Deletion of the BH1 Domain of Bcl-2 Accelerates Apoptosis by Acting in a Dominant Negative Fashion*
Makoto Kawatani and
Masaya Imoto
From the
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan
Received for publication, December 20, 2002
, and in revised form, March 3, 2003.
 |
ABSTRACT
|
---|
To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/
BH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/
BH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/
BH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/
BH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL.
 |
INTRODUCTION
|
---|
Apoptosis, a morphologically distinguished form of pro-grammed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases including cancer, autoimmune disease, and neuro-degenerative disorders (1, 2, 3). Mitochondria is the crucial regulatory organelle for the signaling pathway of apoptosis (4, 5). Many death signals cause irreversible dysfunction of mitochondria, leading to the release of several mitochondrial intermembrane proteins. Cytochrome c was the first characterized mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be involved in the activation of caspase by forming apoptosomes (6, 7, 8). Like cytochrome c, other mitochondrial intermembrane proteins such as AIF, Smac/DIABLO, Endo G, and Omi/HtrA2 were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process (9).
The Bcl-2 family of proteins plays a pivotal role in the regulation of mitochondrial integrity and response to apoptotic signals (10). Bcl-2 family proteins can be subdivided into three distinct groups: (i) anti-apoptotic members such as Bcl-2 and Bcl-xL with sequence homology at Bcl-2 homology 1 (BH1),1 BH2, BH3, and BH4 domains; (ii) pro-apoptotic molecules, such as Bax and Bak, with sequence homology at BH1, BH2, and BH3; and (iii) pro-apoptotic proteins that share homology only at the BH3 domain, such as Bid, Bik, Noxa, and Bim (11). These Bcl-2 family members are characterized by their ability to interact and form homo- and heterodimers (10, 12). Structural and mutational analyses have been used to assess the function of each BH domain in anti-apoptotic Bcl-2 family members. The BH4 domain, which is conserved only among anti-apoptotic Bcl-2 family members, has been reported to bind with other proteins regulating apoptosis, including calcineurin (13), Raf-1 (14), and voltage-dependent anion channel (VDAC) (15), and deletion of BH4 from Bcl-2/Bcl-xL has been shown to abrogate their anti-apoptotic ability (16, 17, 18, 19, 20). This indicates that BH4 of anti-apoptotic Bcl-2 family members is crucial for anti-apoptotic events. The BH3 domain plays the role of ligand during dimerization of Bcl-2 family proteins, whereas a combination of the BH1, BH2, and BH3 domains appears to be required for forming an elongated hydrophobic cleft, which can bind the BH3-containing peptides of the death promoters (21, 22, 23).
Earlier studies have indicated that the BH1 and BH2 domains of Bcl-2/Bcl-xL are crucial for dimerization with pro-apoptotic family members (12, 24). Through BH1 and BH2, anti-apoptotic proteins can heterodimerize with the pro-apoptotic protein, and the ratio between anti- and pro-apoptotic proteins determines survival or death following various apoptotic stimuli. Mutation of the highly conserved glycine 145 to alanine of the BH1 domain in Bcl-2 (Bcl-2/G145A) (12), the homologous G138A mutation in Bcl-xL (Bcl-xL/G138A) (24), or tryptophan 188 to alanine of the BH2 domain in Bcl-2 abrogates the ability of Bcl-2 and Bcl-xL to dimerize with Bax. These mutations also fail to inhibit cell death induced by deprivation of interleukin-3 (IL-3) or dexamethasone in the murine prolymphocytic IL-3-dependent cell line, F5.12 (12, 24). In addition, deletion of BH1 or BH2 has been shown to abrogate the death repressor activity of Bcl-2 in staurosporine-induced cell death in human GM701 fibroblasts (17). Contrary to these indications, others have suggested that heterodimerization of Bcl-2/Bcl-xL with Bax through the BH1 and BH2 domains is not required for anti-apoptotic function (25, 26). Moreover, Oh et al. (27) has reported that overexpression of the BH1 or BH2 point mutant Bcl-2 protein suppressed staurosporine-induced apoptosis in a dopaminergic neuronal cell line, MN9D. Therefore, these contradictory findings suggest that the requirement of these dimerization domains of Bcl-2/Bcl-xL may be cell- and/or stress type-specific, and the mechanism of regulating apoptosis by Bcl-2 family member is not fully elucidated.
In the course of examining the role of homo- and heterodimerization of Bcl-2 family members in antitumor drug-induced apoptosis using various Bcl-2 mutant-overexpressing human small cell lung carcinoma (SCLC) Ms-1 cells, we found that overexpression of Bcl-2 lacking a BH1 domain accelerated antitumor drug-induced apoptosis. Here, we show that a deletion mutant of the BH1 domain of Bcl-2 acts as a dominant negative of anti-apoptotic proteins of the Bcl-2 family.
 |
EXPERIMENTAL PROCEDURES
|
---|
MaterialsCamptothecin, colchicine, anti-
-tubulin (clone DM 1A) monoclonal antibody, and anti-Bak (B5929) antibody were purchased from Sigma Chemical Co. Vinblastine sulfate and paclitaxel were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). C2-ceramide was obtained from Biomol (Plymouth Meeting, PA). Anti-poly(ADP-ribose) polymerase (PARP) (H-250), anti-Bcl-2 (
C21), and anti-Bax (N-20) polyclonal antibodies and anti-Myc (9E10) monoclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytochrome c (clone 7H8.2C12) monoclonal antibody was obtained from BD PharMingen (San Diego, CA). Anti-Bcl-2 (clone 124) monoclonal antibody was from DAKO (Glostrup, Denmark).
PlasmidspGEM-T Easy-human bcl-2, -human bcl-xL, and -human bax plasmids and pCI-neo plasmid were kindly provided by Dr. S. Simizu (RIKEN). pCAGGS-human bak plasmid was kindly provided by Dr. Y. Tsujimoto (Osaka University Graduate School of Medicine, Japan). To generate the pCI-myc-neo plasmid, the fragment of 6x Myc epitope in pCS-MT was excised by digestion with BamHI/XbaI, blunted using 1 unit of T4 DNA polymerase (Takara, Tokyo, Japan) and then subcloned into an EcoRI-digested and T4 DNA polymerase-blunted pCI-neo plasmid. Bcl-2 deletion mutants were previously described (28). Bcl-2 point mutants, Bcl-2/G145A and Bcl-2/D140A, were constructed as follows. First, 100 ng of pGEM-T Easy-human bcl-2 plasmid was used for amplification under the following PCR conditions: 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 50 °C, and extension for 2 min at 72 °C. The following specific primers were used: Bcl-2/G145A mutant, 5'-GGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGCAAGGATTGTGGCCTTC-3' (forward) and 5'-TATTTAGGTGACACTATAG-3' (SP-6, reverse); Bcl-2/D140A mutant, 5'-GGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGGGCAGG GGTGAACTGGGGGAGG-3' (forward) and 5'-TATTTAGGTGACACTATAG-3' (SP-6, reverse). These PCR products were digested by BstXI, and then subcloned into a BstXI-digested pGEM-T Easy-human bcl-2 plasmid. These constructs were confirmed by DNA sequence analysis and digested by EcoRI, and then cloned into the mammalian expression plasmid, pCI-neo or pCI-myc-neo.
