From the Division of Biology, California Institute of Technology, Pasadena, California 91125
Received for publication, August 29, 2000
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ABSTRACT |
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In the present work, Jurkat cells
undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited
in increasing proportion a massive release of cytochrome c
from mitochondria, as revealed by double-labeling confocal
immunofluorescence microscopy. The cytochrome c release was
followed by a progressive reduction in the respiratory activity of the
last respiratory enzyme, cytochrome c oxidase (COX), and
with a little delay, by a decrease in overall endogenous respiration
rate, as measured in vivo in the whole cell population.
Furthermore, in vivo titration experiments showed that an
~30% excess of COX capacity over that required to support endogenous
respiration, found in naive cells, was maintained in anti-Fas-treated
cells having lost ~40% of their COX respiratory activity. This
observation strongly suggested that only a subpopulation of
anti-Fas-treated cells, which maintained the excess of COX capacity,
respired. Fractionation of cells on annexin V-coated paramagnetic beads
did indeed separate a subpopulation of annexin V-binding apoptotic
cells with fully released cytochrome c and completely
lacking respiration, and a nonbound cell subpopulation exhibiting
nearly intact respiration and in their great majority preserving the
mitochondrial cytochrome c localization. The above findings
showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step
preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring
anti-Fas-treated cells, but not in naive cells, indicated that the
cells responding to digitonin had already been primed for apoptosis,
and that this treatment bypassed or accelerated the rate-limiting step
most probably at the level of the mitochondrial outer membrane.
Recent studies on apoptosis, a highly controlled form of cell
death triggered by a variety of stimuli, have demonstrated that mitochondria function as a common regulator of apoptotic
self-destruction (1). In particular, mitochondria serve as a reservoir
of apoptogenic molecules, such as cytochrome c (2, 3),
apoptosis-inducing factor (4), and Smac/Diablo (5, 6), and their
release from these organelles is controlled by many of pro- and
anti-apoptotic Bcl-2 family proteins (6-8). Cytochrome c,
when released from the mitochondrial intermembrane space into the
cytosol, participates, together with Apaf-1 and pro-caspase-9, in
activation of the apoptotic protease cascade (9). The mechanism of
cytochrome c release is largely unknown, though several
models have been proposed (1, 10, 11).
As cells progress through apoptosis, mitochondria undergo many changes.
Specifically, mitochondrial alkalization and swelling, loss of
electrochemical potential across the mitochondrial inner membrane,
outer membrane rupture, and permeability transition have been reported
(1, 10, 12). Although cytochrome c release precedes the loss
of the mitochondrial inner membrane potential in many systems (13-15),
very little is known about the relationship of cytochrome c
release with respiratory changes in the apoptotic cell (8, 10, 16).
Since cytochrome c functions as a mobile electron carrier of
the respiratory chain, it seems plausible to predict that the complete
release of cytochrome c from mitochondria would cause loss
of respiration. Very recently, however, it has been reported that HeLa
cells induced to undergo apoptosis by UV irradiation released rapidly
their cytochrome c into the cytosol, but still maintained an
azide-sensitive membrane potential, indicative of a functional
cytochrome c oxidase
(COX),1 if caspase activation
was blocked (17).
In the present work, measurements of respiration and cytochrome
c localization in intact Jurkat cells induced to undergo
anti-Fas antibody (anti-Fas)-mediated apoptosis and cell sorting
experiments have unambiguously shown a massive release of cytochrome
c associated with a complete loss of COX respiratory
activity and of endogenous respiration in a subpopulation of cells.
This subpopulation increased with time after the apoptotic stimulus,
with nearly all remaining cells exhibiting normal cytochrome
c localization and respiration. These observations have
pointed to the existence of a rate-limiting step preceding cytochrome
c release in the apoptotic cascade. Most significantly, we
obtained strong evidence that this rate-limiting step occurred in cells
already primed for apoptosis. Exogenous cytochrome c
restored the COX respiratory activity in digitonin-treated cells nearly
completely, but, surprisingly, the glutamate/malate- or
succinate-dependent respiration only partially.
Cells and Culture Conditions--
Jurkat cells, a
lymphoblastoma-derived T-cell line (TIB 152, ATCC), were grown in RPMI
1640 medium with 10 mM Hepes, 2 mM L-glutamine, and 10% fetal bovine serum. Individual
cultures were maintained at a cell concentration between
105/ml and 106/ml for no longer than 2 months.
For apoptosis induction, cells were transferred to fresh medium, and,
after 16 h, 50 ng/ml anti-Fas IgM (clone CH-11, Kamiya Biomedical
Co.) was added to a culture containing ~106 cells/ml.
Assessment of Apoptosis--
Cells were fixed on coverslips by
formaldehyde and methanol treatment (as described below), washed in TD
buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM
Tris-HCl, pH 7.4 at 25 °C), and stained with 1 µg/ml dsDNA-binding
fluorochrome 4,6-diamidino-2-phenylindole (DAPI, Sigma) in TD buffer
for 5 min. With DAPI staining, normal cells show homogenous staining of
their nuclei, whereas apoptotic cells show irregular staining as a
result of chromatin condensation and nuclear fragmentation (18). Both
normal and apoptotic nuclei were counted using fluorescence microscopy.
Apoptosis was also determined by fluorescence microscopy of cells
stained with FITC-labeled annexin V (Kamiya Biomedical Co.), according
to the manufacturer's protocol.
Measurements of Respiration Rates in Intact Cells--
Previous
work in this laboratory had shown that the osteosarcoma-derived 143B
TK KCN Titration of COX Activity in Intact Cells and Determination
of COXR(max)--
Cells were resuspended at 1.5-2.0 × 107/ml in TD buffer containing either 17 µM DNP, for KCN titration of "integrated" COX activity, or 17 µM DNP, 20 nM antimycin A, 10 mM ascorbate, and 200 µM TMPD, for KCN
titration of "isolated" COX activity, and transferred into two
chambers connected in parallel, as described (19). If anti-Fas-treated
cells were analyzed, the cell concentration was increased to yield
respiration rates similar to those of naive cells. The KCN titration
measurements and the determination of maximum COX capacity
(COXR(max)), relative to the uncoupled endogenous respiration rate, from the threshold plots (i.e. plots of
relative endogenous respiration rate versus the percentage
of inhibition of isolated COX activity at the same KCN concentration in
DNP-uncoupled intact cells) were performed as described (19).
