From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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Activation of apoptosis is associated with generation of reactive oxygen species. The present research shows that superoxide is produced by mitochondria isolated from apoptotic cells due to a switch from the normal 4-electron reduction of O2 to a 1-electron reduction when cytochrome c is released from mitochondria. Bcl-2, a protein that protects against apoptosis and blocks cytochrome c release, prevents superoxide production when it is overexpressed. The switch in electron transfer provides a mechanism for redox signaling that is concomitant with cytochrome c-dependent activation of caspases. The block of cytochrome c release provides a mechanism for the apparent antioxidant function of Bcl-2.
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INTRODUCTION |
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Reactive oxygen species
(ROS)1 are important
regulators of apoptosis. Pro-oxidants and redox cycling agents, such as
H2O2 (1), diamide (2), etoposide (3), and
semiquinones (4), can induce apoptosis. Other apoptotic stimuli, such
as treatment with TNF (5), lipopolysaccharide (6), ceramide (7),
growth factor withdrawal (8), and human immunodeficiency virus
infection (9), can stimulate the ROS production. Studies with TNF
and ceramide have shown that these agents activated ROS generation by
mitochondria (7). The recent proposed model of p53-induced apoptosis
also placed ROS as a central signaling event (10, 11). Antioxidants and
thiol reductants, such as NAC (12), overexpression of thioredoxin (13),
and MnSOD (14), can block or delay apoptosis. On the other hand, ROS
can provide protective mechanisms under some conditions, such as the
activation of NF
B by TNF-induced ROS production (15, 16). Thus ROS,
and the resulting cellular redox change, can be part of the signal
transduction pathway during apoptosis. Yet the mechanisms of ROS
generation and its relationship with the well studied caspase
activation have not been resolved.
A role of ROS during apoptosis was initially proposed based upon the
observation that Bcl-2, a general inhibitor of apoptosis in mammalian
cells, has an apparent antioxidant function (17, 18). Bcl-2 is largely
localized to the mitochondrial outer membrane, and when overexpressed,
it protects cells from lipid peroxidation and thiol oxidation induced
by menadione and hydrogen peroxide (17, 18). Bcl-2 also protects
0 cells from apoptosis under conditions that ROS
generation is limited (19, 20). Thus, its anti-apoptotic function is
not limited to the antioxidant property. Nonetheless, this apparent
antioxidant property of Bcl-2 has not been explained.
When human myeloid leukemia HL 60 and U-937 cells undergo apoptosis
induced by staurosporine (21), etoposide (21), cytosine -D-arabinofuranoside (22), and ionizing radiation (23),
there is an early release of cyt c from mitochondria, and
the released cyt c participates in the activation of caspase
3. Bcl-2 family proteins prevent cyt c from entering the
cytosol either by blocking cyt c release (21, 22) or by
binding directly with cyt c (23). Cyt c is
localized in the mitochondrial intermembrane space and is part of the
mitochondrial electron transport chain. A variety of mitochondrial
poisons are well known to stimulate ROS generation by a mechanism that
involves inhibition of electron transfer, accumulation of reducing
equivalents in the middle portion of the electron transfer chain, and
direct one-electron transfer to O2 to produce superoxide
(for review, see Ref. 24). This switch appears to occur principally at
the level of coenzyme Q (ubiquinone/ubiquinol) and thus could also
occur as a consequence of cyt c loss.
In this study, we used staurosporine-treated HL 60 cells to study the mechanism of ROS generation during apoptosis. Results show that the cellular redox change occurred following mitochondrial release of cytochrome c and was in parallel to the caspase activation. The release of cytochrome c was associated with inhibited mitochondrial respiration and stimulated mitochondrial superoxide production, and overexpression of Bcl-2 inhibited all of these processes.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- HL 60 cells transfected with bcl-2 (bcl-2) or vector alone (neo) were cultured as described (21). Overexpression of Bcl-2 was monitored by Western blot. Cells were passaged every 2-3 days and seeded at 0.25 × 106/ml, and apoptosis was induced by adding 1 µM staurosporine.
Measurement of Intracellular and Mitochondrial Redox
Change--
After treatment, cells were pelleted and resuspended in
5% perchloric acid/saturated boric acid, supplemented with 5 µM -glutamylglutamate as internal standard.
