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* Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA; and
Department of Pathology and Laboratory Medicine Service, Veterans Administration Hospital, University of Wisconsin Medical School, Madison, Wisconsin 55705, USA
1Correspondence: Graduate Center for Toxicology, 360 Health Sciences Research Bldg., University of Kentucky, Lexington, KY 40536-0305, USA. E-mail: dstcl00{at}pop.uky.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSWestern analysisRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • reactive oxygen species • caspase 3 • antimycin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSWestern analysisRESULTSDISCUSSIONREFERENCES |
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. Release of cytochrome
c from mitochondria has been shown to be an important factor
in staurosporine, 1-ß-arabinofuranosylcytosine, and ionizing
radiation-induced apoptosis (2
3
4)
. Several lines of
evidence suggest that the release of apoptotic factors from the
mitochondria may be a result of respiratory inhibition, reactive oxygen
species
(ROS)2
formation, and membrane permeability transition (1
, 5
6
7)
.
Overexpression of bcl-2, a protein found within the mitochondrial
membrane, has been shown to prevent tumor necrosis factor 
(TNF-), staurosporine, ionizing radiation, and adriamycin- or
thapsigargin-induced apoptosis, suggesting an important role for this
organelle in apoptosis (8
9
10
11)
. Recently,
bcl-xL, a member of the bcl-2 family of proteins,
was shown to block apoptosis by binding directly with cytochrome
c and preventing its release from the mitochondria
(10)
.
Generation of ROS has been implicated in apoptotic cell death
pathways upon treatment with a variety of agents including TNF-
(12)
, ceramide (13)
, glutamate
(14)
, amyloid ß-peptide (15
, 16)
, alkaline
conditions (17)
, and nerve growth factor withdrawal
(18
, 19)
. With many of the apoptotic inducers, the
specific subcellular source of the ROS generation is unknown. Some
studies using TNF, ceramide, or glutamate have implicated the
mitochondria as the source of ROS generation (13
, 20)
.
This is not surprising since mitochondria constitute a major cellular
source of ROS, primarily as byproducts of aerobic respiration.
Components of the electron transport chain such as the NADH–coenzyme Q
reductase complex and the reduced form of coenzyme Q leak electrons
onto oxygen, producing a univalent reduction to generate superoxide
radicals (21)
.
Within the mitochondrial matrix, manganese superoxide dismutase
(MnSOD) is an essential antioxidant enzyme that catalyzes the
conversion of superoxide radical to hydrogen peroxide and molecular
oxygen. In addition, generation of cytosolic superoxide radicals can be
scavenged by the mitochondria via a polarized inner mitochondrial
membrane. It has been hypothesized that superoxide radicals can diffuse
into the mitochondrial intermembranous space by presentation of a
localized proton-rich environment. Within the intermembranous space,
superoxide radicals can be protonated, followed by diffusion into the
matrix where dismutation by MnSOD could occur (22)
.
Previous studies have shown that overexpression of MnSOD in various
cell lines can prevent TNF- (23)
, alkaline
(17)
, or peroxynitrite-mediated apoptosis
(24)
. Thus, it has been hypothesized that upon treatment
with these agents, radicals may be generated within one or more
cellular compartments. Therefore, to `directly' test the role of
mitochondria derived-superoxide radicals in apoptotic cell death, we
treated a murine fibrosarcoma cell line (FSa) with specific respiratory
chain inhibitors that are known to increase superoxide levels within
that organelle. Here we demonstrate that overexpression of MnSOD in
murine FSa cell lines specifically protected against cell death upon
treatment with either complex I or complex III, but not complex IV,
inhibitors. Our results demonstrate that MnSOD protects against
antimycin-induced caspase-3 activation, nuclear condensation, DNA
fragmentation, and PARP cleavage independent of bcl-xL and
cytochrome c release. Taken together, these findings
demonstrate that mitochondrial-mediated ROS can directly signal the
execution of cell death through activation of cellular proteases.
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MATERIALS AND METHODS |
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. The FSa-II
cells were transfected with p2V2-NEO plasmid or cotransfected with the
sense MnSOD expression plasmid (pHßAPR-1), as described previously
(26)
. After 48 h, the cells were exposed to 400
µg/ml of geneticin. Colonies originating from a single transfected
cell were selected. Two clones that stably expressed different levels
of MnSOD activity [i.e., low (SOD-L) and (SOD-H)] were selected
together with a control clone transfected with only the pSV2-NEO
plasmid (NEO).
