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From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) in the presence of 0.5 µg/ml
actinomycin D (Act D) is prevented by inhibition of the mitochondrial
permeability transition (MPT) with cyclosporin A (CyA) in combination
with the phospholipase A2 inhibitor aristolochic acid
(ArA). The MPT is accompanied by the release of cytochrome
c from the mitochondria, caspase-8 and caspase-3
activation in the cytosol, cleavage of the nuclear enzyme
poly(ADP-ribose)polymerase (PARP), and DNA
fragmentation, all of which were inhibited by CyA plus ArA. The
caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone
(Z-DEVD-FMK) did not prevent the loss of viability or the
redistribution of cytochrome c, but it did
prevent caspase-3 activation, PARP cleavage, and DNA
fragmentation. Inhibition of the MPT reduced the activation of
caspase-8 to the level occurring with TNF- alone (no ActD). The
caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone
(Z-IETD-FMK) did not prevent the cell killing and decreased only
slightly the translocation of Bid to the mitochondria. These data
indicate that induction of the MTP by TNF- causes a release of
cytochrome c, caspase-3 activation with PARP
cleavage and DNA fragmentation. The loss of viability is dependent on
the MPT but independent of the activation of caspase-3. The activation
of caspase-8 is not dependent on the MPT. There is no evidence linking
this enzyme to the loss of viability. Thus, the killing of L929
fibroblasts by TNF- can occur in the absence of either caspase-3 or
caspase-8 activity. Alternatively, cell death can be prevented
despite an activation of caspase-8.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mitochondrial permeability transition (MPT) is a well known alteration implicated as a mechanism of cell injury. The MPT refers to the regulated opening of a large, nonspecific pore in the inner mitochondrial membrane.5-7 Although the molecular elements that form this pore have not been definitively established, they are presumed to derive from well known membrane constituents, including the adenine nucleotide translocator, porin molecules, and the complex forming the peripheral benzodiazepine receptor.8,9 The MPT is a critical event in the killing of cells that follows an inhibition of mitochondrial electron transport by anoxia.10 The MPT has also been implicated as a mechanism of mitochondrial dysfunction in apoptosis.11
We have shown that the overexpression of Bax in Jurkat lymphocytes results in the killing of cells by a process that displays many of the features associated with apoptosis.12 In particular, the overexpression of Bax in the Jurkat cells induces the MPT, an event that is responsible for the loss of viability. The MPT is accompanied by the release of cytochrome c and, in turn, caspase-3 activation with the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) and the fragmentation of DNA. Inhibition of the MPT by cyclosporin A (CyA) prevented all manifestations of apoptosis, whereas caspase-3 inhibition prevented PARP cleavage and DNA fragmentation. The caspase-3 inhibitor was without effect on induction of the MPT and its functional consequences, namely cell death and the release of cytochrome c.
Several important concerns were left unresolved by this earlier study.
In particular, one needs to know how relevant the findings that
occurred with Bax overexpression are to cell death occurring in other
models. The killing of L929 mouse fibroblasts by tumor necrosis
factor- (TNF-) also depends on induction of the
MPT.13
In this model, both the MPT and the cell killing
induced by TNF- are inhibited by CyA. Thus, the killing of L929
fibroblasts by TNF- provides a very convenient system with which to
further explore the nature and consequences of mitochondrial
dysfunction in lethal cell injury. In the present study, we demonstrate
that TNF- causes a redistribution of cytochrome c from
the mitochondria to the cytosol that is dependent on the induction
of the MPT. In addition, the functional consequences of this
redistribution of cytochrome c and their relationship to the
loss of cell viability are defined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The L929 line of mouse fibroblasts (ATCC-CCL-1, American Type Culture Collection, Manassas, VA) was maintained in 25-cm2 polystyrene flasks (Corning Costar Corp., Oneonta, NY) with 5 ml of Dulbecco’s modified Eagle’s medium (DMEM; high glucose, without pyruvate; MediaTech), containing 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (complete DMEM) and incubated under an atmosphere of 95% air and 5% CO2. For all experiments, cells were plated at a density of 200,000/cm2 in complete DMEM. After overnight incubation the cells were washed twice with phosphate-buffered saline (PBS) and placed in DMEM without serum.
