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Dipartimento di Biologia, Universita' di Roma Tor Vergata, 00133 Roma, Italy;
* Istituto di Genetica Biochimica Evoluzionistica, CNR, 27100 Pavia, Italy; and
Dipartmento di Scienze Biomediche, Università di Chieti G. D'Annunzio, 66013 Chieti, Italy
1Correspondence: Dipartimento di Biologia, Università di Roma Tor Vergata, via Ricerca Scientifica, 00133 Roma, Italy. E-mail: ghibelli{at}uniroma2.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: caspase 3 • redox modulation • PARP • apoptotic cytochrome c release • puromycin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in healthy (stressed?) cells whereas in apoptosing
cells, it may activate the cascade of caspases (2)
, a set
of cysteine proteases that are responsible for the ultimate degradation
of key structural and regulatory proteins during apoptosis
(3)
. Cytosolic cytochrome c catalyzes the
proteolytic activation of caspase 9, which in turn activates caspase 3
(4)
, considered to be the key regulator of the proteolytic
events that lead to the morphofunctional changes occurring in the
execution phase of apoptosis.
A crucial role for cytochrome c release in apoptosis was
stressed by some recent studies demonstrating that cytochrome
c directly microinjected in the cytoplasms of a variety of
cell types is capable of triggering apoptosis on its own
(5)
, while APAF-1 is needed as a cofactor in cell free
extracts (2)
. In contrast, examples of apoptosis without
cytochrome c release (6)
indicate that this
event may not be necessary, at least in some apoptotic pathways.
Further investigations are required in order to elucidate the role of
cytochrome c in apoptosis.
Another open matter is the mechanism that triggers cytochrome
c relocalization to the cytosol, which requires the passage
through the outer mitochondrial membrane. A novel mechanism of protein
export from mitochondria that is dependent on Bax (7
8
9)
and/or Bid (10)
, two members of the Bcl-2 family of
proteins that regulate apoptosis, has recently been postulated. It
involves the translocation of Bid or Bax from the cytosol to
mitochondria (10
, 11)
, where they may possibly form a
pore, as shown for the related protein Bcl-XL
(12)
; Bax translocation requires homodimerization
(13)
, achieved by disulfide bond formation
(14)
. As an alternative model, it has been proposed that
cytochrome c may be released via the opening of the
mitochondrial permeability transition pore (PTP) (15
, 16)
,
possibly as a result of mitochondrial swelling-induced outer membrane
damage (17)
; PTP opening is a complex phenomenon,
controlled by the mitochondrial redox potential (18)
.
Glutathione is the key regulator of intracellular redox status; it
performs an antioxidant cell-protective action, cycling between its
reduced (GSH) and oxidized (GSSG) forms. We (19)
and
others (20)
have demonstrated that upon apoptogenic
stimuli that do not directly elicit an oxidative stress, glutathione is
extruded from the apoptosing cells in its reduced form. More recently,
we reported that prevention of GSH extrusion prevents apoptosis,
showing that GSH extrusion is necessary for the downstream events of
apoptosis, probably via the alteration of the intracellular redox
signaling (21)
.
Redox alterations are involved in both the proposed models of
cytochrome c export by facilitating PTP opening
(15)
, on the one hand, and on the other by promoting
disulfide bonds formation leading to Bax homodimerization
(14)
. This led us to hypothesize that GSH depletion and
the consequent redox disequilibrium might directly cause cytochrome
c release; this might possibly explain how GSH extrusion
triggers apoptosis. We could demonstrate that the release of mature
cytochrome c from mitochondria is indeed a cellular response
to the depletion of glutathione, independently from the destiny of the
cells (i.e., apoptosis or survival). This implies that the extremely
common redox imbalance occurring during apoptosis may be the trigger
for cytochrome c export.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. All the experiments were performed in complete
medium on log phase cells at a cell density of 7 x
105 cells/ml (U937) and 2 x
106/25 cm2 flasks (HepG2).
