From the Division of Cellular Immunology, La Jolla
Institute for Allergy and Immunology, San Diego, California 92121 and
the ¶ Department of Biochemistry, University of Alberta, Edmonton,
Alberta T6G 2H7, Canada
Received for publication, October 3, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Cytotoxic T lymphocytes kill
virus-infected and tumor cell targets through the concerted action of
proteins contained in cytolytic granules, primarily granzyme B and
perforin. Granzyme B, a serine proteinase with substrate specificity
similar to the caspase family of apoptotic cysteine proteinases, is
capable of cleaving and activating a number of death proteins in target
cells. Despite the ability to engage the death pathway at multiple
entry points, the preferred mechanism for rapid induction of apoptosis
by granzyme B has yet to be clearly established. Here we use time lapse
confocal microscopy to demonstrate that mitochondrial cytochrome
c release is the primary mode of granzyme B-induced
apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event.
Caspase activation is not required for cytochrome c
release, an activity that correlates with cleavage and activation of
Bid, which we have found to be cleaved more readily by granzyme B than
either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of
targets by granzyme B by blocking mitochondrial involvement in the process.
Cytotoxic T lymphocytes
(CTL)1 and natural
killer cells induce apoptosis in their targets through the
concerted action of effector molecules contained in cytolytic granules
that engage the death pathway (1, 2). Granzyme B enters the target cell
and, with another granule protein, perforin, triggers all of the
characteristic manifestations of apoptosis, providing the principal
mechanism of killing by CD8+ CTL and natural killer cells (3). Granzyme B is a serine protease that shares substrate specificity with many
members of the caspase family of cysteine proteases (4). In fact,
granzyme B cleaves and activates the apical caspase, caspase-8, as well
as caspases-3 (5, 6), -6, and -7 (7, 8). Granzyme B can directly
activate caspase-3 and is capable of triggering apoptosis at multiple
points of the caspase-dependent pathway (9, 10) and
therefore is not absolutely dependent on caspase-8 cleavage. This
pathway differs from another common death pathway utilized by CTL,
signaling through the Fas surface receptor by Fas ligand expressed on
the surface of the CTL. Apoptotic signaling through Fas requires an
obligate activation of caspase-8 (11) and can proceed via
mitochondria-dependent or -independent pathways (12). The
mitochondrial pathway involves the release of cytochrome c
for caspase activation and apoptosis (13, 14).
Release of mitochondrial cytochrome c is a pivotal event in
the apoptosis of many cell types induced by many stimuli (15, 16). Upon
release, cytochrome c binds Apaf-1 and promotes the formation of an oligomeric Apaf-1 apoptosome that recruits and activates the effector caspase, caspase-9 (17-19). In
receptor-mediated apoptosis the requirement for cytochrome c
release is dependent on the type of cell triggered to die, and the
decision to utilize the mitochondrial route appears to rely primarily
on the concentration of caspase-8 (20). Although the same pathways are
used in granule-mediated apoptosis, the conditions governing the
premitochondrial events remain to be clearly established.
Granule-driven CTL killing represents a very effective means to induce
apoptosis largely because of the ability of granzyme B to engage the
death pathway at multiple entry points. Despite recent advances
outlining the mechanism(s) of action of granzyme B within the target
cell, there are still numerous conflicting reports regarding the
requirements and dependence of granzyme B-mediated apoptosis on
caspases and the potential for inhibition by the anti-apoptotic
proto-oncogene, Bcl-2. It is clear that granzyme B is capable of
cleaving and activating a number of key enzymes in the caspase cascade,
notably caspases-8 and -3 (10, 21-23), but it is unclear what
preference, if any, exists for each potential event. In this report we
address the contribution of some of these aspects of granzyme-mediated
apoptosis. Also, because granzyme B can activate the effector caspases
directly, we sought to determine the necessity for cytochrome
c release and its contribution to the efficient death in
target cell apoptosis.
In early studies involving Bcl-2 in CTL-mediated apoptosis, it appeared
that target cell expression of the proto-oncogene could confer some
protection from CTL (24-26). Cytochrome c release in target
cells has been shown to occur, but it is not clear whether it is a
requirement for death. In this report we follow single cells within
populations to ascertain the order and extent of cytochrome
c release in the context of its (in)dependence on caspase activation. We have also utilized our experimental system to assess the
effects of Bcl-2 on both cytochrome c release and subsequent apoptosis. In this way we determined that: 1) Bcl-2 inhibits cytochrome c release and the ultimate death of the target cell; and 2)
granzyme B induces target cell cytochrome c release in a
caspase-independent manner via Bid proteolysis. Because apoptotic
Bcl-2-expressing cells do not release cytochrome c, we also
conclude that when necessary, granzyme B bypasses the mitochondria via
direct activation of effector caspases rather than overriding Bcl-2 by
proteolytic degradation or some other mechanism.
