* Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom; and Department of Medicine, (Renal and Inflammatory Disease), University Hospital, Nottingham NG7 2UH, United Kingdom
There is increasing evidence for a central
role in mammalian apoptosis of the interleukin-1-
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.
Apoptosis is an innate cell suicide mechanism invoked in disparate situations, both physiological
and pathological, to ablate unwanted, damaged,
or potentially neoplastic cells. Apoptosis is classically defined by a characteristic series of morphological changes in
the doomed cell, including membrane blebbing, cell shrinkage, chromatin condensation, and DNA cleavage,
and culminating in fragmentation of the cell into membrane-bound apoptotic bodies whose surfaces express potent triggers for phagocytosis. A cell suicide program appears to have arisen early in metazoan evolution and its
basal machinery has been substantially conserved. The key
genetic protagonists of programmed cell deaths in the
nematode worm, Caenorhabditis elegans, are two "killer"
genes, ced-3 and ced-4, that are required for the execution
of the death program and ced-9, the "protector" gene,
which mitigates the actions of the killer genes. ced-9 and
ced-3 each have numerous mammalian homologues: ced-9
is related structurally and functionally to the family of
death-regulating genes of which the oncogene bcl-2 is the
prototype (Hengartner and Horvitz, 1994 ICE was originally described as the cysteine protease required for the cleavage of pro-interleukin-1 All members of the ICE-related protease family share a
predilection for cleavage of their substrates after an aspartate residue at the P1, usually followed by a small residue
at the P1 Some clues as to how activated IRPs trigger apoptosis
come from identification of potential IRP targets. CPP32 In the nematode, genetic evidence favors the notion that
the IRP Ced-3 is part of the "execution machinery" of
nematode cell death since, in ced-3-deficient mutants, no
cell deaths occur (Ellis and Horvitz, 1986 One way to approach these questions is to use inhibitors of
IRPs based upon the preferred peptide substrate specificity
of these enzymes. In this paper, we use two broad-spectrum
cell permeable IRP inhibitors Cell Culture and Cell Lines
Rat-1 fibroblasts expressing the Reagents and Antibodies
4-OHT was obtained from SEMAT (St. Albans, UK). A 1 mM stock solution in 100% ethanol was prepared, stored at Polyclonal antibody to adenovirus E1A protein was the gift of Dr. N. Jones, Imperial Cancer Research Fund, London, UK (ICRF). Polyclonal
antibody to PARP (C-2-10) was the gift of Dr. W. Earnshaw (Edinburgh
University, Scotland, UK). The anti-lamins A and C antibody (131C3)
was the gift of Dr. Y. Raymond (Institut du Cancer de Montreal, Montreal, Canada), together with the control antibody MOPC 21C. The actin
antibody was the gift of Dr. Fulvia Verde, ICRF.
Time-Lapse Videomicroscopy
Time-lapse videomicroscopic images were acquired on an inverted phase
contrast microscope (Diaphot; Nikon, Inc., Mellville, NY) equipped with
epifluorescence and collected on either BetaCam or sVHS video tape under the control of an external animation controller (EOS Electronics AV
Ltd., Barry, Wales, UK) as described (Evan et al., 1992 Western Blotting
Cleavage of poly(ADPribose) polymerase, nuclear lamins A and C, and
actin was analyzed by SDS-PAGE and subsequent transfer to nitrocellulose membrane (Immobilon P, Amersham International). Membranes
were blocked in TNT buffer (1% TBS, 0.5% Tween 20, and 5% skimmed
milk) for 30 min and then incubated in antibodies to PARP (Lamarre et al.,
1986 Electron Microscopy and Immunocytochemistry
Standard electron microscopic techniques were used as follows. Cells were
pelleted, fixed for 1 h at RT in 2.5% glutaraldehyde in Sorensens buffer,
postfixed in osmium tetroxide in the same buffer, and embedded in
Araldite resin. Sections were cut on a Reichert ultracut, stained with uranyl acetate and lead citrate, and viewed on an electron microscope (EM
10 CR; Carl Zeiss, Inc., Thornwood, NY). PARP expression and localization was determined immunocytochemically as previously described
(Lamarre et al., 1986 Assays of Cell Viability
Mitochondrial dehydrogenase activity, used as a marker of cell survival,
was assayed by the ability of cells to convert soluble MTT into an insoluble formazan reaction product (Mosmant, 1983 Cell survival was also assayed by a direct microscopic assessment of the
proportion of cells able to exclude the vital dye, trypan blue. Rat-1/cMycERTM cells were grown in 3-cm tissue culture dishes, changed to serum-free medium for 48 h, and then 4-OHT ± ZVAD.fmk/BD.fmk was
added (time 0). At the time points indicated, cells were trypsinized and
washed in DME, and the number of trypan blue-positive cells was assessed by counting a minimum of 200 cells on a hemocytometer.