Confocal Immunofluorescence Microscopy--
Cell culture samples
(in some experiments after digitonin treatment, see below) were
centrifuged onto glass coverslips, and then sequentially incubated in
2% formaldehyde in PBS (140 mM NaCl, 3.8 mM
NaH2PO4, 16.2 mM
Na2HPO4), PBS, anhydrous methanol, PBS, 2%
horse serum in PBS (HSPBS) containing 0.5% Triton X-100 (20). The
coverslips were then incubated with mouse anti-cytochrome c
monoclonal antibody 6H2.B4 (PharMingen), diluted 1:15 in HSPBS, and
rabbit anti-Hsp60 antiserum (StressGen Biotechnologies Corp.), diluted
1:50, for 1 h at 37 °C in a humidified chamber. After three
washes in HSPBS, the coverslips were incubated with 1:50-diluted FITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch
Laboratories) and 1:100-diluted lyssamine-rhodamine-conjugated goat
anti-rabbit IgG (Jackson Immunoresearch Laboratories) for 1 h at
room temperature. After three washes in PBS, the coverslips were
mounted onto microscope slides in FluoroGuard antifade reagent
(Bio-Rad), and analyzed on a Zeiss 310 laser-scanning microscope
equipped with 488-nm argon and 543-nm helium neon lasers. Cells with
diffuse cytosolic cytochrome c staining (green) and punctate
mitochondrial Hsp60 staining (red) were counted as cells carrying
cytochrome c released from mitochondria into cytosol. Cells
with punctate cytochrome c staining that overlapped with
Hsp60 staining were counted as cells with mitochondrial cytochrome
c staining.
Magnetic Cell Sorting--
Apoptotic cells were separated from
nonapoptotic cells by magnetic enrichment using the Apoptotic Cell
Isolation Kit (Miltenyi Biotec), according to the manufacturer's
protocol. Cells (4 × 107) were incubated with 80 µl
of annexin V-Microbeads in 400 µl of binding buffer for 15 min at
12 °C and, after a 20-fold dilution with binding buffer,
centrifuged. The cells, resuspended in the same buffer, were then
passed through the magnetic separation column for positive selection
(VS+), which was placed in the magnetic field of the Vario
MACS magnetic separator. The flow-through fraction was centrifuged and
resuspended in TD buffer. After removal of the column from the magnetic
field, the magnetically retained cells were eluted, centrifuged, and resuspended in TD buffer. In a separate experiment, the flow-through fraction of the cell population, manipulated as described above, but
without addition of annexin V-microbeads, was also collected to obtain
the mock-fractionated cell fraction.
Substrate-dependent Respiration in
Digitonin-permeabilized Cells--
In a typical experiment, cells were
resuspended in a measurement buffer at ~1.2 × 107/ml, transferred into the 1.9-ml oxygraphic chamber, and
the cell number was then determined by counting. After taking four
aliquots from the chamber for protein determination, the oxygen
consumption in the presence of DNP (endogenous uncoupled respiration)
was measured. Then, digitonin was added from 5% stock solution in Me2SO to permeabilize the cells. The concentration of
digitonin that, in preliminary tests (data not shown), produced the
highest stimulation of the glutamate/malate-dependent
respiration rate in naive Jurkat cells (5 µg/106 cells)
was used. If not indicated otherwise, two min after the addition of
digitonin a substrate was added to support the respiration. The
glutamate/malate-dependent respiration was measured, in the presence of 17 µM DNP, 5 mM glutamate, and 5 mM malate, in respiration medium I (21) (75 mM
sucrose, 20 mM D-glucose, 5 mM
KPi, 40 mM KCl, 0.5 mM EDTA, 3 mM MgCl2, 30 mM Tris, pH 7.4), as
oxygen consumption that was sensitive to 0.2 µM rotenone.
The respiration medium I was chosen because the
glutamate/malate-dependent respiration rate in
digitonin-permeabilized naive cells in this measurement buffer was 1)
almost constant during the course of experiment, and 2) similar to the
endogenous respiration rate. The succinate-dependent respiration was measured, in the presence of 17 µM DNP or
0.5 mM ADP, 0.2 µM rotenone, and 5 mM succinate, in respiration medium I as oxygen consumption
that was sensitive to 20 nM antimycin A. The
TMPD-dependent respiration was measured in respiration medium II (250 mM sucrose, 20 mM Hepes, 10 mM MgCl2, 2 mM KPi, pH
7.1), since in this measurement buffer only a low rate of
ascorbate/TMPD autooxidation occurred. In this medium, the respiration
rate was determined in the presence of 17 µM DNP, 20 nM antimycin A, 10 mM ascorbate, and 400 µM TMPD, and then corrected by subtracting the
nonspecific oxygen consumption rate due to autooxidation of ascorbate/TMPD in the same buffer. Cytochrome c (Sigma) was
added from a 10 mM stock solution. In this form, cytochrome
c was fully oxidized, since addition of potassium
ferricyanide did not decrease the A550. Reduced
cytochrome c was prepared by addition of few crystals of
sodium hydrosulfite into a 10 mM stock solution of cytochrome c, and stirring for 1 h. Full reduction of
cytochrome c was verified by showing that addition of extra
sodium hydrosulfite did not increase the
A550.
Immunoblot Analysis--
Samples (pellet of intact cells or
pellet and supernatant of digitonin-treated cells) derived from the
same number of cells, as estimated from the amount of total cellular
protein (350 µg), were analyzed by 15% SDS-PAGE. Proteins were then
transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad)
at 150 mA for 12 h in a buffer (0.037% SDS, 20 mM
Tris, 150 mM glycine, 20% methanol, pH 8.2 (25 °C)).
After blocking of nonspecific binding in PBST (0.1% Tween 20, 3%
nonfat milk in PBS) containing 3% bovine serum albumin for 3 h at
room temperature, the membranes were incubated with mouse
anti-cytochrome c monoclonal antibody 7H8.2C12 (PharMingen),
diluted 1:500 in PBS containing 0.05% Tween 20 and 3% nonfat milk,
for 17 h at 4 °C. The membranes, washed three times in PBST,
were incubated with sheep anti-mouse IgG peroxidase-linked (Amersham
Pharmacia Biotech), diluted 1:1000 in PBST, for 2 h at room
temperature. The membranes were washed five times in PBST, and specific
protein complexes were identified using the SuperSignal West Pico
chemiluminescence reagent (Pierce) by autoradiography.
Preparation of Digitonin Supernatants--
Cells were washed in
respiration medium I, counted, resuspended in the same medium at
107/ml, treated with digitonin (5 µg/106
cells) for 7 min at 37 °C, and centrifuged at 400 × g for 5 min. The resulting supernatant (digitonin
supernatant) was then transferred to the oxygraphic chamber.
Cellular Mosaicism in Massive Release of Mitochondrial Cytochrome
c--
Since the determination of cytochrome c release from
mitochondria based on immunoblotting of mitochondrial and cytosolic
fractions prepared from cellular homogenates have led to different
conclusions even in the same system (22-27), in the present work,
double-labeling confocal immunofluorescence microscopy was used to
analyze in situ this phenomenon in apoptotic Jurkat cells.
