From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, California 92037
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INTRODUCTION |
Apoptosis comprises a series of events that occur in response to
the activation of a program found in most cells whose purpose is to
kill the cell without releasing its contents into the extracellular environment. The events of apoptosis usually include among other things
the breakdown of the genome into nucleosomal fragments (i.e.
fragments that are integral multiples of ~200 base pairs); the
activation of the caspases, a family of proteases that
cleave a limited set of proteins (e.g. poly(ADP)ribose
polymerase, lamin B, fodrin) on the C-terminal side of an aspartate
residue; the cross-linking of actin, and the appearance of
phosphatidylserine on the cell surface. Ultimately the apoptotic cell
sheds its substance by blebbing, and the released fragments are taken
up and degraded by mononuclear phagocytes.
The involvement of mitochondria in apoptosis was first suggested by the
discovery that the anti-apoptotic protein Bcl-2 is found in the
mitochondrial outer membrane (1, 2). A later report attributed
apoptosis to the release of apoptosis-inducing factor from the
mitochondrial intermembrane space because of the opening of the
mitochondrial permeability transition pore in cells undergoing
apoptosis (3). We ourselves found that cytoplasm from apoptotic cells
contains cytochrome c inactivating factor of apoptosis, a
substance that within minutes eliminates the ability of cytochrome
c to transfer electrons to cytochrome oxidase (4). Most
recently it was shown that cytochrome c, in collaboration with APAF-1, the mammalian homolog of the Caenorhabditis
elegans death protein Ced-4, assists in the activation of the
caspases (5, 6).
There is, however, an unsettled question concerning the participation
of cytochrome c in apoptosis. Several laboratories have reported that during apoptosis, some of the cytochrome is released from
the mitochondria into the cytosol where it exerts its effect on the
caspases (7, 8). We, however, have reported that the cytochrome remains
associated with the mitochondria during apoptosis (4, 9). For our study
we measured cytochrome c spectrophotometrically, but others
measured the cytochrome by immunoblotting. In hopes of reconciling
these findings, we re-examined the distribution of cytochrome
c in apoptotic cells using immunoblotting to quantify the
cytochrome.
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MATERIALS AND METHODS |
Anti-Fas antibody (clone CH-11) was purchased from Kamiya
Biomedicals, Thousand Oaks, CA, anti-cytochrome c antibody
from Pharmingen, San Diego, and F(ab')2 anti-mouse and anti-rabbit (human-adsorbed) alkaline phosphatase-conjugated secondary antibodies from Caltag, Burlingame CA. Jurkat cells were obtained from the American Type Culture Collection. The cells were grown in RPMI 1640, 5% fetal calf serum, 2 mM glutamate to a density of
106 cells/ml. For use, they were pelleted and resuspended
in serum-free medium at 4 × 107 cells/ml, then
incubated at 37 °C with or without 0.5 µg/ml anti-Fas IgM for the
indicated lengths of time. The cells were then washed in ice-cold
Dulbecco's phosphate-buffered saline, then divided into 2 groups, one
for disruption by N2 cavitation and the other for
disruption by homogenization in a Potter-Elvehjem homogenizer with a
Teflon pestle. All subsequent steps were carried out on ice or at
4 °C. For N2 cavitation, the cells were
washed with MA buffer (100 mM sucrose, 1 mM
EGTA, 20 mM MOPS (pH 7.4), bovine serum albumin 1 g/liter)
then resuspended at 2 × 108 cells/ml in MB buffer (MA
buffer plus 10 mM triethanolamine, 5% Percoll, and an
antiproteinase mixture consisting of aprotinin, pepstatin A, and
leupeptin, each at 10 µM, and 1 mM
phenylmethylsulfonyl fluoride). The cells were then disrupted by
N2 cavitation as described elsewhere. For homogenization,
the cells were pelleted and resuspended at 2 × 108
cells/ml in homogenization buffer (250 mM sucrose, 20 mM K+HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride), and disrupted by
homogenization by the method of Yang et al. (8). Lysates
prepared by either method were centrifuged twice at 2500 × g for 5 min to remove nuclei and unbroken cells then at
25,000 × g for 30 min to isolate mitochondria. The
post-mitochondrial supernatant was spun at 100,000 × g
for 30 min to yield cytosol, which was saved, and membrane particles,
which were discarded. The mitochondria were suspended in MC buffer
(identical to MA buffer except that the sucrose concentration was 300 mM and an antiprotease mixture was included that consisted
of aprotinin, pepstatin A, and leupeptin, each at 10 µM)
and used for measuring oxygen consumption and cytochrome c
content. In the preparation of mitochondria, all steps were carried out
at 4 °C.