Determination of cell doubling time
On the basis of the plating efficiencies of the cell lines
(27)
, 1 x 105 cells for the
NEO line and 5 x 104 cells for the
MnSOD-transfected lines were plated in 10–14 dishes. After 24 h,
media was removed and replaced with fresh medium containing either
antimycin (2.5–25 µM), rotenone (10–100 nM), or sodium cyanide
(10–50 mM). Two dishes from each group were trypsinized every day, and
the cells were counted using a hemacytometer. Cell doubling times were
obtained by linear regression analysis after plotting the logarithm of
the number of cells vs. hours after plating.
Colony survival assay
Single cells suspended in supplemented McCoy's medium were
plated onto 60 mm dishes in triplicate for each clone examined. Cells
(500/dish for NEO and 300/dish for SOD-L and SOD-H each) were plated.
After 48 h, media were removed and replaced with medium containing
either antimycin (25 nM) or rotenone (10 nM). Cells were kept for 12
days in the incubator to allow for colony formation. The colonies were
fixed and stained in 0.1% crystal violet and 2.1% citric acid.
Colonies containing more than 50 cells were counted using a dissecting
microscope. All experiments were performed in triplicate. The surviving
fraction was calculated as follows:
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DNA fragmentation
Cells were plated (6x105 NEO;
3x105 SOD-H) in 100 mm dishes and treated with
0–100 µM antimycin for 0–48 h. Cells were washed twice with
phosphate-buffered saline (PBS), resuspended in lysis buffer [50 mM
Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), 200
µg/ml proteinase K], and incubated at 37°C overnight. Crude DNA
preparations were extracted with phenol:chloroform:isoamylalcohol
(25:24:1) and precipitated with 0.1 volume of 3 M sodium acetate and
2.0 volumes of 100% ethanol. The DNA pellet was air-dried and
resuspended in Tris-EDTA (TE) buffer containing 10 mM Tris-HCl (pH
8.0), 1 mM EDTA, and 20 µg/ml DNase-free RNase A. After an incubation
for 1 h at 37°C, the concentration of nucleic acid was
determined by UV absorbance at 260 nm. The same amount of nucleic acid
from each sample was resolved by electrophoresis on a 1% agarose gel
and visualized by UV fluorescence after staining with ethidium bromide.
Electron microscopy
Seventy-two hours after plating in 100 mm dishes, cells were
treated with 100 µM antimycin for 0–48 h, rinsed with PBS, and fixed
with 2.5% glutaraldehyde in Sorensen's phosphate buffer at 4°C.
Cells were postfixed in Caulfield's osmium tetroxide, rinsed with
water, dehydrated in a graded ethanol series with 100% propylene oxide
as a transitional solvent, and embedded in Epon 812. Random areas (1 mm
square) were cut and processed. Sections were cut using an LKB
ultramicrotome and transferred to copper grids that were stained with
lead citrate and uranyl acetate. Using a Hitachi H-300 electron
microscope, 20 cells per treatment group and time period were
photographed and described in a blinded fashion by a pathologist.
Flow cytometry
Cells were plated and treated as described above. Twenty-four to
48 h after treatment with antimycin, cells were incubated with 10
µM 2',7'-dichlorofluorescin diacetate (Molecular Probes Inc., Eugene,
Oreg.) for 30 min at 37°C in the dark. Cells were washed with PBS and
then incubated for 6 min at 37°C with cell dissociation solution
(Sigma, St. Louis, Mo.). Cells were filtered through 35 µM nylon mesh
(Small Parts Inc., Miami, Fla.), counted using a hemacytometer, diluted
to a final concentration of 1 x 106
cells/ml, and analyzed immediately on a FACScan flow cytometer using
excitation at 488 nm and emission at 525 nm.