Treatment Protocols
In all experiments TNF- (Sigma, St. Louis, MO) was added
to a final concentration of 2 ng/ml (22 units/ng). TNF- was
dissolved in PBS and added to the cells in 0.2% volume. Act D (Sigma)
was dissolved in dimethyl sulfoxide (DMSO), further diluted in PBS, and
added in 0.2% volume to a final concentration of 0.5 µg/ml. Where
indicated the cells were pretreated for 30 minutes with the following
reagents before addition of TNF- and Act D. Cyclosporin A (Biomol,
Plymouth Meeting, PA) was dissolved in DMSO and added in a 0.2%
volume to the cell culture media to give a final concentration of 5
µmol/L. Aristolochic acid (Biomol) was dissolved in PBS and added in
a 0.2% volume to give a final concentration of 50 µmol/L. The cell
permeable caspase-3 inhibitor (Z-Asp-Glu-Val-aspartic acid
fluoromethylketone, Z-DEVD-FMK) and the cell permeable caspase-8
inhibitor (Z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone, Z-IETD-FMK;
Kamyia Biomedical Co., Seattle, WA) were dissolved in DMSO and added in
a 0.2% volume to give the concentrations indicated in the text. In all
cases the vehicles used to prepare stock solutions of the reagents had
no effect on the cells or the parameters measured at the concentrations
used.
Measurement of Cell Viability
Cell viability was determined by trypan blue exclusion. After treatment the cells were trypsinized and 10 µl of a 0.5% solution of trypan blue was added to 100 µl of treated cells. The suspension was then applied to a hemocytometer. Both viable and nonviable cells were counted. A minimum of 200 cells was counted for each data point in a total of eight microscopic fields.
Measurement of Caspase-3
The assay is based on the ability of the active enzyme to cleave the chromophore pNA from the enzyme substrate DEVD-pNA. Extracts from treated cells were diluted 1:1 with 2x reaction buffer (10 mmol/L Tris, pH 7.4, 1 mmol/L dithiothreitol, 2 mmol/L EDTA, 0.1% (3-[3-cholamido- propyl]dimethylammonio)-1-propanesulfonate, 1 mmol/L PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin). DEVD-pNA was added to a final concentration of 50 µmol/L, and the reaction was incubated for 1 hour at 37°C. The samples were then transferred to a 96-well plate, and the absorbance measurements were made with a 96-well plate reader at 405 nm.
Measurement of Caspase-8
The activity of caspase-8 was measured with the Apo-Alert Caspase-8 Colorimetric Assay Kit (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer’s instructions. Absorbance measurements were made with a 96-well plate reader at 405 nm.
Isolation of Cytosol and Mitochondrial Fractions and Determination of Bid or Cytochrome c Content
Cells were harvested by trypsinization after treatment. Soybean trypsin inhibitor was used to neutralize trypsin after harvest. Cells were centrifuged at 750 x g for 10 minutes at 4°C. The pellets were washed with SHE-PI (10 mmol/L HEPES-KOH (pH 7.4), 1 mmol/L EDTA, protease inhibitor cocktail (PI), and 250 mmol/L sucrose). The cell pellets were resuspended in SHE-PI. The cell suspension was transferred to a dounce homogenizer and the cells broken open with 20 strokes of the pestle. The homogenate was transferred to a high speed centrifuge tube containing SHE-PI and 0.5% phenol red. HE-PI (10 mmol/L HEPES-KOH, pH 7.4, 1 mmol/L EDTA, protease inhibitor cocktail (PI) containing 750 mmol/L sucrose) was laid below the homogenate. Centrifugation was conducted at 10,000 x g for 30 minutes at 4°C. The resulting mitochondrial pellet was resuspended and centrifuged a second time into 750 mmol/L sucrose in HE-PI. The mitochondria were further concentrated in a Centricon (MW cutoff of 10,000) microconcentrator and the resulting retentate analyzed for cytochrome c. The supernatant from the 10,000 x g spin was centrifuged at 100,000 x g for preparation of cytosol. For the detection of cytochrome c, mitochondrial and cytosolic fractions were separated on 12% SDS-polyacrylamide electrophoresis gels with an equal amount of protein loaded onto each lane as determined by the bicinchoninic acid assay. Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The gels were then electroblotted onto nitrocellulose membranes. Cytochrome c was detected by a monoclonal antibody (Pharmingen, San Diego, CA) at a dilution of 1:5000. Secondary goat-anti-mouse horseradish peroxidase-labeled antibody (1:2000) was detected by enhanced chemiluminescence.
