Dissociation of Phagocyte Recognition of Cells Undergoing
Apoptosis from Other Features of the Apoptotic Program*
Jianguo
Zhuang
,
Yi
Ren§,
Roger T.
Snowden
,
Huijun
Zhu
,
Vladimir
Gogvadze
¶,
John S.
Savill§, and
Gerald M.
Cohen
From the
Medical Research Council Toxicology Unit,
Centre for Mechanisms of Human Toxicity, University of Leicester,
Hodgkin Building, Lancaster Road, Leicester LEI 9HN and the
§ Division of Renal and Inflammatory Disease, Department of
Medicine, University Hospital,
Nottingham NG7 2UH, United Kingdom
 |
ABSTRACT |
Apoptosis is a programmed form of cell death
characterized by biochemical and morphological changes affecting the
nucleus, cytoplasm, and plasma membrane. These changes in various
cellular compartments are widely regarded as mechanistically linked
events in a single "program" in which activation of caspases and
proteolysis of intracellular substrates represent a final common
pathway leading to cell death. To date there has been very limited
exploration of the linkage of this program to the plasma membrane
changes, which bring about swift recognition, uptake, and safe
degradation of apoptotic cells by phagocytes. Using the mitochondrial
inhibitors antimycin A and oligomycin in human monocytic THP.1 cells
triggered into apoptosis, we report the uncoupling of plasma membrane
changes from other features of apoptosis. These inhibitors blocked
increased plasma membrane permeability, externalization of
phosphatidylserine, and recognition by two classes of phagocytes but
not activation of caspase-3, cleavage of poly(ADP-ribose) polymerase
and DNA fragmentation. Externalization of phosphatidylserine in
apoptotic human leukemic U937 cells was also dissociated from
caspase activation. Thus changes governing safe clearance of apoptotic
cells may be regulated by an independent pathway to those bringing
about caspase activation. This finding could have important
consequences for attempts to manipulate cell death for therapeutic gain
in vivo.
 |
INTRODUCTION |
Apoptosis is a fundamentally important and programmed form of cell
death with wide-ranging significance in health and disease (1, 2).
Apoptotic cell death occurs in two phases, an initial commitment phase
followed by an execution phase, which is characterized by a series of
stereotypic changes including cell shrinkage, plasma membrane
alterations, and condensation and fragmentation of chromatin (3).
Internucleosomal cleavage of DNA, demonstrated as a DNA ladder by
agarose gel electrophoresis, was originally considered as a biochemical
hallmark of apoptosis (4), although it is now considered as a late
event in apoptosis, which may be dissociated from early more critical
changes (5). These DNA ladders are derived from large fragments of DNA
of 30-50 and 200-300 kilobase pairs in length (6). A family of at
least 10 interleukin-1
-converting enzymes (now known as caspases)
(7) appear to be responsible for most of the biochemical changes
associated with the execution phase of apoptosis (reviewed in Refs.
8-10). Caspases are cysteine proteases that have an absolute
specificity for an aspartic acid in the P1 position of the
substrate and cleave important cellular substrates including lamins,
poly(ADP-ribose) polymerase
(PARP)1 and
DNA-dependent protein kinase (3, 8-10). Cleavage of intact PARP (116 kDa) to its 85-kDa signature fragment is observed in many
different types of cells undergoing apoptosis and has also been
proposed as a biochemical marker of apoptosis (11). Many if not all
caspases are capable of cleaving PARP in vitro, but in
intact cells it is likely that caspase-3 (CPP32) and caspase-7 (Mch3)
are primarily responsible for the cleavage (8, 10, 12).
In vivo intact cells dying by apoptosis are usually swiftly
recognized and safely cleared by phagocytes. This protects surrounding tissues from exposure to injurious contents leaking from dying cells,
which is inevitable in accidental cell death by necrosis (1, 13).
Currently, there is only limited understanding of the molecular
mechanisms that render apoptotic cells recognizable to phagocytes.
However, exposure of phosphatidylserine (PS), an anionic phospholipid
normally confined to the inner leaflet of the membrane bilayer, serves
as a recognition signal for yet to be characterized PS receptors on
certain phagocyte populations (14-16). The mechanisms mediating PS
exposure are complex but seem to involve activation of caspases (17,
18). Another mechanism by which phagocytes recognize cells undergoing
apoptosis involves the phagocyte adhesion receptor CD36 co-operating
with the
v
3 vitronectin receptor integrin
to bind thrombospondin (TSP), which acts as a molecular bridge between
the apoptotic cell and the macrophage (19).
We have studied the induction of apoptosis in two human tumor cell
lines, THP.1 and U937. Using the mitochondrial inhibitors antimycin A
and oligomycin, we now demonstrate for the first time that in THP.1
cells induction of the apoptotic phenotype, characterized by DNA
fragmentation and the activation of caspases with ensuing cleavage of
PARP, can be dissociated from cell surface changes that result in the
phagocytic recognition of apoptotic cells by both the PS and
CD36/
v
3/TSP mechanisms. Similar
dissociation of externalization of phosphatidylserine from activation
of caspases was also observed in U937 cells.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Treatments--
Media and serum were purchased
from Life Technologies, Inc. (Paisley, UK). All chemicals were obtained
from Sigma Chemical Company (Dorset, UK) except for
N-tosyl-L-phenylalanyl chloromethyl ketone
(TPCK, Boehringer-Mannheim UK, Lewes, UK) and
benzyloxycarbonyl-valinyl-alanyl-aspartyl (OMe) fluoromethyl ketone
(Z-VAD.FMK, Enzyme Systems Inc., Dublin, CA). Both human monocytic
THP.1 cells and human leukemic U937 cells were maintained in suspension
culture as described (20, 2l). Apoptosis was induced in THP.1 cells by
incubating cells for 4 h with either etoposide or TPCK, whereas
U937 cells were incubated for either 3 h with tumor necrosis
factor-
(TNF-
) in combination with cycloheximide or 5 h with
etoposide. To assess the effects of the inhibitors on apoptosis,
antimycin A, oligomycin (65% oligomycin A), and ZVAD.FMK were
added 5 min before the cells were treated with apoptotic
stimuli in all experiments except for apoptosis induced by
TNF-
in U937 cells, which were pre-incubated with Z-VAD.FMK
for 30 min before exposure to TNF-
.
Flow Cytometric Analysis of Apoptosis--
Apoptosis in THP.1
and U937 cells (0.5 × 106 cells) was assessed with
Hoechst 33342 and propidium iodide (20, 21). Cells with a high blue
fluorescence (Hoechst 33342), because of an increase in cell membrane
permeability (22), exhibit an apoptotic morphology together with
internucleosomal cleavage of DNA (20, 21). PS exposure in apoptotic
cells was detected by incubating cells with fluorescein
isothiocyanate-labeled Annexin V (Bender MedSystems, Vienna, Austria)
as described (15, 23). Mitochondrial membrane potential
(
m) was measured following incubation of the cells for 25 min at 37 °C with the fluorochrome
3,3'-dihexyloxacarbocyanine iodide (50 nM) (Molecular
Probes Inc., Eugene, OR), which is retained in mitochondria with a
normal membrane potential (24).
Western Blot Analysis--
THP.1 and U937 cells (0.5 × 106) were prepared for SDS-polyacrylamide gel
electrophoresis as described previously (25). Intact PARP (116 kDa) and
its apoptotic 85-kDa signature fragment were detected with a rabbit
polyclonal (318) or a mouse monoclonal antibody (C2-10) (from Dr. G. Poirier, Laval University, Quebec, Canada). Procaspase-3 and its
catalytically active large subunit were detected with a rabbit
polyclonal antibody raised against the p17 subunit of caspase-3 (from
Dr. D. Nicholson, Merck Frosst, Quebec, Canada) as described (25).
Internucleosomal Cleavage of DNA--
Agarose gel
electrophoresis was used to detect DNA laddering in whole cells (2 × 106) as described previously (5).
Phagocytosis Assay--
Isolation and culture of mouse
macrophages were essentially as described previously (26) except for
minor modifications as follows. Inflammatory macrophages were elicited
into the peritoneal cavity of 8-16-week-old Balb/c mice with 2%
Brewer's thioglycollate. Peritoneal exudate cells were harvested 4 days later by peritoneal lavage with balanced salt solution, plated in
96-well plates at 0.1 × 106 cells/well, and cultured
overnight in complete medium (Dulbecco's modified Eagle's medium
containing 5% fetal calf serum, 50 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin) before use in the phagocytosis assay. Bone
marrow was harvested from Balb/c mice and plated at 0.1 × 106 cells/well in 96-well plates in Dulbecco's modified
Eagle's medium containing 10% conditioned medium from L929 cells as a
source of macrophage colony-stimulating factor. Bone marrow macrophages were used in the phagocytosis assay after being cultured for 5-7 days.
For the phagocytosis assays, macrophage monolayers were washed, and
THP.1 cells (0.5 × 106) in RPMI 1640 containing 5%
fetal calf serum were added to each well. The plates were incubated for
1 h and then washed in ice-cold saline after treatment with
trypsin to remove adherent macrophages. After cytospinning the
macrophages onto microscope slides, cells were fixed with 2%
glutaraldehyde and stained with Giemsa dye. Phagocytosis was evaluated
by counting 500 macrophages/slide of four replicate slides in each
experiment. Results were expressed as the percentages of macrophages
that had phagocytosed THP.1 cells.
 |
RESULTS |
Antimycin A and Oligomycin Inhibit Apoptosis Assessed by PS
Exposure and Hoechst Staining but Not by Decreased Mitochondrial
Membrane Potential (
)--
Treatment of THP.1 cells with
etoposide, a DNA topoisomerase II inhibitor, or TPCK, a
chymotrypsin-like serine protease inhibitor, induces apoptosis,
assessed by an increase in Hoechst 33342 fluorescence (20, 25, 27).
Increase in Hoechst fluoresence results from an increased cell
permeability of apoptotic cells compared with normal cells (22). In
this study we have also assessed apoptosis using two other flow
cytometric methods, i.e. externalization of PS and the
percentage of cells with decreased 
m. Externalization of PS is considered to be a general feature of apoptosis induced by
many different stimuli and is measured by binding of annexin V, a
PS-binding protein (15, 23). A reduction in 
m has
also been reported to be an early event in the induction of apoptosis in many different systems (28, 29). Incubation of THP.1 cells with
etoposide (25 µM) or TPCK (75 µM) resulted
in a time-dependent induction of apoptosis, as assessed by
both PS exposure and decreased 
m (Table
I). Although a decreased

m has been proposed to regulate the externalization
of PS (29), in our studies both of these changes demonstrated a similar
time dependence irrespective of the apoptotic stimulus (Table I). Thus
induction of apoptosis in THP.1 cells is accompanied by an increased
Hoechst 33342 fluorescence, externalization of PS, and a decreased

m.
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Table I
Time-dependent induction of apoptosis by etoposide and TPCK
in THP.1 cells
THP.1 cells were incubated for up to 4 h with either etoposide (25 µM) or TPCK (75 µM). The percentage of
apoptotic cells was then determined by an increase in PS
externalization or a decrease in  m. The data represent
the means (± S.E.) of at least three experiments.
|
|
To investigate the possible relationship between reduction in
mitochondrial 
and other features of apoptosis such as
externalization of PS, we used antimycin A and oligomycin, inhibitors
of the mitochondrial respiratory chain and ATP synthase, respectively
(30). Both antimycin A and oligomycin caused a
concentration-dependent inhibition of apoptosis induced by
either etoposide or TPCK as assessed either by PS exposure or Hoechst
33342 staining, whereas they had little effect on the decreased

m (Fig. 1). Neither
antimycin A nor oligomycin alone induced apoptosis assessed by PS
exposure, Hoechst 33342 staining, or DNA fragmentation (data not
shown). Thus these experiments dissociated those features of the
apoptotic phenotype assessed by PS exposure and Hoechst 33342 staining
from those measured by the changes in 
m.

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Fig. 1.
Antimycin A and oligomycin cause a
concentration-dependent inhibition of externalization of PS
and increase in Hoechst 33342 fluoresence but not of the reduction in
 m. THP.1 cells were incubated for 4 h with
either etoposide (25 µM) (A) or TPCK (75 µM) (B) either alone or in the presence of the
indicated concentrations of antimycin A (1-30 µM) or
oligomycin (0.1-5 µg/ml) or in the presence of Z-VAD.FMK (50 µM). The percentage of apoptotic cells was then measured
flow cytometrically using an increase in externalization of PS
(open bars), an increase in Hoechst 33342 fluoresence
(filled bars), or a decrease in  m
(diagonally hatched bars). The data represent the means (± S.E.) of at least three experiments.
|
|
Z-VAD.FMK (50 µM), a cell membrane-permeable inhibitor of
caspases, inhibited apoptosis induced by both etoposide and TPCK, as
assessed by all three methods (Fig. 1), supporting a role for caspases
in the execution phase of apoptosis in THP.1 cells in agreement with
our previous observations (20, 25, 27). These results demonstrated that
in this model of apoptosis the activation of caspases occurred upstream
of PS exposure, increased Hoechst 33342 staining, and a decrease in

m.
Dissociation of PS Externalization from Caspase-3 Activation and
PARP Cleavage--
During the execution phase of apoptosis, caspases
are activated and cleave cellular substrates, such as PARP (3, 8-12). Therefore we wished to examine whether the mitochondrial inhibitors differentially affected the activation of caspases and the cleavage of
PARP. Using Western blot analysis, control cells contained almost
entirely intact PARP (116 kDa) (Fig.
2A, lane 1). Induction of apoptosis by either TPCK or etoposide was accompanied by the cleavage of PARP to its 85-kDa signature fragment (Fig. 2A,
lanes 2 and 6), which was completely blocked by
Z-VAD.FMK (50 µM) (Fig. 2A, lanes 5 and 9), further supporting the involvement of caspases in
the execution phase of apoptosis in THP.1 cells. Neither oligomycin nor
antimycin A blocked proteolysis of PARP to its 85-kDa fragment following incubation with either TPCK (Fig. 2A, lanes
3 and 4) or etoposide (Fig. 2A, lanes
7 and 8). The cleavage of PARP to its 85-kDa signature
fragment, which occurs at a DEVD
G sequence separating the
amino-terminal DNA binding domain and carboxyl-terminal catalytic
domain of the enzyme (31), suggested the activation of caspase-3
(CPP32) and/or caspase-7 (Mch3). We therefore examined the activation
of caspase-3 using an antibody raised against its p17 large subunit.
Caspase-3 is usually present in intact cells as an inactive p32 proform
(25, 32). On induction of apoptosis it is initially processed at
Asp175 between the large and small subunits yielding a p20
subunit, which is further processed at Asp9 and
Asp28 to yield p19 and p17 large subunits, respectively
(12, 33). Control THP.1 cells showed the presence of the intact proform of caspase-3 (Fig. 2B, lane 1). Induction of
apoptosis with either TPCK or etoposide was accompanied by loss of the
proform of caspase-3 together with formation of the catalytically
active large subunit of caspase-3 (Fig. 2B, LS,
lanes 2 and 6). Cleavage of caspase-3 was more
extensive in response to TPCK compared with etoposide commensurate with
the greater induction of apoptosis induced by this stimulus. Z-VAD.FMK,
which blocked the induction of apoptosis induced by these stimuli (Fig.
1), almost totally blocked the formation of the large subunit of
caspase-3 in cells treated with either TPCK (Fig. 2B,
lane 5) or etoposide (Fig. 2B, lane
9), although a small amount of a slightly larger immunoreactive
fragment (~p20) was also observed (Fig. 2B, lane
9), which was catalytically inactive because of the lack of the
cleavage of PARP (Fig. 2A, lane 9). In contrast,
neither oligomycin nor antimycin A inhibited the activation of
caspase-3 after treatment with either TPCK (Fig. 2B,
lane 3 and 4) or etoposide (Fig. 2B,
lane 7 and 8). Thus oligomycin and antimycin A
inhibited the externalization of PS and the increase in cell membrane
permeability assessed by Hoechst 33342 staining (Fig. 1A)
but did not inhibit the activation of caspase-3 and cleavage of PARP
(Fig. 2).

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Fig. 2.
Z-VAD.FMK but not antimycin A or oligomycin
inhibits PARP cleavage and caspase-3 activation in THP.1 cells.
THP.1 cells were incubated for 4 h with either TPCK (75 µM) or etoposide (25 µM) in the presence or
absence of oligomycin (Oligo, 5 µg/ml), antimycin A
(Ant, 30 µM), or Z-VAD.FMK (Z-VAD,
50 µM). A, to detect the 85-kDa fragment of
PARP, cellular proteins were resolved on an SDS-7% polyacrylamide gel,
transferred onto nitrocellulose membrane, and probed with rabbit
antiserum (318). B, to detect caspase-3 activation, cellular
proteins were resolved on an SDS-15% polyacrylamide gel. After
transferring, the proteins were probed with rabbit polyclonal
antibodies raised against the large subunit (LS) of
caspase-3. Results are representative of three experiments.
|
|
Dissociation of Externalization of PS from DNA
Fragmentation--
To extend these studies, we investigated whether
the treatment with antimycin A or oligomycin had interfered with the
DNA fragmentation, which occurs late in apoptosis. Etoposide alone induced internucleosomal cleavage of DNA compared with control cells
(Fig. 3, compare lanes 1 and
2). Z-VAD.FMK (50 µM) but not antimycin A or
oligomycin inhibited etoposide-induced internucleosomal cleavage (Fig.
3, lanes 3-5). TPCK alone induced fragmentation of DNA to
large fragments of 30-50, 200-300, and 600-700 kilobase pairs in
length in the absence of internucleosomal cleavage (27). Z-VAD.FMK but
not antimycin A or oligomycin inhibited the formation of these large
DNA fragments (data not shown). Thus externalization of PS during
apoptosis can also be dissociated from DNA fragmentation.

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Fig. 3.
Neither antimycin A nor oligomycin inhibits
internucleosomal cleavage of DNA in THP.1 cells. THP.1 cells were
incubated with etoposide (25 µM) in the presence or
absence of oligomycin (Oligo, 5 µg/ml), antimycin A
(Ant, 30 µM), and Z-VAD.FMK (Z-VAD,
50 µM) for 4 h. 2 × 106 cells were
loaded per lane and subjected to conventional agarose gel
electrophoresis to detect internucleosomal cleavage of DNA. Results are
representative of three experiments.
|
|
Cell Surface Changes Resulting in Phagocytic Recognition Can Be
Dissociated from Other Features of the Apoptotic Phenotype--
One of
the key features of the apoptotic process is the rapid recognition and
subsequent phagocytosis of apoptotic cells (1, 13). Because both
antimycin A and oligomycin inhibited externalization of PS but not
other features of the apoptotic phenotype, we wished to assess
whether they also interfered with phagocytic recognition of apoptotic
THP.1 cells. Peritoneal and bone marrow macrophages phagocytose
apoptotic cells utilizing receptors for PS or thrombospondin (i.e.
v
3 and CD36),
respectively (13, 14, 19, 26). Exposure of THP.1 cells to the two
pro-apoptotic stimuli, etoposide and TPCK, resulted in an increase in
the percentage of cells phagocytosed by both peritoneal macrophages and
bone marrow-derived macrophages (Table
II). Z-VAD.FMK, antimycin A, and
oligomycin all inhibited the etoposide- and TPCK-induced increases in
recognition and phagocytosis by both peritoneal and bone marrow-derived
macrophages (Table II). Thus cells displaying many features of
apoptosis including DNA fragmentation, processing of caspase-3, and
PARP cleavage do not display characteristic cell surface changes, which
result in phagocytic recognition and engulfment.
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Table II
Inhibition of phagocytosis of apoptotic THP.1 cells by oligomycin and
antimycin A
Induction of apoptosis in THP.1 cells was essentially as described in
the legend to Table I. Phagocytosis was evaluated by counting 500 macrophages/slide of four replicate slides in each experiment (see
"Experimental Procedures"). Peritoneal and bone marrow macrophages
phagocytose apoptotic cells utilizing receptors for PS and
thrombospondin, respectively. Results were expressed as percentages of
macrophages that had phagocytosed THP.1 cells. The data represent the
means (± S.E.) of three experiments.
|
|
Dissociation of Cell Membrane Changes from Caspase Activation
during Induction of Apoptosis in U937 Cells--
To determine whether
the phenomenon of dissociation of externalization of PS from other
features of the apoptotic phenotype was cell type-specific, we extended
the study to human leukemic U937 cells. These cells were treated with
two commonly used apoptotic stimuli, etoposide (25 µM) or
TNF-
(10 ng/ml) in the presence of cycloheximide (0.9 µM) (17). Apoptosis was induced by both these stimuli, as
assessed by externalization of PS, increase in Hoechst 33342 fluoresence, and increase in cells with a decreased 
m
(Fig. 4A). Both oligomycin (5 µg/ml) and antimycin A (30 µM) largely blocked the
apoptotic changes induced by either TNF-
or etoposide as assessed
either by PS exposure or Hoechst 33342 staining but had little effect
on the decreased 
m (Fig. 4A). Control U937
cells contained almost entirely intact PARP (116 kDa) (Fig.
4B, lane 1). Induction of apoptosis by either
TNF-
or etoposide was accompanied by activation of caspases as
assessed by the cleavage of PARP to its 85-kDa signature fragment (Fig. 4B, lanes 2 and 6). This cleavage was
completely blocked by Z-VAD.FMK (Fig. 4B, lanes 5 and 9), further supporting the activation of caspases during
apoptosis. Apoptosis, assessed by PS exposure, Hoechst 33342 staining,
or PARP cleavage was not induced by either antimycin A or oligomycin
alone (data not shown). Neither antimycin A nor oligomycin blocked
proteolysis of PARP to its 85-kDa fragment following incubation with
either TNF-
(Fig. 4B, lanes 3 and
4) or etoposide (Fig. 4B, lanes 7 and
8). Thus, these results in U937 cells dissociated those
features of the apoptotic phenotype assessed by PS exposure and Hoechst
33342 staining from those measured by the changes in

m and caspase activation.

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Fig. 4.
Dissociation of PS externalization from
caspase activation in apoptotic U937 cells. Apoptosis was induced
in U937 cells following incubation for either 3 h with TNF- (10 ng/ml) in the presence of cycloheximide (0.9 µM)
(lanes 2-5) or 5 h with etoposide (25 µM) (lanes 6-9). U937 cells were incubated
either alone (lane 1) or with the apoptotic stimuli in the
presence of antimycin A (30 µM) (lanes 3 and
7) or oligomycin (5 µg/ml) (lanes 4 and
8). Cells were also incubated with the apoptotic stimuli in
the presence of the caspase inhibitor Z-VAD.FMK, the concentration of
which was either 2 or 20 µM in the case of TNF- or
etoposide, respectively (lanes 5 and 9).
A, the percentage of apoptotic cells was then measured flow
cytometrically using either increase in externalization of PS
(open bars), increase in Hoechst 33342 fluoresence
(filled bars), or decrease in  m
(diagonally hatched bars). The data represent the mean (± S.E.) of at least three experiments except for  m,
which represents the mean of two determinations. B, cellular
proteins were resolved on SDS-10% polyacrylamide gels, transferred
onto nitrocellulose membrane, and probed with a mouse monoclonal
antibody (C2-10) to detect both intact PARP (116 kDa) and its cleaved
85-kDa fragment.
|
|
 |
DISCUSSION |
In this paper we present data demonstrating the dissociation of
cell membrane changes leading to phagocyte recognition of cells
undergoing apoptosis from other features of this programmed form of
cell death. The mitochondrial inhibitors, antimycin A and oligomycin,
not only inhibited surface changes of apoptosis, such as PS exposure
and recognition by PS-dependent elicited murine peritoneal
macrophages but also inhibited surface changes recognized by murine
bone marrow-derived macrophages, which use the
v
3/thrombospondin recognition mechanism
(Table II). However, antimycin A and oligomycin did not inhibit
caspase-3 activation, cleavage of PARP, DNA degradation, and reduction
in 
m (Figs. 1-4). These data imply that the surface changes of apoptosis relate to an independent pathway of events, which
may be differentially regulated from the activation of "effector" caspases, which are believed to trigger the nuclear and some of the
cytoplasmic changes of apoptosis.
It has become a widely held view that PS exposure and associated
changes, such as increased plasma membrane permeability to Hoechst dye,
represent "early" features of a single, coordinately regulated
common pathway leading to cell death, whereas DNA degradation is a
"late" feature. However, temporal dissociation of membrane changes
including PS exposure from other features of apoptosis could also imply
that mechanisms regulating the ability of dying cells to be recognized
by phagocytes are part of a parallel process, which is potentially
independent from other features of the program. This interpretation of
the data is strongly supported by the effects of antimycin A and
oligomycin reported here. Future studies will need to characterize the
pathway by which dying cells display "eat me" signals. The effects
of Z-VAD.FMK reported here support previous data implicating caspase
activation in externalization of PS (17, 18), although effector
caspases such as caspase-3 appear not to be involved.
The blockade of this putative "recognition pathway" by antimycin A
and oligomycin also provides important clues for its further dissection. A critical role for the mitochondrial release of proteins, such as apoptosis-inducing factor (AIF) and cytochrome c,
has been proposed in initiating the apoptotic program including DNA fragmentation and activation of caspase-3 (34, 35). Bcl-2 may prevent
apoptosis in part by blocking the mitochondrial release of both AIF
and/or cytochrome c (34, 36, 37). Although antimycin A and
oligomycin may affect targets other than their known mitochondrial ones, our results suggest that their interference of mitochondrial function prevents the mitochondrial release of some factor(s), other
than cytochrome c or AIF, that directly or indirectly
results in exposure of PS.
Lastly, our findings could have very important consequences for
attempts to manipulate apoptosis for therapeutic gain. In disorders
characterized by unscheduled cell loss by apoptosis, such as
neurodegenerative disease, potential therapies will not only need to
block intracellular pathways leading to engagement of effector enzymes
but will also need to interfere with those governing recognition, lest
potentially rescued cells are undesirably removed by phagocytes.
Conversely, there may be potential for selective triggering of the
surface changes of apoptosis in "undesirable" cells (such as
cancer cells) so that these are removed by innate mechanisms.
 |
ACKNOWLEDGEMENTS |
We thank Dr. G. G. Poirier for PARP antibody
and Dr. D. W. Nicholson for the caspase-3 antibody.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by the European Science Foundation Program in
Toxicology. Present address: Inst. of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino 142292 Russia.
To whom correspondence should be addressed: MRC Toxicology
Unit, Centre for Mechanisms of Human Toxicity, University of Leicester, Hodgkin Bldg., P.O. Box 138, Lancaster Road, Leicester LEI 9HN, UK.
Tel.: 44-116-252-5589; Fax: 44-116-252-5616; E-mail: gmc2{at}le.ac.uk.
1
The abbreviations used are: PARP,
poly(ADP-ribose) polymerase; TPCK,
N-tosyl-L-phenylalanyl chloromethyl ketone;
Z-VAD.FMK, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone; PS,
phosphatidylserine; TSP, thrombospondin; TNF-
, tumor necrosis
factor-
; AIF, apoptosis-inducing factor.
 |
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