From the Division of Biology, California Institute of Technology, Pasadena, California 91125
Received for publication, September 10, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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We have shown here that the apoptosis inducer
staurosporine causes an early decrease in the endogenous respiration
rate in intact 143B.TK It has become clear over the last several years that mitochondria
play a central role in the cell death program initiated by most
inducers of apoptosis. Several key components of the apoptotic machinery, i.e. cytochrome c, AIF
(apoptosis-inducing factor), SMAC/Diablo, and HtrA2/Omi, are released
from mitochondria in cells undergoing apoptosis (1-5). Although the
importance of mitochondrial changes leading to the release of these
apoptogenic molecules is well established, the detailed mechanism(s)
involved in this phenomenon, and in particular, the possible role of
dysfunctions of the oxidative phosphorylation apparatus and of
oxidative stress, have yet to be clarified. The observation that
mitochondrial DNA-less ( Cell Culture--
143B.TK Bcl-2 Transfection--
A human Bcl-2 Nuclear Apoptosis Assay--
The cells were grown in a 6-cm
Petri dish; 0.5 volume of phosphate-buffered saline (PBS; 0.145 M NaCl, 8.1 mM
Na2HPO4, 1.5 mM
KH2PO4, pH 7.65), and formaldehyde, to a final
concentration of 4%, was added directly to the medium in the dish.
After 10 min at room temperature, bovine serum albumin was added to
0.1%, and the cells were collected by scraping them with a rubber
policeman and then transferred into a centrifuge tube. The cells were
washed once with PBS, 0.1% bovine serum albumin and then resuspended in 1 ml of the same solution and stained with 1 µg/ml of the
double-stranded DNA-binding fluorochrome 4,6-diamidino-2-phenylindole
(DAPI) for 10 min at room temperature, with occasional agitation. The
cells were collected by centrifugation, resuspended in 10 µl of PBS, mixed with 10 µl of a 50:50 mixture of glycerol and 2× PBS, and mounted on a slide for viewing in a fluorescence microscope.
Oxygen Consumption Measurements in Intact Cells--
The
respiration rate was measured in an oxygraph (Yellow Springs
Instruments, model 5300) in a suspension of naive or
staurosporine-treated cells at 3-6 × 106 cells/ml in
TD buffer (25 mM Tris-HCl, pH 7.4-7.5 (25 °C), 137 mM NaCl, 10 mM KCl, 0.7 mM
Na2HPO4) at 37 °C. Previous work from this
laboratory had shown that 143B cells respire in TD buffer at the same
rate as in Dulbecco's modified Eagle's medium lacking glucose (19).
After obtaining a stable rate, the uncoupler dinitrophenol (DNP) was
added at a concentration of 17-25 µM, and the resulting respiration rate was recorded. After blocking the electron flux upstream of cytochrome c oxidase with 20 nM
antimycin A, the cytochrome c oxidase-dependent
oxygen consumption rate isolated from the upstream segment of the
respiratory chain was measured by using the membrane-permeant electron
donor
N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at 400 µM and ascorbate, as primary reducing
agent, at 10 mM (19). The rate of TMPD-driven
O2 consumption was corrected for the autooxidation rate of
TMPD, measured on the same day, as determined in a separate run in TD
in the absence of cells. Oxygen consumption rates were expressed in
nmol of oxygen consumed/min/mg of cellular protein. The rates of the
treated samples were calculated as percentages of the rates of
untreated samples, determined on the same day.
Oxygen Consumption Measurements in Digitonin-permeabilized
Cells--
Digitonin permeabilization was carried out by a
modification of a previously described procedure (20-22). For
measurements of glutamate/malate- and succinate-driven respiration
rates, 2-10 × 106 cells were harvested by
trypsinization, washed in 5 ml of Respiration Buffer I (modified from
Ref. 23: 30 mM HEPES, pH 7.1, 75 mM sucrose, 20 mM glucose, 5 mM potassium
Pi, 40 mM KCl, 0.5 mM EDTA, 3 mM MgCl2), centrifuged, and resuspended in 0.15 ml of the same buffer per 106 cells. A fresh 1000-fold
dilution of 10% (w/v) digitonin was then made in Respiration Buffer I,
an equal volume of the 0.01% digitonin was added to the cell
suspension, and the mixture was incubated for 2 min at room
temperature. A concentration of 0.005% digitonin permeabilized, in
general, 100% of untreated 143B cells and 97-100% of cells treated
with staurosporine. Permeabilization was stopped by the addition of
8-10 volumes of Respiration Buffer I containing 0.3% bovine serum
albumin. After confirming by trypan blue uptake that at least 95% of
the cells were permeabilized, the cells were centrifuged, resuspended
in 150 or 300 µl of Respiration Buffer I, and transferred into one or
two oxygraph chambers, respectively, each containing about 1.4 ml of
the same buffer. Respiration measurements were carried out in the
presence of 1 mM ADP. Glutamate and malate were added at 5 mM each. Alternatively, 40-200 nM rotenone and 10 mM succinate were added. Both the coupled and 17-25
µM DNP-uncoupled rates were determined. Cytochrome
c, when used, was 115 µM. To measure
TMPD-driven rates, permeabilization was carried out, and respiration
rates, measured in an alternative buffer, Respiration Buffer II (0.25 M sucrose, 20 mM Hepes-KOH, pH 7.1, 2 mM potassium phosphate, 10 mM
MgCl2, 1 mM ADP (20)) because the autooxidation rate of TMPD is lower in Respiration Buffer II than in Respiration Buffer I. For these measurements, 20 nM antimycin, 0.4 mM TMPD, and 10 mM ascorbate were used. The
TMPD-dependent respiration rate was determined after
subtraction of the autooxidation rate, measured on the same day.
Respiration rates were expressed as nmol/min/mg. Rates of treated
samples were compared with those of untreated samples determined on the
same day.
In one experiment aimed at testing the possibility of transactivation
(from cell to cell), the cells were suspended at one-seventieth of the
usual concentration during permeabilization. Because of the high
dilution, 10 times as much digitonin per cell (but only one-seventh the usual concentration) had to be used to effect permeabilization of the plasma membrane. After 2 min of incubation at
room temperature and the addition of bovine serum albumin to quench the
action of the digitonin, the cells were centrifuged, resuspended, and
added to the oxygraph chamber.