Nuclear extract preparation
The cell lines were seeded at a density of either 6.0 x
105 cells/100 mm dish (NEO) or 3.0 x
105 cells/100 mm dish (SOD-H). During the
exponential growth phase, nuclear extracts were isolated as described
by Dignam and Roeder (28)
with the inclusion of 35%
glycerol and protease inhibitors (pepstatin, leupeptin, aprotinin) at 1
µg/ml in the extraction buffer. Protein concentration was determined
by a colorimetric assay (Bio-Rad Laboratories, Richmond, Calif.).
Subcellular fractionation
The mitochondrial fraction of FSa cells was prepared by washing
the cells twice in ice-cold PBS, followed by resuspension in 5 ml of
0.25 M sucrose, 1 mM EGTA, and 10 mM Tris-HCl (pH 7.4) and centrifuging
at 500 x g for 2 min at 5°C. The supernatant was
discarded and the cells were resuspended in 5 ml of the same buffer.
The cells were homogenized in a glass Teflon homogenizer using 10
up-and-down strokes at 500 rpm. The homogenate was centrifuged at
1500 x g for 10 min at 5°C. The crude mitochondrial
pellet was resuspended in 100 µl of buffer. The supernatant was
recentrifuged at 100,000 x g (4°C, 1 h) to
generate the S-100 fraction. Protein concentration of both fractions
was determined by a colorimetric assay (Bio-Rad Laboratories).
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Western analysis |
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Cytochrome c and Bcl-xL
Mitochondrial proteins (35–50 µg/lane) were electrophoresed
in a 12.5% gel, transferred to nitrocellulose, and blocked as
described previously. For bcl-xL analysis, an
affinity-purified rabbit anti-bcl-xL antibody
(1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.)
was used. For cytochrome c analysis, a monoclonal antibody
purchased from PharMingen (San Diego, Calif.) was used at a final
concentration of 1 µg/ml.
Bax
Cells were washed in PBS and solubilized in lysis buffer (50 mM
Tris-HCl pH 7.4, 125 mM NaCl, 0.1% SDS, 5 mM NaF, 1 mM PMSF, 1 ng/ml
leupeptin, 1 ng/ml aprotinin, 1 ng/ml pepstatin) for 1 h on ice.
Lysates were centrifuged at 2500 x g for 5 min. The
protein concentration in the supernatants was determined as described
previously. One hundred micrograms of total cell lysate was resolved on
a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and probed with
an affinity-purified rabbit anti-bax antibody (1 µg/ml; Santa Cruz
Biotechnology). The antibody was raised against a peptide corresponding
to amino acids 43–61, mapping at the amino terminus of the murine bax
protein.
PARP cleavage products
Antimycin-induced apoptosis was examined by proteolytic cleavage
of PARP (29)
. Briefly, cells were treated with 100 µM
antimycin for 0–48 h at 37°C, then washed with PBS and sonicated
(10% power-60 s continuous) on ice. Protein concentration was
determined by a colorimetric assay (Bio-Rad Laboratories). Next, 40
µl of sample buffer (50 mM Tris, pH 6.8, 6 M urea, 6%
ß-mercaptoethanol, 0.003% bromphenol blue, and 3% SDS) was added to
the samples (200 µg) and heated at 80°C for 5 min. Samples were
loaded and run on an 8% SDS-PAGE gel according to the method of
Laemmli (30)
. Transfer and blocking of the membrane were
performed as described previously. The membrane was incubated with a
rabbit anti-PARP antibody (1:2,000; Boehringer Mannheim, Mannheim,
Germany) in Blotto at 4°C overnight. Protein bands were visualized
using the enhanced chemiluminescence detection system (Amersham).
Caspase-3 protease activity
Activation of caspase-3 (CPP-32) was determined using the
ApoAlert assay kit (Clontech Laboratories, Palo Alto, Calif.), which
fluorometrically detects CPP-32 activity using proteolytic cleavage of
the fluorophore 7-amino-4-trifluoromethyl coumarin (AFC) from the
substrate conjugate DEVD-AFC. Cells were plated
(6x105 NEO; 3x105 SOD-H)
in 100 mm dishes and treated with 100 µM antimycin for 0–48 h. Cells
(1x106) were collected at various time points
after treatment, lysed on ice for 10 min, centrifuged (12,000 rpm for 3
min), and the supernatants were collected and stored at -70°C until
further use. Cell lysates were incubated with DEVD-AFC in the presence
of DTT for 1 h at 37°C. Detection of AFC was measured using a
400 nm excitation filter and a 530 nm emission filter in a Cytofluor
2300 fluorometer (Millipore, Bedford, Mass.). Caspase-3 activity is
expressed as percent control.