Flow Cytometric Analysis of Cells
Adherent cells were trypsinised, fixed in 70% ethanol, and stained with
propidium iodide. Flow cytometric analysis was carried out on a BectonDickinson (Mountain View, CA) FACSstar plus.
Annexin V Binding to External Phosphatidyl Serine
Cells undergoing apoptosis in the presence or absence of ZVAD.fmk or
BD.fmk were observed by time-lapse videomicroscopy. At the time that a
significant proportion of the cells were either apoptoptic (no ZVAD.fmk/
BD.fmk) or blebbing (plus ZVAD.fmk/BD.fmk), the culture was flooded
with FITC-annexin V. The same microscopic field as had been observed
thus far was then examined by fluorescence and phase microscopy to detect bound annexin V and examine cell morphology, respectively.
ZVAD.fmk and BD.fmk, Inhibitors of
ICE-related Proteases, Delay but Do Not Prevent Cell
Death Induced by Multiple Signals
Apoptosis can be triggered by a wide range of influences,
including oncogene deregulation, DNA damage, and expression of killer genes. To determine the general relevance of IRPs to apoptosis, we examined the effect of the
cell-permeable IRP inhibitors, ZVAD.fmk and BD.fmk,
upon apoptosis induced in Rat-1 fibroblasts by a number of agents. Apoptosis was induced by activation of a deregulated conditional allele of c-Myc (Littlewood et al., 1995 The Rat-1 fibroblast cell lines were induced, activated,
or treated with drug, as appropriate, in the presence or absence of ZVAD.fmk or BD.fmk. Cells were monitored by
time-lapse videomicroscopy and scored as dead when they
became detached from the substratum. Both ZVAD.fmk
and BD.fmk afforded excellent protection from apoptosis in all cases, regardless of the trigger (data for ZVAD.fmk
shown in Fig. 1). Thus, all these triggers of apoptosis act
via a ZVAD.fmk- or BD.fmk-sensitive agent, presumably
one or more of the IRPs.
As shown in Fig. 1, the degree of protection afforded by
ZVAD.fmk or BD.fmk is profound. Nonetheless, under
the sustained pro-apoptotic influence of c-Myc, E1A, Bak,
or continuous DNA damage, all cells were eventually observed to detach from the substratum, undergo partial
chromatin condensation, and eventually become permeable, even in the sustained presence of ZVAD.fmk or BD.fmk (see below). Neither higher levels of ZVAD.fmk
or BD.fmk nor regular 24 hourly readdition of ZVAD.fmk
or BD.fmk to the culture medium was able to delay the
eventual demise of cells (data not shown), suggesting
ZVAD.fmk and BD.fmk inhibition of cell death is eventually overwhelmed or bypassed. In all cases tested, the effects of ZVAD.fmk and BD.fmk were identical. Accordingly, primarily data for ZVAD.fmk is shown henceforth.