Confocal Immunofluorescence Microscopy--
Cells grown on
coverslips were fixed and stained for immunofluorescence as detailed
(24). In brief, the cells were double-labeled with mouse
anti-cytochrome c monoclonal antibody 6H2.B4 (Pharmingen) and rabbit anti-Hsp60 antiserum (Stressgen Biotechnologies Corp.). The
secondary antibodies were fluorescein isothiocyanate-conjugated goat
anti-mouse IgG and lyssamine-rhodamine-conjugated goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories). The preparations were analyzed
on a Zeiss 310 laser scanning confocal microscope. In some experiments,
the cells were centrifuged onto glass coverslips and then fixed
and treated as described above. Cells with punctate cytochrome
c staining (green) that overlapped with Hsp60 staining (red)
were counted as cells with mitochondrial cytochrome c
staining, whereas cells with diffuse nuclear and cytosolic cytochrome
c staining and punctate mitochondrial Hsp60 staining were
counted as cells with extramitochondrial cytochrome c.
Preparation of Mitochondrial Fraction--
Cells were harvested
from ~40 T-175 flasks by trypsinization and washed thrice in PBS. For
cell breakage, the cells were resuspended in mitochondria isolation
medium consisting of 0.25 M sucrose, 10 mM
Hepes-KOH, pH 7.4, 1 mM EGTA, and 0.5% bovine serum
albumin. The cells were disrupted with a motor-driven Potter-Elvehjem
glass-Teflon homogenizer until ~80% of the nuclei had been released.
The mitochondrial fraction was isolated by differential centrifugation
(25) and subsequently washed and resuspended in mitochondria isolation medium and stored at Enzyme Activity Assays--
All assays were carried out at room
temperature in a split-beam spectrophotometer. The
mitochondria-enriched fraction was thawed, adjusted to a mitochondrial
protein concentration of 1 mg/ml, and frozen and thawed two more times.
For assays of complex I, complex III, and complex I + III activities,
the mitochondria were sonicated in a microcentrifuge tube,
immersed in ice water, with a stepped microtip using a Branson Digital
Sonifier for 60 s in 2-s bursts at 8-s intervals at 20%
amplitude. The output was 3 watts.
Complex I activity was assayed in samples containing 30 µg of
mitochondrial protein in 50 mM potassium phosphate, pH 7.5, 2 mM KCN, 50 µM NADH. The reaction was
started by the addition of 10 µM decylubiquinone, and the
change in absorbance at 340 nm was recorded. For both complex III and
complex I-III activity measurements, the assay mixtures were as
described (26), and the increase in absorbance at 550 nm was recorded.
Cytochrome c oxidase activity was measured at 550 nm in
samples containing 10 µg of mitochondrial protein, using unsonicated mitochondria, in 40 mM potassium phosphate, pH 6.65, with
0.5% Tween 80 (low peroxide, Sigma) in the cuvette. The initial
concentration of reduced cytochrome c was 0.04% w/v. The
aforementioned assays measured inhibitor-sensitive activities as the
reference cuvette in the split beam spectrophotometer contained the
reaction mixture plus inhibitor. The inhibitors for complex I, complex
III, complex I-III, and cytochrome c oxidase activity
assays were 2 µM rotenone, 5 µg/ml antimycin, 5 µg/ml
antimycin, and 1 mM KCN, respectively.
Citrate synthase activity was measured in unsonicated mitochondria by
using buffer and reagent concentrations as described (26). In addition,
the cuvette contained 0.5% Triton X-100 to lyse the mitochondria. The
change in absorbance at 412 nm was recorded. The reference cuvette
contained the reaction mixture lacking oxaloacetate.
Reagents--
Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone
(zVADfmk) was purchased from Kamiya Biomedicals; bisindolylmaleimide GF 109203X (Bis-I) (27, 28) was purchased from Calbiochem. Cyclosporine A
(CsA) was a gift from Sandoz Research Institute, East Hanover, NJ. All
other reagents were obtained from Sigma.
Miscellaneous--
Protein concentration was determined as
described (29).
Early Decrease in Endogenous Respiration Rate in
Staurosporine-treated 143B Cells--
143B osteosarcoma cells
induced to undergo apoptosis by exposure to 1 µM
staurosporine exhibited a rapid decline in their respiration rate in
the first hour, to ~80% of the normal rate, followed by a slower
decline over the next 15 h, to 35% of normal (Fig.
1). This rate was measured in intact
cells suspended in buffered saline solution. Thus, respiration in these
cells was driven by endogenous substrates. The rate of respiration
uncoupled by the addition of DNP decreased with staurosporine exposure
in a way that paralleled closely the decrease in endogenous
respiration. To measure the respiratory activity of the terminal enzyme
of the respiratory chain, cytochrome c oxidase, the upstream
segment of the respiratory chain was first blocked by the addition of antimycin. Respiration was then started by the addition of a
cell-permeant reductant of cytochrome c, TMPD, and the
primary reductant, ascorbate. The TMPD-driven respiration rate, which
was typically 1.4-1.7 times higher than the endogenous
substrate-dependent rate, was minimally affected ( The Decrease in Respiration Rate Precedes Cytochrome c
Release--
Cytochrome c was released from mitochondria in
cells treated with staurosporine, as shown in Fig.
2. The anti-cytochrome c antibodies revealed a diffuse extramitochondrial localization of
cytochrome c in a progressively increasing fraction of the cells during their treatment with the drug, whereas in all untreated cells, cytochrome c had a punctate distribution and
co-localized with Hsp60, a mitochondrial protein. It appears from Fig.
2 that in many cells, the extramitochondrial cytochrome c
had a nuclear localization. This phenomenon was reported previously in
breast carcinoma cells induced to apoptosis by tumor necrosis
factor-
For the first 8 h after staurosporine addition to 143B cells, the
TMPD-driven respiration rate did not decrease significantly (Fig. 1),
indicating that cytochrome c oxidase activity remained high.
On the other hand, by 8 h after staurosporine addition, mitochondria had already massively released cytochrome c in
~40% of 143B cells. This fact indicated that, even after cytochrome c had been released, the level of cytochrome c
available to cytochrome c oxidase was still sufficient to
maintain normal cytochrome c oxidase activity. This is
consistent with the finding of Waterhouse et al. (32) that
the mitochondrial membrane potential could be maintained by respiration
after cytochrome c release (provided that caspase activity
was inhibited).