To detect Bid, the mitochondrial fraction was electrophoresed on 15% SDS-polyacrylamide gels. The gel was then electroblotted onto nitrocellulose membranes. Bid was detected with a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. The secondary anti-goat horseradish peroxidase-labeled antibody (1:20,000) was visualized by enhanced chemiluminescence.
Detection of DNA Fragmentation
Cells (1.0 x 106) were trypsinized and collected by centrifugation at 750 x g for 10 minutes. The cell pellet was washed in PBS and then lysed in 200 µl of 10 mmol/L Tris, pH 8.0, 10 mmol/L EDTA, 0.5% Triton X-100. The lysate was centrifuged at 13,000 x g for 20 minutes at 4°C. RNase (0.2 mg/ml) was added, and the lysate was incubated for 30 minutes at 37°C. Proteinase K (0.1 mg/ml) and SDS (final concentration 1%) were added, followed by incubation at 50°C for 16 hours. DNA was extracted with phenol/chloroform and then with chloroform, precipitated with ethanol and sodium acetate, and electrophoresed on 1.2% agarose gels.
Determination of PARP Content
Cells (1.0 x 105) were trypsinized and then pelleted at 750 x g, resuspended in 20 µl of SDS sample buffer, and boiled for 10 minutes. The samples were run on an 8% SDS-polyacrylamide electrophoresis gel. Protein content was determined by BCA, and molecular weight by external standards. The gels were electroblotted onto nitrocellulose membranes and probed with PARP rabbit antiserum (PA3–950, Affinity Bioreagents, Golden, CO) at 1:500 dilution. A secondary horseradish peroxidase-labeled goat-anti-rabbit antibody at 1:10,000 was detected using enhanced chemiluminesence.
Electron Microscopy
After treatment the cells were trypsinized and pelleted. The cells were then suspended and fixed in 2% gluteraldehyde with 1% tannic acid in 0.1 mol/L sodium cacodylate at pH 7.3 overnight at 4°C. The cells were rinsed three times in the sodium cacodylate buffer and then incubated in 2% osmium tetroxide in the same buffer for 2 hours at room temperature. The cells were rinsed three times in distilled water and then exposed to 1% uranyl acetate in water for 15 minutes at room temperature. The cells were then rinsed twice in distilled water, after which they were spun down into 3% agarose at 45°C and cooled to form blocks. The agarose blocks were dehydrated in graded steps of acetone and embedded in Spurr’s low viscosity media. After polymerization overnight at 65°C, 80-nm sections were cut on a Reighert-Jung Ultra Cut E ultramicrotome and picked up on copper grids. The grids were poststained in uranyl acetate and bismuth subnitrate. The sections were observed in a Hitachi 7000 STEM and micrographs recorded on Kodak 4489-sheet film.
