From the Interdisciplinary Center for Clinical
Research (IZKF) and the § Department of Pharmacology and
Toxicology, Westphalian Wilhelms-University,
D-48149 Münster, Germany
Received for publication, October 22, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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The temporal relationship between mitochondrial
membrane permeabilization and reactive oxygen species production during
apoptosis remains unknown. We analyzed the rate of superoxide
production of human breast carcinoma cells expressing a cytochrome
c-green fluorescent protein (cyt-c-GFP) fusion
protein at the single-cell level during apoptosis. In cells treated
with the proapoptotic agents staurosporine (3 µM) or
tumor necrosis factor- The release of soluble proteins from the mitochondrial
intermembrane and intracristal space is a central, coordinating step in
many apoptosis pathways (1, 2). The release of cytochrome c
(cyt-c)1 and
Smac/DIABLO triggers or potentiates the activation of caspases, a
family of cytosolic cysteine proteases (3-5). Activation of executioner caspases 3, 6, and 7 is responsible for the majority of the
biochemical and morphological changes during apoptosis. Mitochondria
also release other factors that trigger caspase-independent apoptotic cell death, in particular the apoptosis-inducing factor AIF
(6).
However, mitochondrial membrane permeabilization is also able to
activate additional cell death pathways. A decrease in mitochondrial ATP generation, mitochondrial membrane potential ( Materials--
Recombinant human tumor necrosis factor- Cell Culture and Transfection--
Generation and
characterization of human breast carcinoma MCF-7 cells stably
expressing a cytochrome c-green fluorescent protein (cyt-c-GFP) fusion protein have been described (18). We have previously shown that cyt-c-GFP is imported into
mitochondria and co-released with endogenous cyt-c after
selective outer membrane permeabilization with digitonin. Subcellular
fractionation experiments confirmed the concomitant release of
endogenous cyt-c and cyt-c-GFP from mitochondria
during apoptosis (18).
Time-lapse Confocal Fluorescence
Microscopy--
Cyt-c-GFP and ethidium fluorescence were
monitored using an inverted Olympus IX70 confocal microscope equipped
with a 488-nm argon laser (Fluoview; Olympus, Hamburg, Germany). HEt is
a nonfluorescent compound that is oxidized by superoxide to its
fluorescent product Et (19), allowing a semiquantitative determination
of superoxide production (12, 20). HEt was used at an extracellular
concentration of 1 µM. Measurement of
Fluorescence transmitted the first dichroic mirror above 505 nm, was
divided with a second dichroic mirror at 550 nm, and detected after
transmission of a 510-540-nm bandpass filter (GFP) or a 590-nm high
pass emission filter (Et and TMRM). There was no Et or TMRM
fluorescence detectable in the GFP channel. The cross-talk between the
average pixel intensity of GFP in the Et or TMRM channel was also
negligible. Fluorescence was detected from a 0.7-µm-thick confocal
section (full width half-maximum). Horizontal resolution was 0.2 µm
per pixel. Image data were obtained using Fluoview 2.0 software
(Olympus) and Kalman-filtered from two scans (2.2 s/512 × 512 pixel) for each image.
For time-lapse imaging, culture dishes were mounted onto the microscope
stage that was equipped with a temperature-controlled inlay (HT200,
Minitüb, Tiefenbach, Germany). In control experiments constant
fluorescence values were monitored for 24 h in case of cyt-c-GFP. Et fluorescence increase with constant slope was
detected for 4 h under control conditions.
For induction of apoptosis, cells were incubated with 3 µM STS or 100 ng/ml TNF- Image Processing--
The quantitative analysis of the
fluorescence images was performed using UTHSCSA ImageTool
program (University of Texas Health Science Center at San Antonio, TX).
After background subtraction the average fluorescence intensity per
pixel was calculated. The release kinetics of cyt-c-GFP of
individual cells are shown as S.D. of the average pixel intensities
(22). A compartmentalized cyt-c-GFP fluorescence contributes
to a high S.D. of the pixel intensities, and a homogenous distribution
after release of cyt-c-GFP is represented by a drop in S.D.
