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First published online 22 November 2005
doi: 10.1242/jcs.02676

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Bax activation and translocation to mitochondria mediate EGF-induced programmed cell death

¹ Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA
² Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA

View larger version (64K): [in a new window]   Fig. 1. Limited role of caspase 3 and 7 activation in EGF-induced cell death. (A) A431/ErbB2 or SKBr3/EGFR cells were grown until 70-80% confluency and then treated with EGF (20 ng/ml) or vehicle in the presence or absence of caspase inhibitors (Z-VAD-FMK, 30 µM; caspase inhibitors 3 and 9, 20 µM) for the indicated times. Staurosporin-treated cells were used as a positive control. The cells were lysed in CHAPS buffer as indicated in Materials and Methods. The levels of activated caspases were detected by western blotting using antibodies against cleaved caspase 3 and caspase 7. (B) MCF7 cells, which are devoid of caspase 3, were transfected with EGFR*GFP and ErbB-2*RFP constructs and stable cell lines were selected. The cells were incubated with or without EGF for 4 days and images were prepared using fluorescence microscopy.

View larger version (86K): [in a new window]   Fig. 2. Bax overexpression accelerates the onset of EGF-induced apoptosis. A431/ErbB2 cells were transfected with a construct encoding a Bax*GFP fusion protein and stable cell lines were selected. The addition of EGF (20 ng/ml) to the parental A431/ErbB2 cells resulted in the majority of cells dying in 3-5 days (A, upper row). Expression of the Bax*GFP fusion protein dramatically accelerates the rate of apoptosis in an EGF-dependent fashion, as nearly all cells died during the first 14-18 hours of incubation with EGF (A, lower row). (B) Quantitative differences in viability of A431/ErbB2 and A431/ErbB2/Bax cells after incubation with EGF for the indicated times.

View larger version (56K): [in a new window]   Fig. 3. EGF-induces Bax translocation to the mitochondria. (A) A431/ErbB-2, SKBr3/EGFR or SKBr3 cells were incubated in the presence or absence of EGF (20 ng/ml) for the indicated times. The cells were homogenized and subjected to subcellular fractionation as described in Materials and Methods. The presence of Bax in the mitochondrial fractions was determined by western blotting (W.B.) using an antibody against Bax. (B) A431/ErbB2/Bax cells were incubated with EGF (20 ng/ml) for 0, 8 or 24 hours. Then, the cells were fixed in 4% paraformaldehyde and the cell examined using confocal microscopy.

View larger version (54K): [in a new window]   Fig. 4. EGF-induced Bax oligomerization coincides with Bax translocation from the cytosol into subcellular foci. (A) SKBr3/EGFR or SKBr3 cells were stimulated with EGF (20 ng/ml) for the indicated times. The cell homogenates were subjected to subcellular fractionation and western blotting (W.B.) using 6A7 antibody against activated Bax. (B) A431/ErbB2/Bax cells were treated with EGF (20 ng/ml) for the indicated times and fixed in 4% paraformaldehyde. The cells were stained with 6A7 antibody against activated Bax (red) and examined using confocal microscopy.

View larger version (70K): [in a new window]   Fig. 5. Colocalization of Bax with mitochondria in cells treated with EGF. (A-D) A431/ErbB2-mito cells, which express RFP targeted to the mitochondria (red), were incubated with EGF or vehicle for 24 or 48 hours and then stained with 6A7 antibody (green). (A) Control cells show weak background staining with antibody evenly distributed throughout the cytosol. (B) In contrast, in the cells treated with EGF for 24 hours, 6A7 staining is clearly colocalizes with the mitochondrial network. In some cells the mitochondria, which still appears to have activated Bax evenly distributed on the membrane, form stacks that accumulate in the perinuclear area (arrow). (C) A431/ErbB2-mito cells treated with EGF for 24-48 hours undergo a loss of cell volume to become rounded. Although activated Bax still shows an even distribution associated with the mitochondrial membrane, many individual mitochondria have bright Bax foci at fission sites (arrows). (D) In dying cells, identified by morphological criteria, there is no colocalization of activated Bax with mitochondrial membrane because Bax predominantly forms aggregates at mitochondrial fission sites (arrow). (E,F) A431/ErbB-2/Bax-mito cells stably express GFP*Bax and RFP targeted to the mitochondria. The cells treated with EGF for 16-24 hours show Bax redistribution in the cytosol (E). At the beginning of Bax relocation the pattern of Bax redistribution closely resembles the pattern of mitochondrial network (F). (G) Later, Bax forms aggregates coalescing to the mitochondrial fission sites (left). Eventually, all Bax is accumulated at fission sites at the time of cell death (right). Bars, 20 µm.

View larger version (75K): [in a new window]   Fig. 6. Loss of MMP after Bax translocation to foci in EGF-treated cells. A431/ErbB2/Bax cells were incubated in the presence of EGF or vehicle for 16 hours. Next, the cells were incubated for 30 minutes with MitoTracker CMTMRos (100 nM) and examined by fluorescence confocal microscopy. The mitochondrial network visualized by MitoTracker CMTMRos (red) is present in all control cells, which express Bax*GFP, and is evenly distributed in the cytosol (A). In contrast, cells incubated with EGF show a redistribution of Bax to foci (B, arrow; C, arrowheads), which coincides with a loss of MMP. A few cells, where a translocation of Bax can be observed in one part of the cell, still retain MMP in another region of the same cell that does not show Bax relocation (C, arrow). Bars, 20 µm.

View larger version (67K): [in a new window]   Fig. 7. Expression of Bcl-xL prevents EGF-induced apoptosis. (A) The level of Bcl-xL expression decreased in A431/ErbB2 cells treated with EGF. A431/ErbB2 cells were incubated in the presence of EGF (20 ng/ml) or vehicle for the indicated times. Antibody was used to immunoprecipitate Bcl-xL from cell lysates and to detect its levels in cells by western blotting (W.B.). (B) A431/ErbB2 cells were transfected with a pIRES2-EGFP vector containing the Bcl-xL cDNA sequence. Cell sorting was used to select stable cell lines expressing Bcl-xL using GFP as a marker. Cells treated with EGF are morphologically different from control. However, they do not form dome-like clusters, as do A431/ErbB-2 cells (see Fig. 2), and continue to grow in the presence of EGF. (C) The different responses of A431/ErbB2 and A431/ErbB2/Bcl-xL cells to EGF (proliferation or cell death) are shown quantitatively.

View larger version (83K): [in a new window]   Fig. 8. Influence of EGF on the cytochrome c release from mitochondria. (A) The SKBr3/EGFR cells were incubated in the presence of EGF (20 ng/ml) or vehicle for indicated times. The cells were then homogenized and subjected to subcellular fractionation and fractions containing mitochondria or cytosol were analyzed by western blot (W.B.) for the presence of cytochrome c. The cytosol fractions are about 50 times more dilute than the mitochondrial fractions, as indicated in the Materials and Methods. (B) A431/ErbB2-mito cells were treated with EGF for 48 hours, fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and stained with cytochrome c antibody. The two thinner arrows indicate cells that show colocalization of cytochrome c (green) with the mitochondrial network (red). A thick arrow indicates a cell with cytochrome c released in the cytosol. Notably, this cell has a compressed mitochondrial network still entrapping most of the cytochrome c. Bar, 20 µm.

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