The majority of the cells treated for 4 h with anti-Fas antibody
exhibited diffused cytosolic cytochrome c staining, while
staining of Hsp60, located in the mitochondrial matrix, was punctate
(Fig. 1). In contrast, in untreated
(naive) cells or in cells treated with anti-Fas antibody and the
caspase inhibitor z-Val-Ala-Asp(OMe)-CH2F (z-VADfmk,
Kamiya), cytochrome c staining was punctate and colocalized
with the Hsp60 staining. These results confirmed earlier observations
(23, 24, 26), made by immunoblotting experiments, that, in Jurkat cells
treated with anti-Fas antibody, cytochrome c is released in
a caspase-dependent manner from mitochondria into the
cytosol. Furthermore, the physical integrity of the inner mitochondrial membrane was preserved under these conditions, as indicated by the
behavior of Hsp60. In addition, an analysis of multiple optical sections of individual cells anti-Fas-treated for 4 h showed that the majority of the cells displayed either only punctate mitochondrial cytochrome c staining, or diffused cytosolic cytochrome
c staining without detectable mitochondrial staining (data
not shown). These observations indicated the existence of a mechanism
for a rapid cytochrome c release from all mitochondria of
individual cells, as recently reported for HeLa cells induced to
undergo apoptosis by UV irradiation or staurosporine treatment (17).
Furthermore, there was clearly a marked cellular heterogeneity in
cytochrome c release from mitochondria (Fig. 1).
Decrease in Endogenous and TMPD-dependent Respiration
in Vivo--
To investigate the possible effects of cytochrome
c release on respiration, the rate of oxygen consumption in
naive Jurkat cells and in cells induced to undergo apoptosis by
anti-Fas antibody was measured both in the absence and in the presence
of the uncoupler DNP. Fig. 2A
presents the results of a representative experiment showing that Jurkat
cells, treated with anti-Fas antibody for increasing time periods,
exhibited a progressively lower rate of both uncoupled and coupled
endogenous respiration. The uncoupled respiration rate decreased to
~62% of the rate of naive cells after 4 h of treatment, ~53%
after 6 h, and ~43% after 8 h. The uncoupled endogenous
respiration of both naive and anti-Fas-treated cells was 98% antimycin
A-sensitive. The progressive decrease in endogenous respiration rate
correlated well with an increase in number of apoptotic cells, which
exhibited a characteristic shrunk and fragmented appearance of their
nuclei, as determined by staining with the dsDNA-binding fluorochrome
DAPI (Fig. 2A).
To dissect further the anti-Fas-induced changes in respiration, the
effect of anti-Fas antibody on COX respiratory activity of intact cells
was measured. The rate of oxygen consumption, in the presence of the
membrane-permeant electron donor TMPD, of ascorbate as primary reducing
agent, and of antimycin A to block the electron flux upstream of COX,
is known to depend on both cytochrome c and COX, providing a
measure of COX-dependent oxygen consumption that is
isolated from the upstream segment of the respiratory chain (19). Fig.
2A shows that the TMPD-dependent respiration
rate decreased progressively with increasing time of cell treatment
with anti-Fas antibody. The kinetics of decrease in
TMPD-dependent respiration rate during the first 8 h
of treatment was very similar to the kinetics of decrease in endogenous
uncoupled respiration rate. However, it was apparently faster in the
first few hours. In fact, a comparison of the kinetics of decrease in the endogenous and TMPD-dependent respiration rates in the
first 3 h after anti-Fas-induction revealed that the reduction in
endogenous respiration rate was slightly, but significantly, delayed
with respect to the decrease in TMPD-dependent respiration
rate. In particular, as shown by a representative experiment in Fig.
2B, after 1 h of anti-Fas-induction, the endogenous
respiration rate was almost unchanged, whereas the
TMPD-dependent respiration rate was decreased by ~12%
relative to the control. It was also found that z-VADfmk fully
prevented the anti-Fas-induced decrease in endogenous coupled,
endogenous uncoupled, and TMPD-dependent respiration rates
(Fig. 2C), indicating a requirement for caspase activation in anti-Fas-triggered loss of respiration in intact cells.
Release of Cytochrome c Precedes Loss of Respiration--
In Fig.
3, the changes in respiration in
apoptotic Jurkat cells were correlated with the kinetics of cytochrome
c release. Quantification of cytochrome c release
from images such as those shown in Fig. 1 revealed that about 25% of
the cells released massively cytochrome c into the cytosol
after less than 1 h of treatment with anti-Fas antibody (Fig. 3).
After 2 h of treatment, 58% of the cells had released cytochrome
c, while the endogenous uncoupled and
TMPD-dependent respiration rates had decreased only by 20 and 22%, respectively, relative to those measured in naive cells.
After 4 h of treatment, 71% of cells had released cytochrome c, while the endogenous uncoupled and
TMPD-dependent respiration rates had decreased only by 40%
and 39%, respectively. Cytochrome c remained
mitochondria-localized in ~20% of the cells even after 8 h of
treatment with anti-Fas antibody, when the uncoupled and TMPD-dependent respiration rates were decreased by ~60%
and ~57%, respectively (Fig. 3). The faster kinetics of increase in
the percentage of cells with released cytochrome c and the
faster kinetics of respiration loss during the first 4 h indicated
the presence of a subpopulation of cells responding faster to the apoptotic stimulus. These results also suggested a sequence of events
in which loss of cytochrome c from a cell would precede a
decrease in COX-dependent oxygen consumption and endogenous respiration. The observation mentioned above of a slight, although apparently significant, delay in the kinetics of decrease in the endogenous respiration rate relative to the kinetics of decrease in
TMPD-dependent respiration rate could reflect an excess of COX capacity over that required to support the normal endogenous respiration. In fact, recent studies have demonstrated that, in a
variety of human cell types analyzed, including fibroblasts and
myoblasts, there is in vivo a relatively low excess of COX capacity (19, 21). In the present work, the TMPD-dependent respiration rate had been measured in naive uncoupled Jurkat cells, and
found to be ~33% higher than the endogenous uncoupled respiration rate (Fig. 2, A and B, and data not shown). Thus,
it seemed plausible to assume that the release of cytochrome
c from mitochondria would cause initially a decrease in
COX-dependent oxygen consumption, without affecting the
endogenous respiration rate.
Excess of in Vivo COX Capacity of Naive Cells Is Maintained in
Anti-Fas-treated Cells--
To obtain a deeper insight into the role
of the respiratory flux control by COX in the apoptosis-related events,
the relative COX capacity in intact naive Jurkat cells was determined.
For this purpose, the COX activity in DNP-uncoupled intact cells was titrated with the specific COX inhibitor KCN both as isolated step, in
the presence of antimycin A, ascorbate, and TMPD, and as respiratory
chain-integrated step (endogenous respiration) (19). In the low range
of KCN concentrations, the integrated COX activity was less sensitive
to KCN inhibition than the isolated COX activity (Fig.
