 40 nM CMX-Ros 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).
Cell survival assay
To quantitatively measure the cell survival, cell cultures were stained with trypan blue and counted under microscopy in triplicates. To determine the anti-apoptotic effect of NF-B, 293/53BP2 cells were cultured without pon A to 50% confluence, stimulated with pon A (5 µM), and after 24 h in culture they were treated with IL-1ß (20 ng/mL). The cell survival was assessed every 24 h for an additional 4 days after the stimulation with IL-1ß.
Electrophoretic mobility shift assay (EMSA)
293/53BP2 cells were pretreated with or without IL-1ß (20 ng/mL) and nuclear extracts were prepared as previously described (Takada et al. 2002). The double-stranded oligonucleotide probe for NF-B was synthesized and end-labeled by 
-[32P]-dATP. The B sequence was taken from the human immunodeficiency virus long-terminal repeat (HIV-LTR). The B sequence used was forward (5'-TTT CTA GGG ACT TTC CGC CTG GGG ACT TTC CAG-3') and complement (5'-TTT CTG GAA AGT CCC CAG GCG GAA AGT CCC TAG-3'). Nuclear extracts were incubated in 10 µL EMSA buffer containing the radiolabelled B oligonucleotide probe. The samples were analyzed by 6% non-denaturing PAGE. For NF-B supershift assay, antibodies to NF-B p65 and/or p50 subunits were added (30 min, 4 °C).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tokamoto{at}med.nagoya-cu.ac.jp
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References |
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Received: 27 January 2005
Accepted: 5 May 2005
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transfection with pEGFP (control); 
transfection with pEGFP-53BP2. (C) Nuclear morphology of apoptotic cells in 293/53BP2 upon 53BP2S/Bbp induction. The pon A-induced cells were stained with Hoechst-33258 and observed under a fluorescent microscopy. The apoptotic cells were identified by their fragmented and/or condensed nuclear morphology (indicated by arrowheads). (D) Induction of cell death by 53BP2S/Bbp. 293/53BP2 and 293/LZ (control) cells were stimulated with pon A (5 µM) for the indicated periods (in days) and stained with trypan blue. The surviving cells, not stained with trypan blue, were counted.
). Control (
m was observed under the confocal microscope. Figures on the left represent merged images of GFP (53BP2S/Bbp) staining (green) and CMX-ROS (