Apoptosis was induced with 10 µg/ml purumycin (PMC) or 3–5 mM
dithiothreitol (DTT) for the time indicated. For detection of
apoptosis, cells were stained with the DNA-specific, cell-permeable dye
Hoechst 33342; apoptotic cells were recognized according to their
nuclear morphology (different stages of nuclear fragmentation)
(22
, 23)
. Apoptosis was quantitated as described in ref
21
; briefly, the fraction of U937 or HepG2 cells with
fragmented nuclei among the total cell population is calculated in the
Hoechst 33342 stained cells, counting at least 300 cells in at least 10
random selected fields. Glutathione depletion was achieved by inhibiting glutathione neosynthesis with 1 mM buthionine sulfoximine (BSO) for the indicated time.
Glutathione determination
U937 and HepG2 cells were harvested, lysed, and proteins were
precipitated from lysates as described previously (21)
.
The clear supernatant was used for GSH and GSSG determination by
high-performance liquid chromatography (21)
. Basal values
of intracellular GSH content were 50.64 ± 13.67 (HepG2) and
46.54 ± 13.98 (U937) nmol of GSH/mg of protein. The values of GSH
loss are calculated as the percent of reduction of GSH in treated
samples with respect to control values.
Caspase 3 activation
For sample preparations, 107 cells were
washed and collected by centrifugation (preceded by trypsinization for
HepG2) at 200 x g for 5 min. The cell pellet was
resuspended in 100 µl lysis buffer (40 mM sucrose, 50 mM NaCl, 2 mM
MgCl2, 5 mM EGTA, 10 mM HEPES, pH=7) and
subjected to three cycles of freeze and thaw. The supernatants
recovered after 30 min 15,000 x g spin were stored at
-70°C until analysis. Assay: 10 µl of extract was added to caspase
assay buffer (10% sucrose, 0.1% Nonidet P40, 10 mM DTT, 100 mM HEPES,
pH 7.25) in a total volume of 1 ml in the presence of 40 µM
florogenic substrate specific for caspase 3 z-DEVDafc. The increase in
fluorescence due to substrate cleavage was monitored in a fluorometer
tuned at 400 nm (excitation) and 505 nm (emission). The values are
expressed as arbitrary units obtained by monitoring the increase in
fluorescence of treated samples with respect to controls.
The specificity of caspase 3 activation was always controlled with the immunoblotting assay (see below), which allows detection of the disappearance of the 32 kDa band as a sign of proteolytic activation of caspase 3. This assay always confirmed the results obtained with the fluorometric assay.
Preparations of cytosolic extracts and immunoblotting
Cells were washed and collected by centrifugation (preceded by
trypsinization for HepG2) at 200 x g for 5 min and at
4°C. The cell pellet was resuspended in 600 µl of extraction buffer
containing 220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50
mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and
protease inhibitors. After 30 min incubation on ice, cells were
homogenized with an Eppendorf pestle. Cell homogenates were spun at
14,000 x g for 15 min; supernatants were removed and
stored at -80°C until analysis by gel electrophoresis. Fifty
micrograms of cytosolic protein extracts was loaded onto each lane of a
12% sodium dodecyl sulfate (SDS) -polyacrylamide gel, separated, and
blotted to nitrocellulose membrane (Bio-Rad). Anti-cytochrome
c mouse monoclonal antibody (PharMingen) or anti-caspase 3
(CPP32) goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif.) was used as primary antibodies. The specific protein
complexes formed upon appropriate secondary antibody treatment were
identified using the `SuperSignal' substrate chemiluminescence
reagent (Pierce, Rockford, Ill.). The quantitation was obtained with
densitometric scanning with a LKB ultrascan XL laser densitometer
coupled with a LKB 2400 GelScan XL software package; the values are
referred to the total cellular cytochrome c.
