Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak

Abstract

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode "death" gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage-induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing.

Figure 1

The cell-permeable inhibitors of ICE-related proteases, ZVAD.fmk and BD.fmk, suppress apoptosis induced by oncogenes, DNA damage, and Bak. Rat-1 cell cultures were observed by time-lapse videomicroscopy and images were collected at the rate of 12 frames/h. At the end of each 5-h period, the total number of apoptotic events (as determined by cell detachment) thus far was summed and plotted against time. Rates of apoptotic events in each culture are shown in the presence or absence of ZVAD.fmk (100 μM). Both ZVAD.fmk and tamoxifen (where applicable) were added at time 0. (A) Rat-1 cells expressing c-Myc-ER™ in the presence (▴) or absence (•) of ZVAD.fmk. c-Myc was activated by addition of 4-OHT (100 nM). (B) Rat-1 cells expressing Bak under the control of a GalER-VP16 promotor in the presence (▴) or absence (•) of ZVAD.fmk. The GalER-VP16 chimeric transcription factor was activated by addition of 4-OHT (to 100 nM), so inducing expression of Bak. (C) Rat-1 cells constitutively expressing the adenoviral E1A protein in the presence (▴) or absence (•) of ZVAD.fmk. (D) Rat-1 cells expressing c-MycER™ and treated with etoposide/ VP16 (100 nM) in the presence (▴) or absence (•) of ZVAD.fmk. c-Myc was again activated by addition of 4-OHT (to 100 nM).

Figure 2

ZVAD.fmk and BD.fmk do not delay the onset of cell death, but block completion of apoptosis after the onset of membrane blebbing. (A) ZVAD.fmk does not delay onset of cell death but extends the kinetics of individual apoptotic events. Timelapse videomicroscopic quantitation of onset of apoptosis induced in Rat-1/c-MycER™ cells by 4-OHT (100 nM) in the presence (▴) or absence (•) of ZVAD.fmk (to 100 μM). Time-lapse videomicroscopy was performed as before, but cells were scored for initiation of membrane blebbing. (B) Both ZVAD.fmk and BD.fmk show profound cytoplasmic blebbing 24 h after c-Myc induction in the absence of serum. Rat-1/c-MycER™ cells were serum starved for 48 h before addition of 4-OHT (100 nM) with and without 100 μM ZVAD.fmk or 5 μM BD.fmk. After 24 h, cells were examined by light microscopy using Hoffman optics. Both photographs show a representative field of blebbing cells treated with either ZVAD.fmk or BD.fmk.

Figure 2

ZVAD.fmk and BD.fmk do not delay the onset of cell death, but block completion of apoptosis after the onset of membrane blebbing. (A) ZVAD.fmk does not delay onset of cell death but extends the kinetics of individual apoptotic events. Timelapse videomicroscopic quantitation of onset of apoptosis induced in Rat-1/c-MycER™ cells by 4-OHT (100 nM) in the presence (▴) or absence (•) of ZVAD.fmk (to 100 μM). Time-lapse videomicroscopy was performed as before, but cells were scored for initiation of membrane blebbing. (B) Both ZVAD.fmk and BD.fmk show profound cytoplasmic blebbing 24 h after c-Myc induction in the absence of serum. Rat-1/c-MycER™ cells were serum starved for 48 h before addition of 4-OHT (100 nM) with and without 100 μM ZVAD.fmk or 5 μM BD.fmk. After 24 h, cells were examined by light microscopy using Hoffman optics. Both photographs show a representative field of blebbing cells treated with either ZVAD.fmk or BD.fmk.

Figure 2

ZVAD.fmk and BD.fmk do not delay the onset of cell death, but block completion of apoptosis after the onset of membrane blebbing. (A) ZVAD.fmk does not delay onset of cell death but extends the kinetics of individual apoptotic events. Timelapse videomicroscopic quantitation of onset of apoptosis induced in Rat-1/c-MycER™ cells by 4-OHT (100 nM) in the presence (▴) or absence (•) of ZVAD.fmk (to 100 μM). Time-lapse videomicroscopy was performed as before, but cells were scored for initiation of membrane blebbing. (B) Both ZVAD.fmk and BD.fmk show profound cytoplasmic blebbing 24 h after c-Myc induction in the absence of serum. Rat-1/c-MycER™ cells were serum starved for 48 h before addition of 4-OHT (100 nM) with and without 100 μM ZVAD.fmk or 5 μM BD.fmk. After 24 h, cells were examined by light microscopy using Hoffman optics. Both photographs show a representative field of blebbing cells treated with either ZVAD.fmk or BD.fmk.