Measurement of Malate Dehydrogenase (MDH)
MDH activity was measured in L929 cytosolic extracts according to the method of Ochoa.14 Briefly, cytosolic extracts (2 mg of protein) were added to 300 µl of MDH buffer (259 mmol/L glycylglycine, pH 7.4, 1.5 mmol/L NADH). To this, 100 µl of 7.6 mmol/L oxalacetate was added to initiate the reaction. Readings were taken in a spectrophotometer at 340 nm and made against a blank containing all components except NADH. The decrease in the optical density between 30 and 45 seconds after the start of the reaction was used to calculate enzyme activity. The concentration of MDH in the cytosolic extracts was calculated by comparison to a standard curve. MDH and NADH were both from Sigma.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in L929
Fibroblasts
TNF- induces the mitochondrial permeability transition (MPT) in
L929 fibroblasts and, in turn, the death of virtually all of the
cells.13
On treatment with TNF- in the presence of
either actinomycin D (ActD) or cycloheximide (CHX), the MPT (as
measured by a loss of mitochondrial energization) is evident within 4
hours.13
As shown in Figure 1
, TNF- produced over a similar time
course an increase in caspase-3 activity in the cytosol of L929
fibroblasts. Within 4 hours caspase-3 activity increased by eightfold
and 16-fold by 6 hours, a result that was entirely dependent on
induction of the MTP. Cyclosporin A (CyA) together with the
phospholipase A2 inhibitor aristolochic acid
(ArA) prevents the development of the MPT in L929 cells treated with
TNF-.13
Figure 2
shows
that CyA + ArA inhibited the increase in caspase-3 activity induced by
TNF-. CyA + ArA lowered caspase-3 activity in TNF--treated cells
to that in the control cells. Importantly, direct addition of CyA + ArA
to cellular extracts of TNF--treated cells did not inhibit caspase-3
activity (data not shown), a result demonstrating that CyA + ArA does
not directly inhibit caspase-3.
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. As
shown in Figure 2, Z
-DEVD-FMK produced a dose-dependent inhibition of
the increase in caspase-3 activity, with 100 µmol/L reducing
caspase-3 activity to that of controls.
Whereas the data in Figure 2
indicate that activation of caspase-3 is
entirely a consequence of the MPT, there are potential, alternative
mechanisms for caspase activation in response to TNF-. In
particular, caspase-8 can be activated as a direct consequence of the
binding of TNF- to the 55-kd receptor (TNFR-1) with recruitment of
TRADD (TNFR-1-associated death domain protein) and, in turn, FADD
(Fas-associated death domain protein).16
Caspase-8 can
then activate downstream caspases, such as caspase-3.17,18
Figure 3
details the activation of
caspase-8 in L929 fibroblasts treated with TNF-. Within 2 hours,
TNF- alone (no ActD) produced a threefold increase in caspase-8
activity. It deserves emphasis that there is no loss of viability with
TNF- alone.13
In the presence of TNF- and ActD, a
10-fold activation of caspase-8 occurred (Figure 3)
. CyA + ArA reduced
the activation of caspase-8 by TNF- and ActD to that with TNF-
alone (Figure 3)
. Thus, the additional activation of caspase-8 above
that with TNF- alone that occurred with TNF- and ActD is a
consequence of the MPT. Importantly, Z-IETD-FMK, a specific inhibitor
of caspase-8,19
prevented both components (that with
TNF- alone plus that with TNF- and ActD) of the activation of
this enzyme, thereby reducing its activity to the control level (Figure 3)
.
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-Induced Translocation of Bid
Activation of Bid and its translocation to the mitochondria have
been attributed to activation of caspase-8.20,21
Treatment
of L929 fibroblasts with TNF- and ActD caused the translocation of
Bid to the mitochondria (Figure 4)
.
Interestingly, despite the complete inhibition of caspase-8 activation
by Z-IETD-FMK (Figure 3)
, the translocation of Bid is only partially
prevented by this specific caspase inhibitor (Figure 4)
. This result is
consistent with the previous report that, under conditions where the
cell killing is not entirely dependent on caspase activation, Bid
translocation is similarly not prevented by caspase
inhibitors.22
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-Induced PARP Depletion Is Inhibited by Z-DEVD-FMK and CyA
Cleavage of PARP is a prominent manifestation of the activation of
caspase-3 in many models of apoptosis.23
Similarly, the
treatment of L929 fibroblasts (in the presence of ActD) causes a loss
of the 116-kd PARP (Figure 5
, land 2).