ZVAD.fmk Does Not Delay the Onset
of Membrane Blebbing but Blocks Completion of the
Apoptotic Program
Time-lapse videomicroscopy permits observation both of
the onset and the execution of individual apoptotic events
within a cell culture. The onset of apoptosis is characterized by the sudden initiation of surface blebbing, together
with cytoplasmic fragmentation and exfoliation, from
which the name apoptosis derives. In fibroblastic cells, onset of blebbing is rapidly followed by nuclear condensation and collapse and total cell fragmentation, a process that
typically takes between 30-60 min (Evan et al., 1992 Using time-lapse videomicroscopy to observe cells undergoing c-Myc-induced apoptosis in the presence of either ZVAD.fmk or BD.fmk, we were surprised to find
that neither inhibitor appears to delay the initiation of apoptosis within the cultures, as judged by the onset of
blebbing. Indeed, the kinetics of onset of apoptotic events
within ZVAD.fmk-treated cultures were superimposable
on those of untreated cell cultures (Fig. 2 A). However,
unlike untreated cells, ZVAD.fmk- or BD.fmk-treated cells
did not rapidly condense and fragment but continued to
bleb and boil for extended periods (Fig. 2 B). During this
extended blebbing, often lasting days, cells remain adherent, exclude trypan blue, and exhibit physical activity, all
suggestive of sustained metabolism (see Note Added in
Proof for Web Information). This latter notion was supported by assaying mitochondrial activity of ZVAD.fmktreated cells using the MTT assay, which showed continued mitochondrial dehydrogenase activity in such cells
(Fig. 3 A).
Thus, ZVAD.fmk-treated cells appear to initiate apoptosis normally but then become arrested within the apoptotic program. Identical results were obtained when apoptosis was induced by E1A, etoposide, or Bak expression
(data not shown).
Characterization of Apoptotic Cells Blocked
with ZVAD.fmk
To investigate further the ZVAD.fmk-mediated block in the
death program, we examined a number of biochemical and
morphological parameters of apoptosis in the ZVAD.fmktreated blebbing cells. First, we examined the efficacy with
which ZVAD.fmk inhibits intracellular IRPs by examining
cleavage of known IRP substrates by a combination of Western blotting and confocal immunofluorescence microscopy. Cells were harvested from ZVAD.fmk-treated cultures in
which 50-60% of cells exhibited the continuously blebbing
phenotype as judged by time-lapse videomicroscopy (typically 24-36 h after induction of apoptosis), and cell lysates
fractionated by SDS-PAGE and electroblotted. Blots were
probed with antibodies to actin, lamins A+C, and PARP, all known IRP substrates (Lazebnik et al., 1994
Next, electron microscopy was performed on Rat-1/cMycERTM cells induced to undergo apoptosis in the presence
or absence of ZVAD.fmk (Fig. 5). 24 h after c-Myc activation in low serum, many cells in the non-ZVAD.fmktreated population exhibited features of classical apoptosis,
i.e., chromatin condensation, cell shrinkage, cytoplasmic
blebbing, and cell fragmentation. In contrast, although cells from ZVAD.fmk-treated populations exhibited gross
cytoplasmic blebbing and some dilatation of the endoplasmic reticulum, their organelle structure was otherwise
well preserved. More remarkably, nuclei of ZVAD.fmktreated cells resembled those of nonapoptotic cells, with
reticular chromatin pattern bounded by intact nuclear lamina and membrane.
We next examined integrity of DNA in ZVAD.fmktreated blebbing cells. Flow cytometric analysis demonstrated that ZVAD.fmk suppresses the appearance of cells
with sub-G1 content of DNA characteristic of apoptotic
cells (Fig. 3 B). Consistent with this, no degradation of
DNA into typical oligonucleosomal fragments (Wyllie,
1980 Apoptosis is classically associated with loss of membrane phospholipid asymmetry: inactivation of a critical
"flippase" results in expression of phosphatidylserine on
the cell surface, a signal for recognition and ingestion of
apoptotic cells by macrophages (Fadok et al., 1992
As discussed above, ZVAD.fmk-treated cells induced to
undergo apoptosis and exhibiting the blebbing phenotype
for several days do eventually detach and become permeable
to vital dyes. Analysis of these "late-stage" ZVAD.fmktreated cells revealed a ragged surface membrane, vacuolated
cytoplasm, partial chromatin condensation (although less
than in "normal" apoptotic cells), and eventual membrane
permeability (data not shown). We conclude that these
cells appear to have undergone a partial apoptotic cell death,
although this will require further characterization.