Et fluorescence kinetics of individual cells were fitted with linear
regression, and the ratios of the slopes after and before the release
of cyt-c-GFP or after and before addition of menadione were
calculated. Control ratios were calculated from Et fluorescence slopes
during incubation with 1 µM HEt from 120 to 180 min
divided by slopes from 60 to 120 min in cells treated with vehicle.
Cell Lysate Assay--
Under conditions of mitochondrial
depolarization, mitochondrially generated Et may be released into the
cytoplasm, resulting in an artificial fluorescence enhancement (23). We
therefore additionally quantified Et production of whole cell cultures
using a previously described cell lysate assay (12).
Statistics--
Data are given as means ± S.E. For
statistical comparison, analysis of variance and subsequent Tukey test
were employed (SPSS 10.0, SPSS Inc., Chicago, IL). p values
smaller than 0.05 were considered to be statistically significant.
Rapid Increase in Superoxide Production after Mitochondrial
Membrane Permeabilization--
MCF-7 cells stably expressing a
cyt-c-GFP fusion protein were exposed to the protein kinase
inhibitor STS (activation of the mitochondrial pathway) or to
TNF-
The release of cyt-c triggers
The above experiments also suggested that mitochondrial depolarization
per se was not sufficient to acutely trigger an increase in
superoxide production. Indeed, even prolonged treatment with FCCP (10 µM) neither induced cyt-c-GFP release from
mitochondria nor triggered the generation of superoxide (Fig. 2,
E and F).
The Broad-spectrum Caspase Inhibitor z-VAD-fmk Inhibits the
Increase in Superoxide Production after Mitochondrial Membrane
Permeabilization--
A treatment with the SOD mimetic MnTBAP (100 µM) potently blocked the increase in superoxide
production after STS-induced cyt-c-GFP release (Fig.
3A). Interestingly, treatment
with the broad-spectrum caspase inhibitor z-VAD-fmk did not inhibit
cyt-c-GFP release or alter its kinetics (18) but also
potently inhibited the increase in superoxide production after the
cyt-c-GFP release (Fig. 3B). Quantification of
the Et fluorescence slopes before and after the STS-induced
cyt-c-GFP release demonstrated a significant, more than
3-fold slope increase. This increase was significantly reduced in STS-
plus z-VAD-fmk-treated cells (Fig. 3C). Quantification of Et
production of whole cell cultures treated for 6 h with 3 µM STS also showed a 21.5 ± 1.9% increase in Et
fluorescence per µg of protein compared with vehicle-treated controls
(p < 0.05; n = 12 and 10 cultures,
respectively). Et fluorescence in whole cell lysates was significantly
reduced in 3 µM STS- plus 200 µM z-VAD-fmk-treated cells to a level of 12.6 ± 1.5% above control (p < 0.05 compared with the STS treatment;
n = 12 cultures). The level of Et production in these
bulk analyses was significantly smaller than expected from the
single-cell measurements, suggesting that not all cells had released
cytochrome c or that superoxide production had ceased in
severely damaged cells.
Menadione-induced Superoxide Production Is Sensitive to
z-VAD-fmk--
Treatment with 100 µM menadione acutely
increased the superoxide production of MCF-7/cyt-c-GFP cells
in the absence of any detectable cyt-c-GFP release (Fig.
4A). Surprisingly, cells
pretreated with 200 µM z-VAD-fmk and then treated with
menadione did not show such increase (Fig. 4B), again in the
absence of any detectable changes in cyt-c-GFP
redistribution. Quantification of the Et fluorescence slopes in
individual cells before and after the addition of menadione confirmed
the inhibitory effect of z-VAD-fmk quantitatively (Fig.
4C).