Intracellular GSH and GSSG were measured with high pressure liquid
chromatography as described (25), and the amount of acid-insoluble
protein was measured by the Bradford method. Cell volume and pH were
measured radiochemically (26), and the redox potential was calculated
using the Nernst equation with E0 adjusted to
cell pH (
59 mV/pH unit). E0 at pH 7.0 was taken as
240 mV (27). Mitochondrial GSH and GSSG were measured similarly, except cells were first permeabilized with digitonin as
described below.
Measurement of Mitochondrial Generation of ROS-- After treatment with 1 µM staurosporine for 4 h, cells were stained with 15 µM dihydrorhodamine (DHR) for 20 min, washed with phosphate-buffered saline, and loaded onto a Becton-Dickinson FACStation. FL-1 fluorescence was recorded, and cell debris was electronically gated out based on the forward light scatter. 300 µM tert-butylhydroperoxide treatment was used as a positive control. The DHR fluorescence was also recorded with confocal microscopy. Cells double-stained with 60 nM DiOC6 and 15 µM DHR were washed and resuspended in 15 µl of phosphate-buffered saline and placed on a no. 1 thickness coverslip. The green and red fluorescence were simultaneously recorded with a Bio-Rad MRC 1024 laser scanning confocal microscopy system, with the excitation wavelength set at 488 nm. Images were acquired and processed with Laser-Sharp software, using identical background and gain settings. Six to eight areas were randomly picked, and representative ones are presented in Fig. 2.
Measurement of Mitochondrial Membrane Potential
(mt)--
After treatment, cells were loaded with either 2 µM rhodamine 123 (28) or 60 nM
DiOC6 (29) for 10 min, and mt
was measured by
FACScan.
Measurement of Substrate-stimulated Oxygen Consumption-- After treatment with staurosporine for the indicated periods of time, 2 × 106 cells were washed with phosphate-buffered saline and resuspended in mitochondrial respiration buffer (30). O2 consumption was measured in a 2.0-ml chamber with a Clark-type oxygen electrode (Yellow Springs) calibrated with air and sodium dithionite (30). Cells were permeabilized with 0.01 mg/ml digitonin for 1 min, and substrate-stimulated respiration was recorded by adding 2.5 mM succinate and 0.25 mM ADP. The substrate stimulated O2 consumption rate over the rate after digitonin permeabilization was calculated as the respiratory control ratio.
Measurement of Superoxide Generation in Isolated Mitochondria-- Mitochondria were isolated from neo and bcl-2 cells with nagarse-digitonin treatment (31). Measurements were performed in a SLM/Aminco DW-2000 spectrophotometer at 30 °C. 40 µM acetylated cytochrome c was included in the incubation buffer, and the absorbance change was monitored at 550 nm (32). Reaction was initiated by adding 3 mM succinate. KCN (0.5 mM) was used to inhibit normal electron flow and stimulate superoxide production (positive control), and bovine erythrocyte superoxide dismutase (SOD) (Sigma; 100 units) was added to verify that the reduction of acetylated cyt c was due to superoxide generation.
Western Blot Analysis of Mitochondrial cyt c Release-- Mitochondrial and cytosolic fractions were prepared with digitonin-nagarse treatment. Each lane was loaded with 50 µg of protein, and Western blot was performed as described (21), using a gift antibody from Dr. R. Jemmerson (University of Minnesota).
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RESULTS AND DISCUSSION |
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To determine whether an oxidant signal precedes or follows
mitochondrial release of cyt c, we measured the redox state
of the intracellular GSH pool as a function of time following treatment of neo cells with 1 µM staurosporine (Fig.
1). Under these conditions, cyt
c release was detectable already at 1 h (Fig.
1B), but oxidation of cellular GSH/GSSG did not occur until
after 2 h (Fig. 1A). By 6 h, the redox potential
Eh was oxidized by 72 mV (from 239 ± 6 to
167 ± 9 mV), a change similar to the 86 mV oxidation in apoptotic HT29 cells (data not shown). In cells overexpressing Bcl-2,
cyt c release was blocked (Fig. 1B), and the
redox change was inhibited (Fig. 1A). The measured
mitochondrial GSH/GSSG ratio changed with a similar time course (Fig.