ZVAD.fmk Inhibits Apoptosis Downstream of
the Antiapoptotic Cytokine IGF-1 or the Apoptosis
Suppressor, Bcl-2
Both cytokines (Harrington et al., 1994
Finally, we asked whether ZVAD.fmk-blocked, blebbing cells might be rescued by removal of 4-OHT from the
growth medium, thus inactivating c-Myc protein, which, in
the Rat-1/c-MycERTM cells, is dependent upon the continuous presence of 4-OHT (Littlewood et al., 1995 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, ced-9
and ced-3, encode polypeptides that have multiple mammalian homologues: the Bcl-2 (Hengartner and Horvitz, 1994 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 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 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 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.
) and the ced-3 product is structurally and functionally homologous to a
class of mammalian cysteine proteases, of which the prototype is the interleukin-1
-converting enzyme (ICE)1 (Yuan
et al., 1993
). To date, no vertebrate homologue for ced-4 has been identified.
at Asp116Ala117 to generate the active cytokine (Cerretti et al., 1992
;
Thornberry et al., 1992
). ICE is but one of an emerging
family of ICE-related proteases (IRPs), whose known
members include Ich-1/Nedd-2 (Kumar et al., 1994
; Wang
et al., 1994
), Ich-2/TX/ICErelII (Faucheu et al., 1995
; Kamens et al., 1995
; Munday et al., 1995
), CPP32/Apopain/ Yama (Fernandes-Alnemri et al., 1994
; Nicholson et al.,
1995
; Tewari et al., 1995
), Mch2 (Fernandes-Alnemri et al.,
1995a
), Mch3/ICE-LAP-3 (Duan et al., 1996a
; FernandesAlnemri et al., 1995b), ICE-LAP-6 (Duan et al., 1996b
),
ICErelIII/TY (Faucheu et al., 1995
; Munday et al., 1995
)
and FADD-like ICE/MORT1-associated CEP-3 homologue (FLICE/MACH) (Boldin et al., 1996
; Muzio et al., 1996
). Of these, ICE, CPP32
, Mch2, and FLICE/MACH
have been directly implicated in apoptosis: FLICE/MACH
(Boldin et al., 1996
; Muzio et al., 1996
) and ICE (Enari et al.,
1995
; Kuida et al., 1995
) are required for induction of apoptosis through the TNF-R1 and CD95/Fas signaling pathways. FLICE/MACH is directly activated upon ligand
binding to either TNF-R1 or CD95/Fas receptors (Boldin et al., 1996
; Muzio et al., 1996
), and ICE activity is required (Enari et al., 1995
; Los et al., 1995
) for downstream
activation of a CPP32
-like protease necessary for apoptosis to occur (Enari et al., 1996
). CPP32
has also been
implicated as a general component of the apoptotic program (Nicholson et al., 1995
; Tewari et al., 1995
) and is a
component of prICE, a proteolytic extract derived from
apoptotic chick cells that induces condensation and fragmentation of interphase nuclei (Lazebnik et al., 1995a
).
Mch2 cleaves nuclear lamins (Orth et al., 1996
; Takahashi
et al., 1996
), an activity also present in prICE (Lazebnik
et al., 1995b
).
position, and all are synthesised as proenzymes
that are activated by cleavage at critical aspartate residues
that themselves conform to the substrate consensus for
ICE family proteases. Thus, the IRPs are presumed to exist within hierarchies of auto- and trans-cleavage. For example, proICE can be activated by auto-cleavage (Thornberry et al., 1992
), and proCPP32
can be activated by
ICE (Tewari et al., 1995
) as well as by the cytotoxic T cell
granule serine protease granzyme B (Darmon et al., 1995
).