Activation of death receptors or activation of the mitochondrial
apoptosis pathway is known to increase the production of reactive
oxygen species, and this has been demonstrated both during TNF- Interestingly, z-VAD-fmk also inhibited superoxide production triggered
by an acute administration of menadione. Menadione is a prototype of a
redox cycling compound that is reduced by complex I of the respiratory
chain and subsequently generates superoxide (25). Under our
experimental conditions, release of cyt-c-GFP could not be
detected in response to menadione, even in cells monitored up to 120 min after the addition of menadione. Cells also exhibited normal
morphology,2 suggesting that
apoptotic pathways were not activated. These results suggest that the
widely used broad spectrum caspase inhibitor z-VAD-fmk also exerts
direct or indirect antioxidative effects and that inhibition of caspase
activation is not solely responsible for the effects of z-VAD-fmk on
superoxide production during apoptosis.
At least two separate processes may account for potential
caspase-independent superoxide production after mitochondrial membrane permeabilization: (i) increased superoxide production due to reverse electron flow after the loss of cyt-c and (ii) decreased
antioxidant defense capacity in the mitochondrial intermembrane space.
Cyt-c may function as a direct antioxidant in living cells
(26). It is known that a large fraction of cyt-c does not
participate in electron transport (27) and hence that cyt-c
is present in excess in the mitochondrial intermembrane space. Electron
microscopy studies have indicated that the release of cyt-c
during apoptosis is a complete process, leading to a total depletion of
cyt-c in affected mitochondria (28). There is also evidence
that cyt-c is released specifically out of apoptotic
cells prior to a loss of plasma membrane integrity (18, 29). It is
therefore possible that the loss of cyt-c out of apoptotic
mitochondria or cells increases their oxidative stress burden.
However, this view is challenged by a recent study performed in
isolated mitochondria that suggests that increased superoxide production during apoptosis may be related to the
cyt-c-linked electron transport activity rather than the
direct radical scavenging activity of cyt-c (14).
Mitochondria that are energized with malate and glutamate have been
shown to generate superoxide via the flavin mononucleotide group of
complex I in response to inhibition of mitochondrial electron flow (14,
30, 31). Superoxide generation due to reversed electron transport to
complex I has been shown to be dependent on In conclusion, our study demonstrates that increased superoxide
production and hence the activation of additional cell death pathways
starts immediately after mitochondrial membrane permeabilization during
STS- and TNF- (100 ng/ml), the release of
cyt-c-GFP was individually set for each cell, and the
majority of the fusion protein was released in less than 10 min. Prior
to the release of the fusion protein, cells demonstrated a constant
rate of superoxide production determined with the probe hydroethidine. After the release was completed, the superoxide concentration increased rapidly to a level more than 3-fold above baseline. Treatment with the broad spectrum caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk; 200 µM) did not alter the kinetics of the
cyt-c-GFP release but significantly reduced superoxide
concentration after the release of cyt-c-GFP. Interestingly, treatment with z-VAD-fmk also reduced the increase in
superoxide concentration in response to menadione in the absence of
mitochondrial cyt-c-GFP release. Mitochondrial
depolarization with the protonophore carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone per se did
not trigger cyt-c-GFP release or an increase in superoxide production. Our data suggest that mitochondria increase their superoxide production during apoptosis directly after the
quantitative release of soluble intermembrane proteins and demonstrate
novel antioxidative effects of the commonly used caspase inhibitor
z-VAD-fmk.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m)
depolarization, activation of the permeability transition pore complex,
and increased reactive oxygen species production are features of
necrotic but also apoptotic cell death (7-13). Loss of
cyt-c directly causes a respiratory inhibition and
m depolarization (13). The increase in superoxide
production during apoptosis has been shown to require mitochondrial
respiratory chain activity and may also be a direct consequence of the
loss of cyt-c (10, 12, 14). In neurons and other cell types
susceptible to pro-oxidants, superoxide formation may play a prominent
role in cell death execution during apoptosis (8, 15, 16). In some
settings, inhibition of superoxide formation may exert stronger
protective effects than inhibition of executioner caspases (12, 17).