Cell Culture and TransfectionHuman SCLC Ms-1 and human embryonic kidney 293T cells were cultured in RPMI 1640 medium containing 5% fetal bovine serum, penicillin G (100 units/ml), and kanamycin (0.1 mg/ml) at 37 °C in 5% CO2, 95% air. For establishing stable cell lines, Ms-1 cells (2 x 106) were transfected with 10 µg of plasmid pCI-neo alone or pCI-neo containing various constructs using LipofectAMINETM reagent (Invitrogen) according to the manufacturer's instructions. These transfectants were selected by supplementing the medium with 700 µg/ml G418 (Sigma Chemical Co.). Single cell clones were isolated from the bulk transfectants by limited dilution cloning in 96-well plates and characterized by Western blot analysis as described below. 293T cells were transiently transfected with 1 µg of plasmid DNA per 105 cells using the LipofectAMINETM reagent according to the manufacturer's instructions.
Western Blot AnalysisThe cells were lysed in radioimmune precipitation assay buffer (25 mM HEPES, 1.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.5 M NaCl, 5 mM EDTA, 50 mM NaF, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml leupeptin, pH 7.8) at 4 °C with sonication. The lysates were centrifuged at 15,000 x g for 15 min, and the concentration of the protein in each lysate was determined with Coomassie Brilliant Blue G-250. Loading buffer (42 mM Tris-HCl, 10% glycerol, 2.3% SDS, 5% 2-mercaptoethanol, and 0.002% bromphenol blue) was then added to each lysate, which was subsequently boiled for 3 min and then electrophoresed on an SDS-polyacrylamide gel. Proteins were transferred to Hybond-P membrane (Amersham Biosciences) and immunoblotted with anti-Bcl-2 (clone 124 or
C21), anti-Bax (N-20), anti-PARP (H-250), anti-Bak (B5929) or anti-Myc (9E10) antibody. Detection was performed with enhanced chemiluminescence reagent (PerkinElmer Life Sciences).
Cell Viability AssayDrug-treated or untreated Ms-1 cells or transiently transfected 293T cells were stained with trypan blue (Sigma Chemical Co.), and the percentage of viable cells was determined using a hemocytometer. Cell viability (%) means the ratio of the number of trypan blue-impermeable cells in total cell counts (trypan blue-impermeable cell number/total cell number).
Apoptosis AssayCells were seeded onto glass coverslips. Detection of apoptotic cells were performed by annexin V and propidium iodide (PI) staining using the annexin V-EGFP apoptosis detection kit (MBL, Nagoya, Japan) according to the manufacturer's instructions. Apoptotic cells (annexin V-positive and PI-negative cells) were scored under a fluorescence microscope.
Evaluation of Cytosolic Cytochrome cDrug-treated or untreated cells (1 x 107) were harvested and washed with ice-cold phosphate-buffered saline. Cells were resuspended in 600 µl of buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol) containing 250 mM sucrose and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 1 mM leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml chymostatin). The cells were homogenized with 20 strokes of a glass Pyrex homogenizer. Unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifuging the homogenates at 1,000 x g for 10 min at 4 °C. The supernatant was centrifuged at 100,000 x g for 1 h at 4 °C to prepare cytosol. The resulting supernatant was used as the soluble cytosolic fraction. Western blot analysis for cytochrome c (anti-cytochrome c (clone 7H8.2C12) monoclonal antibody) or
-tubulin (anti-
-tubulin (clone DM 1A) monoclonal antibody) was performed.
ImmunoprecipitationTransiently transfected 293T cells were resuspended in Extraction buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol, 0.1% Tween 20, 10 mM sodium-
-glycerophosphate, 10% glycerol, 1 mM NaF, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml leupeptin, pH 7.5), sonicated on ice and centrifuged at 15,000 x g for 20 min at 4 °C. The supernatants were precleared by using protein G-agarose resin suspension (Oncogene Research Products, Boston, MA), and then immunoprecipitated using 10 µl of anti-Bax (N-20), anti-Bak (B5929), or anti-Myc (9E10) antibody overnight at 4 °C. Next, 10 µl of protein G-agarose resin suspension was added to each of the immunocomplexes, and the mixtures were rotated for 5 h at 4 °C. After 4 washes with Extraction buffer, the immunocomplexes were eluted with SDS sample buffer. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis with the antibodies.
 |
RESULTS
|
---|
Overexpression of Bcl-2/
BH1 Accelerates Camptothecin-induced Apoptosis in Ms-1 CellsStable clones of human SCLC Ms-1 cells, overexpressing wild-type (wt) Bcl-2, lacking the BH1 domain of Bcl-2 (Bcl-2/
BH1) or vector only (neo), were exposed to camptothecin, and their cell viabilities were assessed by trypan blue dye exclusion assay. As shown in Fig. 1A, camptothecin decreased the viability of vector control clone (Ms-1/neo 13) in a time-dependent manner and failed to induce cell death in the wt Bcl-2-overexpressing Ms-1 clone (Ms-1/wt Bcl-2 7). On the other hand, camptothecin-induced cell death was markedly accelerated in the Bcl-2/
BH1-overexpressing Ms-1 clone (Ms-1/Bcl-2/
BH1 72) when compared with the Ms-1/neo 13 clone (Fig. 1A). When Ms-1/Bcl-2/
BH1 72 was treated with 0.1 or 0.3 µg/ml camptothecin for 18 h, cell viability decreased about 60 or 40%, respectively, under the conditions where significant cell death was not observed in Ms-1/neo 13 (Fig. 1B), indicating that expression of Bcl-2/
BH1 reduced the dosage of camptothecin required to induce cell death. Similar results were obtained from independently derived clones (Fig. 1C). Moreover, overexpression of Bcl-2/
BH1 also accelerated cell death induced by vinblastine, paclitaxel, or colchicine, as shown in Fig. 1D, suggesting that the acceleration of cell death by Bcl-2/
BH1 is not specific to camptothecin-induced cell death. On the other hand, Bcl-2/
BH1 and wt Bcl-2 inhibited C2-ceramide-induced cell death (Fig. 1D) as well as cytochrome c release and caspase-3(-like) protease activation (data not shown).