PARP proteolysis
Western blot analysis was carried out essentially as
described in ref 24
. Briefly, cells were washed twice with
ice-cold PBS and resuspended at the concentration of 5 x
106/ml in a buffer containing 62.5 mM Tris-HCl pH 6.8, 4 M
urea, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.003%
bromophenol blue, as described in ref 25
. Cells were then
disrupted by sonication on ice, twice for 30 s (60 W). Equal
volumes (corresponding to 30 µl) of each sample were incubated for 15
min at 65°C before loading on SDS-polyacrylamide gel. Samples were
electrophoresed in a 7.5% SDS-PAGE minigel and transferred onto a
nitrocellulose filter (Bio-Rad, Hercules, Calif.) for 3 h at 4°C
under a constant voltage of 120 V (26)
. The membrane was
saturated overnight with PTN (PBS containing 0.1% Tween-20 and 10%
newborn calf serum) and then incubated for 3 h with the monoclonal
antibody C-2–10 kindly provided by Dr. G. Poirier (diluted 1:10,000 in
PTN), which recognizes an epitope located between the zinc finger
region and the automodification domain, at the carboxyl end of the
DNA-binding domain of poly(ADP-ribosyl)polymerase (PARP)
(26)
. After washings with PBS containing 0.2%
Tween 20, the membrane was incubated for 2 h with anti-mouse IgG
conjugated with peroxidase. Visualization of immunoreactive peptides
was achieved by ECL system (NEN Dupont, Boston, Mass.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In Fig. 1
we show that cytochrome c release and GSH loss occur in
tight concomitance, both preceding caspase 3 activation and nuclear
apoptosis. Notably, glutathione is depleted by 38% at 4 h of
treatment (Fig. 1A
) and cytochrome c is released
into the cytosol (Fig. 1A, B
), but no caspase 3 activation
or apoptosis is detectable (Fig. 1A
); thus, despite having
released cytochrome c, these cells are not dead. As a matter
of fact, they are not even committed to death: upon removal of PMC,
they escape apoptosis. After 14 h of additional culture in
PMC-free medium, they were still viable: GSH levels were restored to
control values and any trace of cytochrome c had disappeared
from the cytosol (Fig. 1C
). This set of experiments shows
that cytochrome c is released by preapoptotic cells
concomitant with GSH extrusion and that both events occur before the
irreversible commitment to death; this urged us to investigate a
possible link between glutathione extrusion and cytochrome c
release.
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BSO-induced depletion of GSH causes cytochrome c
release without affecting cell viability
To investigate the latter assumption, we asked whether
cytochrome c moves to cytosol when GSH is depleted without
apoptosis. We took advantage of the possibility of uncoupling
glutathione depletion and apoptosis. Indeed, HepG2 and U937 cells
treated with 1 mM BSO, a compound that inhibits glutathione
neosynthesis, had their glutathione content decreased without
undergoing apoptosis (19)
. We found that these
glutathione-depleted cells do release a substantial amount of
cytochrome c in their cytosolic fraction (Fig. 2A, B
) already in the first few hours of treatment. This is not accompanied
by caspase 3 activation or apoptosis (Fig. 2A
). In fact, no
loss of viability occurs even for longer incubation times, and
GSH-depleted cells remained viable and capable of replicating for
several days in the continuous presence of BSO (Fig. 2C
).
These results show that cytochrome c is released from
mitochondria as a consequence of the diminution of intracellular
glutathione content and that these events per se are not necessarily
leading to apoptosis.
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DTT-induced apoptosis occurs without GSH depletion, cytochrome
c release or caspase 3 activation
So far we have shown that GSH depletion, a common apoptotic
event, is sufficient to cause cytochrome c translocation to
the cytosol. Is GSH depletion or the consequent oxidative status also
necessary for cytochrome c release? To answer to this
question, we attempted to uncouple apoptosis from GSH extrusion.
We found that high doses (1–5 mM) of the reducing agent DTT induce
apoptosis on U937 and HepG2 cells. This apoptosis consequent to a
`reducing stress' is characterized by regular apoptotic morphology
(i.e., nuclear vesiculation), but it occurs without GSH loss
(Fig. 3A
). We wondered whether cytochrome c would be released in
cells induced to apoptosis by DTT, as generally occurs in apoptotic
U937, or whether in this case the lack of an oxidative environment
would prevent cytochrome c export. Figure 3A, B
shows that no cytosolic cytochrome c is detectable. This
suggests that GSH extrusion or the redox imbalance consequent to it is
necessary for cytochrome c translocation in apoptosis.