Data analysis
Data were evaluated using analysis of variance for multiple
comparisons of each dependent variable. A P value <0.05
was considered to be statistically significant.
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RESULTS |
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shows the results of experiments performed in triplicate with each of
the respiratory inhibitors. As previously reported, the doubling time
between the MnSOD transfectants and the control (NEO) line was not
significantly different (31)
. However, upon treatment with
25 µM antimycin, the doubling time of the MnSOD transfectants
increased ~1.4 fold whereas the doubling time of the NEO line
increased by a factor of 2. Rotenone treatment (100 nM) had no effect
on the growth of the MnSOD transfectants whereas the doubling time of
the control cells increased ~1.5 fold. Sodium cyanide (NaCN) was
equally toxic to all three lines. Concentrations of NaCN above 20 mM
were lethal to all cell lines.
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Colony formation analysis (Fig. 1
A, B) show increased protection against inhibition of complex
I or III with MnSOD overexpression. With 25 nM antimycin or 10 nM
rotenone, the surviving fraction for SOD-L and SOD-H were significantly
greater compared with the nontransfected control (P<0.05).
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It should be noted that both antimycin and rotenone toxicities were dependent on cell density. Therefore, lower concentrations of both inhibitors were used for the survival assay in which only 300 (SOD-L, SOD-H) or 500 (NEO) cells were plated/60 mm dish as compared to the growth kinetics experiment.
Electron microscopy and DNA fragmentation analysis
To determine whether cells were dying by apoptosis, we examined
cellular morphology by electron microscopy after antimycin treatment.
In addition, we extracted cellular DNA and used electrophoresis to
analyze for fragmentation patterns characteristic of apoptotic cell
death. Figure 2
, an electron micrograph of FSa cells after exposure to 100 µM
antimycin, shows morphological changes characteristic of apoptosis,
such as multiple areas of heterochromatin condensation, are not present
in the control cells (NEO) at 36 h after treatment (Fig. 2a
), but appear by 48 h (Fig. 2b
). In
addition, at 48 h the mitochondria of the NEO cells were swollen
with loss of cristae. No evidence of apoptosis was seen in the SOD-L
(Fig. 2c, d
) or SOD-H (Fig. 2e, f
) cell lines
at either 36 or 48 h after treatment. In Fig. 3
, neither NEO or MnSOD transfectants showed evidence of DNA
fragmentation with 25 µM antimycin treatment after 48 h (lanes
2–4). Increasing the concentration of antimycin to 100 µM resulted
in DNA fragmentation in the NEO cell line at 48 h, suggesting a
time- and dose-dependent effect on cell death (lane 5).
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Generation of ROS by antimycin
To estimate the level of ROS production by antimycin in the FSa
cell lines, we used 2',7'-dichlorofluorescin diacetate (DCFH-DA), a
membrane-permeable nonfluorescent probe that is sequestered in living
cells after deacetylation by endogenous esterases. Reactive oxygen
species oxidize the diesterified product to generate a fluorescent
compound, 2',7'-dichlorofluorescein (DCF), which can be analyzed by
flow cytometry (32
, 33)
. Analysis of fluorescence of
untreated cells served as a baseline for assessment of increased ROS
levels as a result of antimycin treatment. Upon treatment with 100 µM
antimycin, both the control (NEO) and SOD-H cell lines exhibited a
time-dependent increase in DCF fluorescence (Fig. 4
).
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Mitochondrial bcl-xL is increased upon treatment with
antimycin; cellular bax remains unchanged
We previously reported a decrease in mitochondrial
bcl-xL content in the murine FSa cell line, which
overexpressed MnSOD (31)
. Since
bcl-xL is known to be an antiapoptotic protein
and bax is a proapoptotic protein, we used Western analysis to
examine the cellular content of these two proteins after treatment with
antimycin. Within 24 h, antimycin treatment lead to an increase in
mitochondrial bcl-xL protein, most noticeably in
the NEO cell line (Fig. 5
A). The increase in mitochondrial
bcl-xL was apparent with both 25 (lane 2) and 100
µM (lane 3) antimycin in the NEO cells. Bax levels did not change in
either cell line upon treatment with antimycin (Fig. 5B
).