Bcl-2/
BH1, but Not Bcl-2/
BH2, Bcl-2/
BH3, and Bcl-2/
BH4, Accelerates the Step of Cytochrome c Release from Mitochondria Induced by CamptothecinNext, we examined whether other BH domain deletion mutants of Bcl-2/
BH2, Bcl-2/
BH3, and Bcl-2/
BH4 also accelerated the cell death induced by camptothecin. As shown in Fig. 2, although all deletion mutant clones tested expressed similar amounts of mutant Bcl-2 protein (Fig. 2A), significant cell death was observed in the Ms-1/Bcl-2/
BH1 no. 72 clone but not the Ms-1/Bcl-2/
BH2 no. 109, Ms-1/Bcl-2/
BH3 no. 55, or Ms-1/Bcl-2/
BH4 no. 35 clone 10 h following camptothecin treatment (Fig. 2B). Fig. 2C shows that 18 h after treatment with 1 µg/ml camptothecin, the cell viability of the Ms-1/Bcl-2/
BH3 no. 55 and Ms-1/Bcl-2/
BH4 no. 35 clones was reduced, and the rate of cell death in these clones was quite similar to that in Ms-1/neo no. 13; however, the Ms-1/Bcl-2/
BH2 no. 109 clone was resistant to camptothecin. Similar results were obtained from at least two independently derived clones from each transfectant (data not shown).
We next examined the effect of Bcl-2/
BH1 on camptothecin-induced cytochrome c release. As shown in Fig. 3A, cytosolic cytochrome c was detected in Ms-1/Bcl-2/
BH1 no. 72 at 1 h after camptothecin treatment, under the conditions where cytochrome c release was not yet detected in camptothecin-treated Ms-1/neo no. 13 or the other transfectants. Moreover, at 2 h after the treatment with camptothecin, activation of caspase-3(-like) proteases, as estimated by the cleavage of PARP, was also markedly induced only in the Ms-1/Bcl-2/
BH1 no. 72 clone (Fig. 3B). These results indicate that Bcl-2/
BH1 accelerates the release of cytochrome c leading to caspase-3-(-like) protease activation induced by camptothecin, thereby enhancing the cell death. On longer exposure to camptothecin, the release of cytochrome c (4 h) and cleavage of PARP (6 h) occurred in Ms-1/Bcl-2/
BH3 no. 55 and Ms-1/Bcl-2/
BH4 no. 35 as well as in Ms-1/neo no. 13 (Fig. 3, A and B). In contrast, the Ms-1/Bcl-2/
BH2 no. 109 clone showed significant resistance to camptothecin, when compared with the Ms-1/neo no. 13 clone (Fig. 3, A and B).
Mutant Bcl-2/G145A but Not Bcl-2/D140A Accelerates Camptothecin-induced Cell DeathMutational and structural analyses have indicated that the BH1 domain of Bcl-2/Bcl-xL is crucial for dimerization with pro-apoptotic family members (12, 25). Therefore, to investigate whether the promotional effect on apoptosis of Bcl-2/
BH1 was due to a failure of interaction with pro-apoptotic members, Bax and Bak, we generated two point mutations in the BH1 domain of Bcl-2: Bcl-2/G145A, which had been unable to bind Bax or Bak (12); Bcl-2/D140A, which corresponds with Bcl-xL/D133A that had been able to bind Bak, but not Bax (25). Each of these mutant Bcl-2 products was first tested for the ability to heterodimerize with Bax or Bak. 293T cells were transiently co-transfected with pCI-neo plasmids encoding Bax or Myc-tagged Bak and wt Bcl-2 or these mutants, and co-immunoprecipitation was performed with anti-Bax or anti-Myc antibody. In agreement with a previous report (12), Bcl-2/G145A bound neither Bax nor Myc-tagged Bak (Fig. 4, A and B). We found that Bcl-2/D140A failed to interact with Bax, but still interacted with Myc-tagged Bak (Fig. 4, A and B). Then, we established stable Ms-1 cells overexpressing Bcl-2/G145A or Bcl-2/D140A (Fig. 5A) and analyzed them for the ability to enhance camptothecin-induced cell death. Two clones overexpressing Bcl-2/G145A (Ms-1/Bcl-2/G145A nos. 1 and 4) displayed increased camptothecin-induced cell death compared with Ms-1/neo no. 13 with similar kinetics observed in Ms-1/Bcl-2/
BH1 no. 72, as shown in Fig. 5B. In contrast, camptothecin-induced cell death was completely inhibited in two clones overexpressing Bcl-2/D140A (Ms-1/Bcl-2/D140A nos. 25 and 17) (Fig. 5C). Similarly, Ms-1/Bcl-2/G145A no. 4 displayed increased cytosolic cytochrome c and apoptosis induced by camptothecin compared with Ms-1/neo no. 13, however, Ms-1/Bcl-2/D140A no. 25 as well as Ms-1/wt Bcl-2 no. 7 was resistant to camptothecin (Fig. 5, D and E). Thus, Bcl-2/G145A but not Bcl-2/D140A showed similar activity to Bcl-2/
BH1, suggesting that the promotional effect on apoptosis of Bcl-2/
BH1 might require Bcl-2 to lose the interaction with both Bax and Bak. On the other hand, cytochrome c release and apoptosis induced by C2-ceramide were not observed in Ms-1/Bcl-2/G145A no. 4 and Ms-1/Bcl-2/D140A no. 25 (Fig. 5, D and E).