4A, left
panel), indicating an excess of COX capacity over that
required to maintain a normal endogenous respiration rate. The COX
excess was determined from the threshold plot, i.e. the plot
of the relative endogenous respiration rates of the cells against the
percentages of inhibition of the isolated COX activity at the same KCN
concentrations (Fig. 4A, right panel).
From this plot it could be determined that the maximum COX capacity of
Jurkat cells, relative to the uncoupled endogenous respiration rate
(COXR(max)), was ~1.28. Unexpectedly, the
COXR(max) of Jurkat cells treated for 2 h with
anti-Fas antibody was found to be 1.34 (Fig. 4B), and
remained 1.34 even after 4 h of treatment (data not shown), when
both the endogenous and the TMPD-dependent absolute
respiration rates had decreased to ~60% of the rates found in naive
cells (Fig. 3).
Fractionation of Anti-Fas-treated Cells Reveals a Rate-limiting
Step Preceding Cytochrome c Release--
The kinetics of loss of
cytochrome c and of respiration (Fig. 3), on one hand, and
the maintenance of an excess of COX capacity even after 4 h of
anti-Fas treatment, on the other hand, strongly suggested that
anti-Fas-treated Jurkat cells consist of two subpopulations changing in
relative proportions during the treatment. One of these would have
released cytochrome c massively, and probably completely,
from mitochondria into the cytosol, and consequently would have lost
entirely the capacity to respire. The other subpopulation would, on the
contrary, exhibit normal mitochondrial cytochrome c
localization and respire at a rate similar to that of untreated cells.
To obtain direct evidence for this model, advantage was taken of the
fact that annexin V, a Ca2+-dependent
phospholipid-binding protein, binds to apoptotic cells as a result of
the phosphatidylserine redistribution in the cell membrane (28-30). In
particular, annexin V-coated paramagnetic beads and magnetic sorting on
a column were used to separate cells treated for 4 h with anti-Fas
antibody into two populations, bead-bound and flow-through. These two
populations, two other portions of the anti-Fas-treated cells either
unfractionated or passed through a column without prior exposure to
beads (mock-fractionated), and a naive cell population passed through a
column after exposure to beads were then analyzed for their ability to
bind FITC-labeled annexin V (31), for cytochrome c
localization, for nuclear apoptosis, and for endogenous respiration
(Fig. 5). About 53% and 52% of the
cells in the unfractionated and mock-fractionated anti-Fas-treated cell
populations released cytochrome c into cytosol (Fig.
5A, upper panel), indicating that the
passage of cells not exposed to beads through a column had
substantially no effects on the release of cytochrome c.
Passage of naive cells exposed to beads through a column reduced by
~22% the endogenous respiration rate (Fig. 5A,
lower panel). The observation that an
anti-Fas-treated cell population not exposed to beads and passed
through a column (mock-fractionated) exhibited only a slight reduction
in endogenous respiration rate (~5%) relative to the unfractionated
population (Fig. 5A, lower panel)
strongly suggests that exposure to the beads was responsible for the
effect observed in naive cells.
Significantly, the population of anti-Fas-treated cells that was
retained by the beads was 99% annexin V-positive; 98% of these cells
released cytochrome c into the cytosol, and 94% had apoptotic nuclei (Fig. 5A, upper
panel). Neither endogenous respiration (Fig. 5A,
lower panel) nor TMPD-dependent
respiration (data not shown) was detected in this cell population. By
contrast, the cell population that was not retained by the beads
consisted of only 4% annexin V-positive cells, 13% of cells that
released cytochrome c into the cytosol, and 2% of cells
with apoptotic nuclei. In this population, about ~20% of the cells
that released cytochrome c into cytosol contained some
remaining mitochondria-localized cytochrome c (data not
shown); this is, however, a very small fraction of the total cells
releasing cytochrome c (<3%), supporting the conclusion
that the cytochrome c release from mitochondria in
individual cells is a rapid and, in general, complete process (17).
Interestingly, the endogenous respiration rate of this fraction was
maintained at 87% of the level found in naive cells exposed to the
beads and passed through the column. In the fractionation experiments
described above, the cytochrome c release and loss of
respiration only in a subpopulation of cells clearly revealed the
occurrence of a rate-limiting step preceding cytochrome c release in the anti-Fas-treated cell population.
The recovery of only a portion of the original cell population in the
combined bead-bound fraction (~23%) and flow-through fraction
(~52%), as estimated from the amount of protein associated with
these fractions relative to the protein in the total unfractionated anti-Fas-treated cell population, indicated that about 25% of the
cells were lost during the fractionation (Fig. 5B). When the percentages of cells releasing cytochrome c in the
bead-bound (~23%) and flow-through fractions (~6%) of the
anti-Fas-treated cell population, expressed relative to the total cells
in the unfractionated population (Fig. 5B), are combined and
compared with the percentage of cells releasing cytochrome c
in the unfractionated anti-Fas-treated cell population (~53%), one
can calculate that about 24% of the cells with released cytochrome
c were lost during fractionation on the column. This loss
corresponds well to the overall loss of cells during fractionation of
the original cell population, as estimated from the protein content
(~25%, see above), strongly suggesting that the loss of cells
occurred mainly in the bead-bound fraction. This loss could be due to
degradation of the apoptotic cells during elution and subsequent loss
of the fragments in the subsequent centrifugation, or to incomplete
elution. Fig. 5B also shows that, when the overall
endogenous respiration rates, i.e. not normalized for cell
content (nmol of O2 min Digitonin-enhanced Loss of Respiration and Cytochrome c Release in
Anti-Fas-treated Cells--
To investigate the substrate-specific
respiration in anti-Fas-treated cells, these were treated with the
minimal amount of digitonin that just allowed the maximum
glutamate/malate-dependent, rotenone-sensitive respiration
in naive cells, and respiration was then measured. Surprisingly, we
found that cells treated for 4 h with anti-Fas antibody, which
maintained ~65% of the uncoupled endogenous respiration rate of
naive cells (Fig. 6A), lost
rapidly, after the addition of digitonin, the respiration promoted by
glutamate/malate (Fig. 6A). In fact, 7 min after digitonin
addition, no glutamate/malate-dependent respiration was
detected. In contrast, digitonin-treated naive cells maintained a
nearly constant glutamate/malate-dependent respiration
rate. In another experiment, anti-Fas-treated cells, which maintained
~65% of the uncoupled endogenous respiration rate of naive cells
(Fig. 6B), lost rapidly, after the addition of digitonin,
the respiration promoted by succinate (Fig. 6B). In fact, 7 min after digitonin addition, the succinate-dependent respiration rate was decreased to ~1% of the naive cell rate. In
contrast, digitonin-treated naive cells maintained a nearly constant
succinate-dependent respiration rate. In still other experiment, anti-Fas-treated cells, which maintained ~64% of the uncoupled endogenous respiration rate of naive cells (Fig.