Tests of Possible Role of Caspases, Permeability Transition, and
Protein Kinase Inhibition in Respiration Decrease--
To gain some
insight into the mechanisms underlying the decrease in endogenous
respiration rate in staurosporine-treated cells, several inhibitors
were tested for the ability to modify this effect of staurosporine. The
caspase inhibitor zVADfmk failed to prevent the decrease in respiration
rate (Fig. 3a) and, as had
been found previously (33), the release of cytochrome c from
mitochondria (Fig. 2), whereas apoptosis was completely inhibited in
these cells (not shown). Thus, it appeared that the respiration decrease was not dependent on the activity of caspases. An inhibitor of
the permeability transition, CsA (34-36), present at 10 mM
during the staurosporine treatment, also had no significant effect on the decrease in respiration (Fig. 3b). Treatment of the
cells with 20 mM CsA for 6 h or with 10 mM
CsA added twice during the staurosporine treatment likewise had no
effect (not shown). These results indicated that, barring an unusual
failure of the cells to take up the drug or a high capacity to export
it, permeability transition did not influence the decrease in
respiration rate.
Lastly, an attempt was made to dissociate the respiration rate decline
from the apoptotic program. Specifically, we used an analog of
staurosporine, Bis-I (27, 28), which, like staurosporine, inhibits
protein kinase C (28, 37), but unlike staurosporine, does not
induce apoptosis (37). Fig. 3c shows that 1 µM
Bis-I did not cause a decrease in respiration rate. It was also
confirmed that Bis-I did not induce apoptosis in 143B cells (data not
shown). Thus, these experiments did not provide any evidence that the respiration decline is not a part of the apoptotic program.
Bcl-2-overexpressing 143B Cells Also Exhibit the Early Endogenous
Respiration Decrease--
Experiments were carried out to determine
whether overexpression of the antiapoptotic protein Bcl-2 (16) would
prevent the decrease in respiration rate. 143B cells were stably
transfected with a cDNA clone for Bcl-2, and an overexpressing
clone, B15, was analyzed for its response to staurosporine. As observed
previously for Bcl-2-overexpressing staurosporine-treated HL-60 cells
(38), Bcl-2-overexpressing 143B cells did not release cytochrome
c from mitochondria upon exposure to staurosporine (Fig. 2).
Also, as expected, the cells did not undergo apoptosis, as assayed by
nuclear fragmentation (Fig. 4).
Nevertheless, as Fig. 4 shows, B15 cells exhibited a decrease in both
coupled and uncoupled respiration rates of a degree comparable with
that observed in 143B cells. Thus, it appeared that the site of Bcl-2
activity was downstream of the respiration rate decrease or in an
independent pathway.
Hypersensitivity of the Outer Mitochondrial Membrane to Digitonin
is an Early Staurosporine-induced Effect--
The endogenous
substrate-dependent respiration rate depends on three other
respiratory chain complexes besides complex IV: namely, complex I, or
respiratory NADH dehydrogenase; complex III, or ubiquinone-cytochrome
c oxidoreductase; and complex II, or succinate
dehydrogenase. Electrons pass from NADH, generated by the oxidation of
metabolites, sequentially to complex I, ubiquinone, complex III,
cytochrome c, complex IV, and finally, oxygen. Electrons from fatty acid oxidation can also enter the respiratory chain via
succinate and succinate dehydrogenase. This enzyme transfers electrons
to ubiquinone, and subsequently, electron transfer continues as
described above. In 143B cells, the endogenous
substrate-dependent respiration has been found to be
By using digitonin-permeabilized cells and by providing mitochondria
with exogenous substrates that feed into complex I or that bypass
complex I, one can determine which respiratory enzyme activity is
decreased. Fig. 5a shows the
results of exogenous substrate-driven respiration assays performed on
digitonin-permeabilized 143B cells. Surprisingly, all activities,
whether driven by glutamate and malate (electrons entering the
respiratory chain at complex I), succinate (electrons entering at
complex II), or TMPD and ascorbate (electrons entering at complex IV),
were reduced by at least 75% within 4 h after staurosporine
addition. The relative rates shown in Fig. 5a represent the
initial rates after the addition of DNP. For each substrate, the
respiration rate decreased, in staurosporine-treated cells, during the
measurement, over the course of minutes. The difference observed
between the dramatic decreases in respiration rates in permeabilized
cells and the moderate decreases in intact cells suggested the
possibility that digitonin treatment had removed an electron carrier or
some other component(s) of the respiratory chain in cells treated with
staurosporine. The addition to the oxygraph chamber of 10 µM ubiquinone (oxidized or reduced) had no effect (not
shown). On the contrary, the addition of cytochrome c
restored the ability of the permeabilized staurosporine-treated cells
to respire (Fig. 6a),
suggesting that the electron carrier cytochrome c had been
lost from mitochondria. In fact, in the presence of added cytochrome
c, respiration driven by glutamate and malate, by succinate,
or by TMPD/ascorbate attained the rates observed in permeabilized
untreated cells. To confirm that digitonin treatment of
staurosporine-treated 143B cells caused the premature loss of
cytochrome c from mitochondria, permeabilized treated and
untreated cells were incubated with anti-cytochrome c and anti-Hsp60 antibodies and examined by confocal fluorescence microscopy. Fig. 7 shows that cytochrome c
was virtually absent in the permeabilized staurosporine-treated cells,
but it was readily visible and co-localized with Hsp60 in the
digitonin-permeabilized untreated cells.
The most plausible explanation for the loss of cytochrome c
from mitochondria of staurosporine-treated and permeabilized 143B cells
was that the mitochondrial outer membrane had lost integrity. However,
an alternative explanation was that the digitonin permeabilization of
the plasma membrane allowed the diffusion of transactivating factors
from cells advanced in the apoptotic program to cells less advanced. To
test this hypothesis, digitonin permeabilization of
staurosporine-treated 143B cells was performed at a very low cell
concentration, i.e. at one-seventieth of the normal
concentration, to dilute any possible transactivating factors, thereby
presumably reducing their activity. In this experiment, exogenous
substrate-driven respiration rates were found to be decreased as much
as in a parallel experiment in which permeabilization was carried out
at the usual cell concentration (not shown). This result strongly
suggested that the loss of cytochrome c from mitochondria
was not due to the action of transactivating factors.
Lack of Hypersensitivity to Digitonin in Bcl-2-overexpressing
Staurosporine-treated Cells--
Quite different results from those
described above were obtained with B15 Bcl-2-overexpressing cells
treated with staurosporine and permeabilized with digitonin (Fig.