This loss of PARP protein was not apparent with TNF- in the absence
of ActD (data not shown). Despite the fact that the antibody used here
(PA3–590) recognizes the carboxyl terminal of mouse PARP, it was not
possible to detect the 85-kd carboxyl terminal cleavage fragment of
PARP in the L929 cell extracts. However, it could be demonstrated that
the loss of the native PARP protein produced by TNF- depended on
caspase-3 activity. The TNF--induced PARP depletion was inhibited by
pretreatment of the cells with Z-DEVD-FMK (Figure 5
, lane 3).
Importantly, the loss of PARP also depended on induction of the MPT as
shown by the similar ability of CyA (in the presence of ArA) to
maintain the control content of this enzyme (Figure 5
, lane 4).
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-Induced DNA Fragmentation Is Inhibited by Z-DEVD-FMK and
CyA
The cleavage of DNA to oligonucleosome size fragments is another
prominent feature of apoptosis. Treatment of L929 fibroblasts with
TNF- results in DNA fragmentation within 6 hours (Figure 6a)
. DNA fragmentation was not detected
at the same times with TNF- in the absence of ActD (data not shown).
This DNA fragmentation induced by TNF- is caspase-3-dependent. As
shown in Figure 6b
, the caspase-3 inhibitor Z-DEVD-FMK completely
prevented the evidence of DNA fragmentation at 6 hours produced by
TNF-. As with the increase in caspase-3 activity itself and the
depletion of PARP protein, DNA fragmentation was also dependent on the
MPT. CyA + ArA completely prevented the DNA fragmentation evident 6
hours after treatment with TNF-.
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The activation of caspases in some models of apoptosis has been
attributed to the release and redistribution of cytochrome c
from the mitochondria to the cytosol.24,25
To determine
the role of cytochrome c release in the activation of
caspase-3 by TNF-, cytosolic and mitochondrial fractions were
derived from homogenates of TNF--treated L929 cells. Figure 7a
shows that TNF- (in the presence of
ActD) produced a decrease in the mitochondrial content of cytochrome
c that was evident after 4 hours and more prominent at 6
hours. This depletion of cytochrome c in the mitochondrial
fraction was mirrored by a concomitant increase of the protein in the
cytosolic fraction. There was no change in the cellular distribution of
cytochrome c in cells treated with TNF- in the absence of
ActD (data not shown). The small amount of cytosolic cytochrome
c evident at time zero was owing presumably to the trauma
incurred by the mitochondria on homogenization and isolation of the
respective subcellular fractions.
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is dependent
on induction of the MPT. CyA + ArA prevented the release and
redistribution of cytochrome c from mitochondria to the
cytosol at 6 hours (Figure 7b)
. By contrast, Z-DEVD-FMK had no effect
on either the mitochondrial loss or cytosolic accumulation of
cytochrome c in L929 cells treated TNF- (Figure 7b)
.
Similarly, Z-DEVD-FMK did not prevent induction of the MPT as measured
by loss of mitochondrial membrane potential (data not shown). In
keeping with the conclusion that the release of cytochrome c
is a consequence of the MPT, Figure 8
shows that 20 µmol/L Z-IETD-FMK, a dose that prevents caspase-8
activation (Figure 3)
, is without effect on either the loss of
cytochrome c from the mitochondria (Figure 8
, top) or its
accumulation in the cytosol (Figure 8
, bottom).