Figure 3

ZVAD.fmk delays the onset of characteristic markers of apoptosis. (A) ZVAD.fmk delays destruction of mitochondrial integrity. Rat-1/c-MycER™ cells were plated into 96-well plates in DME containing 10% FCS at a density of approximately 3,000 cells per well. After 48 h of culture, the medium was replaced with serum-free medium and the cells left a further 48 h. Cells were washed again, and the medium was replaced with either growth medium containg 100 nM 4-OHT or 4-OHT plus ZVAD.fmk, as indicated. Mitochondrial integrity was then assayed at various times by a photometric MTT assay. Bars represent means of quadruplicate culture wells ± SDs. Data shown are from a single representative experiment. (B) Flow cytometric analysis of Rat-1/c-MycER™ cells undergoing c-Myc–induced apoptosis in cells in the presence or absence of ZVAD.fmk. At time 0, 4-OHT ± ZVAD.fmk was added to serum-deprived cells. Cells were then harvested, at time points indicated, by trypsinization. Cells were fixed in ethanol, stained with propidium iodide, and examined by flow cytometry. The sub-G1 peak characteristic of apoptotic cells is absent from the ZVAD.fmk-treated populations.

Figure 3

ZVAD.fmk delays the onset of characteristic markers of apoptosis. (A) ZVAD.fmk delays destruction of mitochondrial integrity. Rat-1/c-MycER™ cells were plated into 96-well plates in DME containing 10% FCS at a density of approximately 3,000 cells per well. After 48 h of culture, the medium was replaced with serum-free medium and the cells left a further 48 h. Cells were washed again, and the medium was replaced with either growth medium containg 100 nM 4-OHT or 4-OHT plus ZVAD.fmk, as indicated. Mitochondrial integrity was then assayed at various times by a photometric MTT assay. Bars represent means of quadruplicate culture wells ± SDs. Data shown are from a single representative experiment. (B) Flow cytometric analysis of Rat-1/c-MycER™ cells undergoing c-Myc–induced apoptosis in cells in the presence or absence of ZVAD.fmk. At time 0, 4-OHT ± ZVAD.fmk was added to serum-deprived cells. Cells were then harvested, at time points indicated, by trypsinization. Cells were fixed in ethanol, stained with propidium iodide, and examined by flow cytometry. The sub-G1 peak characteristic of apoptotic cells is absent from the ZVAD.fmk-treated populations.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 4

ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycER™ cells expressing activated c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycER™ cells in which c-Myc has been activated in low serum in the presence or absence of ZVAD.fmk. (i) Inhibition of cleavage of lamins A and C by ZVAD.fmk. ZVAD.fmk treatment inhibits the appearance of the characteristic 46-kD IRP cleavage product in apoptosis (Oberhammer et al., 1994) at both 24 and 48 h. Lane 1, time-0 cells; lane 2, 24-h control cells; lane 3, 24-h ZVAD.fmk-treated cells; lane 4, 48-h control cells; lane 5, 48-h ZVAD.fmk-treated cells. (ii) Inhibition of PARP cleavage by ZVAD.fmk. IRP cleavage of PARP produces a characteristic 85-kD fragment in apoptotic cells (Lazebnik et al., 1994). In non– ZVAD.fmk-treated Rat-1/cMycER™ cells, the 85-kD PARP fragment is visible by 24 h and further increased at 48 h. In the ZVAD.fmktreated cells, the 85-kD fragment is absent at 24 h and only faintly seen at 48 h. Lane 1, time-0 control cells; lane 2, time-0 ZVAD.fmk-treated cells; lane 3, 24-h control cells; lane 4, 24-h ZVAD.fmk-treated cells; lane 5, 48-h control cells; lane 6, 48-h ZVAD.fmk-treated cells. (iii) ZVAD.fmk inhibits actin cleavage in apoptotic cells, characterized by the appearance of a 15-kD fragment recognized by the antibody used. Appearance of this fragment is inhibited by ZVAD.fmk. Lane 1, time-0 cells; lane 2, 48-h control cells; lane 3, 48-h ZVAD.fmk-treated cells; lane 4, 96-h control cells; lane 5, 96-h ZVAD.fmk-treated cells. (B) Confocal immunofluorescence microscopy of distribution of known IRP substrates. (i) Viable Rat-1/c-MycER™ cells stained with anti–lamin A+C antibody demonstrate characteristic lamin staining in the nuclear periphery. In apoptotic cells, such staining is absent because of degradation and dispersal of lamins. In contrast, ZVAD.fmktreated blebbing cells exhibit near-normal lamin A+C staining when compared to controls. (ii) Rat-1/c-MycER™ cells stained with phalloidin-FITC to examine the distribution of actin. Viable cells exhibit normal actin fibers; apoptotic cells are condensed with no actin filaments visible. In ZVAD.fmk-treated cells, actin is clumped in blebs on the cell surface. Note that blebbing cells are markedly reduced in size compared with their viable counterparts. (iii) Both viable and blebbing Rat-1/c-MycER™ cells exhibit nuclear PARP staining, which is absent in apoptotic cells. Bars, 10 μm.