View larger version (22K):
[in this window]
[in a new window]
|
FIG. 4. Bcl-2/D140A fails to interact with Bax but interacts with Bak. A, 293T cells were transiently co-transfected with pCI-neo plasmid encoding Bax and pCI-neo empty plasmid (neo) or pCI-neo plasmids encoding wt Bcl-2 or the indicated BH1 domain point mutants. After 24 h of transfection, lysates were immunoprecipitated with anti-Bax antibody and immunoprecipitates were immunoblotted with anti-Bcl-2 (clone 124) or anti-Bax antibody. In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) antibody. The asterisk represents the immunoglobulin light chain. B, 293T cells were transiently co-transfected with pCI-neo plasmid encoding Myc-tagged Bak and pCI-neo empty plasmid (neo) or pCI-neo plasmids encoding wt Bcl-2 or the indicated BH1 domain point mutants. After 24 h of transfection, lysates were immunoprecipitated with anti-Myc antibody, and immunoprecipitates were immunoblotted with anti-Bcl-2 ( C21) or anti-Myc (to detect Bak) antibody. In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) antibody. The data shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
FIG. 5. Overexpression of Bcl-2/G145A but not Bcl-2/D140A accelerates camptothecin-induced apoptosis in Ms-1 cells. A, Western blot analysis of wt Bcl-2 and BH1 domain point mutanto-verexpressing Ms-1 cell lines. Equal aliquots of protein extracted from stable transfectants of the indicated Ms-1 cells were immunoblotted with anti-Bcl-2 (clone 124) or anti-Bax antibody. B, stable transfectants of the indicated Ms-1 cells were treated with 1 µg/ml camptothecin for the periods indicated. C, stable transfectants of the indicated Ms-1 cells were treated with 1 µg/ml camptothecin for 18 h. Cell viability was assessed by trypan blue dye exclusion assay. Values are means ± S.D. of quadruplicate determinations. D, stable transfectants of the indicated Ms-1 cells were treated with 1 µg/ml camptothecin for 8 h or 30 µM C2-ceramide for 8 h. Apoptotic cells (annexin V-positive and PI-negative cells) were scored under a fluorescence microscope. Values are means ± S.D. of quadruplicate determinations. E, stable transfectants of the indicated Ms-1 cells were treated with 1 µg/ml camptothecin for 4 h or 30 µM C2-ceramide for 6 h. Cytosolic proteins were isolated and immunoblotted with anti-cytochrome c or anti- -tubulin antibody, as described under "Experimental Procedures."
|
|
The BH2 domain as well as BH1 domain of Bcl-2/Bcl-xL has been reported to be crucial for dimerization with pro-apoptotic family members (12, 25). However, overexpression of Bcl-2/
BH2 did not accelerate camptothecin-induced apoptosis, but rather, significantly inhibited it (Figs. 2 and 3). In order to verify this difference, we confirmed the interaction between Bcl-2/
BH2 and Bax or Bak. The pCI-neo plasmid encoding wt Bcl-2, Bcl-2/
BH1, Bcl-2/G145A, or Bcl-2/
BH2 was transiently co-transfected with the pCI-neo plasmid encoding Bax or Myc-tagged Bak into 293T cells and co-immunoprecipitation was performed with anti-Bax or anti-Myc antibody. As shown in Fig. 6A and B, although the binding activity was reduced, Bcl-2/
BH2 still retained the ability to bind to Bax or Myc-tagged Bak, under the conditions where Bcl-2/
BH1 or Bcl-2/G145A failed to interact with Bax or Myc-tagged Bak, respectively. Therefore, these results suggest that the different activity between Bcl-2/
BH1 and Bcl-2/
BH2 for regulating apoptosis induced by camptothecin is caused by the difference between Bcl-2/
BH1 and Bcl-2/
BH2 in interacting with pro-apoptotic members, Bax and Bak.
Bcl-2/G145A as Well as Bcl-2/
BH1 Accelerates Bax- or Bak-induced ApoptosisBecause Bcl-2/G145A and Bcl-2/
BH1 failed to bind to Bax and Bak, we next examined whether Bax- or Bak-induced apoptosis is also accelerated by these two mutants. The pCI-neo plasmid encoding wt Bcl-2 or Bcl-2/G145A, or control vector was transfected into 293T cells with or without the pCI-neo plasmid encoding Bax. As shown in Fig. 7A, expression of Bax induced cell death in a time-dependent manner, and wt Bcl-2 completely blocked the Bax-induced cell death. In contrast, Bcl-2/G145A significantly accelerated Bax-induced cell death (Fig. 7A). A similar result was obtained when cells were transfected with Bak instead of Bax (Fig. 7B). Acceleration of Bax- or Bak-induced cell death was also observed by Bcl-2/
BH1 expression (Fig. 7, C and D). Moreover, Bcl-2/G145A accelerated Bax-induced caspase-3(-like) protease activation under the conditions where wt Bcl-2 completely suppressed it (Fig. 8). Thus, Bcl-2/G145A and Bcl-2/
BH1 showed a promotional effect on apoptosis induced not only by camptothecin but also by Bax and Bak.

View larger version (25K):
[in this window]
[in a new window]
|
FIG. 7. Bcl-2/G145A as well as Bcl-2/ BH1 accelerates Bax- or Bak-induced cell death in 293T cells. A and B, 293T cells were transiently transfected with 0.5 µg of pCI-neo plasmids encoding Bax (A), 0.5 µg of Myc-tagged Bak (B), 0.3 µg of wt Bcl-2 and 0.3 µg of Bcl-2/G145A alone, or in combination. Note that neo was transfected with 0.8 µg of pCI-neo empty plasmid alone. The total amounts of DNA were normalized to 0.8 µg with pCI-neo plasmid. C and D, 293T cells were transiently transfected with 0.5 µg of pCI-neo plasmids encoding Bax (C), 0.5 µg of Myc-tagged Bak (D), 0.3 µg of wt Bcl-2 and 1.25 µg of Bcl-2/ BH1 alone, or in combination. Note that neo was transfected with 1.75 µg of pCI-neo empty plasmid alone. The total amounts of DNA were normalized to 1.75 µg with pCI-neo plasmid. After the indicated times of transfection, cell viability was assessed by trypan blue dye exclusion assay. Values are means ± S.D. of quadruplicate determinations. Transfection efficiencies were >80% for all samples, as determined by co-transfection with 0.3 µg of pEGFP-N1 plasmid.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
FIG. 8. Bcl-2/G145A accelerates Bax-induced caspase-3(-like) protease activation in 293T cells. 293T cells were transiently transfected with pCI-neo plasmids encoding Bax, wt Bcl-2, and Bcl-2/G145A alone, or in combination. Note that (-) was transfected with pCI-neo plasmid. After 16 h of transfection, lysates were immunoblotted with anti-PARP or anti-Bcl-2 (clone 124) antibody.