1A, inset). Thus, cyt c loss preceded the
generation of an oxidant signal and as previously reported, Bcl-2
blocked cyt c release (21) and had an apparent antioxidant
function (17, 18), as measured by Eh of the
GSH/GSSG pool and the mitochondrial GSH/GSSG ratio.
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Antioxidants, such as NAC, can inhibit apoptosis in some systems (12). Although the staurosporine-induced caspase activation and subsequent DNA fragmentation could be completely inhibited by overexpression of Bcl-2, they were not sensitive to NAC (Fig. 1C). Thus in staurosporine-induced apo-ptosis, the caspase activation seems to be redox-insensitive, and the effects of Bcl-2 cannot be attributed to its apparent antioxidant function.
Because the redox signal was not observed until after cyt c
release, we then examined whether the mitochondrion is a possible source of the redox signal. For this, we used DHR, a non-fluorescent compound that accumulates in the mitochondria and can be oxidized to
the fluorescent rhodamine (33). FACScan showed that DHR was oxidized in
staurosporine-treated neo cells but not in cells overexpressing Bcl-2
(Fig. 2, A and B).
Confocal microscopy confirmed that this fluorescence was mainly
localized to the mitochondria, together with the mt-driven dye
DiOC6 (Fig. 2C). Thus, the results suggest that
ROS may be generated in the mitochondria upon cyt c release and that this is prevented by Bcl-2 in association with its inhibition of cyt c release.
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Babior and co-workers (30, 34) found that substrate-dependent mitochondrial O2 consumption was inhibited with loss of cyt c in a Fas-activated model of apoptosis. Measurements in mitochondria of neo cells treated with staurosporine also showed that substrate-stimulated O2 consumption was inhibited (Fig. 3A). Thus, the loss of cyt c is associated with an interruption of normal electron flow that could divert electron transfer to the generation of superoxide.
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Activation of the mitochondrial permeability transition (MPT) is a
major controlling mechanism in some apoptotic systems (for review, see
Ref. 35), and this could also contribute to cyt c release
(data not shown) and superoxide production (36, 37). To determine
whether the MPT had occurred in neo cells treated with staurosporine,
we used FACS analysis with rhodamine 123 or DiOC6. Results
showed that was largely retained under conditions where cyt
c was released and the redox signal was generated (Fig. 3,
B and C). At later times, the
was lost
(Ref. 21 and data not shown), indicating that the redox change preceded
the MPT and was not a result of MPT.
To determine whether mitochondria that have lost cyt c during apoptotic signaling have increased substrate-dependent superoxide production, we measured stimulated superoxide production in mitochondria isolated from staurosporine-treated cells. Results showed that superoxide generation was enhanced in mitochondria isolated from staurosporine-treated neo cells. By 4 h, the rate was 5.28 ± 0.33 nmol/mg protein per min (Fig. 4A). This was dependent upon respiratory substrate and was inhibited by superoxide dismutase.
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To determine whether superoxide production could be blocked by
preventing cytochrome c release, superoxide production by
mitochondria from staurosporine-treated bcl-2 cells was measured.
Results showed that mitochondria from cells overexpressing Bcl-2 had no
increase in superoxide production following staurosporine treatment
(Fig. 4B). Addition of cyanide, a direct mitochondrial
electron transport inhibitor, resulted in superoxide generation in
mitochondria from Bcl-2-overexpressing cells at a rate similar to that
from neo cells. Thus, the results show that the effect of Bcl-2 was not due to a direct electron-scavenging or superoxide-metabolizing activity
of Bcl-2 itself but rather to the prevention of O2
production.
These observations demonstrate that the antioxidant activity of Bcl-2
is indirect and clarify an important issue concerning Bcl-2 function.
However, this finding also raises a fundamental question about the
redox signaling in cells. The redox state of the GSH/GSSG pool is
substantially oxidized (239 mV) compared with the NADPH pool (
394
mV) (38) that drives GSSG reduction. If the steady state
Eh of GSH/GSSG reflects a balance between the
rate of GSH oxidation by mitochondrially derived oxidants (e.g. superoxide and H2O2) and the
rate of GSSG reduction by NADPH, then it places Bcl-2 at a pivotal
point in regulation of the cellular thiol disulfide redox state by
controlling the rate of mitochondrial generation of ROS
(Fig. 5).