Figure 5

Electron microscopic analysis of apoptosis in serum-deprived Rat-1/c-MycER™ cells in the presence and absence of ZVAD.fmk. Electron microscopical analysis of individual Rat-1/c-MycER™ cells undergoing 4-OHT–induced apoptosis in low serum. (A) Normal, viable Rat-1/c-MycER™ cells. (B) A typical apoptotic cell. (C and D) Early and late stages, respectively, of the morphological changes where apoptosis is induced in the presence of ZVAD.fmk, showing dramatic cytoplasmic blebbing but absence of chromatin condensation. Bars, 1 μm.

Figure 6

Expression of cell surface phosphatidyl serine. ZVAD.fmk delays the emergence of annexin V binding. Serum-deprived Rat-1/cMycER™ cells undergoing c-Myc–induced apoptosis in the presence or absence of ZVAD.fmk were examined by phase-contrast microscopy to determine their morphologies (a and c). FITC-labeled annexin V was then added to the culture dishes to a final concentration of 2.5 μg/ml, and the same cells were examined by fluorescence microscopy (b and d). In the absence of ZVAD.fmk, apoptotic cells stain with annexin V (a and b). In contrast, ZVAD.fmk-treated blebbing cells do not bind annexin V (c and d).

Figure 7

IGF-I and Bcl-2 delay the initiation of the apoptotic program but not its execution. Serum-deprived Rat-1/c-MycER™ cells were treated with 100 nM 4-OHT to activate c-Myc, and this was followed by time-lapse videomicroscopy. Onset of apoptosis was scored at the start of blebbing. End points of cell death were scored at the point of cell detachment, and the time between these two is represented by the length of the horizontal line. (A) Apoptotic events in the presence or absence of IGF-1. The effect of IGF-1 on the kinetics of Myc-induced apoptosis. (B) The effect of Bcl-2 expression on the kinetics of Myc-induced apoptosis. Apoptotic events in the presence or absence of co-expressed Bcl-2.

Figure 7

IGF-I and Bcl-2 delay the initiation of the apoptotic program but not its execution. Serum-deprived Rat-1/c-MycER™ cells were treated with 100 nM 4-OHT to activate c-Myc, and this was followed by time-lapse videomicroscopy. Onset of apoptosis was scored at the start of blebbing. End points of cell death were scored at the point of cell detachment, and the time between these two is represented by the length of the horizontal line. (A) Apoptotic events in the presence or absence of IGF-1. The effect of IGF-1 on the kinetics of Myc-induced apoptosis. (B) The effect of Bcl-2 expression on the kinetics of Myc-induced apoptosis. Apoptotic events in the presence or absence of co-expressed Bcl-2.

Figure 8

Serum survival factors do not rescue ZVAD.fmkblocked apoptotic cells once they have initiated blebbing. Apoptosis was induced in serum-deprived Rat-1/c-MycER™ fibroblasts by addition of 4-OHT, and cells were observed by timelapse videomicroscopy. 40 h after addition of 4-OHT, FCS was added back to the growth medium to a final concentration of 10%. Cells that had initiated membrane blebbing were then followed to determine their fates. The figure shows a representative study of 18 independent cells fates. Initiation of membrane blebbing (•) and death of the cell (|) were determined as before, and the time interval between the two is given by the horizontal line. All cells that initiated membrane blebbing before serum readdition eventually died. In contrast, all observed cells that had not yet initiated blebbing at the time of serum readdition survived and went on to divide (not shown).