5b). In fact, in B15 cells permeabilized after 6 h of
exposure to staurosporine, the glutamate- and malate-driven
respiration rate was decreased by 40%, and the succinate-driven rate
was decreased by 37% (Fig. 5b). The decrease in the
glutamate/malate-driven respiration rate was thus very similar to the
decrease, 30-35%, which had been observed in the endogenous
substrate-dependent respiration rate of intact B15 and 143B
cells. The TMPD-driven respiration rate in the digitonin-permeabilized B15 cells was not decreased by the staurosporine treatment, as had been
observed in intact cells. Furthermore, the addition of cytochrome
c had no effect on the glutamate/malate- or succinate-driven respiration rates of staurosporine-treated permeabilized B15 cells (Fig. 6b). These results thus led to the conclusion that
Bcl-2 protected staurosporine-treated cells from the dramatic loss of cytochrome c and respiration caused by permeabilization with
digitonin. Thus, presumably, Bcl-2 helped to maintain the integrity of
the outer mitochondrial membrane.
Respiratory Enzyme Defects Are Not Involved in the Early Endogenous
Respiration Decrease--
The polarography experiments carried out on
B15 cells suggested that both complex I and complex III activities were
affected by staurosporine treatment of the cells (Fig. 5b).
To validate this suggestion and to extend it to 143B cells,
spectrophotometric enzyme assays were performed on disrupted
mitochondria from 143B cells treated with staurosporine for 6 h,
from untreated 143B cells, and likewise, from naive and
staurosporine-treated B15 cells. The surprising results shown in Fig.
8 revealed that the activities of complex
I, complex III, and complex I + III were not significantly different
between untreated and staurosporine-treated cells. This important
observation, combined with the finding of complete restoration by
cytochrome c of the capacity of digitonin-permeabilized staurosporine-treated 143B cells to respire on exogenous oxidizable substrates, indicated that the early decline in endogenous respiration in staurosporine-caused apoptosis was due to an apoptosis-induced outer mitochondrial membrane permeability change, resulting in a block
in respiratory substrate uptake.
We have reported here an early and progressive inhibition of
endogenous substrate-dependent respiration in 143B cells
during the first 6-8 h of treatment with the apoptosis inducer
staurosporine. TMPD-driven (complex IV-dependent)
respiration was not significantly affected during the same period. We
have no information on the distribution within the cell population of
this respiration loss, i.e. whether respiration was affected
partially in every cell or whether a fraction of the cells had
completely lost endogenous substrate-dependent respiration.
The latter alternative seems, however, more likely, on the basis of the
available evidence on the intercellular mosaicism of apoptosis-related
phenomena (24, 39). In any case, the data clearly showed that this loss
of respiration in staurosporine-treated cells preceded the release of
cytochrome c from mitochondria and the fragmentation of
nuclei. Our results differ from those of Hájek et al.
(24), who reported that, in anti-Fas antibody-treated Jurkat cells, the
loss of TMPD-dependent and of endogenous respiration
occurred nearly simultaneously and followed cytochrome c
release from mitochondria. This difference in the sequence of events
could be due to the different pathway taking place in Fas-induced apoptosis.
It should be mentioned that an early decline in endogenous
substrate-dependent respiration, which appeared to be
independent of cytochrome c release, has also been observed
in a rat cell line of neuronal derivation, PC12, treated with
staurosporine.3 In yet
another system, Schulze-Osthoff et al. (40) reported a
specific decrease in complex I- and complex III-dependent
respiration rates in mouse fibrosarcoma cells treated with tumor
necrosis factor- A valuable insight into the mechanism underlying the early decrease in
endogenous respiration in staurosporine-treated cells was provided by
the observation that, although respiration measurements in B15 cells
suggested that complex I and complex III activities were decreased,
spectrophotometric assays of the respective enzyme activities,
namely NADH-ubiquinone oxidoreductase activity and ubiquinol-cytochrome
c oxidoreductase activity, showed them to be normal in
sonicated mitochondria from both 143B and B15 cells. We propose that
the basis for this apparent discrepancy in our results is that the
outer mitochondrial membrane, during staurosporine-induced programmed
cell death, becomes impermeable to metabolites such as glutamate,
succinate, and ADP. Consequently, respiration is halted in these cells
because of the inability of mitochondria to take up oxidizable
substrates. Fig. 9 depicts in schematic form our views of the early changes in mitochondria that have been
detected in our experiments. Thus, we interpret our finding that, in
intact 143B cells, the overall endogenous
substrate-dependent respiration rate was decreased by about
30% in a cell population exposed to staurosporine for 6 h as
indicating that, in 30% of the cells, the outer mitochondrial membrane
was impermeable to substrates.
cells. On the other hand,
the activity of cytochrome c oxidase is unchanged for the
first 8 h after staurosporine treatment, as determined by oxygen
consumption measurements in intact cells. The decrease in the
endogenous respiration rate precedes the release of cytochrome
c from mitochondria. Moreover, we have ruled out caspases,
permeability transition, and protein kinase C inhibition as being
responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in
respiration rate. The last finding suggests that Bcl-2 acts downstream
of the perturbation in respiration. The evidence of normal enzymatic
activities of complex I and complex III in staurosporine-treated 143B.TK
osteosarcoma cells indicates that the cause of
the respiration decrease is probably an alteration in the permeability
of the outer mitochondrial membrane. Presumably, the
voltage-dependent anion channel closes, thereby preventing
ADP and oxidizable substrates from being taken up into mitochondria.
This interpretation was confirmed by another surprising finding, namely
that, in staurosporine-treated 143B.TK
cells
permeabilized with digitonin at a concentration not affecting the
mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2
overexpression prevents the staurosporine-induced hypersensitivity of
the outer membrane to digitonin. Our experiments have thus revealed
early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from
mitochondria in intact cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
°) cells can be induced to undergo
apoptosis (6, 7) has led to the conclusion that the apoptotic program
does not require a functional oxidative phosphorylation apparatus.
However, these experiments did not exclude the possibility that, in
normally respiring cells, oxidative phosphorylation alterations do play an important role in the early phase of apoptosis and that this role
can be bypassed by the profound biochemical changes accompanying the
establishment of
° cells. Indeed, several mitochondrial functional changes, such as impaired mitochondrial adenine nucleotide exchange (8)
and permeability transition pore (9) opening with membrane potential
collapse (10), have been suggested to play an important role in
apoptosis. Moreover, the level of cellular ATP can determine whether a
cell undergoes necrosis or apoptosis (11). We considered the
possibility that investigating the respiratory activity in cells
induced to undergo apoptosis might reveal changes in mitochondria that
precede, and may contribute to, the release of cytochrome c
and other apoptogenic molecules. Our investigations on
143B.TK
osteosarcoma cells induced to undergo apoptosis
with staurosporine have indeed revealed two early changes in
mitochondria that result from initiation of the apoptosis program.