|
|
Bcl-2/G145 or Bcl-2/
BH1 Acts as a Dominant Negative Bcl-2Although the BH3 deletion mutant of Bcl-2 also failed to bind to Bax and Bak, this mutant did not accelerate camptothecin-induced cell death (Fig. 2B). Therefore, it seems likely that Bcl-2/
BH1 or Bcl-2/G145A abrogates the ability of endogenous Bcl-2 to inhibit apoptosis by acting in a dominant negative fashion, thereby enhancing cell death induced by camptothecin as well as Bax and Bak. To directly test this hypothesis, 293T cells were transiently transfected with pCI-neo plasmids encoding Bax, Myc-tagged wt Bcl-2 and Bcl-2/G145A alone, or in combination. At 40 h after transfection, expression of Bax induced cell death, and the Bax-induced cell death was inhibited by co-expression of Myc-tagged wt Bcl-2 (Fig. 9A). However, the inhibition of Bax-induced cell death by Myc-tagged wt Bcl-2 was cancelled by the expression of Bcl-2/G145A without affecting the expression levels of Myc-tagged wt Bcl-2 and Bax, suggesting that Bcl-2/G145A abrogates the action of wt Bcl-2 for inhibiting Bax-induced cell death (Fig. 9A). Similarly, Bcl-2/G145A and Bcl-2/
BH1 abrogated the action of wt Bcl-2 for inhibiting Bax- or Bak-induced apoptosis, as judged from annexin V staining (Fig. 9B). Moreover, Bcl-2/G145A or Bcl-2/
BH1 abrogated the anti-apoptotic action of wt Bcl-xL (data not shown). To examine the interaction with wt Bcl-2 and Bcl-2/G145A, we transiently co-transfected pCI-neo plasmids encoding non-tagged wt Bcl-2 or Bcl-2/G145A and Myc-tagged wt Bcl-2 into 293T cells and performed co-immunoprecipitation with anti-Myc antibody. In agreement with a previous report (12), Bcl-2/G145A interacted with Myc-tagged wt Bcl-2 (Fig. 10A). In addition, we found that Bcl-2/
BH1 also interacted with wt Bcl-2 (Fig. 10B). Moreover, Bcl-2/G145A or Bcl-2/
BH1 interacted with other anti-apoptotic member Bcl-xL (Fig. 10, C and D). To examine whether Bcl-2/G145A or Bcl-2/
BH1 interferes with the binding of wt Bcl-2 to Bax, 293T cells were transiently co-transfected with pCI-neo plasmids encoding Bax, Myc-tagged wt Bcl-2 and Bcl-2/G145A and immunoprecipitaion was performed with anti-Bax antibody. As shown in Fig. 11A, Myc-tagged wt Bcl-2 interacted with Bax, but this was not observed on the expression of Bcl-2/G145A, indicating that Bcl-2/G145A abrogates the interaction between Bax and wt Bcl-2, possibly due to the formation of a complex with wt Bcl-2. Bcl-2/
BH1 also abrogated the interaction between Bax and wt Bcl-2 (Fig. 11B). In addition, Bcl-2/G145A and Bcl-2/
BH1 abrogated the interaction between Bak and wt Bcl-2 (Fig. 11C). Therefore, these results suggest that Bcl-2/G145A or Bcl-2/
BH1 acts as a dominant negative form of the wt Bcl-2/Bcl-xL, thereby enhancing cell death.

View larger version (28K):
[in this window]
[in a new window]
|
FIG. 9. Bcl-2/G145A or Bcl-2/ BH1 abrogates the action of wt Bcl-2 for inhibiting Bax/Bak-induced apoptosis. A, 293T cells transiently transfected with 0.5 µg of pCI-neo plasmids encoding Bax, 0.1 µg of Myc-tagged wt Bcl-2 (triangle represents 0.1, 0.03 µg), and 0.3 µg of Bcl-2/G145A alone, or in combination. Note that (-) was transfected with pCI-neo plasmid. The total amounts of DNA were normalized to 1 µg with pCI-neo plasmid. After 40 h of transfection, cell viability was assessed by trypan blue dye exclusion assay. Values are means ± S.D. of quadruplicate determinations. Transfection efficiencies were >80% for all samples, as determined by co-transfection with 0.3 µg of pEGFP-N1 plasmid. Right panel, After 40 h of transfection, the expression levels of Myc-tagged wt Bcl-2, Bcl-2/G145A and Bax were analyzed by Western blotting using anti-Bcl-2 (clone 124) or anti-Bax antibody. B, 293T cells transiently transfected with 0.5 µg of pCI-neo plasmids encoding Bax, 0.5 µg of Bak, 0.05 µg of wt Bcl-2, 0.3 µg of Bcl-2/G145A, and 1.25 µg of Bcl-2/ BH1 alone, or in combination. Note that (-) was transfected with pCI-neo plasmid. The total amounts of DNA were normalized to 1.8 µg with pCI-neo plasmid. After 18 h of transfection, apoptotic cells (annexin V-positive and PI-negative cells) were scored under a fluorescence microscope. Values are means ± S.D. of quadruplicate determinations.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
FIG. 10. Bcl-2/G145A or Bcl-2/ BH1 interacts with wt Bcl-2/Bcl-xL. A and B, 293T cells were transiently co-transfected with pCI-neo plasmid encoding Myc-tagged wt Bcl-2 and pCI-neo empty plasmid (neo) or pCI-neo plasmids encoding wt Bcl-2, Bcl-2/G145A (A) or Bcl-2/ BH1 (B). After 24 h of transfection, lysates were immunoprecipitated with anti-Myc antibody and immunoprecipitates were immunoblotted with anti-Bcl-2 ( C21) or anti-Myc (to detect Myc-tagged wt Bcl-2) antibody. In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) antibody. C and D, 293T cells were transiently co-transfected with pCI-neo plasmid encoding Myc-tagged wt Bcl-xL or wt Bcl-2 and pCI-neo empty plasmid (neo) or pCI-neo plasmid encoding wt Bcl-2, Bcl-2/G145A (C), or Bcl-2/ BH1 (D). After 24 h of transfection, lysates were immunoprecipitated with anti-Myc antibody, and immunoprecipitates were immunoblotted with anti-Bcl-2 ( C21) or anti-Myc (to detect Myc-tagged wt Bcl-xL and wt Bcl-2) antibody. In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) or anti-Myc antibody. The asterisk represents the immunoglobulin light chain. The data shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
FIG. 11. Bcl-2/G145A as well as Bcl-2/ BH1 abrogate the interaction between wt Bcl-2 and Bax. A and B, 293T cells were transiently transfected with pCI-neo plasmid encoding Bax, Myc-tagged wt Bcl-2, Bcl-2/G145A (A), or Bcl-2/ BH1 (B) in the indicated combination. After 24 h of transfection, lysates were immunoprecipitated with anti-Bax antibody and immunoprecipitates were immunoblotted with anti-Myc (to detect Myc-tagged wt Bcl-2) or anti-Bax antibody. In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) antibody. C, 293T cells were transiently transfected with pCI-neo plasmid encoding Bak, Myc-tagged wt Bcl-2, Bcl-2/G145A, or Bcl-2/ BH1 in the indicated combination. After 24 h of transfection, lysates were immunoprecipitated with anti-Bak antibody, and immunoprecipitates were immunoblotted with anti-Myc antibody (to detect Myc-tagged wt Bcl-2). In addition to immune complexes, the total lysates were immunoblotted with anti-Bcl-2 (clone 124) or anti-Bak antibody. The data shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot.
