A cell suicide program appears to have arisen early in metazoan evolution and been substantially conserved. Two of the three principal genes directly involved in control and execution of ontogenetic cell death in the developmentally invariant nematode worm C. elegans
-9 and ced
-3, encode polypeptides that have multiple mammalian homologues: the Bcl-2 (Hengartner and Horvitz, 1994
) and ICE-related cysteine protease (Xue and Horvitz, 1995
) families, respectively. Both the Bcl-2 and IRP families are implicated in vertebrate cell suicide, a process whose morphological manifestation is apoptosis. In contrast to C. elegans
, however, cell suicide in vertebrates is influenced by many diverse factors that include extracellular signals, genotoxic and physical trauma, anoxia, oncogene expression, and immune killing. How all of these differing agents impact upon the underlying cell death mechanism is unclear. Specifically, it is unclear which of the factors that modulate vertebrate cell death are part of the actual execution machinery—the mechanism that physically dismantles the cell—and which are merely regulatory mechanisms that govern the engagement of the execution process.
The best candidates for components of the execution machinery of vertebrate apoptosis are the Ced-3 homologues, the ICE-related proteases. Consistent with this, many diverse studies implicate IRPs in apoptosis induced by insults such as growth factor deprivation (Milligan et al., 1995
; Nicholson et al., 1995
), loss of contact with extracellular matrix (Boudreau et al., 1995
), Fas/TNF (Enari et al., 1995
; Los et al., 1995
; Duan et al., 1996a
; Schlegel et al., 1996
), and cytotoxic T cell killing (Darmon et al., 1995
). We have therefore investigated the requirement of IRPs in mammalian apoptosis using the IRP inhibitors ZVAD.fmk and BD.fmk. ZVAD.fmk and BD.fmk are particularly useful inhibitors for studying IRP action in intact cells because their intrinsic hydrophobicity permits their entry into cells, whereupon intracellular esterases convert the aspartyl methyl ester to aspartate to generate irreversible inhibitors of IRPs. Both ZVAD.fmk and BD.fmk have been shown to be effective inhibitors of apoptosis in intact cells with broad reactivities amongst tested ICE family proteases (Fearnhead et al., 1995
; Slee et al., 1996
). In our studies, we observed no differences in effect between ZVAD.fmk and BD.fmk. Consequently, most of the data we show is confined to studies with just one of these inhibitors, ZVAD.fmk.
We investigated the ability of ZVAD.fmk to inhibit apoptosis triggered by diverse promoters of apoptosis: c-Myc and E1A oncogene expression, DNA damage induced by the topoisomerase II inhibitor etoposide, and ectopic expression of the killer member of the Bcl-2 family Bak. In all cases, ZVAD.fmk provided substantial protection from apoptosis that correlated with inhibition of cleavage of the known IRP substrates, actin, lamins, and PARP. Thus, oncogenes, p53 (after DNA damage), and pro-apoptotic members of the Bcl-2 family all induce apoptosis via a ZVAD.fmk-sensitive activity, most probably one or more IRPs. These findings extend the critical role of IRPs in mammalian apoptosis to include stimuli of fundamental importance in the genesis and progression of neoplasia.
However, detailed time-lapse videomicroscopic analysis of cells protected by ZVAD.fmk revealed an unexpected and novel phenotype. An early manifestation of apoptosis in most cells is the onset of vigorous membrane blebbing. Membrane blebbing is a well-described characteristic of apoptosis in many cell types that, in the normal course of apoptosis in fibroblastic cells, is rapidly (20–60 min) followed by cell shrinkage, chromatin condensation, and nuclear and cellular fragmentation. Surprisingly, in cultures of ZVAD.fmk-treated cells induced to initiate apoptosis by the action of c-Myc, E1A, or Bak or the genotoxic agent etoposide, we found that the onset of membrane blebbing was not delayed by the presence of ZVAD.fmk in the culture medium. However, whereas untreated blebbing apoptotic cells rapidly proceed through the latter stages of chromatin condensation and cell fragmentation, ZVAD.fmk-treated cells continue to bleb vigorously for greatly extended periods of time, typically several days. Such blebbing cells exhibit few characteristics of apoptotic cells: cell nuclei remain intact with little evidence of chromatin condensation or nuclear fragmentation, plasma membrane integrity and mitochondrial function are preserved, and phosphatidylserine is not expressed on the cell surface. Thus, ZVAD.fmk appears not to inhibit the onset of membrane blebbing, although it effectively suppresses classical late-stage apoptotic events.