ICE-related protease processing releases an NH2-terminal
prodomain of varying length in different IRPs and cleaves
the remaining polypeptide to yield two subunits (p20 and
p10 for human ICE) that form the active (p20:p10)2 enzyme (Walker et al., 1994
; Gu et al., 1995b
). The larger
subunit contains the catalytic cysteine, but both subunits
are required for enzyme activity.
cleaves poly-(ADP-ribose) polymerase (PARP) (Lazebnik et al., 1994
; Gu et al., 1995a
) and DNA-dependent protein kinase (Casciola Rosen et al., 1995), both involved in
aspects of DNA damage sensing and repair, whereas Mch2
cleaves nuclear lamins (Orth et al., 1996
; Takahashi et al., 1996
). Other targets of various IRPs are the sterol regulatory element-binding proteins SREBP-1 and SREBP-2
(Wang et al., 1995
), the 70-kD protein of the U1-snRNP
(Casciola Rosen et al., 1994), PKC
(Emoto et al., 1995
),
and various components of the cytoskeleton such as actin
(Mashima et al., 1995
) and Gas2, a component of the microfilament system (Brancolini et al., 1995
). However, it is
unclear which, if any, of these targets is responsible for the
cell blebbing, condensation, and fragmentation that characterizes apoptosis.
). However, the
increased complexity and potential hierarchical relationships between mammalian IRPs make it unclear which
proteases might act as executors of apoptosis and which
might serve to regulate those executors. It is also unclear
whether certain mammalian IRPs comprise a "final and
obligate common pathway" activated by all triggers of
mammalian apoptosis.
benzyloxycarbonyl-ValAla-Asp(O-methyl)-fluoromethylketone (ZVAD.fmk) and
t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk)
to demonstrate that IRPs are involved in mammalian apoptosis induced by disparate signals. Most importantly, however, we show that inhibition of IRPs blocks only part of
the classical apoptotic program, generating doomed cells
that are arrested before the program has completed.
Materials and Methods
-oestradiol and the 4-hydroxytamoxifen
(4-OHT)-dependent conditional alleles of c-Myc (Rat-1/c-MycER and
Rat-1/c-MycERTM , respectively) were isolated as described (Littlewood et al.,
1995
). Rat-1 fibroblasts expressing Bak from a Gal-4 element under the
control of the 4-OHT-activatable chimeric GalER-VP16 transcription
factor (Braselmann et al., 1993
) have also been described (Rat-1/Bak
cells) (Chittenden et al., 1995
), as have Rat-1/c-MycER cells constitutively
expressing Bcl-2 (Rat-1/c-MycER/Bcl-2) (Fanidi et al., 1992
). A further
Rat-1 cell line, constitutively expressing the adenovirus E1A (12S) protein
(Rat-1/E1A), was isolated by infection of Rat-1 cells with the recombinant
retrovirus pBabeNEO containing the entire open reading frame of 12S
E1A under the control of the virus LTR and subsequent selection and
cloning. Expression of 12S E1A was verified by immunoblotting. Rat-1
cells expressing E1A (12S) or empty vector controls were washed twice in
serum-free medium and subsequently cultured in serum-free conditions. Cells were observed for 48 h using time lapse videomicroscopy. Rat-1/E1A
cells exhibited marked apoptosis compared to controls demonstrating the
ability of E1A to induce apoptosis in the absence of serum. Rat-1/cMycERTM , Rat-1/c-MycER/Bcl-2, and Rat-1/E1A cell lines were maintained in DME supplemented with 10% FCS and 1 mg/ml gentamicin.
Rat-1/Bak cells were maintained in phenol-red-free DME supplemented
with 10% charcoal-dextran-stripped FCS and 1 mg/ml gentamicin. Cells
were selected using puromycin at 5 µg/ml for Rat-1/c-MycERTM , neomycin
at 1 mg/ml for Rat-1/E1A cells, and with puromycin at 5 µg/ml and neomycin at 1 mg/ml for Rat-1/Bak cells. Cells were passaged by standard
trypsinization and seeded directly onto tissue culture plastic. Cells were
serum-depleted by culture in 0.05% FCS for 48 h. Both Myc and Bak expression were induced by addition of 4-OHT to the cell cultures to a final concentration of 100 nM.