6C), lost rapidly, after the addition of digitonin, the
respiration promoted by TMPD (Fig. 6C). In fact, 12.5 min
after digitonin addition, the TMPD-dependent respiration
rate was decreased to ~9% of the naive cell rate. In contrast,
digitonin treatment had no effect on the TMPD-dependent
respiration rate of naive cells (data not shown).
The unexpected finding of a rapid and complete loss of
glutamate/malatedependent, succinate-dependent, and
TMPD-dependent respiration in digitonin-treated,
anti-Fas-induced cells led us to examine the distribution of cytochrome
c in equally treated cells. Double-labeling confocal
immunofluorescence microscopy of digitonin-treated cells pretreated for
4 h with anti-Fas antibody revealed that nearly all cells had lost
substantially all of their cytochrome c, while the Hsp60
staining intensity did not change (Fig.
7A). There was no major
heterogeneity in cytochrome c and Hsp60 staining among these
cells, in contrast with the obvious cellular heterogeneity in
cytochrome c distribution observed in anti-Fas-treated
intact cells (compare Fig. 7A with Fig. 1). The cytochrome
c staining of digitonin-treated naive cells remained punctate and colocalized with Hsp60 staining in all cells, and quantitatively unaltered. The very low mitochondrial cytochrome c staining of anti-Fas-treated cells remaining 7 min after
the addition of digitonin (Fig. 7A) correlated well with the
undetectable glutamate/malate-dependent respiration rate
and the very low succinate-dependent respiration rate
(~1%) (Fig. 6, A and B). The rapid and
near-complete release of cytochrome c from nearly all
digitonin-treated, anti-Fas-treated cells was confirmed by immunoblot
analysis of the pellet and supernatant fractions of the
digitonin-treated cells. (Fig. 7B). It appears that all
digitonin-treated cells, pretreated for 4 h with anti-Fas antibody, had released most of their cytochrome c outside
the cell, while the digitonin-treated naive cells retained most of their cytochrome c within the mitochondria.
The very rapid and complete loss of cytochrome c and
respiration induced by digitonin in the subpopulation of
anti-Fas-treated cells which still respired normally and maintained a
mitochondrial localization of cytochrome c indicated that
digitonin treatment bypassed or accelerated a rate-limiting step in the
mitochondrial apoptotic process. In the present work, the occurrence of
this rate-limiting step had indeed been suggested by the kinetics of cytochrome c release and respiration loss in the cell
population (Fig. 3) and confirmed by the magnetic sorting experiment
(Fig. 5). In previous studies (20, 22, 32), it had been shown that a
cellular extract or the cytosolic fraction of anti-Fas-treated Jurkat
cells was able to induce mitochondrial apoptotic changes, including a
decrease in TMPD-dependent respiration, in mitochondria isolated from naive cells or in digitonin-treated naive cells. These
observations raised the question of whether, in the present experiments, the proposed bypassing or acceleration of the observed rate-limiting step by digitonin treatment did require priming for
apoptosis of the apparently "normal" anti-Fas-treated cells. To
obtain some information on this question, an experiment was carried out
in which 2.3 × 107 cells anti-Fas-treated for 4 h were mixed with 0.9 × 107 naive Jurkat cells in the
oxygraph chamber and digitonin-treated, and glutamate/malate-promoted
respiration was then measured. As shown in Fig. 6D, the
glutamate/malate-promoted respiration of the mixture of naive and
anti-Fas-treated cells decreased rapidly, although, with an apparently
slower kinetics, when compared with the kinetics of respiration loss
observed in anti-Fas-treated cells alone (Fig. 6A). In fact,
7 min after digitonin addition, the
glutamate/malate-dependent respiration rate was decreased only to ~30% of the naive cell rate (Fig. 6D), if one
considers that anti-Fas-treated cells did not respire at this time
(Fig. 6A). This experiment strongly suggested that some
factor(s) released by the digitonin-treated anti-Fas-induced cells was
able to elicit a loss of glutamate/malate-dependent
respiration in digitonin-treated naive cells.
Reconstitution Experiments Using Magnetic Bead Cell Sorting Reveal
Priming Event in Anti-Fas-treated Cells with Normal
Respiration--
The experiments described above indicated the
possibility that the rate-limiting step of the apoptotic cascade, which
was shown above to occur in the still respiring subpopulation of
anti-Fas-treated cells, may have been bypassed or accelerated, after
digitonin treatment, by a transactivating factor(s) generated in the
apoptotic cells and acting on the still respiring subpopulation of
cells. To distinguish whether the observed loss of respiration after the addition of digitonin was caused by transactivation of nonprimed cells or by some action of digitonin on cells already primed for apoptosis by anti-Fas-treatment, succinate-dependent
respiration was measured in separated, annexin V-nonbinding
(flow-through) subpopulations of cells treated for 4 h with
anti-Fas antibody or of naive cells, either in the absence (Fig.
8A) or in the presence of
digitonin supernatant from naive cells or from the flow-through subpopulation of anti-Fas-treated cells (Fig. 8, B and
C). The flow-through subpopulation of anti-Fas-treated
cells, which exhibited initially ~87% of the endogenous coupled
respiration rate of naive cells treated identically (Fig.
5A, and data not shown) and ~84% of the naive cell
succinate-dependent respiration rate (Fig.
8A), lost rapidly, after the addition of digitonin,
the respiration promoted by succinate both in the absence (Fig.
8A) and in the presence (Fig. 8B) of digitonin
supernatant from naive cells. In fact, 7 min after digitonin addition,
no succinate-dependent respiration was detected (Fig. 8,
A and B). By contrast, the flow-through subpopulation of naive cells maintained, both in the absence (Fig. 8A) and in the presence of digitonin supernatant from the
flow-through subpopulation of anti-Fas-treated cells (Fig.
8B), a nearly constant succinate-dependent
respiration rate during first 10 min after digitonin addition. Thus,
this experiment clearly showed that the apparently normal
anti-Fas-treated cells were already primed, and that these primed cells
underwent, in the presence of digitonin, a rapid and complete loss of
succinate-dependent respiration.
After a prolonged incubation with digitonin supernatant from the
flow-through subpopulation of anti-Fas-treated cells, also the
flow-through subpopulation of naive cells underwent a complete loss of
succinate-dependent respiration. In fact, 21 min after digitonin addition, no succinate-dependent respiration was
measured in these cells (Fig. 8, B and C). By
contrast, the same cells, in the presence of supernatant from naive
cells, maintained throughout a nearly constant
succinate-dependent respiration rate (Fig. 8C). These results showed that nonprimed cells can undergo loss of succinate-dependent respiration by the mechanism of
transactivation, confirming the results described earlier (Fig.