Moreover, although ZVAD.fmk substantially ameliorates the pro-apoptotic effects of c-Myc, DNA damage, and Bak in Rat-1 fibroblasts, most cells nonetheless do eventually detach from the substratum. Such cells exhibit some chromatin condensation (although far less than in normal late-stage apoptotic cells) but preserve intact nuclear lamina; they also have extensively fragmented and vacuolated cytoplasm. Eventually, however, late-stage ZVAD.fmk-treated cells lose their integrity and take up vital dyes—indisputable evidence of their ultimate death. We conclude that late-stage ZVAD.fmk-treated cells undergo a delayed “partial” apoptotic process, although a more detailed characterization of these late-stage cells will be required to establish their precise phenotype. We have investigated whether the delay to cell death afforded by ZVAD.fmk might be extended either by addition of higher initial concentrations of ZVAD.fmk to the growth medium or by its repeated readdition. However, neither alters the kinetics of cell death within Rat-1 cell cultures. It is difficult to assess the significance of this observation because of uncertainty as to the effective intracellular concentration of ZVAD.fmk administered to culture medium, or of the inhibitor's half-life in culture media or within cells. Nonetheless, it is possible that a continued pro-apoptotic stimulus eventually overrides the ZVAD.fmk block or bypasses the ZVAD.fmk-sensitive components of the apoptotic program.
Why should apoptotic cells exhibit prolonged blebbing when treated with ZVAD.fmk? One possibility is that the blebbing we observe in ZVAD.fmk-treated cells is different from the normal blebbing that accompanies apoptosis and has nothing to do with apoptosis. Three factors argue against this. By time-lapse videomicroscopy and light and electron microscopy, the blebbing observed in the presence of ZVAD.fmk appears identical to that seen in control cells undergoing normal apoptosis. In addition, the onset of blebbing in ZVAD.fmk-treated cell populations is identical to that in control populations; it merely continues for far longer as it is not accompanied by cell and nuclear fragmentation. Finally, time-lapse videomicroscopy shows that blebbing ZVAD.fmk-treated cells adhere to live cells in the same way as true apoptotic bodies (Whyte, M., N. McCarthy, and G.I. Evan, unpublished data) (see Note Added in Proof for Web Information), indicating that at least some of the pro-phagocytic processes triggered during normal apoptosis (although not surface display of phosphatidylserine) also occur in ZVAD.fmk-blocked cells. A second possibility is that ZVAD.fmk fails to prevent membrane blebbing because blebbing is not part of the basal apoptotic program but merely a dispensable epiphenomenon that commonly accompanies the process. However, the notion that membrane blebbing is not part of apoptosis is difficult to reconcile with our time-lapse videomicroscopic data, which show that once blebbing is initiated, it is not reversed by the action of antiapoptotic cytokines or by removal of the original pro-apoptotic trigger. Thus, blebbing appears to be concomitant with commitment to death. We also dislike this explanation because it seeks to explain the ZVAD.fmk phenotype by redefining apoptosis so as to exclude a characteristic of the process that is so unique and ubiquitous as to give the phenomenon its name. A third possibility is that membrane blebbing is part of the bona fide apoptotic process but triggered by IRPs that are not inhibited by ZVAD.fmk. This possibility needs to be investigated using a range of IRP inhibitors with differing spectrums of specificity. However, at present the possibility remains that membrane blebbing is triggered by a completely different mechanism that might not involve IRP action at all.