Despite the importance of mitochondrial superoxide production for cell
death execution during apoptosis, the relationship between
mitochondrial membrane permeabilization and superoxide production have
not yet been determined at the single-cell level. In the present study,
we investigated superoxide production during apoptosis by confocal
time-lapse imaging of human breast carcinoma cells expressing a
cyt-c-green-fluorescent-protein (GFP) fusion protein (11,
18).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
), cycloheximide (CHX), carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (FCCP), menadione, and
embryo-tested paraffin oil were purchased from Sigma.
Staurosporine (STS) and manganese tetrakis (4-benzoyl acid) porphyrin
(MnTBAP) were from Alexis (Grünberg, Germany). The broad-spectrum
caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) was purchased from Bachem (Heidelberg, Germany).
Hydroethidine (HEt) and tetramethylrhodamine methyl ester (TMRM)
were from Molecular Probes (Leiden, The Netherlands).
m
was performed after equilibration of MCF-7/cyt-c-GFP cells
with 30 nM of the cationic, voltage-sensitive dye TMRM
(21).
plus 1 µg/ml CHX
(TNF-
/CHX). The medium was enriched with 10 mM HEPES (pH
7.4) and thoroughly mixed to ensure a proper distribution of the drugs.
To prevent evaporation the media was covered with embryo-tested
paraffin oil.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/CHX (activation of death receptors). After 120 min, the culture
medium was supplemented with 1 µM HEt. The kinetics of
cyt-c-GFP release and HEt oxidation were monitored by
confocal fluorescence microscopy. Control cells treated with vehicle
demonstrated a constant rate of HEt oxidation in the absence of
cyt-c-GFP release (Figs.
1A and 2B). In
cultures exposed to STS, individual cells released the
cyt-c-GFP fusion protein at different time points after
onset of the treatment (Fig. 1, B and C). The
release was characterized by a sudden redistribution from a punctate,
filamentous to a diffuse, homogeneously distributed GFP signal (Fig.
1B) (18, 22). The cyt-c-GFP redistribution was indicated by
a rapid (<10 min) decrease in the S.D. of the average GFP pixel
intensity (Fig. 1C). Confocal imaging of the kinetics of
cyt-c-GFP release and the rate of superoxide production demonstrated that the rate of HEt oxidation increased significantly after the release of the cyt-c-GFP fusion protein. Similar
results were obtained in cells exposed to TNF-
/CHX (Fig.
1D).
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Fig. 1.
The release of cyt-c-GFP
during apoptosis increases HEt oxidation. A,
MCF-7/cyt-c-GFP cell incubated with vehicle for 120 min.
After addition of 1 µM HEt, GFP and Et fluorescence were
monitored confocally at the times indicated. Scale bar, 5 µm. B, MCF-7/cyt-c-GFP cell incubated with the
apoptosis-inducing kinase inhibitor STS (3 µM) for 120 min. After addition of 1 µM HEt, GFP and Et fluorescence
were monitored confocally. Note the rapid redistribution of the
cyt-c-GFP signal and the rapid increase in HEt oxidation
after the release. Scale bar, 5 µm. C,
traces of two cells treated with 3 µM STS. HEt
was added 120 min into the STS exposure, and cells were monitored
subsequently. The release of cyt-c-GFP was detected by a
change in the S.D. of the GFP fluorescence pixel intensity. Note the
rapid increase in HEt oxidation after the cyt-c-GFP
redistribution. Squares and diamonds indicate
corresponding cyt-c-GFP and Et changes of the two cells,
respectively. Similar traces were recorded from 22 cells in three
independent experiments. D, traces of two cells treated
similarly with 100 ng/ml TNF- and 1 µg/ml CHX and analyzed as
described above. Similar traces were recorded from 10 cells in two
independent experiments.
m
depolarization (13), a process that has also been observed during the
release of cyt-c-GFP (11).
m
depolarization can lead to an artificial Et fluorescence enhancement
when large amounts of mitochondrially generated Et are released into
the cytosol ("unquenching" effect) (23). To avoid this artifact,
our study was performed using a low HEt concentration (1 µM). Under these conditions, rapid and complete
mitochondrial depolarization with the protonophore FCCP (10 µM) did not result in an increased Et fluorescence
signal, even after prolonged pre-exposure to HEt (Fig.