6D). However, the observation that the loss of
succinate-dependent respiration by transactivation in naive
cells was clearly delayed, when compared with the loss of respiration
in the flow-through subpopulation of anti-Fas-treated cells caused by
the action of digitonin in already primed cells, justifies the
conclusion that, in the observed effects of digitonin treatment on the
still respiring subpopulation of anti-Fas-treated cells, the
transactivation phenomenon played only a minor role.
Restoration of Respiration by Exogenous Cytochrome c--
If the
loss of mitochondrial cytochrome c and the resulting
subsequent limitation of this electron carrier in the respiratory chain
of anti-Fas-treated cells was the only cause of the respiration loss,
it should have been possible to restore the respiration by introduction
of exogenous cytochrome c into the organelles. To test this
possibility, anti-Fas-treated cells were treated with limiting amounts
of digitonin in an attempt to introduce exogenous cytochrome
c into the mitochondria. Fig. 6A shows that exogenous cytochrome c (oxidized) partially restored the
glutamate/malate-dependent (rotenone-sensitive)
respiration; in particular, addition of 80 µM cytochrome
c increased the glutamate/malate-dependent
respiration rate from undetectable levels to ~57% of the
glutamate/malate-dependent respiration rate measured in
naive cells. An increase in exogenous cytochrome c up to 300 µM had no further effect on the restoration of
glutamate/malate-dependent respiration (data not shown).
The stimulation of the glutamate/malate-dependent
respiration rate in anti-Fas-treated cells by reduced cytochrome
c was identical to that observed with oxidized cytochrome
c (data not shown). Fig. 6B shows that exogenous
cytochrome c partially restored also the
succinate-dependent (antimycin A-sensitive) respiration; in particular, addition of 80 µM cytochrome c
increased the succinate-dependent respiration rate from
~1% to ~58% relative to the succinate-dependent respiration rate in naive cells. Fig. 6C shows that, in
contrast to the partial restoration of the glutamate/malate- or
succinate-dependent respiration, exogenous cytochrome
c restored nearly completely the KCN-sensitive
TMPD-dependent respiration of anti-Fas-treated cells; in
particular, addition of 80 µM cytochrome c
increased the TMPD-dependent respiration rate from ~9%
to ~92% relative to the TMPD-dependent respiration rate
in naive cells. In contrast to the results obtained with
anti-Fas-treated cells, the glutamate/malate-dependent or
succinate-dependent or TMPD-dependent
respiration in digitonin-treated naive cells was almost unaffected by
the addition of cytochrome c (Fig. 6, A-C).
The glutamate/malate-dependent respiration rate recovered
in digitonin-treated anti-Fas-induced cells in the presence of
exogenous cytochrome c (~57% of the rate in naive cells)
was comparable to the endogenous respiration rate measured in intact
cells anti-Fas-treated for 4 h (corresponding to ~65% of the
rate in naive cells) (Fig. 6A). This observation strongly
suggests that the exogenous cytochrome c was able to restore
completely the portion of respiration that was lost rapidly as a result
of digitonin treatment of anti-Fas-induced cells. If this
interpretation is correct, it seems very plausible that the portion of
respiration lost as a result of the anti-Fas treatment was not
restorable. In addition, the glutamate/malate-dependent or
succinate-dependent respiration restored in
anti-Fas-treated cells in the presence of exogenous cytochrome
c was fully sensitive to antimycin A (Fig. 6, A
and B). This observation indicated that electrons must have
been shuttled to complex IV from complex III, and that, therefore, the
restored respiration was presumably glutamate/malate or, respectively,
succinate-dependent.
In the present work, we have characterized the changes in
respiration occurring in intact Jurkat cells undergoing Fas-mediated apoptosis and correlated these changes with the release of cytochrome c from mitochondria into the cytosol. We found that, in
these cells, the release of cytochrome c from the organelles
preceded and was, presumably, the limiting factor underlying the loss
of respiration. The discrepancy between the kinetics of loss of
cytochrome c and the kinetics of decrease in
TMPD-dependent and endogenous respiration rates (Fig. 3)
could, at least in part, be accounted for by a molar excess of
cytochrome c over COX, such as that which has been shown to
occur in certain cell types (33, 34). The distribution of cytochrome
c in different compartments in mitochondria (35), which has
been proposed on the basis of tomographic studies carried out on these
organelles using high voltage electron microscopy (36), could possibly
also play a role in the present observations.
A dominant feature that has emerged from the present studies is the
cellular heterogeneity that characterizes the involvement of
mitochondria in the apoptotic process. The use of confocal fluorescence
microscopy and cell sorting on annexin V-coated paramagnetic beads and
the determination of COX capacity have allowed the identification and
quantification of a marked cellular mosaicism at the level of 1)
release of cytochrome c from mitochondria, 2) decrease in COX-dependent oxygen consumption and in endogenous
respiration, 3) control of respiration by COX, and 4) cell membrane and
nuclear changes. Thus, we found that loss of cytochrome c
and consequent loss of COX-dependent oxygen consumption and
endogenous respiration in Fas-mediated apoptosis were fairly rapid and
massive processes in individual cells, which occurred heterogeneously
in the Jurkat cell population. These observations argue against the
alternative possibility that the observed progressive decrease in
respiration rate was a consequence of the slow release of cytochrome
c from the mitochondria of every cell in the population.
Although the endogenous and TMPD-dependent respiration
rates were found to decrease as the time of anti-Fas induction
increased, the excess of COX capacity in vivo was shown not
to change significantly with the time of induction for at least 4 h. Of the two subpopulations of anti-Fas-treated cells separated on the
basis of their ability to bind to annexin V-coated beads, that one that
did not bind to these beads maintained almost intact its original
endogenous respiration rate and included only a small percentage of
cells with cytosolic cytochrome c staining. Virtually all
cells that were bound to annexin V-coated beads had released their
cytochrome c from mitochondria and exhibited no endogenous
or TMPD-dependent respiration. These results suggested an
interesting interpretation of the kinetics of the respiratory changes
in individual anti-Fas-treated cells, namely that the cells that kept a
near-to-normal respiration rate maintained also a normal excess of COX
capacity. Furthermore, the observed inability to consume oxygen (even
in the presence of ascorbate and TMPD) of the cells with released
cytochrome c that bound to the annexin V-coated beads
suggested that the cytochrome c released into the cytosol
could not serve as an electron donor for COX. This observation argues
against the possibility, in the present system, of cytosolic cytochrome
c re-entering into the intermembrane space and supporting respiration.