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p53 is not detectable in the FSa cell line
Protein expression of p53 in nuclear extracts of antimycin-treated
cells was below the level of detection using Western analysis (data not
shown).
Cytochrome c is retained by the mitochondria
after antimycin treatment
Cytochrome c is a mitochondrial protein released upon
exposure to a variety of agents. Interaction of cytochrome c
with various cytoplasmic proteins has been shown to activate caspases,
resulting in apoptotic cell death (34)
. To determine
whether antimycin leads to release of cytochrome c, we
isolated the mitochondria from cells treated with antimycin for 0–48
h. Figure 6
shows retention of cytochrome c within the mitochondria of
both the NEO (lanes 1–3) and SOD-H (lanes 4–6) cell lines after
treatment with antimycin for either 24 or 48 h.
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Caspase 3 (CPP-32) activity is increased in NEO cells after
antimycin treatment
The role of CPP-32 in antimycin-induced apoptosis in FSa cells was
measured using the ApoAlert assay kit by Clontech. Cells were treated
with 100 µM antimycin and samples were collected at various time
points from 0 to 48 h. Figure 7
shows a time-dependent increase in CPP-32 activity. Within 48 h,
CPP-32 activity had increased > 200% in the NEO cells. Only a
slight increase (140%) was noted in the SOD-H clone, suggesting that
MnSOD reduces antimycin-induced activation of CPP-32.
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Poly(ADP-ribose) polymerase (PARP) cleavage in antimycin-treated
cells
PARP cleavage has been identified as an early event in programmed
cell death. PARP has also been shown to serve as a substrate for
CPP-32. In the present study we used Western analysis to detect the
parental enzyme (116 kDa) as well as its proteolytically cleaved
fragment(s). We were able to detect degradation of the parental enzyme
in nuclear extracts obtained from NEO cells treated with antimycin
(Fig. 8
A, lane 3). In addition, we were able to detect accumulation
of a 64 kDa fragment as early as 24 h in both nuclear extracts
(Fig. 8A
, lanes 2 and 3) and total cell lysates (Fig. 8B
, lanes 2 and 3) from NEO cells. The accumulation of the
64 kDa fragment paralleled the degradation of the parental enzyme.
Furthermore, minor accumulation of the 64 kDa fragment was seen in the
SOD-H cell line at 24 (Fig. 8B
, lane 5) and 48 h (Fig. 8B
, lane 6) after antimycin treatment, consistent with the
slight increase in CPP-32 activity (Fig. 7)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSWestern analysisRESULTSDISCUSSIONREFERENCES |
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. MnSOD is the primary
antioxidant defense against superoxide radicals within the
mitochondria. In the presence of MnSOD, superoxide radicals are rapidly
converted to hydrogen peroxide and molecular oxygen within the
mitochondrial matrix. The objective of the present work was to
investigate the role of mitochondrial-derived ROS in programmed cell
death. To accomplish this, we treated an MnSOD-transfected murine
fibrosarcoma cell line (FSa-II) with a series of respiratory chain
inhibitors; neomycin-transfected cells (NEO) were used as the control.
Overexpression of MnSOD significantly protected cells against rotenone-
and antimycin-mediated cytotoxicity; however, no advantage in cellular
resistance to cyanide-mediated cell death was observed in either the
SOD-L or SOD-H cell lines (see Table 1
and Fig. 1
). The reason for
selectivity of protection by MnSOD upon inhibition of complexes I and
III, but not IV, is currently unknown. Possible explanations may be due
to the reaction rate of superoxide generation vs. ATP depletion upon
treatment with cyanide.