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The present demonstration of a mechanism for ROS generation during apoptosis does not resolve whether ROS has a role in apoptosis signaling. Despite the fact that an oxidant signal occurs during apoptosis by diverse stimuli, so far the available pieces of evidence suggest that caspase activation is irrelevant to the redox state. In vitro data using cell-free systems indicated that caspase 3 activation was independent of the redox state of cyt c (39, 40). Similarly, when we added 5 mM NAC to the neo cells, we obtained no inhibition of caspase activation, as measured by DNA fragmentation (Fig. 1C) and phosphatidylserine translocation (data not shown). Thus when ROS is generated concomitantly with a direct activating mechanism of caspases, it might be only a side effect that is unrelated to the key signaling events leading to the caspase-executed cell death.
However, a range of other possible functions for ROS could exist beyond
the activation of death proteases. Caspases are inhibited by oxidants
(41, 42). The superoxide generated could function as a mechanism to
inactivate caspases and prevent their destruction of cells that
phagocytose apoptotic bodies (41). ROS may also counteract the lethal
effects of caspases in non-apoptotic cells. Caspase 3 has recently been
found to function in the processing of pro-interleukin 16, a
non-apoptotic function that would appear to require some low level of
constitutive activation (43). If low levels of cyt c release
are involved in this process, a concomitant superoxide signal may
activate protective mechanisms, such as the activation of the
redox-sensitive transcription factor NF-B (44, 45). In
TNF-
-induced apoptosis, NF-
B has been shown to control the
expression of several protective genes, such as c-IAP2 and MnSOD (13,
14). Despite the phosphorylation of I-
B, the intracellular redox
state is known to be another regulatory mechanism of NF-
B (46).
Alternatively, the switch of mitochondrial electron transport from its
normal pathway through cyt c, which does not result in free
radical production, to one in which electrons are transferred to
O2 with production of superoxide, may provide a fail-safe
mechanism that complements cyt c-dependent
caspase activation for the execution of cell death. The dramatic 75-mV
oxidation of the cellular GSH/GSSG pool (Fig. 1A) is a
sufficient signal to result in a 500-fold change in the function of
systems regulated by vicinal dithiols with Eo in
an appropriate range (47). Thus, global oxidation due to the electron
transfer switch triggered by cyt c release could provide a
general backup mechanism for a redox-activated molecular machinery of
apoptosis. One example of such a mechanism could be the activation of
MPT. The MPT pore has critical thiols, which upon oxidation can trigger
its opening (48) and release the apoptosis-inducing factor (35, 49).
Although not purified yet, apoptosis-inducing factor seems also to be a
cysteine protease and can induce nuclear apoptosis in cell-free
systems. Oxidants, such as diamide and
tert-butylhydroperoxide, can induce its release from
isolated mitochondria (49). The time course of mt change (Fig. 3)
indicated that MPT does not contribute significantly to the generation
of superoxide in staurosporine-induced apoptosis in HL 60 cells, but
rather superoxide production may subsequently activate the MPT in a
final common pathway of cell death.
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ACKNOWLEDGEMENTS |
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We thank Prof. Sten Orrenius for critical reading and helpful comments; Dr. Ronald Jemmerson (University of Minnesota) for kindly providing the anti-cytochrome c antibody; Nyoka Roberts for manuscript preparation; and Dr. Jie Yang and Kasey Nelson for technical support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants ES 09047 and EY 07892.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.
To whom correspondence should be addressed. Tel.: 404-727-5970;
Fax: 404-727-3231; E-mail: dpjones{at}emory.edu.
1 The abbreviations used are: ROS, reactive oxygen species; cyt c, cytochrome c; SOD, superoxide dismutase; NAC, N-acetylcysteine; TNF, tumor necrosis factor; MnSOD, manganese superoxide dismutase; DHR, dihydrorhodamine; Eh, GSH redox potential; MPT, mitochondrial permeability transition.
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REFERENCES |
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