These are 1) a decrease in the permeability of the outer membrane,
pointing to a closure of the voltage-dependent anion
channel (VDAC),1 and 2) a
hypersensitivity of the outer mitochondrial membrane to digitonin,
suggesting that a change in lipid organization or composition has taken place.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a human
osteosarcoma-derived cell line (ATCC CRL 8303) (12-15), and hereafter
referred to as 143B, was grown in Dulbecco's modified Eagle's medium
supplemented with 5 or 10% fetal bovine serum. For induction of
apoptosis, semiconfluent dishes of cells were exposed for different
times to 1 µM staurosporine. For experiments involving
respiration measurements, cells were plated 3 days before the
experiment, and the medium was changed in the afternoon or evening of
the day before.
cDNA (16)
was subcloned from SFFV-Bcl-2 n1 into the neomycin resistance
marker-containing plasmid pcDEF3 (17) and then used for transfection of
143B cells by calcium phosphate precipitation (18). Stably transfected
clones were selected in medium containing 0.5 mg/ml G418 sulfate
(Geneticin; Invitrogen).
80°.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5%)
during the first 6 h of staurosporine treatment and then slowly
declined to about 70% of the control rate by 16 h of cell
exposure to the drug.
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Fig. 1.
Decrease in endogenous respiration rate
precedes cytochrome c release in intact
staurosporine-treated 143B cells. Respiration rates of intact 143B
cells treated with 1 µM staurosporine for different
lengths of time were measured as described under "Experimental
Procedures" and normalized to the protein content of the samples.
Values (averages of 2-11 independent experiments ± one standard
deviation) are given as percentages of the rates in untreated
cells measured on the same day. (Filled squares, endogenous
substrate-dependent respiration; open squares,
uncoupled endogenous substrate-dependent respiration;
filled circles, TMPD-driven respiration.) The increase with
time of the percentage of cells having released cytochrome c
from mitochondria after staurosporine addition was determined by
confocal laser scanning microscopy (filled triangles,
percentage of cells with extramitochondrial cytochrome c).
In other experiments, cells stained with DAPI were viewed by
fluorescence microscopy, and total cells and cells with fragmented
nuclei were counted in several adjacent fields (open
diamonds, percentage of cells with fragmented nuclei).
or staurosporine (30) and in NIH-3T3 cells induced to
apoptosis by actinomycin D (31), and it was observed in PC12
cells treated with
staurosporine.2 The
proportion of cells that showed release of cytochrome c was quantified at various times after staurosporine addition. Fig. 1 shows
that the percentage of cells having released cytochrome c
was less than the percentage of decrease in endogenous respiration rate
for the first 6 h after staurosporine addition, indicating that
the respiration decrease was not dependent on cytochrome c
release from mitochondria. Furthermore, the fraction of cells that had
reached the late apoptosis stage of nuclear fragmentation was
determined in DAPI-stained cells (Fig. 1). Under the reasonable assumption that the fraction of cells having released cytochrome c included those undergoing nuclear fragmentation, it
appears that cells reached this stage only 2-3 h after their
mitochondria had released cytochrome c.
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Fig. 2.
Distribution of cytochrome
c in intact staurosporine-treated and untreated cells
and B15 Bcl-2-overexpressing cells. Untreated 143B and
Bcl-2-overexpressing 143B cells (B15) and the same cells
exposed to staurosporine (sts) for 8 h were fixed and
double-immunolabeled with antibodies directed against cytochrome
c and against Hsp60. The panels in the
third column show the merged images of green
(cytochrome c) and red (Hsp60) fluorescence. In
one experiment, 143B cells were exposed to zVADfmk during the
staurosporine treatment.
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Fig. 3.
Staurosporine-induced decline in respiration
rate is not prevented by inhibitors of caspases (zVADfmk) or
permeability transition (CsA) or mimicked by protein kinase C inhibitor
(Bis-I). As shown in a, 100 µM zVADfmk
(zVAD) was added to cultures 1 h prior to the
addition of 1 µM staurosporine. As shown in b,
10 mM CsA was added at the time of staurosporine addition.
As shown in c, 1 µM Bis-I instead of
staurosporine was added to cultures at time 0. Cultures were harvested
and assayed for respiration activity 6 h after staurosporine
(a and b) or Bis-I (c) addition.
Black bars represent endogenous
substrate-dependent respiration; stippled bars
represent DNP-uncoupled endogenous substrate-dependent
respiration; and hatched bars represent TMPD-driven
respiration. Values are given as percentages of the rates in untreated
cells. Data in panels b and c are averages of 2 independent experiments ± one standard deviation.
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Fig. 4.
Staurosporine-treated B15
Bcl-2-overexpressing cells also exhibit the early decline in endogenous
respiration rate. The respiration rates of intact B15 cells
treated with 1 µM staurosporine were measured as
described under "Experimental Procedures," normalized to the
protein content of the samples, and presented as percentages of the
rates in untreated cells, measured on the same day. Except for the
16 h data, values given are averages of 2-4 independent
experiments ± one standard deviation. (Filled squares,
endogenous substrate-dependent respiration; open
squares, uncoupled endogenous substrate-dependent
respiration; filled circles, TMPD-driven respiration.) The
proportions of cells with extramitochondrial cytochrome c
(cyt c) (triangles) and with fragmented nuclei
(diamonds) are indicated as well.
90%
inhibitable by rotenone, an inhibitor of complex I (not shown); thus,
electrons from endogenous substrates enter the chain almost
exclusively through complex I, and very few electrons enter through
complex II. Therefore, the observed early decrease in respiration rate
in staurosporine-treated cells could be due to a decrease in either
complex I or complex III activity or both.
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Fig. 5.
Respiration rates in digitonin-permeabilized
143B and B15 cells treated with 1 µM staurosporine. Respiration
measurements were carried out in the presence of DNP. The
glutamate/malate- (squares), succinate-
(diamonds), and TMPD/ascorbate- (circles) driven
rates in staurosporine-treated cells were normalized to the protein
content of the samples and expressed as percentages of the rates in
untreated cells. The data presented are averages ± one standard
deviation of 2-7 independent experiments. a, 143B cells;
b, B15 cells.
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Fig. 6.