20°C, and diluted in DME
as required. ZVAD.fmk and BD.fmk were obtained from Enzyme Systems Products (Dublin, CA). Stock solutions in DMSO were prepared at
50 mM, stored at
80°C, and used as required. Etoposide/VP16, phalloidin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were obtained from Sigma (Poole, UK). FITC-labeled annexin V
was obtained from Bender & Co. GmbH (Vienna, Austria). Secondary
antibodies and enhanced chemiluminescence were obtained from Amersham International (Amersham, UK).
). Apoptotic cell
deaths were scored at the point when they were fully detached and
rounded. Cells with the "blebbing" phenotype were deemed to be alive
while they remained adherent and actively blebbing and similarly scored
as dead once fully detached and rounded.
), actin, or lamins A and C antibody at 4°C for 12 h in TNT buffer.
Membranes were washed twice in TNT and then incubated for 1 h at room
temperature (RT) with anti-mouse or anti-rabbit HRP-conjugated secondary antibody as appropriate. Finally, membranes were washed twice in
TNT buffer and once in TBS and 0.5% Tween 20, and bound antibodies
were visualized by enhanced chemiluminescence.
). Briefly, cells were grown on 8-well chamber slides
and fixed with 100% methanol for 10 min at
20°C. Fixed cells were incubated with primary antibody for 60 min at RT in PBS followed by five
washes in PBS buffer containing 0.1% Tween 20. After incubation in appropriate FITC-labeled secondary antibodies, cells were viewed using a
confocal microscope (model MRC 1000; BioRad Labs, Hercules, CA).
Lamins were identified immunocytochemically using anti-lamin A+C antibody (1:100), incubated with cells previously fixed as described above,
and preblocked in 10% FCS and PBS. After three washes in PBS, cells
were incubated with secondary antibody as described above. For actin
staining, cells were fixed in 4% paraformaldehyde in PBS for 10 min,
washed in PBS at RT, and then permeabilized with 0.2% Triton in PBS
for 5 min. Cells were then rinsed in PBS and incubated with 0.1 µg/ml
FITC-phalloidin in PBS for 20 min at RT. Finally, cells were washed several times in PBS, mounted, and viewed as above.
). 10 µl of MTT solution (5 mg/ml in PBS) was added to each well of a 96-well plate containing 3,000 cells in 100 µl of medium. The reaction was stopped after 2 h at 37°C by
removal of the supernatant and addition of 200 µl of acid propan-2-ol.
The plates were left at RT for 15 min in the dark, and absorbance difference at 570-630 nm was measured using a microplate reader and % survival calculated as (Experiment
Blank)/(Control
Blank) × 100%,
where the blank is medium + MTT without cells and the Control was the
signal from wells with maximum cell survival, i.e., with serum and no 4-OHT.
Results
),
expression of the adenovirus protein E1A (Rao et al.,
1992
), DNA damage in combination with c-Myc expression using the topoisomerase II inhibitor etoposide
(Fanidi et al., 1992
), or expression of the killer member of
the Bcl-2 family, Bak (Chittenden et al., 1995
). Both E1A
(Debbas and White, 1993
) and etoposide (Lowe et al.,
1993
) induce apoptosis via a p53-dependent mechanism;
c-Myc may act partly via p53 (Littlewood et al., manuscript in preparation) and Bak killing is p53 independent (T. Chittenden, unpublished data).
Fig. 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-ERTM 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-MycERTM 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).
[View Larger Version of this Image (23K GIF file)]
) (see
Note Added in Proof for Web information). However,
whereas an individual apoptotic event is rapid, individual
cells within a culture, undergoing apoptosis induced by activation of c-Myc in low serum, demonstrate asynchronous
initiation of apoptosis over a prolonged period. Since such
apoptosis is cell cycle independent, the basis of this asynchronicity is unclear, although its incidence is greatly affected by levels of c-Myc protein and the presence of survival factors in the culture medium (Harrington et al., 1994
).