|
|
 |
DISCUSSION
|
---|
In the present study, we investigated the role of the BH1 domain of Bcl-2 in the regulation of antitumor drug-induced apoptosis. The BH1 deletion mutant of Bcl-2 fails to inhibit cell death induced by camptothecin, paclitaxel, vinblastine, and colchicine, but rather, significantly enhances it through accelerating cytochrome c release and casapase-3(-like) protease activation (Figs. 1 and 2). This enhanced effect on cell death induced by several antitumor drugs is also observed in the clones expressing Bcl-2 bearing a G145A mutation within the BH1 domain (Bcl-2/G145A), but not in the clones expressing Bcl-2 bearing a D140A mutation within BH1 (Bcl-2/D140A), which inhibit camptothecin-induced apoptosis (Fig. 5). Pro-apoptotic Bax and Bak have been known to be activated in response to a wide variety of apoptotic stimuli, such as antitumor drugs including camptothecin (29, 30, 31, 32, 33, 34). Because Bcl-2/G145A and Bcl-2/
BH1 do not bind to Bax or Bak (Ref. 12 and Fig. 4), we demonstrated that Bcl-2/G145A and Bcl-2/
BH1 cannot inhibit antitumor drug-induced cell death, possibly due to a failure to suppress the pro-apoptotic function of Bax and Bak activated by antitumor drugs. Indeed, these two mutants do not inhibit Bax- or Bak-induced cell death (Fig. 7). However, this interpretation still does not explain why Bcl-2/G145A as well as Bcl-2/
BH1 enhances cell death induced by antitumor drugs such as camptothecin. Ms-1 cells express low levels of Bcl-2 and Bcl-xL. Therefore, it is likely that endogenous Bcl-2 and Bcl-xL inhibit camptothecin-induced apoptosis by binding to endogenous Bax and Bak, which are activated by camptothecin treatment, and expression of Bcl-2/G145A as well as Bcl-2/
BH1 interferes with the binding of endogenous Bcl-2 or Bcl-xL to activated Bax and Bak by forming a complex with endogenous Bcl-2 and Bcl-xL. This idea is supported by the following findings. 1) Bcl-2/G145A and Bcl-2/
BH1 enhance Bax- or Bak-induced cell death (Fig. 7). 2) suppression of Bax/Bak-induced apoptosis by wt Bcl-2/Bcl-xL is cancelled out by the expression of Bcl-2/G145A as well as Bcl-2/
BH1 (Fig. 9 and data not shown). 3) Bcl-2/G145A as well as Bcl-2/
BH1 still retains the capacity to interact with wt Bcl-2/Bcl-xL (Ref. 12 and Fig. 10). 4) Bcl-2/G145A as well as Bcl-2/
BH1 inhibit the binding of wt Bcl-2/Bcl-xL to Bax/Bak (Fig. 11 and data not shown). Thus, we clearly show here that Bcl-2/G145A as well as Bcl-2/
BH1 functions as a dominant negative of Bcl-2/Bcl-xL, and accelerates apoptosis induced by antitumor drugs as well as Bax/Bak.
On the other hand, Bcl-2/D140A, which still retains the ability to interact with Bak, but not Bax, inhibits antitumor drug-induced apoptosis (Figs. 4 and 5 and data not shown). This result indicates that suppression of Bak by Bcl-2 is enough to inhibit antitumor drug-induced apoptosis. This is consistent with the findings that the anti-apoptotic ability of Bcl-xL/ D133A, which corresponds with Bcl-2/D140A is comparable with that of wt Bcl-xL (6), that the Bak-deficient T leukemic cells were resistant to apoptosis induced by various apoptotic stimuli including antitumor drugs (32) and that Bax and Bak may not have fully overlapping functions in all cell types (35). In contrast, others have reported that fibroblasts from mouse embryos doubly deficient for both Bax and Bak are defective in apoptosis mediated by various apoptotic stimuli, and MEFs singly deficient for either Bax or Bak appear to exhibit a normal apoptotic response to these agents, suggesting that both Bax and Bak functionally overlap, and these multidomain proapoptotic molecules constitute the "gateway" for apoptosis (36, 37). Therefore, we cannot exclude the possibility that a mechanism other than interaction of Bcl-2/D140A with Bak is responsible for the inhibition of antitumor drug-induced apoptosis by Bcl-2/D140A.
We also demonstrated that Bcl-2/
BH3 and Bcl-2/
BH4 do not suppress camptothecin-induced apoptosis (Figs. 2 and 3), suggesting that the BH3 and BH4 domains of Bcl-2 are a requirement for the inhibitory effect on camptothecin-induced apoptosis. Our findings are consistent with the finding that BH3 and BH4 are the principal domains of Bcl-2 for full multifunctional ability (16, 38). On the other hand, a BH4-deleted Bcl-2 has been reported to convert Bcl-2 into a dominant negative molecule (17). However, unlike Bcl-2/
BH1 and Bcl-2/G145A, Bcl-2/
BH4 did not accelerate apoptosis induced not only by camptothecin (Figs. 2 and 3) but also by C2-ceramide and adriamycin (data not shown), suggesting that Bcl-2/
BH4 is not a dominant negative inhibitor of Bcl-2. In this regard, another group also has shown that deletion of BH4 from Bcl-2 does not create a dominant negative mutant (16).
Unlike Bcl-2/
BH1, Bcl-2/
BH2 retains its anti-apoptotic ability upon camptothecin treatment, as judged from cell viability, cytochrome c release and caspase-3(-like) protease activation (Figs. 2 and 3). Anti-apoptotic ability of Bcl-2/
BH2 was also observed in cells treated with adriamycin, C2-ceramide (data not shown) and inostamycin (28). Earlier studies indicated that hetero- and homodimerization through domains BH1 and BH2 are important for the regulation of cell death by Bcl-2 family members (12, 24). However, our immunoprecipitation experiments showed that Bcl-2/
BH2 retained the ability to interact with Bax and Bak (Fig. 6). Therefore, we conclude that the difference in activity between Bcl-2/
BH1 and Bcl-2/
BH2 for regulating apoptosis is due to the difference between Bcl-2/
BH1 and Bcl-2/
BH2 in interacting with pro-apoptotic members, Bax and Bak.
In contrast to camptothecin-induced apoptosis, C2-ceramide-induced apoptosis is suppressed by the expression of Bcl-2/
BH1 or Bcl-2/G145A in Ms-1 cells (Figs. 1D, 5D, and 5E), suggesting that Bcl-2 also regulates apoptosis by a Bax or Bak heterodimerization-independent mechanism. Our findings are consistent with previous studies showing that Bcl-2/Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms (25, 39, 40). Recent studies have more clearly shown that Bcl-2/Bcl-xL and Bax/Bak act independently (15, 41, 42, 43, 44). Although the Bax/Bak-independent mechanism of Bcl-2 has remained unclear, one possibility is that other dimerization partners are involved in the ability of Bcl-2 to regulate cell survival. Anti-apoptotic Bcl-2 family members have been shown to interact with various other death agonists of the Bcl-2 family and with non-Bcl-2 family proteins, including Raf-1 (14), calcineurin (13), Apaf-1 (45, 46), caspases (47), and voltage-dependent anion channels (VDAC) (15). This extensive network of protein-protein interactions between Bcl-2 family members and non-family members makes the relative contribution of each of these interactions to the regulation of apoptosis difficult to address. A recent study demonstrated that Bax is a key activator of the ceramide-mediated death pathway (34). Therefore, it is likely that Bcl-2/
BH1 and Bcl-2/G145A as well as wt Bcl-2 interact with unidentified protein(s), which act(s) upstream of Bax/Bak in the process of ceramide-induced apoptosis.
In summary, we have shown that Bcl-2/
BH1 and Bcl-2/G145A act in a dominant negative fashion, enhancing apoptosis induced by antitumor drugs, and that this dominant negative activity requires both a failure of interaction of both Bax and Bak through the BH1 but not BH2 domain of Bcl-2 and retention of the interaction of Bcl-2 and Bcl-xL. However, the effect of the Bcl-2 mutant on the BH3 domain-only pro-apoptotic molecules now remains to be determined. In a number of independent studies, Bcl-2 and Bcl-xL blocked the conformational change and/or multimerization (36, 48, 49, 50). One recent study provides evidence that inhibitory Bcl-2-type proteins may inhibit the cooperation between BH3 domain-only proteins and Bax or Bak by sequestering the BH3 domain-only partner of the complex; a Bcl-xL mutant that failed to bind Bim, Bad, or Bid, as well as a Bcl-2 mutant that failed to bind Bid in vivo, could no longer protect cells from apoptosis induced by overexpression of these BH3 domain-only proteins (51). Additional mutational and structural analyses will provide further insights into the detailed biochemical functions of Bcl-2 and how Bcl-2 regulates apoptosis by heterodimerization-dependent and -independent mechanisms.
An antisense approach to decrease the expression of bcl-2 is in various stages of preclinical development for cancer chemotherapy (52). Experiments in vitro and in vivo provide proof-of-principle that such approaches may work. Therefore, gene therapy using bcl-2/
BH1 or bcl-2/G145, or chemotherapy using a small molecule acting near glycine 145 in the BH1 domain of Bcl-2 might be useful in combination with known antitumor drugs.
 |
FOOTNOTES
|
---|
* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and was performed using Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyohi, Kohoku-ku, Yokohama 223-8522, Japan. Tel. and Fax: 81-45-566-1557; E-mail: imoto{at}bio.keio.ac.jp.
1 The abbreviations used are: BH1, Bcl-2 homology 1; SCLC, small cell lung carcinoma; PARP, poly(ADP-ribose) polymerase; wt, wild-type; PI, propidium iodide; EGFP, enhanced green fluorescent protein. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. S. Simizu (RIKEN) for providing pGEM-T Easy-human bcl-2, -human bcl-xL, and -human bax plasmids and pCI-neo plasmid. We thank Dr. Y. Tsujimoto (Osaka University Graduate School of Medicine, Japan) for the generous gift of pCAGGS-human bak plasmid.
 |
REFERENCES
|
---|
- Martin, S. J., and Green, D. R. (1995) Crit. Rev. Oncol. Hematol. 18, 137153[CrossRef][Medline]
[Order article via Infotrieve]
- Thompson, C. B. (1995) Science 267, 14561462[Medline]
[Order article via Infotrieve]
- White, E. (1996) Genes Dev. 10, 115[CrossRef][Medline]
[Order article via Infotrieve]
- Desagher, S., and Martinou, J. C. (2000) Trends Cell Biol. 10, 369377[CrossRef][Medline]
[Order article via Infotrieve]
- Wang, X. (2001) Genes Dev. 15, 29222933[Free Full Text]
- Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Cell 86, 147157[Medline]
[Order article via Infotrieve]
- Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405413[Medline]
[Order article via Infotrieve]
- Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479489[Medline]
[Order article via Infotrieve]
- Van Loo, G., Saelens, X., Van Gurp, M., MacFarlane, M., Martin, S. J., and Vandenabeele, P. (2002) Cell Death Differ. 9, 10311042[CrossRef][Medline]
[Order article via Infotrieve]
- Wang, H-G., and Reed, J. C. (1998) Histol. Histopathol. 13, 521530[Medline]
[Order article via Infotrieve]
- Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Genes Dev. 13, 18991911[Free Full Text]
- Yin, X. M., Oltvai, Z. N., and Korsmeyer, S. J. (1994) Nature 369, 321323[CrossRef][Medline]
[Order article via Infotrieve]
- Shibasaki, F., Kondo, E., Akagi, T., and McKeon, F. (1997) Nature 386, 728731[CrossRef][Medline]
[Order article via Infotrieve]
- Wang, H. G., Rapp, U. R., and Reed, J. C. (1996) Cell 87, 629638[Medline]
[Order article via Infotrieve]
- Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483487[CrossRef][Medline]
[Order article via Infotrieve]
- Huang, D. C. S., Adams, J. M., and Cory, S. (1998) EMBO J. 17, 10291039[Abstract/Free Full Text]
- Hunter, J. J., Bond, B. L., and Parslow, T. G. (1996) Mol. Cell. Biol. 16, 877883[Abstract]
- Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J. C., and Tschopp, J. (1994) J. Cell Biol. 126, 10591068[Abstract]
- Hanada, M., Aime-Sempe, C., Sato, T., and Reed, J. C. (1995) J. Biol. Chem. 270, 1196211969[Abstract/Free Full Text]
- Lee, L. C., Hunter, J. J., Mujeeb, A., Turck, C., and Parslow, T. G. (1996) J. Biol. Chem. 271, 2328423288[Abstract/Free Full Text]
- Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S. L., Ng, S. L., and Fesik, S. W. (1996) Nature 381, 335341[CrossRef][Medline]
[Order article via Infotrieve]
- Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J., Thompson, C. B., and Fesik, S. W. (1997) Science 275, 983986[Abstract/Free Full Text]
- Reed, J. C. (1997) Nature 387, 773776[CrossRef][Medline]
[Order article via Infotrieve]
- Sedlak, T. W., Oltvai, Z. N., Yang, E., Wang, K., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 78347838[Abstract]
- Cheng, E. H., Levine, B., Boise, L. H., Thompson, C. B., and Hardwick, J. M. (1996) Nature 379, 554556[CrossRef][Medline]
[Order article via Infotrieve]
- Kelekar, A., Chang, B. S., Harlan, J. E., Fesik, S. W., and Thompson, C. B. (1997) Mol. Cell. Biol. 17, 70407046[Abstract]
- Oh, Y. J., Uhland-Smith, A., Kim, J. E., and O'Malley, K. L. (1997) Mol. Brain Res. 51, 133142[Medline]
[Order article via Infotrieve]
- Kawatani, M., Uchi, M., Simizu, S., Osada, H., and Imoto, M. (2003) Exp. Cell Res., in press
- Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., and Youle, R. J. (1997) J. Cell Biol. 139, 12811292[Abstract/Free Full Text]
- Hsu, Y. T., Wolter, K. G., and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 36683672[Abstract/Free Full Text]
- Gross, A., Jockel, J., Wei, M. C., and Korsmeyer, S. J. (1998) EMBO J. 17, 38783885[Abstract/Free Full Text]
- Wang, G. Q., Gastman, B. R., Wieckowski, E., Goldstein, L. A., Gambotto, A., Kim, T. H., Fang, B., Rabinovitz, A., Yin, X. M., and Rabinowich, H. (2001) J. Biol. Chem. 276, 3430734317[Abstract/Free Full Text]
- Godlewski, M. M., Motyl, M. A., Gajkowska, B., Wareski, P., Koronkiewicz, M., and Motyl, T. (2001) Anticancer Drugs 12, 607617[CrossRef][Medline]
[Order article via Infotrieve]
- von Haefen, C., Wieder, T., Gillissen, B., Starck, L., Graupner, V., Dorken, B., and Daniel, P. T. (2002) Oncogene 21, 40094019[CrossRef][Medline]
[Order article via Infotrieve]
- Theodorakis, P., Lomonosova, E., and Chinnadurai, G. (2002) Cancer Res. 62, 33733376[Abstract/Free Full Text]
- Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J. (2001) Science 292, 727730[Abstract/Free Full Text]
- Zong, W. X., Lindsten, T., Ross, A. J., MacGregor, G. R., and Thompson, C. B. (2001) Genes Dev. 15, 14811486[Abstract/Free Full Text]
- Choi, W. S., Yoon, S. Y., Chang, I. I., Choi, E. J., Rhim, H., Jin, B. K., Oh, T. H., Krajewski, S., Reed, J. C., and Oh, Y. J. (2000) J. Neurochem. 74, 16211626[CrossRef][Medline]
[Order article via Infotrieve]
- St. Clair, E. G., Anderson, S. J., and Oltvai, Z. N. (1997) J. Biol. Chem. 272, 2934729355[Abstract/Free Full Text]
- Minn, A. J., Kettlun, C. S., Liang, H., Kelekar, A., Vander Heiden, M. G., Chang, B. S., Fesik, S. W., Fill, M., and Thompson, C. B. (1999) EMBO J. 18, 632643[Abstract/Free Full Text]
- Knudson, C. M., and Korsmeyer, S. J. (1997) Nat. Genet. 16, 358363[Medline]
[Order article via Infotrieve]
- Shimizu, S., Narita, M., and Tsujimoto, Y. (2000) Oncogene 19, 43094318[CrossRef][Medline]
[Order article via Infotrieve]
- Priault, M., Chaudhuri, B., Clow, A., Camougrand, N., and Manon, S. (1999) Eur. J. Biochem. 260, 684691[Abstract/Free Full Text]
- Roucou, X., Prescott, M., Devenish, R. J., and Nagley, P. (2000) FEBS Lett. 471, 235239[CrossRef][Medline]
[Order article via Infotrieve]
- Hu, Y., Benedict, M. A., Wu, D., Inohara, N., and Nunez, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 43864391[Abstract/Free Full Text]
- Pan, G., O'Rourke, K., and Dixit, V. M. (1998) J. Biol. Chem. 273, 58415845[Abstract/Free Full Text]
- Clem, R. J., Cheng, E. H., Karp, C. L., Kirsch, D. G., Ueno, K., Takahashi, A., Kastan, M. B., Griffin, D. E., Earnshaw, W. C., Veliuona, M. A., and Hardwick, J. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 554559[Abstract/Free Full Text]
- Nechushtan, A., Smith, C. L., Hsu, Y-T., and Youle, R. J. (1999) EMBO J. 18, 23302341[Abstract/Free Full Text]
- Antonsson, B., Montessuit, S., Sanchez, B., and Martinou, J. C. (2001) J. Biol. Chem. 276, 1161511623[Abstract/Free Full Text]
- Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 1836118374[Abstract/Free Full Text]
- Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001) Mol. Cell 8, 705711[CrossRef][Medline]
[Order article via Infotrieve]
- Jansen, B., Wacheck, V., Heere-Ress, E., Schlagbauer-Wadl, H., Hoeller, C., Lucas, T., Hoermann, M., Hollenstein, U., Wolff, K., and Pehamberger, H. (2000) Lancet 356, 17281733[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.