Addition of cytochrome c
(cyt c) restores respiration in
digitonin-permeabilized 143B cells treated with staurosporine but not
in B15 cells. Cells were exposed to 1 µM
staurosporine (sts) for 6 h or left untreated and then
permeabilized with digitonin. Respiration was stimulated by the
addition of glutamate and malate, of succinate, or of TMPD, in all
cases in the presence of DNP. Rates (averages of 2-4 independent
experiments ± one standard deviation) are given as percentages of
rates in untreated permeabilized cells. a, 143B cells;
b, B15 cells. Light gray bars, no added
cytochrome c; dark gray bars, added cytochrome
c.
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Fig. 7.
Distribution of cytochrome c
(Cyt.c) in digitonin-permeabilized 143B
cells. Unexposed cells or cells exposed to staurosporine for
4 h were permeabilized, centrifuged onto round glass coverslips,
fixed, and stained with antibodies directed against cytochrome
c and against Hsp60. The panels on the
right show the merged images of green fluorescence
(cytochrome c) and red fluorescence (Hsp60).
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Fig. 8.
Enzyme activities in mitochondria isolated
from staurosporine-treated or untreated 143B cells (a)
and B15 cells (b). Dark gray bars
represent data after no staurosporine treatment, and light gray
bars represent data after staurosporine treatment for 6 h at
1 µM. Two mitochondria preparations each were made from
control and staurosporine-treated 143B cells, and two preparations each
were made from control and staurosporine-treated B15 cells.
Measurements were made from both preparations for all assays except for
complex IV in 143B cells. Activities were first normalized to citrate
synthetase (CS) activity in the mitochondria preparations.
The activity for each enzyme assay (averages of 4-7 measurements
(a) or 3-11 measurements (b) ± one
standard deviation) is presented as a percentage of the activity in
mitochondria from untreated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
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Fig. 9.
Scheme of a model proposed for the
interpretation of the experimental results. In 143B intact cells,
respiration is sustained by uptake of ADP and endogenous oxidizable
substrates into mitochondria from the cytosol (pink),
presumably through VDAC (or porin) (brown). Digitonin
(DIG) permeabilization of naive cells causes endogenous ADP
and substrates to diffuse out of the cytosol and allows exogenous ADP
and oxidizable substrates to pass through the plasma membrane
(pink) to drive respiration. Staurosporine (STS)
treatment causes the permeability of the outer mitochondrial membrane
to ADP and oxidizable substrates to become dramatically curtailed,
presumably via VDAC closure. Staurosporine also causes an alteration in
the structure of the outer membrane (shown as a change to
green) so that now, digitonin treatment of
staurosporine-treated cells not only permeabilizes the plasma membrane
but also disrupts the outer mitochondrial membrane, thereby allowing
cytochrome c (red dot) to leave mitochondria and
allowing substrates and ADP to be taken up into the mitochondrial
matrix. In the presence of adequate exogenous cytochrome c
and oxidizable substrates, respiration is fully restored. In B15 cells,
the outer mitochondrial membrane permeability change produced by
staurosporine occurs as in 143B cells, but digitonin treatment does not
cause disruption of the outer membrane, presumably because this
membrane is stabilized by Bcl-2 (in dark blue). Oxidizable
substrates and ADP still cannot be taken up by mitochondria.
The interpretation that we propose to explain the apparent discrepancy between the results of enzyme activity measurements and those of the polarographic measurements has been fully confirmed by the observation of another unusual phenomenon associated with staurosporine-induced apoptosis. As reported previously by Hájek et al. (24) for Jurkat cells induced to undergo apoptosis by anti-Fas antibodies, we found that the outer mitochondrial membrane in cells undergoing the apoptotic program was disrupted by the digitonin treatment used to permeabilize cells, in such a way as to allow cytochrome c to be released from mitochondria (Fig. 9). In our experiments, the first evidence for this disruption of the outer membrane was that cytochrome c added back to digitonin-treated 143B cells stimulated all of the substrate-driven respiration rates. In fact, mitochondria of staurosporine-treated and digitonin-permeabilized 143B cells exhibited a recovery of ~100% of the exogenous substrate- and TMPD-driven respiration rates of digitonin-permeabilized naive cells, when cytochrome c was provided. In this situation, the removal of the outer membrane also removed the barrier to substrate uptake. Therefore, there was no evidence of any defect in complex I- or complex III-dependent respiration.
The loss of cytochrome c from mitochondria in digitonin-permeabilized cells was subsequently directly confirmed by confocal immunofluorescence microscopy as had been done previously for Jurkat cells treated with anti-Fas antibodies and digitonin (24). This loss of outer mitochondrial membrane integrity occurred at a concentration of digitonin that had no effect on normal mitochondria, indicating that the phenomenon took place in cells in which the outer mitochondrial membrane had been "primed" for apoptosis.
At 4 h after the addition of staurosporine, when permeabilization of 143B cells with digitonin led to loss of cytochrome c from mitochondria in at least 75% of the cells, no other parameter of apoptosis progression was positive in such a large proportion of the cell population (Fig. 1). In particular, at 4 h after staurosporine addition, only 12% of intact cells showed an extramitochondrial localization of cytochrome c.
The most likely interpretation of the digitonin hypersensitivity observed in this work is that the outer membrane of mitochondria underwent, very early in apoptosis, a structural change. Such a structural change might have involved a change in lipid composition or distribution that made its integrity sensitive to 0.005% digitonin. This structural change may be the "priming" event for apoptosis referred to by Hájek et al. (24). An alternative interpretation of the digitonin effect, namely that the digitonin permeabilization may have caused the release of transacting apoptogenic factors from cells more advanced in the apoptotic program, thereby triggering cytochrome c release from mitochondria in the less advanced cells, was substantially excluded in the present work by the result of an experiment we performed in which the digitonin permeabilization was carried out in a highly diluted cell suspension.
An important observation in the present work was that Bcl-2-overexpressing 143B cells exhibited the same early decrease in endogenous respiration after staurosporine treatment. This finding indicated that the change in outer membrane permeability occurred upstream of the step affected by Bcl-2 activity or in an independent pathway. In Bcl-2-overexpressing cells, there was no release of cytochrome c from mitochondria, underlining the fact that the decrease in endogenous respiration is not dependent on cytochrome c release.
In contrast to 143B cells, mitochondria of the B15 Bcl-2-overexpressing cells exposed to staurosporine for 6 h and permeabilized with digitonin exhibited a 40% decrease in the rate of glutamate/malate-driven respiration, very similar to the ~35% decrease in the rate of endogenous respiration observed in intact cells. These results are consistent with the interpretation that the outer mitochondrial membrane in digitonin-permeabilized B15 cells is intact, and that in ~40% of the cells, it is refractory to uptake of substrates (Fig. 9). The lack of effect of added cytochrome c on the respiration rates in digitonin-permeabilized B15 cells provided further support for this interpretation.