Fig. 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-MycERTM 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-MycERTM 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.
[View Larger Versions of these Images (111 + 132 + 13K GIF file)]
Fig. 3.
ZVAD.fmk delays the onset of characteristic markers
of apoptosis. (A) ZVAD.fmk delays destruction of mitochondrial
integrity. Rat-1/c-MycERTM 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-MycERTM 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.
[View Larger Versions of these Images (76 + 35K GIF file)]
, 1995b; Gu
et al., 1995a
; Mashima et al., 1995
; Oberhammer et al.,
1994
), and compared with equivalent lysates from control
cultures without ZVAD.fmk. ZVAD.fmk inhibits cleavage
of all proteins for up to 48 h (Fig. 4 A). We also examined
the immunocytochemical distribution of actin, PARP, and
lamin staining in cells with and without ZVAD.fmk treatment by immunofluorescence confocal microscopy (Fig. 4
B). The effect of ZVAD.fmk on actin distribution within
cells was examined by staining permeabilized cells with
phalloidin-FITC. This showed ZVAD.fmk-treated cells to
be morphologically distinct from either log-phase viable or
apoptotic cells, exhibiting very obvious cytoplasmic
blebbing (Fig. 4 B). ZVAD.fmk treatment prevented the
dispersal and reduction of PARP and lamin staining that is typically observed in apoptotic cells. Thus, as reported
elsewhere (Fearnhead et al., 1995
; Fletcher et al., 1995
;
Slee et al., 1996
), ZVAD.fmk effectively inhibits intracellular cleavage of known IRP substrates.
Fig. 4.
ZVAD.fmk inhibits cleavage of known substrates of ICE-related proteases in Rat-1/c-MycERTM
cells expressing activated
c-Myc in low serum. (A) Immunoblot analysis of cleavage of three known IRP substrates in Rat-1/c-MycERTM
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/cMycERTM 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-MycERTM 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-MycERTM 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-MycERTM cells exhibit nuclear PARP
staining, which is absent in apoptotic cells. Bars, 10 µm.
[View Larger Versions of these Images (36 + 21 + 27 + 56 + 60 + 34K GIF file)]
Fig. 5.
Electron microscopic analysis of apoptosis in serum-deprived Rat-1/c-MycERTM cells in the presence and absence of
ZVAD.fmk. Electron microscopical analysis of individual Rat-1/c-MycERTM cells undergoing 4-OHT-induced apoptosis in low serum.
(A) Normal, viable Rat-1/c-MycERTM 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.
[View Larger Version of this Image (168K GIF file)]
) was detected in DNA extracted from ZVAD.fmktreated cells up to 48 h after c-Myc activation (data not
shown).
). Surface phosphatidylserine expression was assessed by binding of FITC-labeled annexin V and visualized by fluorescence microscopy (Koopman et al., 1994
). Rat-1/c-MycERTM cells undergoing normal apoptosis exhibited annexin V
binding, whereas no binding was detected on blebbing cells
blocked with ZVAD.fmk or live cells (Fig. 6). Thus,
ZVAD.fmk blocks apoptosis before destruction of membranes, mitochondria, or loss of phosphatidylserine asymmetry.
Fig. 6.
Expression of cell surface phosphatidyl serine. ZVAD.fmk delays the emergence of annexin V binding. Serum-deprived Rat-1/cMycERTM 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).
[View Larger Version of this Image (88K GIF file)]
) and Bcl-2 (Fanidi
et al., 1992
) are effective at suppressing fibroblast apoptosis, which is most evident as a delay in onset of apoptosis,
with the extent of the delay depending upon levels expression of c-Myc and the abundance of IGF-1 or Bcl-2 (Evan
et al., 1992
; Fanidi et al., 1992
; Harrington et al., 1994
). Intriguingly, time-lapse videomicroscopic analysis reveals
that, while IGF-1 and Bcl-2 delay the onset of apoptosis
(as judged by the start of membrane blebbing), they do not
inhibit completion of the program once initiated. Cells
proceed from blebbing to final fragmentation within 20-60
min, essentially the same kinetics as cells cultured in the absence of IGF-1 or overexpression of bcl-2 (Fig. 7, A and B).