Very little is known about the cellular properties that underlie the
heterogeneous behavior of Jurkat cells in Fas-mediated apoptosis and
about the early biochemical changes leading to this phenotypic cellular
mosaicism. In the present work, the detailed analysis of this mosaicism
has led to the recognition that a step in the apoptotic cascade
preceding cytochrome c release was rate-limiting in the
subpopulation of anti-Fas-treated cells that did not bind to annexin
V-coated beads, which respired normally and had mitochondrial cytochrome c localization. However, we found that controlled
digitonin treatment dramatically enhanced the anti-Fas-triggered
release of mitochondrial cytochrome c and loss of
respiration. The near-to-complete loss of cytochrome c
observed in almost every digitonin-treated, anti-Fas-treated cell
coincided with the complete loss of glutamate/malate- and
succinate-dependent respiration and with the
near-to-complete loss of TMPD-dependent oxygen consumption.
As to the mechanism underlying these phenomena, the evidence obtained
in the present work excludes a major role of transactivation.
Therefore, the most plausible interpretation is that the subpopulation
of apparently normal anti-Fas-treated cells was already primed for
apoptosis, and that, upon digitonin treatment, it acquired the capacity
to rapidly undergo apoptosis-related changes, such as cytochrome c release and loss of respiration. Since these changes were
very rapid, it is likely that the rate-limiting step detected in the present work occurred at the level of primed mitochondria, and that
digitonin accelerated this process by targeting the outer mitochondrial
membrane. It should be mentioned, in this connection, that digitonin
binds to cholesterol, which is present also in the mitochondrial outer
membrane. On the other hand, the immunofluorescence data indicated the
intactness of the inner membrane in digitonin-treated cells.
Digitonin-permeabilized cells have been previously used by others to
study the apoptotic process (17, 22, 32, 37). However, the question of
possible specific effects of digitonin on mitochondria of cells
pre-exposed to an apoptotic stimulus has not been addressed. Our
findings indicate that a re-evaluation should be made of some
conclusions that have been drawn from experiments using cell
permeabilization by digitonin as a tool for the study of cells induced
to apoptosis.
Our experiments on digitonin-treated, anti-Fas-induced cells have also
shown that exogenous cytochrome c restored the
TMPD-dependent oxygen consumption almost to the rate
observed in digitonin-treated naive cells. Considering that the
concentration of added cytochrome c (80 µM)
was lower than the estimated concentration of cytochrome c
in the mitochondrial intermembrane space (100-700 µM)
(38), these observations indicated a full functionality of COX in
Jurkat cells undergoing anti-Fas-triggered apoptosis, in agreement with previous findings (22). In the present work, a significant observation was that, in digitonin-treated cells, in contrast to the near-to-full restoration of the TMPD-dependent oxygen consumption, the
exogenous cytochrome c restored only partially the
glutamate/malate-dependent respiration, which utilizes
complexes I, III, and IV, and the succinate-dependent
respiration, which utilizes complexes II, III and IV. Several
explanations for these observations can be entertained. However, an
intriguing possibility is that either complex III and/or the quinone
pool or the substrate producing machinery are affected during the
apoptotic process. Whether and how the incomplete restoration of
glutamate/malate- or succinate-dependent respiration is
related to the rate-limiting step in the apoptotic cascade identified
in the present work is not known. This question and the other problems
discussed above are presently being further investigated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell line respires in TD buffer at the same rate as in
Dulbecco's modified Eagle's medium lacking glucose (19). Therefore,
the respiration rate was continuously measured in an oxygraph (Yellow Springs Instruments, model 5300) in a suspension at 107
cells/ml in TD buffer, before and after each of the following sequential additions: 17 µM dinitrophenol (DNP), 20 nM antimycin A (Sigma), 10 mM ascorbate + 400 µM N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD, Fluka), essentially as described (19). The concentration of DNP
specified above was one that, in preliminary tests (data not shown),
produced the highest stimulation of the endogenous respiration rate in
naive Jurkat cells. Since ascorbate/TMPD autooxidation causes
significant oxygen consumption, the oxygen consumption rate of
ascorbate and TMPD in the absence of cells was subtracted from the
oxygen consumption rate measured in the presence of cells, DNP,
antimycin A, ascorbate, and TMPD. Oxygen consumption rate was expressed
in nanomoles of oxygen consumed per min and mg of cellular protein, as
determined by the Bradford procedure (Bio-Rad).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
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Fig. 1.
Mosaic pattern of z-VADfmk-sensitive release
of cytochrome c from mitochondria in anti-Fas-treated
Jurkat cells. Double-labeling confocal immunofluorescence
microscopy of cells either untreated (naive), or
treated for 4 h with anti-Fas antibody, without pretreatment
(Fas), or with 30 min z-VADfmk (20 µM)
pretreatment Fas/z-VADfmk Patterns of cytochrome c (in
green), Hsp60 (in red), and merged patterns of
the same representative fields are shown.
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Fig. 2.
z-VADfmk-sensitive decrease in endogenous and
TMPD-dependent respiration rates in intact anti-Fas-treated
Jurkat cells. Oxygen consumption rates were measured in TD buffer
(endogenous coupled, ), in TD buffer containing DNP (endogenous
uncoupled,
), and in TD buffer containing DNP, antimycin A,
ascorbate, and TMPD (TMPD-dependent,
). A and
B, representative experiments involving multiple
measurements on samples of the same cell culture pretreated for the
indicated times with anti-Fas antibody. In A, the
dashed line represents the percentage of nuclear
apoptosis determined by DAPI staining (taken from Fig. 3). Note that,
in B, the ordinate scale starts at 5.5 nmol
min
1 mg
1.
C, relative oxygen consumption rates of untreated cells
(light shaded columns), cells treated
for 4 h with anti-Fas antibody (dark shaded
columns), or cells treated with 10 µM z-VADfmk
for 5 h, in the last 4 h in the presence of anti-Fas antibody
(filled columns), expressed as percentages of the
values obtained for untreated cells. In all panels, the data represent
the means ± S.E. (standard error of the mean) (n = 3-4). The S.E. bars within the symbols are not shown.
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Fig. 3.
The release of cytochrome c
from mitochondria precedes the decrease in respiration rate.
Quantification of percentage of cells with cytosolic cytochrome
c staining ( ), as analyzed by double-labeling
immunofluorescence microscopy, or nuclear apoptosis (
), as analyzed
by DAPI staining, among cells treated for the indicated time with
anti-Fas antibody. The cytochrome c and DAPI staining data
represent the means ± S.E. (n = 3-4). The
corresponding curves of relative endogenous uncoupled (
) and
TMPD-dependent (
) respiration rates (from the data of
Fig. 2 (A and B) and additional data not shown),
as a function of apoptosis induction time, are also shown
(n = 4-11).
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Fig. 4.