What is the change in the outer mitochondrial membrane that occurs early in the program of apoptosis and renders the membrane refractory to uptake of substrates? The passage of substrates, other metabolites, and small molecules through the outer membrane takes place through the VDAC (or porin) (41, 42), an abundant mitochondrial protein. Furthermore, the permeability of VDAC is regulatable (43-45). Thus, the closure of VDAC in a fraction of the cells exposed to staurosporine would restrict respiration driven by oxidizable substrates, such as malate and succinate. On the other hand, the entry of TMPD, which is membrane-permeant, into mitochondria would not be inhibited by a closed VDAC, and TMPD-driven respiration would be expected to be unaffected by VDAC closure. Indeed, in 143B and B15 cells treated with staurosporine for 2-6 h, which exhibited a 20-35% decrease in endogenous respiration rate, the TMPD-driven respiration rate was found to be the same as in untreated cells.
Thompson and colleagues (8, 46) have proposed previously that VDAC closes in apoptosis, on the basis of experiments showing that the outer mitochondrial membrane becomes less permeable to ADP in lymphoblastoid cells that have been deprived of growth factor IL-3. Our results are in agreement with theirs on this point. However, they reported that overexpression of Bcl-2 or Bcl-XL prevents this decrease in outer membrane permeability, whereas we find that overexpression of Bcl-2 has no such effect. This discrepancy may be due to differences in the level of expression of Bcl-2 or to differences in the apoptotic pathway in the two systems.
The evidence that VDAC closure, as concerns ADP/ATP exchange, occurs in
lymphoblastoid cells induced to undergo apoptosis by interleukin
withdrawal (46) provides strong support for the idea that the early
loss of exogenous substrate-dependent respiration in
staurosporine-treated cells represents another manifestation of VDAC
closure. The early decrease in respiration in a different cell
type treated with staurosporine (PC12)3 and the decrease in
complex I- and complex III- dependent respiration rates in a mouse cell
line treated with tumor necrosis factor- (40) may likewise be due to
VDAC closure.
As concerns the question of whether the hypersensitivity to digitonin detected here in staurosporine-treated cells is related to apoptosis, the previous evidence of a similar phenomenon in a completely different apoptotic system (Fas-induced Jurkat cells) strongly supports the conclusion that this hypersensitivity is an important indicator of an apoptosis-induced priming of the outer mitochondrial membrane. On a technical note, the hypersensitivity to digitonin that we have observed indicates that caution should be used in interpreting results from experiments in which digitonin has been used to permeabilize or lyse cells undergoing apoptosis or in the preparation of a mitochondrial fraction from such cells.
In summary, we have provided evidence that points to changes in the
outer mitochondrial membrane that occur very early in the apoptotic
program. A comparison of the data in Figs. 1 and 5a gives an
idea of the order of these changes relative to the hallmark events of
apoptosis. After 4 h of exposure of 143B cells to staurosporine,
the outer mitochondrial membrane in at least 75% of the cells has
undergone a change in structure that makes it hypersensitive to
digitonin (Fig. 5a). At the same time, endogenous substrate-dependent respiration has decreased by 20-25%,
12% of the cells have released cytochrome c from
mitochondria, and fewer than 10% of the cells have progressed to the
stage of having fragmented nuclei (Fig. 1). These data suggest the
following sequence. Very early, after the apoptotic stimulus, the outer
membrane of mitochondria undergoes a structural change, becoming primed
for apoptosis. This change is detectable so far only in
vitro, as a hypersensitivity to digitonin. Next, the outer
membrane becomes impermeable to oxidizable substrates and ADP,
presumably because VDAC closes. This step is followed by cytochrome
c release from mitochondria, and lastly, by nuclear
degradation. It seems likely that staurosporine-treated cells that have
released cytochrome c from mitochondria represent a subgroup
of those in which the outer membrane permeability has been altered
(presumably those in which the effect of VDAC closure are more
advanced). Vander Heiden et al. (46) have suggested that
VDAC closure in lymphoblastoid cells deprived of IL-3 leads to
hyperpolarization of the mitochondrial inner membrane, swelling of the
matrix, and consequent rupture of the outer membrane and release of
cytochrome c. Further work is needed to clarify which effects of VDAC closure in staurosporine-treated cells may be crucial
for the release from mitochondria of apoptogenic molecules, in
particular, of cytochrome c.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Stanley Korsmeyer and
David Hockenbery for the gift of the Bcl2- cDNA and to Sandoz
Research Institute for the gift of cyclosporine A. We are grateful also
to Dr. Svetlana Lyapina for advice on calcium phosphate-mediated
transfection of cells, to Dr. Elisabetta Ferraro for suggesting the
experiment with Bis-I, and to Dr. Gaetano Villani for helpful
discussions. We also thank Benneta Keeley, Arger Drew, Rosie Zedan,
Elisa Chan, and Huamei Xu for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM-11726 (to G. A.) and a Gosney Fellowship (to P. H.).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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: E-mail: chomyn@caltech.edu.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M209269200
2 E. Ferraro and G. Attardi, personal communication.
3 E. Ferraro and G. Attardi, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
VDAC, voltage-dependent anion channel;
Bis-I, bisindolylmaleimide
GF 109203X;
CsA, cyclosporine A;
DAPI, 4,6-diamidino-2-phenylindole;
DNP, dinitrophenol;
PBS, phosphate-buffered saline;
zVADfmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone;
TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine;
143B, 143B.TK cells;
TD, Tris deficient (in
Ca2+, Mg2+).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Cell 86, 147-157[Medline] [Order article via Infotrieve] |
2. | Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., and Kroemer, G. (1999) Nature 397, 441-446[CrossRef][Medline] [Order article via Infotrieve] |
3. | Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve] |
4. | Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve] |
5. | Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R. (2001) Mol. Cell. 8, 613-621[Medline] [Order article via Infotrieve] |
6. | Jacobson, M. C., Burne, J. F., King, M. P., Miyashita, T., Reed, J. C., and Raff, M. C. (1993) Nature 361, 365-369[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Dey, R.,
and Moraes, C. T.