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-Induced Release of MDH from the Mitochondrial Matrix
It has been hypothesized that the release of cytochrome
c during apoptosis is the specific consequence of an as yet
poorly characterized mechanism that disrupts the outer mitochondrial
membrane without any loss of the mitochondrial membrane
potential.25
By contrast, induction of the MPT in isolated
mitochondria results in the release of several proteins in addition to
cytochrome c.26
Similarly, we show here that
cytochrome c is not the only protein whose redistribution is
affected on induction of the MPT by TNF- in intact L929 cells. MDH
is a soluble protein present in the mitochondrial
matrix.26
Table 1
shows that
cytosolic extracts from control L929 cells exhibited some detectable
MDH. This baseline activity is attributable to the MDH normally present
in the cytosol as part of the acetyl CoA cycle and used to transport
acetyl CoA across the mitochondrial membrane for utilization in fatty
acid synthesis. However, the MDH activity more than doubled within 4
hours and was threefold greater than the control level after 6 hours in
cytosolic extracts from cells treated with TNF- (in the presence of
ActD). This increase in the activity of MDH in the cytosol represents a
change from 15% to 65% of the total cellular enzyme (Table 1)
, a
result that would suggest that, at least, half of the mitochondria have
undergone the MPT. There was no increase in MDH in cytosolic extracts
from cells treated with TNF- alone (data not shown). Importantly,
pretreatment of the cells with CyA + ArA prevented the accumulation of
MDH in the cytosol produced by TNF- (Table 1)
.
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Produces Mitochondria Swelling That
Is Inhibited by CyA
It has also been argued that cytochrome c is
released from mitochondria by a mechanism distinct from the MPT that is
inhibited by CyA but not accompanied by swelling of these
organelles.27
Examination of intact L929 cells by electron
microscopy 4 hours after treatment with TNF- (in the presence of
ActD) indicated prominent mitochondrial swelling (Figure 9a)
. Clearing of the matrix space with
loss of cristae was accompanied by increases in the size and
configuration of the mitochondria. It deserves emphasis that these
mitochondrial alterations occurred at a time when the nuclear
membrane was still intact and the chromatin was not condensed or
clumped (Figure 9a)
. At this time (4 hours), the plasma membrane was
also intact (Figure 9a)
. Mitochondrial swelling was prevented by
pretreatment with CyA + ArA (Figure 9b)
.
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Figure 10
demonstrates that
inhibition of the activation of caspase-3 by Z-DEVD-FMK afforded no
protection against the cytotoxicity of TNF-. TNF- (in the
presence of ActD) caused a progressive loss of cell viability with
almost 70% of the cells dead within 6 hours. Despite its ability to
inhibit activation of caspase-3 (Figure 2)
, the depletion of PARP
(Figure 5)
, and the fragmentation of DNA (Figure 6)
, 100 µmol/L
Z-DEVD-FMK had no affect on the rate or extent of cell killing by
TNF- (Figure 10)
. By contrast, inhibition of the MPT by CyA + ArA
totally prevented the loss of cell viability induced by TNF- (Figure 10)
.
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also considers the role of the activation of caspase-8 in the
cell killing by TNF-. Whereas 20 µmol/L Z-IETD-FMK completely
prevented the activation of caspase-8 (Figure 3)
, no effect of this
specific inhibitor on the extent of cell killing was evident. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is accompanied by a redistribution of
cytochrome c from mitochondria to the cytosol. In turn,
cytochrome c release is accompanied by activation of
caspase-3, PARP degradation, and DNA fragmentation. Inhibition of the
MPT by cyclosporin A (in the presence of aristolochic acid) prevented
in parallel with the prevention of cell killing 1) the release of
cytochrome c; 2) the activation of caspase-3; 3) the degradation of
PARP, and 4) the fragmentation of DNA. By contrast, Z-DEVD-FMK, a cell
permeable, irreversible inhibitor of caspase-3, prevented only the DNA
fragmentation and PARP degradation produced by TNF-. Z-DEVD-FMK did
not prevent the redistribution of cytochrome c or the loss
of cell viability. Thus, the present report documents for the first
time that cytochrome c release with caspase activation and
action is a consequence of the MPT induced by a physiological stimulus
(TNF- in this case) under conditions that kill cells. The cell
death, however, is clearly the consequence of the MPT and not of the
release of cytochrome c nor the activation of caspase-3.