Since expression of MnSOD protects against rotenone and antimycin
similarly, we examined the mode of cell death upon exposure to
antimycin. Two distinct modes of cell death, apoptosis and necrosis,
have been described in a variety of cell types exposed to respiratory
chain inhibitors. Endonuclease activation with subsequent DNA
fragmentation was observed in LLC-PK1 cells upon
exposure to antimycin (36)
. Treatment of a series of human
lymphoblastoid cell lines (BM 13674, BMX 13674, Daudi, BL29, K562),
human melanoma cell lines (MEL 3, MEL28, MM96), and a mouse fibroblast
cell line (L929) with antimycin lead to apoptotic cell death visualized
by DNA fragmentation analysis and Hoechst 33258 staining
(37)
. In the present study, apoptotic cell death was
confirmed by DNA fragmentation and ultrastructural analysis using
electrophoresis and electron microscopy (Figs. 2
, 3)
. The control (NEO)
cells exhibited DNA fragmentation and morphological changes
characteristic of apoptosis at 48 h. Cells overexpressing MnSOD
showed no evidence of apoptosis. This finding strongly suggests a role
for the superoxide radical in the antimycin-mediated death cascade.
Antimycin-mediated ROS production was confirmed using DCFH-DA, which is
an indicator of intracellular oxidant production (38)
.
Figure 4
shows a time-dependent increase in DCF fluorescence in both
the NEO and SOD-H cell line; however, a consistently higher level of
DCF fluorescence was noted in the NEO cell line at 48 h. We were
not surprised to see an increase in DCF fluorescence in the MnSOD
overexpressing cells after antimycin treatment because overexpression
of MnSOD should not prevent the increase in superoxide radicals formed
as a result of inhibition of respiration, and dismutation of superoxide
radicals leads to the formation of hydrogen peroxide, which can
contribute to DCF fluorescence (38)
. This study confirms
that treatment with antimycin leads to an increase in ROS production
and suggests that cells overexpressing MnSOD can compensate for the
increase in superoxide radical production, thereby protecting the cells
from ROS-mediated cell death.
To identify a pathway that might be involved in the antimycin-mediated
cell death, we examined the antiapoptotic protein
bcl-xL and the proapoptotic protein bax after
antimycin treatment. Bcl-xL, the predominant
bcl-2 family member in mice (39)
, has been shown to
protect cells against a variety of insults that cause apoptosis.
Previously we reported an increase in the mitochondrial level of
bcl-xL in the control (NEO) cells compared to the
MnSOD overexpressing cells (31)
. However, in this study
the control cells were not protected by their
bcl-xL-enriched mitochondria, suggesting a
bcl-xL-independent cell death pathway.
Furthermore, upon treatment with antimycin, an increase was
consistently noted in the mitochondrial content of
bcl-xL in the NEO cell line (Fig. 5A
,
lanes 2, 3). This finding is consistent with results by Wolvetang et
al. (37)
, who reported apoptotic cell death in a series of
human tumor lines upon treatment with antimycin independent of their
bcl-2 levels. Bax is a cytosolic protein that redistributes to the
mitochondria in cells undergoing apoptosis (40)
. Although
the mechanism is still unclear, studies have shown that overexpression
of bax can accelerate apoptosis after a death signal (41)
.
Bax can form homodimers or heterodimerize with bcl-2 proteins, leading
to speculation that it may function as an inhibitor of bcl-2. In
addition, bax can regulate cell death independent of bcl-2, perhaps by
forming ion channels or inducing the release of apoptogenic factors
from the mitochondria (42)
. In the present study, we saw
no change in bax levels upon exposure to antimycin in either cell line
(Fig. 5B
).
Next, we tried to establish whether the antimycin-mediated cell death
pathway in FSa-II cells involved p53. Polyak et al. (43)
have shown that p53 can induce apoptosis through a multistep process
involving transcriptional induction of p53-induced genes, formation of
ROS, and the oxidative degradation of mitochondrial components.
However, the p53 levels in both the NEO and SOD-H cell line were below
the limits of detection. This was not too surprising since the FSa-II
cell line is highly aggressive. Miyashita et al. (44)
identified p53 as a regulator of bcl-2 and bax gene expression. In a
murine leukemia cell line (M1), a temperature-sensitive p53 induced a
temperature-dependent decrease in bcl-2 while simultaneously
stimulating an increase in bax. In addition, mice deficient in p53
showed elevated levels of bcl-2 and decreased levels of bax in several
tissues. In Fig. 5
the levels of bcl-xL actually
increased with antimycin treatment, whereas no change was observed in
bax expression. Taken together, these results indicate that p53 may not
play a major role in this cell death model.