To quantify apoptosis, cells were fixed with 4% formalin in
phosphate-buffered saline, stained with acridine orange, and then evaluated for nuclear condensation and fragmentation under a
fluorescent microscope as described previously (10). Apoptosis was
determined before the cells were divided into 2 groups for disruption.
Integrity of the outer mitochondrial membrane was determined by
measuring cytochrome oxidase activity using reduced cytochrome c as substrate as described elsewhere (4, 11) but in the absence and presence of digitonin. Cytochrome c oxidation
was followed for the first 3 min of the reaction, an interval during which the reaction rate was constant.
The cytochrome c content of the supernatants and
mitochondria was analyzed by immunoblotting. Cytosol (100 µg) and
mitochondria (25 or 50 µg) were resolved by SDS-polyacrylamide gel
electrophoresis (15% gels, using Laemmli buffers) (12), blotted onto
nitrocellulose, and blocked with BLOTTO. Blots were probed with a
1:1000 dilution of anti-cytochrome c antibody followed by a
secondary antibody conjugated to alkaline phosphatase, and developed
with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
and nitro blue tetrazolium chloride. The blots were then scanned using
a Hewlett-Packard Scan-Jet 4C/T, and the cytochrome c bands
on the scans quantified on a Molecular Dynamics PhosphorImager SI using
ImageQuant software.
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RESULTS |
We felt that it was important not only to try to repeat our own
results, but also to see if we could reproduce the results obtained in
other laboratories. One difference between our work and that of other
groups was the method used for preparing mitochondria. Our method
involved disrupting the cells by nitrogen cavitation in a buffer free
of potassium, whereas the other groups used homogenization in a
potassium-containing buffer to prepare their mitochondria. To see if
these methodological differences might account for the differences in
results among the various laboratories, we disrupted control and
apoptotic cells by both methods and examined the cytochrome c content of the particulate and cytosolic fractions by
immunoblotting. The results (Fig. 1 and
Table I) indicated that at 2 h, when almost half the anti-Fas-treated cells showed apoptotic nuclei, there
was no significant difference between the cytochrome c
content of control and apoptotic cytosols obtained by nitrogen
cavitation, whereas apoptotic cytosols from homogenized cells
consistently contained more cytochrome c than the
corresponding control cytosols.

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Fig. 1.
A representative immunoblot of cytochrome
c in mitochondria and cytosol from normal and apoptotic
cells disrupted by cavitation or homogenization. The experiment
was carried out as described in the text. These results are
representative of three separate experiments performed on different
days. MIT, mitochondria; CSL, cytosol.
Fas, treated with anti-Fas IgM; Con,
control.
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Table I
Cytochrome c in cytosols from control and apoptotic cells
disrupted by N2 cavitation or homogenization
Apoptosis was measured in the cell populations before they were divided
and subjected to disruption. Results are presented as the mean ± S.D. of n experiments. Volumes of the control bands ranged between
234-1725 and 21-116 PhosphorImager units for cytosols and
mitochondria, respectively. Statistical significance of the differences
in cytochrome c distribution in control vs.
apoptotic samples was calculated by Student's t test. On
the scans, the amount of cytochrome c in the homogenized
control mitochondria exceeded the amount in the cavitated control
mitochondria by a factor of 1.5 ± 0.9 (n = 3;
p > 0.1).
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To determine whether the release of cytochrome c from
mitochondria prepared from homogenized cells was a result of the
homogenization procedure or of the relatively minor differences in the
composition of our buffer versus the buffers used in the
other studies, we examined cytochrome c release in cells
homogenized in each of the two buffers. The results (Table
II) showed that the amounts of cytochrome
c released into the cytosol of anti-Fas-treated cells were
similar in the two buffers, suggesting that the difference in
cytochrome c release between the cavitated and homogenized samples was because of the cell disruption procedure rather than the
buffers.
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Table II
Cytochrome c in cytosols from control and apoptotic cells disrupted
by homogenization in two different buffers
The experiments were carried out as described above, and are presented
as the mean ± S.E. of three separate determinations using cytosol
from different cells. Volumes of the control bands ranged from 316 to
2,025 PhosphorImager units.