Similar events and conclusions were reported recently on the
overexpression of Bax in Jurkat cells.12
Accordingly, it
deserves emphasis that the MPT has now been shown to mediate both cell
killing and cytochrome c release in, at least, two distinct
models of cell killing in which loss of viability is accompanied by
other typical manifestations of apoptosis, namely PARP cleavage and DNA
fragmentation. A role for cytochrome c in, at least, the nuclear events of apoptosis was first suggested in studies with cell free extracts by the requirement for an organelle fraction enriched in mitochondria.28 The mitochondrial agent responsible for the degradation of DNA in vitro was subsequently identified as cytochrome c.1 Subsequently, cytochrome c was shown to bind to the cytosolic protein Apaf-1, the human homologue of the C. elegans protein, CED-4. In vitro reconstitution experiments detailed that cytochrome c forms a complex consisting of procaspase-9 and Apaf-1.29 On the addition of dATP, the complex activates procaspase-3.
More controversial has been the mechanism mediating cytochrome c release. Previous studies have both implicated and denied a role for the MPT in cytochrome c release. With isolated mitochondria, induction of the MPT released cytochrome c, along with other mitochondrial proteins.26,30,31 In intact thymocytes, the development of the MPT was inhibited by Bcl-2.32 Moreover, mitochondria isolated from cells that overexpress Bcl-2 were resistant to agents that induce the MPT. We have shown that the overexpression of Bax in Jurkat cells caused the release of cytochrome c as a consequence of the induction of the MTP.12
By contrast, in other studies the data were interpreted to indicate that the release of cytochrome c was not dependent on an induction of the MPT. Recombinant Bax protein induced isolated mitochondria to release cytochrome c, a result that was completely inhibited by CyA.31 Nevertheless, it was concluded that Bax-induced cytochrome c release occurred by a mechanism other than the MPT, because the mitochondria did not swell. In HL-60 cells treated with staurosporine, there was a redistribution of cytochrome c from the mitochondria to the cytosol, a change that preceded a loss of the mitochondrial membrane potential.24 Jurkat cells undergoing Fas-induced apoptosis were reported to exhibit swollen mitochondria and cytochrome c release.33 In this instance, rupture of the outer mitochondrial membrane was said to occur and to account for the cytochrome c release. Interestingly, the mitochondria still maintained a membrane potential, despite the almost complete loss of cytochrome c and gross deformation of their structure. The maintenance of the membrane potential was cited as evidence that the MPT was not the mechanism mediating cytochrome c release.
The electron micrographs presented here (Figure 9)
demonstrate that
CyA-sensitive mitochondrial swelling is a prominent feature in L929
cells treated with TNF-. This change in the morphology of the
mitochondria and its prevention by CyA is further evidence for
induction of the MPT. As evidence for the MPT, we have previously
reported that TNF- causes CyA-sensitive mitochondrial
depolarization.13
An additional finding that indicates the
induction of the MPT is the release of MDH (Table 1)
. The release of
MDH is interpreted as a specific consequence of induction of the MPT,
as its was prevented by CyA (Table 1)
.
It can be argued that during the initial phases of the evolution of the cell injury in apoptosis, normal mitochondria may coexist with those that have developed the MPT. Under such circumstances, fluorescent cationic dyes may redistribute from depolarized mitochondria to mitochondria that are still energized, an effect that may explain the inability to detect a decrease in the mitochondrial membrane potential at early time points in some models of apoptosis.5 Furthermore, early in the process of apoptosis, the release of cytochrome c from a small fraction of mitochondria that have undergone the MPT may be sufficient to activate procaspase-3. In turn, caspase-3 can further cleave procaspase-3 molecules, thereby initiating an autocatalytic cycle.