The excess of relative COX capacity of intact
naive cells is maintained in cell populations undergoing
anti-Fas-mediated apoptosis. Cells were either untreated
(A) or treated with anti-Fas antibody for 2 h
(B), and analyzed as follows. Left
panels, inhibition by KCN of endogenous respiration rate in
TD buffer in the presence of DNP ( ), or of
TMPD-dependent respiration rate in TD buffer in the
presence of DNP and antimycin A (
). Right
panels, percentages of endogenous uncoupled respiration rate
as a function of percentage of COX inhibition (
) by the same KCN
concentrations, and determination of maximum COX capacity
(COXR(max)). The least-square regression lines through the
filled squares beyond the inflection point in
each curve (arrow) are extended to zero COX inhibition. The
equations describing these extrapolated lines are shown in panels (see
"Results"). The data represent the means ± S.E.
(n = 3).
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Fig. 5.
Cellular mosaicism of cytochrome c
release and loss of respiration. Jurkat cells were either
untreated (naive), or treated for 4 h with anti-Fas
antibody (Fas), and fractionated on a column using annexin
V-coated paramagnetic beads. A, the upper panel shows the
results obtained when the fraction of naive cells that did not bind to
the beads (naive FT (flow-through)), the
anti-Fas-treated unfractionated population (Fas
unfraction.), the flow-through fraction of treated cells
mock-fractionated, i.e. not exposed to beads (Fas
mock-fraction.), the fraction of treated cells
that was retained by the beads (Fas bound), and
the fraction of treated cells that did not bind to the beads
(Fas FT) were analyzed by fluorescence
microscopy, using DAPI staining to determine the extent of nuclear
apoptosis (light shaded columns), or
annexin V-FITC staining to determine the extent of apoptosis-caused
plasma membrane changes (dark shaded
columns), or by double immunofluorescence microscopy to
determine the extent of cytochrome c release
(filled columns). The lower
panel shows the parallel analysis of the oxygen consumption
rate in the individual fractions measured in TD buffer
(striped columns). The data represent the
means ± S.E. (n = 3-4). B, the
percentage of cells with released cytochrome c (relative to
the anti-Fas-treated unfractionated cells) and the total endogenous
respiration rate (expressed as percentage of the value for naive
unfractionated cells) in the bead-bound fraction, in the flow-through
fraction, and in the unfractionated anti-Fas-treated cell population
shown in A are normalized on the basis of the total cellular
protein in each fraction relative to the unfractionated cell population
(abbreviations as in A). The percentage of cellular proteins
recovered in each fraction is indicated below the symbols on
the abscissa axes. See "Results" for
details.
1), in the
bead-bound and flow-through cell fractions, expressed relative to the
overall respiration rate in unfractionated naive cells (~0% and
~35%, respectively), are combined and compared with the relative
overall respiration rate in the unfractionated cell population
(~52%), they reveal a 17% decrease. This decrease is presumably due
to a reduction in respiration rate caused by exposure of the cells to
beads in the flow-through fraction, which is similar to that observed
in naive cells exposed to beads and passed through a column (~22%,
see above).
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Fig. 6.
Digitonin-induced loss of respiration and its
restoration by exogenous cytochrome c.
A-C, oxygen consumption was measured in parallel in naive
Jurkat cells (dashed lines) and in cells treated for
4 h with anti-Fas antibody (solid black lines) in
respiration medium I (A and B) or in respiration
medium II (C) after each of the indicated sequential
additions (see `Experimental Procedures`). The relative respiration
rates in anti-Fas-treated cells, expressed as percentages of the rates
in naive cells under the same conditions, determined from tracings such
as those shown, are shown in table form in panels
A-C. The data represent the means ± S.E.
(n = 5-7). The endogenous uncoupled respiration rate
was measured in respiration medium I (A and B) or
in respiration medium II (C). The
glutamate/malate-dependent (A) or
succinate-dependent (B) respiration rates were
determined 7 min after the addition of digitonin and immediately
afterward, following the addition of 80 µM cytochrome
c (+ cyt. c). The
TMPD-dependent respiration rate (C) was
determined 12.5 min after the addition of digitonin and immediately
afterward, following the addition of 80 µM cytochrome
c. D, oxygen consumption was measured in parallel
in 3.2 × 107 naive Jurkat cells (dashed
line) and in a mixture of 0.9 × 107 naive cells
and 2.3 × 107 cells treated for 4 h with
anti-Fas antibody (solid gray line) in respiration medium I
after each of the indicated sequential additions.
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Fig. 7.
Digitonin causes loss of cytochrome
c from all respiring anti-Fas-treated cells.
Untreated cells (naive) or cells treated for 4 h with
anti-Fas antibody (Fas), either intact or treated for 7 min
with digitonin (5 µg/106 cells) in respiration medium I,
were analyzed as follows. A, double-labeling confocal
immunofluorescence microscopy of digitonin-treated cells. Patterns of
cytochrome c (in green), mitochondrial Hsp60 (in
red), and merged patterns of the same representative fields
are shown. B, cytochrome c immunoblot analysis of
17,000 × g pellet of intact cells (C) or
pellet (P) and supernatant (S) of
digitonin-treated cells. The samples were derived from the same number
of cells (see `Experimental Procedures`).
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Fig. 8.
The rapid loss of
succinate-dependent respiration in digitonin-treated
annexin V-nonbinding subpopulation of anti-Fas-treated cells reflects a
preceding priming event. 2 × 107 cells of the
flow-through subpopulation of naive Jurkat cells (dashed
lines) and 2 × 107 cells of the
flow-through subpopulation of cells treated for 4 h with anti-Fas
antibody (solid lines) were each separated on a column using
annexin V-coated paramagnetic beads. Succinate-dependent
respiration was measured in respiration medium I (A) or, as
indicated, in the digitonin supernatants (B and
C) generated from either naive cells (Naive
supern.) or the flow-through subpopulation of 4 h
anti-Fas-treated cells (Fas flow-through
supern.). See "Experimental Procedures" for details.
ADP, rotenone, succinate, and cells were added sequentially (not
indicated), and the chamber was then closed prior to addition of
digitonin. All other additions are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Anne Chomyn, Elisabetta Ferraro, and Jordi Asin-Cayuela for helpful discussion, and Benneta Keeley, Arger Drew, and Rosario Zedan for expert technical assistance.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM11726 (to G. A.) and by 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.
Permanent address: Dept. of Medical Biochemistry and Biology,
University of Bari, 70124 Bari, Italy.
§ To whom correspondence should be addressed. Tel.: 626-395-4930; Fax: 626-449-0756; E-mail: attardig@seqaxp.bio.caltech.edu.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007871200
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ABBREVIATIONS |
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The abbreviations used are: COX, cytochrome c oxidase; anti-Fas, anti-Fas antibody; DAPI, 4,6-diamidino-2-phenylindole; DNP, dinitrophenol; TMPD, N,N,N',N'-tetramethyl-1,4-phenylenediamine; COXR(max), maximum COX capacity; z-VADfmk, z-Val-Ala-Asp(OMe)-CH2F; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween 20; HSPBS, horse serum in phosphate-buffered saline.
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