This is consistent with the notion that Bcl-2, serum and survival factors like IGF-I signaling act upstream of the
onset of membrane blebbing, effectively reducing the
probability that the apoptotic program will be initiated in
any individual cell. In complete contrast, ZVAD.fmk
acts downstream of membrane blebbing but before the
later stages of apoptosis. To investigate further the temporal relationship between serum survival factors and
ZVAD.fmk inhibition of apoptosis, we induced blebbing
in ZVAD.fmk-treated Rat-1/c-MycERTM cells by addition of
4-OHT in the absence of serum and then examined
whether readdition of serum could reverse the blebbing
phenotype and allow cell recovery. Cultures were observed by time-lapse videomicroscopy. At the time serum
was added, some cells within the population had just begun to bleb, whereas others had been blebbing for hours.
In none of >500 cells examined was there any reversal of
blebbing, and all cells that had initiated blebbing eventually went on to die. A study of a representative population
is depicted in Fig. 8. From examination of time-lapse video
data, it was also apparent that ZVAD.fmk-treated blebbing cells do not proliferate. Of some 1,000 cells examined,
no division was ever observed of any cell that had started
blebbing. However, this is not due to a cytostatic effect of
ZVAD.fmk per se since nonapoptotic cells in ZVAD.fmktreated cultures divide at normal rates, as judged by timelapse videomicroscopy (see Note Added in Proof for Web
Information) and DNA profile (Fig. 3 B). Thus, we conclude the onset of blebbing is coincident with loss of clonagenic potential. Interestingly, cells that had not yet initiated blebbing at the time of serum readdition remained
viable, never blebbed, and, in the presence of ZVAD.fmk,
proliferated to eventually repopulate the culture (not
shown). Thus, readdition of survival factors before the onset of membrane blebbing can completely protect both the
viability and proliferative potential of cells.
Fig. 7.
IGF-I and Bcl-2 delay the initiation of the apoptotic program but not its execution. Serum-deprived Rat-1/c-MycERTM 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.
[View Larger Versions of these Images (13 + 7K GIF file)]
Fig. 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-MycERTM 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).
[View Larger Version of this Image (15K GIF file)]
). Of some
500 cells examined, we have observed no recovery of any
ZVAD.fmk-treated blebbing cell upon removal of 4-OHT
(data not shown), affirming the commitment of these cells to eventual death.
Discussion
) 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.
; 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.
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.
; 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 CPP32
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.
Moira K.B. Whyte's current address is the Department of Medicine and Pharmacology, Section of Respiratory Medicine, Floor M, Royal Hallamshire Hospital, Sheffield S10 2JF, UK.
Received for publication 11 July 1996 and in revised form 22 October 1996.
Part of this work was supported by an MRC-DTI LINK award to G.I. Evan and by a Concerted Action grant (No. BMH1-C794-1471) to G.I. Evan and N.J. McCarthy. M. Whyte was supported by an Advanced Clinical Training Fellowship from the Wellcome Trust (Fellowship No. 041759). We are also indebted to the students of Bishop Stopford School for their generous donations in memory of Elliot Smith, Simon Railton, Joanne Briellat, and to whom this work is dedicated.We thank Derek Davies for flow cytometric analysis, Dr. Andrew Edwards and Peter Jordan for help with confocal microscopy, Dr. Nasser Hajibagheri for the electron micrographs, and our colleagues at the ICRF for advice, comment, and support.
4-OHT, 4-hydroxytamoxifen;
BD.fmk, t-butoxy carbonyl-Asp.fluoromethylketone;
FLICE, FADD-like ICE;
ICE, interleukin-1-converting enzyme;
IGF, insulin-like growth factor;
IRP, ICE-related protease;
MACH, MORT1-associated CED-3 homologue;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PARP, poly-(ADP-ribose) polymerase;
RT, room temperature;
ZVAD.fmk, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone.