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Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

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Inhibition of rBax-induced apoptosis by injection of anti-VDAC antibodies. (A) Induction of apoptosis by microinjection of rBax. rBax (1 μg/μl) or the equivalent amount of irrelevant control protein was microinjected into the cytoplasm of HeLa cells with GFP (3 μg/μl). After 8 h, cell morphology was assessed by transmission microscopy (TM) and fluorescence microscopy (GFP). Cells were also stained with annexin V and Hoechst 33342, and observed under a fluorescence microscope. The color photographs were taken from the same field. The arrowhead indicates an example of cells at the terminal stage of apoptosis, showing weak annexin V staining with no Hoechst 33342 staining. (B–E) Inhibition of rBax-induced apoptosis by microinjection of Ab#20 and Ab#25. (B) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, 2 μg/μl of rBax was microinjected into the same cells, and after 15 h, cells were examined under a transmission microscope. All of the cells shown were microinjected. Data are representative of seven independent experiments. (C) The same procedure shown in B was followed, except that 3 μg/μl of GFP was coinjected with rBax. Cell morphology was assessed at the indicated times under a fluorescence microscope. Data are representative of three independent experiments. (D) HeLa cells were microinjected with Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) at the indicated concentrations. After 1 h, rBax at the indicated concentrations was microinjected into the same cells, and apoptosis was investigated under a transmission microscope. More than 100 injected cells were analyzed. Data are representative of two or seven independent experiments. (E) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, the cells were injected with 1 μg/μl of rBax, and apoptosis was assessed at 12 h under a transmission microscope. NMI indicates normal mouse IgG used as a control for 31HL. Data are representative of two independent experiments.
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Figure 4: Inhibition of rBax-induced apoptosis by injection of anti-VDAC antibodies. (A) Induction of apoptosis by microinjection of rBax. rBax (1 μg/μl) or the equivalent amount of irrelevant control protein was microinjected into the cytoplasm of HeLa cells with GFP (3 μg/μl). After 8 h, cell morphology was assessed by transmission microscopy (TM) and fluorescence microscopy (GFP). Cells were also stained with annexin V and Hoechst 33342, and observed under a fluorescence microscope. The color photographs were taken from the same field. The arrowhead indicates an example of cells at the terminal stage of apoptosis, showing weak annexin V staining with no Hoechst 33342 staining. (B–E) Inhibition of rBax-induced apoptosis by microinjection of Ab#20 and Ab#25. (B) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, 2 μg/μl of rBax was microinjected into the same cells, and after 15 h, cells were examined under a transmission microscope. All of the cells shown were microinjected. Data are representative of seven independent experiments. (C) The same procedure shown in B was followed, except that 3 μg/μl of GFP was coinjected with rBax. Cell morphology was assessed at the indicated times under a fluorescence microscope. Data are representative of three independent experiments. (D) HeLa cells were microinjected with Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) at the indicated concentrations. After 1 h, rBax at the indicated concentrations was microinjected into the same cells, and apoptosis was investigated under a transmission microscope. More than 100 injected cells were analyzed. Data are representative of two or seven independent experiments. (E) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, the cells were injected with 1 μg/μl of rBax, and apoptosis was assessed at 12 h under a transmission microscope. NMI indicates normal mouse IgG used as a control for 31HL. Data are representative of two independent experiments.