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Caspase 3 is not activated in DTT-induced apoptosis even though
PARP is cleaved
It is known that caspase 3 is activated as a result of cytochrome
c release (4)
. Thus, we wondered whether
caspase 3 is active in U937 induced to apoptosis by DTT despite the
absence of cytosolic cytochrome c. As shown in Fig. 3A
, in this case caspase 3 is not activated. Apoptosis
without caspase 3 activation has recently been described in the
epithelial cell line MCF7 (27)
. Indeed, MCF7 are able to
undergo apoptosis in a canonical way, i.e., the nuclei vesiculate
normally and PARP, a known target of caspase 3 action
(28)
, is regularly cleaved, conceivably by other
proteases. Thus, we analyzed PARP status in DTT-induced apoptosis; we
also observed a caspase 3-independent PARP cleavage (Fig. 3C
). This means that DTT induces a canonical apoptosis
(nuclear vesiculation, caspase activation, etc.) by eliciting a
noncanonical pathway that bypasses glutathione extrusion, cytochrome
c release, and caspase 3 activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20
21)
.
We have recently shown that the forced maintenance of GSH inside cells
pushed to apoptosis by PMC or etoposide increases cell survival,
implying that GSH depletion is a necessary step in order to trigger
apoptosis by affecting some crucial intracellular target via redox
disequilibrium (21)
. The results presented here indicate
that the modality through which GSH depletion triggers the downstream
events of apoptosis is indeed by promoting cytochrome c
release.
The redox disequilibrium consequent to GSH depletion is likely to be
the direct cause for cytochrome c translocation, since the
mechanisms proposed to be responsible for cytochrome c
release (PTP opening and Bax dimerization/translocation to
mitochondria) are controlled by redox alterations. PTP opening is known
to be facilitated by an oxidant environment (18)
, whereas
Bax homodimerization, which occurs via the formation of disulfide bonds
(14)
, is mostly favored in the absence of GSH.
DTT is a reducing agent that interferes with the formation of protein disulfide bridges. This is also one of the most important functions that GSH exerts in the cytosol, where it contrasts the formation of cystein disulfide bonds. Thus, the inability of cytochrome c to translocate to the cytosol during DTT-induced apoptosis fits with our model, which proposes the requirement of an oxidant environment for cytochrome c release. The finding that upon DTT treatment apoptosis occurs without glutathione depletion, translocation of cytochrome c, or caspase 3 activation indicates that these are three events of the intracellular apoptotic signaling that are tightly linked, and may occur sequentially, delineating a precise apoptotic pathway.
Much effort is currently being expended to study the role of cytochrome
c release in apoptosis, with still confusing and apparently
contradictory results. Indeed, this event has been found to be a
trigger for apoptosis (2
, 5)
, while not occurring at all
in some systems (6)
. Our study might help to solve these
contradictions by suggesting that cytochrome c might play a
role only in some apoptotic pathways, i.e., those involving redox
imbalance.
Reconstruction experiments have shown that cytosolic cytochrome
c needs APAF-1 as a cofactor in the proteolytic activation
of caspase 9, which in turn activates caspase 3 (4)
. This
may explain why cytosolic cytochrome c per se is not
necessarily leading to apoptosis in our experiments. It is possible
that in U937 or HepG2 cells depleted of glutathione by BSO, no APAF-1
is free (ready) to cooperate with cytochrome c to initiate
the apoptotic cascade; likewise, in HepG2 induced to apoptosis by PMC
(Fig. 1)
, the early released cytochrome c might have to wait
until a cofactor is available.
In conclusion, we show that cytosolic cytochrome c is a transient phenomenon, compatible with cell viability, occurring in instances of redox imbalance. In these situations, the release of cytochrome c might be inevitable, but circumstantial. Alternatively, it may respond to some critical requirements of the redox-imbalanced cell, possibly being part of a stress response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem. 273,15540-15545
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