2, A and B).
Parallel measurements of TMRM uptake demonstrated that FCCP depolarized
m within minutes, but it did not lead to the release
of cyt-c-GFP (Fig. 2, C and D).
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Fig. 2.
Depolarization of
m does not increase Et
fluorescence. A, GFP and Et fluorescence images of a
MCF-7/cyt-c-GFP cell exposed to 1 µM HEt for
240 min and then exposed to 10 µM FCCP. Images are shown
before (
5 min) and after (+5 min) addition of FCCP. Scale
bar, 5 µm. B, corresponding traces of a cell exposed
to HEt for 240 min and then exposed to 10 µM FCCP. Note
that the exposure to FCCP did not increase the slope of the Et
fluorescence trace. Similar traces were recorded from 24 cells in three
independent experiments. C, GFP and TMRM fluorescence images
of a MCF-7/cyt-c-GFP cell incubated for 240 min with 30 nM TMRM and then exposed to 10 µM FCCP.
Images are shown before (
5 min) and after (+5 min) addition of FCCP.
Scale bar, 5 µm. D, corresponding traces of a
cell incubated with 30 nM TMRM for 240 min and then exposed
to 10 µM FCCP. Note that the exposure to FCCP caused a
rapid decrease in TMRM fluorescence. Similar traces were recorded from
15 cells in two independent experiments. E, Et fluorescence
trace of a cell recorded over 5 h. FCCP (10 µM) was
added after 120 min (arrow). Note that an increase in
superoxide production or a change in the S.D. of the GFP signal
(indicative of cyt-c-GFP release) could not be detected.
Similar traces were recorded from 26 cells in three independent
experiments. F, confocal images of single
MCF-7/cyt-c-GFP cells treated for 6 h with 10 µM FCCP or vehicle. Scale bar, 5 µm.
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Fig. 3.
The broad spectrum caspase inhibitor
z-VAD-fmk blocks the increase in superoxide production after
mitochondrial membrane permeabilization. A, trace of an
individual MCF-7/cyt-c-GFP cell treated with 3 µM STS and 100 µM of the SOD mimetic
MnTBAP. Cells were incubated with 1 µM HEt 120 min after
treatment with the drugs. Note that the release of cyt-c-GFP
was not followed by an increased HEt oxidation. Similar traces were
recorded from 10 cells in two independent experiments. B,
traces of two MCF-7/cyt-c-GFP cells treated with 3 µM STS and caspase inhibitor z-VAD-fmk (200 µM). Cells were incubated with 1 µM HEt
120 min after treatment with the drugs. Similar traces were recorded
from 20 cells in three independent experiments. C,
quantification of Et fluorescence slopes. The ratio of the Et
fluorescence intensity slopes after and before the release of
cyt-c-GFP was significantly higher in cells treated with 3 µM STS compared with cells treated with 3 µM STS plus 200 µM z-VAD-fmk. Control
ratios were calculated from Et fluorescence slopes during incubation
with 1 µM HEt from 120 to 180 min divided by slopes from
60 to 120 min in cells treated with vehicle. No release of
cyt-c-GFP was observed under these conditions. Data are
means ± S.E. from the n = 20-24 cells in the
three separate experiments per treatment.
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Fig. 4.
Superoxide production generated by menadione
is inhibited by the broad spectrum caspase inhibitor z-VAD-fmk.
A, traces of two MCF-7/cyt-c-GFP cells incubated
with 1 µM HEt during the entire experiment. Menadione
(100 µM) was added after 60 min (arrow).