Mentions: To examine the effect of Ab#20 and Ab#25 on Bax-induced apoptosis, these antibodies or NRI (as a control) were microinjected into the cytoplasm of HeLa cells, and rBax was microinjected into the cytoplasm of the same cells 1 h later to induce apoptosis. After injection of rBax, but not irrelevant protein, nearly all NRI-injected cells underwent apoptosis at 15 h as shown by cellular shrinkage, rounding (Fig. 4 A), and eventual detachment from the culture dishes. Apoptosis was also confirmed by positive annexin V staining and by nuclear shrinkage and fragmentation on Hoechst 33342 staining (Fig. 4 A). Cells in the terminal stage of apoptosis (Fig. 4 A, arrowhead) only showed weak staining with annexin V and lost most of their DNA. On the other hand, depending on the amount of rBax injected (1 or 2 μg/μl), the majority and nearly half of the cells, respectively, remained alive after being preinjected with Ab#20 or Ab#25 (Fig. 4 D, left and middle), as shown by their flat morphology (Fig. 4 B) and by the division of some cells within 24 h (data not shown). Inhibition of apoptosis after injection of Ab#20 and Ab#25 was also confirmed by lack of annexin V staining as well as a normal nuclear morphology on Hoechst 33342 staining (data not shown). Fig. 4 C shows time-lapse photographs of cells injected with Ab#25 or NRI and then with rBax (1 μg/μl) and recombinant GFP (rGFP) to identify injected cells: NRI-injected cells shrank and became fragmented after 3 h, and were detached from the culture dishes by 12 h, whereas these changes were not observed when Ab#20 or Ab#25 was preinjected. Incomplete inhibition of rBax (2 μg/μl)-induced apoptosis by Ab#20 and Ab#25 at 12 μg/μl (Fig. 4 D, middle) was probably due to degradation of injected antibodies, because intracellular concentrations of Ab#20 and Ab#25, as assessed by immunostaining with anti–rabbit antibody conjugated with Alexa568, significantly decreased in a time-dependent manner (75 and 40% at 12 and 24 h after injection, respectively) and because injection of Ab#25 at a little higher concentration, 18 μg/μl, nearly completely inhibited Bax-induced apoptosis (Fig. 4 D, right). The inhibitory effect of these antibodies on Bax-induced apoptosis was not observed at lower concentrations of the antibodies, although the antibodies could still inhibit apoptosis induced by a lower concentration of rBax (data not shown). To exclude the possibility that these antibodies inhibited Bax-induced apoptosis merely by nonspecific binding to the mitochondria, we tested other antibodies, including 31HL and an antibody specific to Tom20, a protein on mitochondrial outer membrane. Both of these other antibodies did not inhibit Bax-induced apoptosis (Fig. 4 E). These results indicated that the VDAC is essential for Bax-induced apoptosis in mammalian cells.


Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Inhibition of rBax-induced apoptosis by injection of anti-VDAC antibodies. (A) Induction of apoptosis by microinjection of rBax. rBax (1 μg/μl) or the equivalent amount of irrelevant control protein was microinjected into the cytoplasm of HeLa cells with GFP (3 μg/μl). After 8 h, cell morphology was assessed by transmission microscopy (TM) and fluorescence microscopy (GFP). Cells were also stained with annexin V and Hoechst 33342, and observed under a fluorescence microscope. The color photographs were taken from the same field. The arrowhead indicates an example of cells at the terminal stage of apoptosis, showing weak annexin V staining with no Hoechst 33342 staining. (B–E) Inhibition of rBax-induced apoptosis by microinjection of Ab#20 and Ab#25. (B) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, 2 μg/μl of rBax was microinjected into the same cells, and after 15 h, cells were examined under a transmission microscope. All of the cells shown were microinjected. Data are representative of seven independent experiments. (C) The same procedure shown in B was followed, except that 3 μg/μl of GFP was coinjected with rBax. Cell morphology was assessed at the indicated times under a fluorescence microscope. Data are representative of three independent experiments. (D) HeLa cells were microinjected with Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) at the indicated concentrations. After 1 h, rBax at the indicated concentrations was microinjected into the same cells, and apoptosis was investigated under a transmission microscope. More than 100 injected cells were analyzed. Data are representative of two or seven independent experiments. (E) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, the cells were injected with 1 μg/μl of rBax, and apoptosis was assessed at 12 h under a transmission microscope. NMI indicates normal mouse IgG used as a control for 31HL. Data are representative of two independent experiments.
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Figure 4: Inhibition of rBax-induced apoptosis by injection of anti-VDAC antibodies. (A) Induction of apoptosis by microinjection of rBax. rBax (1 μg/μl) or the equivalent amount of irrelevant control protein was microinjected into the cytoplasm of HeLa cells with GFP (3 μg/μl). After 8 h, cell morphology was assessed by transmission microscopy (TM) and fluorescence microscopy (GFP). Cells were also stained with annexin V and Hoechst 33342, and observed under a fluorescence microscope. The color photographs were taken from the same field. The arrowhead indicates an example of cells at the terminal stage of apoptosis, showing weak annexin V staining with no Hoechst 33342 staining. (B–E) Inhibition of rBax-induced apoptosis by microinjection of Ab#20 and Ab#25. (B) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, 2 μg/μl of rBax was microinjected into the same cells, and after 15 h, cells were examined under a transmission microscope. All of the cells shown were microinjected. Data are representative of seven independent experiments. (C) The same procedure shown in B was followed, except that 3 μg/μl of GFP was coinjected with rBax. Cell morphology was assessed at the indicated times under a fluorescence microscope. Data are representative of three independent experiments. (D) HeLa cells were microinjected with Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) at the indicated concentrations. After 1 h, rBax at the indicated concentrations was microinjected into the same cells, and apoptosis was investigated under a transmission microscope. More than 100 injected cells were analyzed. Data are representative of two or seven independent experiments. (E) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, the cells were injected with 1 μg/μl of rBax, and apoptosis was assessed at 12 h under a transmission microscope. NMI indicates normal mouse IgG used as a control for 31HL. Data are representative of two independent experiments.
Mentions: To examine the effect of Ab#20 and Ab#25 on Bax-induced apoptosis, these antibodies or NRI (as a control) were microinjected into the cytoplasm of HeLa cells, and rBax was microinjected into the cytoplasm of the same cells 1 h later to induce apoptosis. After injection of rBax, but not irrelevant protein, nearly all NRI-injected cells underwent apoptosis at 15 h as shown by cellular shrinkage, rounding (Fig. 4 A), and eventual detachment from the culture dishes. Apoptosis was also confirmed by positive annexin V staining and by nuclear shrinkage and fragmentation on Hoechst 33342 staining (Fig. 4 A). Cells in the terminal stage of apoptosis (Fig. 4 A, arrowhead) only showed weak staining with annexin V and lost most of their DNA. On the other hand, depending on the amount of rBax injected (1 or 2 μg/μl), the majority and nearly half of the cells, respectively, remained alive after being preinjected with Ab#20 or Ab#25 (Fig. 4 D, left and middle), as shown by their flat morphology (Fig. 4 B) and by the division of some cells within 24 h (data not shown). Inhibition of apoptosis after injection of Ab#20 and Ab#25 was also confirmed by lack of annexin V staining as well as a normal nuclear morphology on Hoechst 33342 staining (data not shown). Fig. 4 C shows time-lapse photographs of cells injected with Ab#25 or NRI and then with rBax (1 μg/μl) and recombinant GFP (rGFP) to identify injected cells: NRI-injected cells shrank and became fragmented after 3 h, and were detached from the culture dishes by 12 h, whereas these changes were not observed when Ab#20 or Ab#25 was preinjected. Incomplete inhibition of rBax (2 μg/μl)-induced apoptosis by Ab#20 and Ab#25 at 12 μg/μl (Fig. 4 D, middle) was probably due to degradation of injected antibodies, because intracellular concentrations of Ab#20 and Ab#25, as assessed by immunostaining with anti–rabbit antibody conjugated with Alexa568, significantly decreased in a time-dependent manner (75 and 40% at 12 and 24 h after injection, respectively) and because injection of Ab#25 at a little higher concentration, 18 μg/μl, nearly completely inhibited Bax-induced apoptosis (Fig. 4 D, right). The inhibitory effect of these antibodies on Bax-induced apoptosis was not observed at lower concentrations of the antibodies, although the antibodies could still inhibit apoptosis induced by a lower concentration of rBax (data not shown). To exclude the possibility that these antibodies inhibited Bax-induced apoptosis merely by nonspecific binding to the mitochondria, we tested other antibodies, including 31HL and an antibody specific to Tom20, a protein on mitochondrial outer membrane. Both of these other antibodies did not inhibit Bax-induced apoptosis (Fig. 4 E). These results indicated that the VDAC is essential for Bax-induced apoptosis in mammalian cells.

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

Show MeSH
Related in: MedlinePlus