The phenotype of continuous membrane blebbing that we observe in ZVAD.fmk-treated apoptotic cells appears to contradict several reports that indicate that chemical and viral IRP inhibitors can afford long term protection against apoptosis. For example, peptide and viral inhibitors of IRPs protect motor neurons against apoptosis after factor withdrawal (Gagliardini et al., 1994
; Martinou et al., 1995
; Tewari et al., 1995
), protect cells from Fas and TNFinduced killing (Beidler et al., 1995
; Enari et al., 1995
, 1996; Los et al., 1995
), and block developmental apoptosis in Drosophila
(Hay et al., 1995
) and C. elegans
(Xue and Horvitz, 1995
). In some of these instances, it is possible that the protected cells have nonetheless initiated a membrane blebbing program that is not easily discernible using static imaging techniques. Furthermore, it is possible that blebbing cells remain capable of sustaining some of the functions of normal living cells within the soma, at least temporarily. In other situations, cell death may be triggered by directly activating or recruiting IRPs, in which case inhibiting IRP activity might be sufficient to prevent completely all manifestations of apoptosis. For example, the DEVD-specific IRP CPP32β is directly cleaved and activated by the cytotoxic T cell granule serine esterase Granzyme B and both the CD95 and TNF-R1 cytotoxic signaling pathways directly recruit (and presumably activate) the IRP FLICE/MACH via their “Death Domains” (Boldin et al., 1996
; Muzio et al., 1996
). By inference, the Drosophila
Reaper, another Death-Domain protein, may act similarly to recruit directly downstream IRPs during developmental apoptosis in the fly. In the cases of oncogene deregulation, DNA damage/p53, and Bak, our data indicate that all these triggers of apoptosis act on some upstream regulator that can independently activate both IRPs and membrane blebbing.
By time-lapse videomicroscopic analysis, we observe a clear difference in the anti-apoptotic actions of the IRP inhibitor ZVAD.fmk, on the one hand, and the well-characterized antiapoptotic factors Bcl-2 and IGF-I, on the other. The actions of both Bcl-2 and IGF-I are to lower the probability of initiation of the apoptotic program, but neither has any effect on the kinetics of each apoptotic event (as defined as the time from onset of blebbing to cell fragmentation) once initiated. In complete contrast, ZVAD.fmk does not inhibit initiation of the apoptotic program, as judged by the onset of membrane blebbing, but prolongs each individual apoptotic event by inhibiting the cellular fragmentation that normally rapidly ensues. Importantly, cells that have initiated membrane blebbing but are blocked in completion of the apoptotic program by ZVAD.fmk cannot be rescued by the action of antiapoptotic survival factors and thus appear to be beyond a point of commitment to die. In addition, ZVAD.fmk-blocked blebbing cells do not divide; they have no clonagenic potential and from the stance of their neoplastic potential may therefore be considered as “genetically dead.” The clear implication from this is that, unlike more upstream inhibitors of apoptosis like Bcl-2 and survival factors, IRP inhibitors (whether viral or chemical) will not act as generic carcinogens because they inhibit the apoptotic program downstream of an irreversible point where replicative potential is lost.
In conclusion, we have shown that oncogene deregulation, DNA damage, and expression of the Bcl-2 family member Bak all induce apoptosis in mammalian fibroblasts via a ZVAD.fmk-inhibitable mechanism, probably one or more IRP. Nonetheless, time-lapse videomicroscopic analysis clearly distinguishes between the antiapoptotic actions of Bcl-2 or the survival factor IGF-I on the one hand and IRP inhibition on the other. Both Bcl-2 and IGF-I signaling act to suppress initiation of the apoptotic program, yet have no effect on the kinetics of each apoptotic event. In contrast, ZVAD.fmk has no effect on initiation of apoptosis, as determined by membrane blebbing, but acts to arrest each apoptotic program before completion. This observation raises the intriguing possibility that membrane blebbing is a discrete subprogram operating during mammalian apoptosis that can lead to cell death via an IRP-independent mechanism. If true, this will constrain the potential therapeutic use of IRP inhibitors. However, such speculations can only be validated by the development of specific inhibitors for each IRP and a more detailed understanding of the intracellular targets and kinetics of these enzymes.