Cytochrome c is a mitochondrial protein that induces
apoptosis when released into the cytosol in response to various stress
inducers. The addition of cytochrome c to cytosolic
fractions from growing cells initiates a cascade, including activation
of caspases and induction of DNA fragmentation (45)
. A
report by Chauhan et al. (4)
showed that apoptotic cell
death could occur in the absence of cytochrome c release
from the mitochondria upon treatment with dexamethasone or anti-Fas
monoclonal antibody in multiple myeloma cells. Therefore, we sought to
determine whether the antimycin-mediated cell death was occurring in
response to cytochrome c release from the mitochondria.
Examination of cytochrome c was performed by Western
analysis over a 48 h time course after antimycin treatment. We
compared levels in the mitochondrial and S-100 fractions and found no
accumulation of cytochrome c in the cytosol after treatment
in either the NEO or SOD-H cell lines (data not shown). Figure 6
shows
no loss of cytochrome c from the mitochondria on antimycin
treatment over the course of 48 h. This suggests that the cell
death pathway initiated in the FSa cells on antimycin treatment does
not require cytochrome c release from the mitochondria.
Studies of the nematode Caenorhabditis elegans have provided
significant insight into the molecular control of apoptosis (46
, 47)
. Mammalian counterparts to numerous C. elegans
genes associated with programmed cell death have been identified.
Caspase 3 (CPP-32), the ced-3 homologue, has been shown to
be one of the major activated cysteine proteases present in cells
undergoing apoptosis, suggesting an important role in the cell death
process (48)
. Recent work by Woo et al. (49)
suggests that the requirement for CPP-32 activation in apoptotic cells
is system and stimulus dependent. In the present study, CPP-32 activity
increased in the NEO cell line as early as 24 h after antimycin
treatment. Exposure for 48 h resulted in a greater than 200%
increase in CPP-32 activity in the NEO cells, which was significantly
higher than the SOD-H cells (P<0.05). These results show an
increase in CPP-32 activity prior to any evidence of DNA fragmentation
or morphological changes consistent with apoptosis. These results also
suggest that the antimycin generated ROS function upstream of CPP-32
activation. Our results compliment studies by Higuchi et al.
(50)
, who treated ML-1a cells with a mixture of xanthine
and xanthine oxidase and showed that the generated ROS act upstream of
the CPP-32 activity. Taken together, these results identify CPP-32 as a
redox-responsive protein.
Activation of the caspase enzymes leads to proteolytic cleavage of a
number of cellular targets including proteins involved in RNA splicing,
DNA repair, and scaffolding of the cytosol and nucleus
(51)
. One DNA repair enzyme, PARP, is a substrate for
proteolytic cleavage by several cellular proteases including CPP-32
(52)
. Cleavage of PARP results in decreased enzyme
activity, which compromises the cell's response to damage.
Furthermore, ROS including peroxides and nitric oxide have been shown
to inactivate PARP, contributing to cell death cascades (53
, 54)
. Using Western analysis of both total cell lysates and
nuclear extracts (Fig. 8A, B
) from antimycin-treated cells,
we were able to detect cleavage of parental PARP (116 kDa) to a 64 kDa
fragment, primarily in the NEO cell line. In both the nuclear extracts
and total cell lysates, the 64 kDa fragment can be seen by 24 h,
with significant accumulation by 48 h. Only minor accumulation of
the 64 kDa fragment was seen in the SOD-H cell line at 24 and 48 h
(Fig. 8A, B
). These results suggest that CPP-32 activation
and PARP cleavage are closely associated events.
In conclusion, this study establishes a role for mitochondrial-derived ROS, primarily superoxide radical and its products in programmed cell death. Inhibition of mitochondrial respiration leads to an increase in cellular ROS, which can cause cell death independent of bcl-2 or cytochrome c release, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death. Furthermore, modulation of a cell's mitochondrial antioxidant status may provide significant protection against toxic insults involving a compromise to mitochondrial respiration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 23, 1999. Accepted for publication without revision April 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSWestern analysisRESULTSDISCUSSIONREFERENCES |
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B by TNF. FEBS Lett 351,45-48[Medline]
B and activated protein-1. J. Biol. Chem. 273,13245-13254This article has been cited by other articles:
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