B, traces of two MCF-7/cyt-c-GFP cells pretreated
for 60 min with 200 µM z-VAD-fmk and subsequently
incubated with 1 µM HEt. Menadione (100 µM)
was added after 60 min (arrow). C, quantification
of changes in Et fluorescence slopes. The ratio of the Et fluorescence
intensity slopes after and before the addition of 100 µM
menadione was significantly higher compared with vehicle-treated
controls and to cells treated with 100 µM menadione plus
200 µM z-VAD-fmk. Data are means ± S.E. from the
n = 15-22 cells in the three separate experiments per
treatment. Control values were calculated as described in the legend to
Fig. 3C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
STS-induced apoptosis (8-10, 17). Our data now demonstrate at the
single-cell level that superoxide production during STS- and
TNF-
-induced apoptosis starts directly after the mitochondrial release of soluble intermembrane proteins. We did not detect a significant increase in superoxide production prior to the
cyt-c-GFP release, and the SOD mimetic MnTBAP did not alter
the kinetics of STS-induced cyt-c-GFP release. These results
suggest that superoxide anions do not significantly modulate the
process of outer mitochondrial membrane permeabilization during
STS-induced apoptosis in MCF-7 cells. However, the increase in
superoxide production may well play an important role in mitochondrial
transition pore opening (24). Interestingly, treatment with the
broad-spectrum caspase inhibitor z-VAD-fmk inhibited the increase in
superoxide production after STS-induced cyt-c-GFP release,
suggesting that this was a caspase-dependent process. Since
the increased superoxide during apoptosis is generated by the
mitochondrial respiratory chain (10, 12), it is possible that caspases
directly target mitochondrial proteins. However, mitochondrial inner
membrane or matrix proteins have not yet been identified among the more
than 250 caspase substrates known to date.
m (30).
Indeed, the qualitatively similar increase in superoxide generation of
isolated mitochondria depleted of cyt-c compared with
mitochondria treated with the complex I inhibitor rotenone, as well as
its dependence on the NADH/NAD+ ratio, suggests that
complex I is a primary site of superoxide production during apoptosis
(14). However, in depolarized mitochondria superoxide is primarily
generated via the ubiquinone in the inner mitochondrial membrane (30,
32).
m depolarizes after the release of
cyt-c or cyt-c-GFP (11, 13, 33). It is therefore possible that ubiquinone is also a major source of superoxide after
mitochondrial membrane permeabilization. Indeed, an electron paramagnetic resonance study performed in digitonin-permeabilized mitochondria has demonstrated that ubiquinone autoxidation at the outer
site of the complex III ubiquinone pool increases superoxide formation
toward the intermembrane space (34).
-induced apoptosis. The increase in superoxide production was sensitive to the broad spectrum caspase inhibitor z-VAD-fmk. However our data also indicate that this commonly used inhibitor may exert antioxidant activities unrelated to caspase inhibition.
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ACKNOWLEDGEMENTS |
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We thank Petra Mech and Christiane Schettler for technical assistance and Drs. A.-L. Nieminen and J. Ma for the cyt-c-GFP construct.
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FOOTNOTES |
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* These experiments were supported by the Deutsche Forschungsgemeinschaft and the Interdisciplinary Center for Clinical Research, University Münster Clinics.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.
¶ Current address and to whom correspondence should be addressed: Experimental Neurosurgery, Klinikum der Johann Wolfgang Goethe-Universität, Haus 25 B, 4. OG, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Tel.: 49-69-6301-6930; Fax: 49-69-6301-5575; E-mail: prehn@em.uni-frankfurt.de.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M210826200
2 H. Düssmann, D. Kögel, M. Rehm, and J. H. M. Prehn, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
cyt-c, cytochrome c;
CHX, cycloheximide;
cyt-c-GFP, cytochrome c green fluorescent protein fusion protein;
Et, ethidium;
FCCP, carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone;
HEt, hydroethidine;
MnTBAP, manganese tetrakis(4-benzoyl
acid)porphyrin;
SOD, superoxide dismutase;
STS, staurosporine;
TMRM, tetramethylrhodamine methyl ester;
TNF-, recombinant human tumor
necrosis factor-
;
z-VAD-fmk, z-Val-Ala-Asp(O-methyl)- fluoromethylketone.
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