(2000)
J. Biol. Chem.
275,
7087-7094 |
8. | Vander Heiden, M. G., Chandel, N. S., Schumacker, P. T., and Thompson, C. B. (1999) Mol. Cell. 3, 159-167[Medline] [Order article via Infotrieve] |
9. | Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467[Medline] [Order article via Infotrieve] |
10. | Hirsch, T., Marzo, I., and Kroemer, G. (1997) Biosci. Rep. 17, 67-76[Medline] [Order article via Infotrieve] |
11. |
Leist, M.,
Single, B.,
Castoldi, A. F.,
Kuhnle, S.,
and Nicotera, P.
(1997)
J. Exp. Med.
185,
1481-1486 |
12. | McBreen, P., Orkwiszewski, K. G., Chern, C. J., Mellman, W. J., and Croce, C. M. (1977) Cytogenet. Cell Genet. 19, 7-13[Medline] [Order article via Infotrieve] |
13. | Orkwiszewski, K. G., Tedesco, T. A., Mellman, W. J., and Croce, C. M. (1976) Somatic Cell Genet. 2, 21-26[Medline] [Order article via Infotrieve] |
14. | Orkwiszewski, K. G., Tedesco, T. A., Mellman, W. J., and Croce, C. M. (1976) Cytogenet. Cell Genet. 16, 427-429[Medline] [Order article via Infotrieve] |
15. | King, M. P., and Attardi, G. (1989) Science 246, 500-503[Medline] [Order article via Infotrieve] |
16. | Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. (1990) Nature 348, 334-336[CrossRef][Medline] [Order article via Infotrieve] |
17. | Goldman, L. A., Cutrone, E. C., Kotenko, S. V., Krause, C. D., and Langer, J. A. (1996) BioTechniques 21, 1013-1015[Medline] [Order article via Infotrieve] |
18. | Chen, C. A., and Okayama, H. (1988) BioTechniques 6, 632-638[Medline] [Order article via Infotrieve] |
19. |
Villani, G.,
and Attardi, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1166-1171 |
20. | Granger, D. L., and Lehninger, A. L. (1982) J. Cell Biol. 95, 527-535[Abstract] |
21. | Hofhaus, G., and Attardi, G. (1993) EMBO J. 12, 3043-3048[Abstract] |
22. | Hofhaus, G., Shakeley, R. M., and Attardi, G. (1996) Methods Enzymol. 264, 476-483[Medline] [Order article via Infotrieve] |
23. |
Villani, G.,
Greco, M.,
Papa, S.,
and Attardi, G.
(1998)
J. Biol. Chem.
273,
31829-31836 |
24. |
Hájek, P.,
Villani, G.,
and Attardi, G.
(2001)
J. Biol. Chem.
276,
606-615 |
25. | Chomyn, A. (1996) in Methods in Enzymology, Mitochondrial Biogenesis and Genetics, Part B (Attardi, G. , and Chomyn, A., eds), Vol. 264 , pp. 197-211, Academic Press, San Diego |
26. | Trounce, I. A., Kim, Y. L., Jun, A. S., and Wallace, D. C. (1996) Methods Enzymol. 264, 484-509[Medline] [Order article via Infotrieve] |
27. |
Harkin, S. T.,
Cohen, G. M.,
and Gescher, A.
(1998)
Mol. Pharmacol.
54,
663-670 |
28. |
Toullec, D.,
Pianetti, P.,
Coste, H.,
Bellevergue, P.,
Grand-Perret, T.,
Ajakane, M.,
Baudet, V.,
Boissin, P.,
Boursier, E.,
and Loriolle, F.
(1991)
J. Biol. Chem.
266,
15771-15781 |
29. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Luetjens, C. M.,
Kogel, D.,
Reimertz, C.,
Dussmann, H.,
Renz, A.,
Schulze-Osthoff, K.,
Nieminen, A. L.,
Poppe, M.,
and Prehn, J. H.
(2001)
Mol. Pharmacol.
60,
1008-1019 |
31. | Ruiz-Vela, A., Gonzalez de Buitrago, G., and Martinez, A. C. (2002) FEBS Lett. 517, 133-138[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Waterhouse, N. J.,
Goldstein, J. C.,
von Ahsen, O.,
Schuler, M.,
Newmeyer, D. D.,
and Green, D. R.
(2001)
J. Cell Biol.
153,
319-328 |
33. |
Bossy-Wetzel, E.,
Newmeyer, D. D.,
and Green, D. R.
(1998)
EMBO J.
17,
37-49 |
34. | Fournier, N., Ducet, G., and Crevat, A. (1987) J. Bioenerg. Biomembr. 19, 297-303[Medline] [Order article via Infotrieve] |
35. | Crompton, M., Ellinger, H., and Costi, A. (1988) Biochem. J. 255, 357-360[Medline] [Order article via Infotrieve] |
36. |
Broekemeier, K. M.,
Dempsey, M. E.,
and Pfeiffer, D. R.
(1989)
J. Biol. Chem.
264,
7826-7830 |
37. | Han, Z., Pantazis, P., Lange, T. S., Wyche, J. H., and Hendrickson, E. A. (2000) Cell Death Differ. 7, 521-530[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Yang, J.,
Liu, X.,
Bhalla, K.,
Kim, C. N.,
Ibrado, A. M.,
Cai, J.,
Peng, T. I.,
Jones, D. P.,
and Wang, X.
(1997)
Science
275,
1129-1132 |
39. | Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I., and Green, D. R. (2000) Nat. Cell. Bio. 2, 156-162[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Schulze-Osthoff, K.,
Bakker, A. C.,
Vanhaesebroeck, B.,
Beyaert, R.,
Jacob, W. A.,
and Fiers, W.
(1992)
J. Biol. Chem.
267,
5317-5323 |
41. | Colombini, M. (1979) Nature 279, 643-645[Medline] [Order article via Infotrieve] |
42. | Colombini, M. (1980) Ann. N. Y. Acad. Sci. 341, 552-563[Abstract] |
43. | Colombini, M. (1987) J. Bioenerg. Biomembr. 19, 309-320[CrossRef][Medline] [Order article via Infotrieve] |
44. | Liu, M. Y., and Colombini, M. (1992) Biochim. Biophys. Acta 1098, 255-260[Medline] [Order article via Infotrieve] |
45. |
Zizi, M.,
Forte, M.,
Blachly-Dyson, E.,
and Colombini, M.
(1994)
J. Biol. Chem.
269,
1614-1616 |
46. |
Vander Heiden, M. G.,
Chandel, N. S., Li, X. X.,
Schumacker, P. T.,
Colombini, M.,
and Thompson, C. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4666-4671 |