The events that follow on cytochrome c release are generally
quite reproducible. In the TNF--treated fibroblasts studied here,
cytochrome c release was accompanied by activation of
caspase-3 activity (Figure 1)
with degradation of PARP (Figure 5)
and
fragmentation of DNA (Figure 6)
. Caspase-3 degrades PARP
directly.15
By contrast, caspase-3 results in DNA
fragmentation by first activating an endonuclease (CAD) and
inactivating its inhibitor, ICAD.34
The increment in
caspase-8 activation that depended on the MPT (Figure 3)
can also be
attributed to the action of caspase-3.35
Similar to the killing of Jurkat cells on the overexpression of
Bax,12
caspase-3 activity, as opposed to the MPT, was not
critical for the loss of viability of L929 fibroblasts (measured by
disruption of plasma membrane integrity) in response to TNF- (Figure 9)
. Interestingly, MCF-7 cells that lack functional caspase-3 failed to
manifest many of the typical morphological manifestations associated
with apoptosis, but they were still sensitive to the cytotoxicity of
TNF-.36
Moreover, MCF-7 cells treated with TNF-
displayed a redistribution of cytochrome c from mitochondria
to cytosol.29
The overexpression of
Bcl-XL in MCF-7 cells inhibited the cytotoxicity
and cytochrome c redistribution produced by
TNF-.29
The microinjection of cytochrome c
into MCF-7 cells treated with TNF- did not overcome the protective
effects of Bcl-XL expression.37
These results can be readily explained by hypothesizing that in MCF-7
cells Bcl-XL prevents the cytotoxicity of TNF-
by inhibiting induction of the MPT. As noted above, Bcl-2, a protein
closely related to Bcl-XL, inhibited induction of
the MPT in both isolated mitochondria and intact cells.
An alternative mechanism for caspase activation in response to TNF-
has been proposed. Activation of the 55-kd TNF- receptor (TNFR-1)
results in receptor aggregation and the recruitment of TRADD (TNFR-1
associated death domain protein). In turn, TRADD binds FADD
(Fas-associated death domain protein). Interaction with FADD unmasks
the amino-terminal death effector domain of FADD, an event that allows
it to recruit caspase-8/FLICE (FADD-like interleukin-1ß-converting
enzyme).16
In turn, caspase-8 activates downstream
caspases, such as caspase-3.17,18
Such a scheme does not
demand the participation of cytochrome c and, thus, should
not depend on changes in mitochondrial structure and function.
Despite the activation of caspase-8 in response to TNF- in L929
fibroblasts (Figure 3)
, a mechanism with which caspase-8 activates
caspase-3 would not seem to be the predominant pathway of caspase-3
activation in TNF--treated L929 cells. In our study, inhibition of
the MPT completely prevented the activation of caspase-3, by preventing
the release of cytochrome c. Our data also document that the
activation of caspase-8 is not necessary for the killing of L929
fibroblasts by TNF-. Inhibition of caspase-8 activation (Figure 3)
had no effect on the extent of cell killing by TNF- (Figure 10)
.
Similarly, caspase 8 was not necessary for the cytotoxicity of TNF-
in MCF-7 cells, which lack caspase-3. As discussed above,
Bcl-XL inhibited the cytotoxicity of TNF-, an
effect that occurred despite the fact that caspase-8 was still
processed proteolytically to its active form.29
The methodology used for assessing apoptosis can clearly affect the
conclusions reached. Janike et al38
found MCF-7 cells
sensitive to the cytotoxicity of TNF- as measured by the uptake of
crystal violet. This assay is similar to the exclusion of trypan blue,
in that only cells with an intact plasma membrane will be scored as
viable. However, in the same study, the typical morphological features
of apoptosis were not found, such as DNA laddering, cell blebbing, and
the appearance of a subG1 peak. Similarly, Oberhammer et
al39
observed that MCF-7 cells treated with TNF-
detached from their substrate but did not manifest the typical
apoptotic morphology or DNA fragmentation. As demonstrated here, in
L929 fibroblasts treated with TNF-, inhibition of caspase-3
prevented DNA fragmentation but not cell killing. Nevertheless,
caspase-3 is an important determinant in apoptosis, in that it is
necessary for the development of the distinguishing features (PARP
cleavage and DNA fragmentation) of this form of cell death.
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Accepted for publication February 15, 2000.
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) for the indicated
times. Alternatively, cells were pretreated with CyA + ArA
(
), DEVD-FMK
(
) or IETD-FMK
(•) followed by TNF-