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At later times, the release of a small amount of cytochrome
c into the cytosol during apoptosis was seen even in cells
disrupted by nitrogen cavitation. Table
III shows the amount of cytochrome c in cavitated cytosols from cells treated for 2 and 4 h with anti-Fas, as compared with cytochrome c from control
cells incubated for similar lengths of time. The results showed that
cytochrome c levels were similar in the 2 h cytosols,
but that at 4 h the amounts of cytochrome c in
apoptotic cytosols exceeded that in the control cytosols. At 6 h,
however, cytochrome c had largely disappeared from the
mitochondria of apoptotic cells and was undetectable in their cytosol,
though the cytochrome c distribution in the control cells
was unchanged from earlier times (not shown).
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Table III
Cytochrome c in cytosols in control and apoptotic cells as a
function of time
Experiments were carried out as described in the text and are presented
as the mean ± S.E. of three independent experiments. Volumes of
the control bands ranged from 235 to 1,178 PhosphorImager units.
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Finally, experiments were carried out to assess the integrity of the
outer membrane in mitochondria prepared from nitrogen-cavitated or
homogenized cells. This was accomplished by determining whether exogenous cytochrome c had access to cytochrome oxidase, as
measured by the ability of the cytochrome to pass electrons to
cytochrome oxidase. The results (Table
IV) showed that the oxidation of reduced cytochrome c by mitochondria from nitrogen-cavitated cells
was barely detectable in the absence of digitonin, a steroid that permeabilizes the outer mitochondrial membrane, but was brisk when
digitonin was present in the assay mixture. In contrast, mitochondria
from homogenized cells were able to oxidize cytochrome c in
the absence of digitonin at more than 50% the rate seen in the
presence of digitonin. These findings indicate that in the nitrogen-cavitated mitochondria the outer membranes were essentially intact, although they were extensively disrupted in the homogenized mitochondria.
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Table IV
Oxidation of cytochrome c by mitochondria in the absence and
presence of digitonin
Assay mixtures contained 2.3 to 21.5 mg of mitochondria protein, 25 nmol of reduced cytochrome c and MES buffer (0.1 M MES, pH 6.0, 10 µM EDTA), plus or minus 5 µl of 10% (w/v) digitonin, in a total volume of 800 µl. The
experiments were carried out as described above. The N2
cavitation experiments are presented as the mean ± S.E. of three
separate determinations using mitochondria from different cells. In two
of the three assays with N2-cavitated cells, cytochrome
c oxidation could not be detected in either the control or
anti-Fas mitochondria in the absence of digitonin. The remaining
experiments are presented as the mean ± S.E. of the averages of
two assays, each carried out in duplicate and each using mitochondria
from different cells. Permeability is presented as the mean ± S.E. of the activity in the absence of digitonin as a fraction of the
activity in the presence of digitonin, expressed as a percent. These
means were determined by averaging the fractions calculated from each
separate experiment.
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DISCUSSION |
The foregoing results suggest that at least in anti-Fas-treated
Jurkat cells, cytochrome c remains associated with the
mitochondria during the first 2 h of apoptosis, by which time
nearly 50% of the cells show the nuclear changes of apoptosis. Despite
these results, our earlier findings and the findings from other
laboratories clearly indicate that the interaction between cytochrome
c and the other mitochondrial components is altered in some
fundamental way in apoptotic cells. This is shown both by the
defunctionalization of the cytochrome in apoptotic mitochondria and by
the greater ease with which the cytochrome is displaced from such
mitochondria as compared with its behavior in control mitochondria, as
indicated by the results obtained when the cells were homogenized
instead of cavitated. Elucidating the basis for this alteration is
likely to lead to novel insights into the mechanism of apoptosis.
It is clear that in a cell-free system under appropriate conditions,
cytochrome c is able to activate caspases, implying that it
can perform this function in intact cells undergoing apoptosis. The
results reported here are not in conflict with this idea. Our results
do suggest, however, that in our system, cytochrome c-dependent caspase activation has to take place
in the mitochondrial intermembrane space. For this to occur, it is
likely that an apoptosis-dependent change in the properties
of the outer mitochondrial membrane must take place so that APAF-1 and
the procaspases can gain access to the cytochrome. Consistent with this
idea, we have reported an increase in the permeability of the outer
membrane of mitochondria isolated from apoptotic cells, whereas Wang
and co-workers found that during apoptosis, the serine-threonine kinase
Raf is transferred to the outer mitochondrial membrane (13). Further
studies of the outer membrane of apoptotic mitochondria are currently
in progress in our laboratory.
We thank Grant Meisenholder and Chris
Yokoyama for superb technical assistance.