Cell Culture and Reagents--
Jurkat and Jurkat-Bcl-2 were
cultured in RPMI with 10% fetal calf serum, penicillin, and
streptomycin. We also employed an HeLa cell line stably transfected
with cytochrome c-GFP (denoted 2H18) as described previously
(27). Cytochrome c-GFP was shown to behave identically to
cytochrome c in 2H18 in apoptosis assays including side by
side immunoblots for cytochrome c and cytochrome c-GFP in subcellular fractions as well as immunofluoresence
assays. A subpopulation of these cells was transfected with the Bcl-2 gene in the plasmid pEFpGKpuro. The transfected cells were selected for
resistance to puromycin, and surviving clones resistant to UV- and
actinomycin D-induced apoptosis were analyzed for Bcl-2 expression by
Western blot analysis (Bcl-2 antibody 65111A; Pharmingen, La Jolla,
CA). The data from one clone (2H18-Bcl-2) are reported here. Both 2H18
and 2H18-Bcl-2 were maintained in Dulbecco's modified Eagle's medium
with 10% fetal calf serum. All cells were grown at 37 °C under 5%
CO2. Granzyme B was purified from the human natural killer
cell line YT-Indy as described previously (28). The broad spectrum
caspase inhibitor zVAD-fmk was purchased from Enzyme Systems Products (Dublin, CA). Antibodies against cytochrome c (1H5.2C12) were purchased from Pharmingen. Rabbit anti-Bid
was raised against the peptide N-RDVFHTTVNFINQNLRTYVRSLARNGMD-C
corresponding to a sequence in the C terminus of Bid. Specificity of
the Bid antiserum was verified by Western blot analysis of purified
recombinant Bid and GST-Bid protein. N-terminal protein sequencing was
performed by core protein facility at the Scripps Research Institute on an excised fragment transferred onto sequencing polyvinylidene difluoride (Amersham Pharmacia Biotech) stained in Coomassie Blue in
the absence of acetic acid.
Apoptosis and Cytochrome c Release Assays--
CTL-free
apoptosis was induced as described previously (28, 29). Briefly, target
cells were incubated in medium containing 0.5% serum with either 1 µg/ml granzyme B and 10 plaque-forming units/ml replication-deficient
adenovirus type V or 100 ng/ml anti-Fas (CH-11) for the times
indicated. Phosphatidylserine (PS) externalization was assayed with
annexin V-fluorescein isothiocyante (CLONTECH; Palo
Alto, CA) labeling and assessment with a Becton-Dickinson FACScan flow
cytometer. Protein lysates from apoptotic cells were produced by direct
lysis into gel loading buffer containing SDS and subjected to
examination by standard SDS-PAGE techniques and immunoblot analysis.
Measurement of the reduction of full-length Bid protein was performed
by densitometric analysis of Western blots. Mitochondria were isolated
from Xenopus laevis according to Kluck et al.
(30). After incubation at 30 °C under experimental conditions noted
in the text, mitochondria were removed from solution by centrifugation
at 14,000 rpm for 10 min at 4 °C. Pellets containing mitochondria
were resuspended in gel sample buffer containing SDS and
2-mercaptoethanol. Mitochondrial and supernatant protein samples were
analyzed for cytochrome c by standard immunoblotting techniques after denaturing PAGE.
Granzyme B Substrate Assays--
The cDNA encoding
full-length human Bid was expressed as a GST fusion protein in the
vector pGEX4T-1 in BL21(DE3) cells as described earlier (31). Bid was
used as a GST fusion protein or on its own after GST was removed by
thrombin digestion followed by purification over GSH-Sepharose
(Amersham Pharmacia Biotech). Radiolabeled Bid, caspase-3, and
caspase-8 were produced by coupled in vitro transcription
and translation (Promega Life Science, Madison, WI) from full-length
clones in the vector pcDNA-3. Specific activity of each translation
product was calculated according to the amount of incorporated
[35S]Met radiolabel relative to the number of
methiothionine residues in each polypeptide. Granzyme B digests were
performed at 30 °C under conditions described previously (32).
Fragments were analyzed by SDS-PAGE, fixed in gel, dried, and exposed
to Kodak X-Omat radiography film. All reactions were carried out with
concentrations of substrate (3 nM) well below the
Km, and first-order kinetics were assumed.
kcat/Km values were
calculated from the linear relation between
log(St/S0) versus
granzyme B concentration where St is the
concentration of substrate at time t, and S0
represents the initial substrate concentration.
FACS Analyses--
2H18 and 2H18-Bcl-2 cells cultured in
flat-bottomed 96-well plates were harvested at the times indicated
using 0.25% trypsin. Floating cells and trypsin-treated cells were
combined and pelleted in a round bottomed 96-well plate. The medium was
aspirated, and the cells were resuspended in 100 µl of ice-cold CLAMI
buffer (80 mM KCl and 25 µg/ml digitonin in
phosphate-buffered saline). The cells were incubated on ice for 5 min
and analyzed directly for GFP content by FACS analysis. Because
cytosolic cytochrome c-GFP is released into the CLAMI
buffer, cells in which mitochondria had not released cytochrome
c-GFP were ~0.5-1 log brighter in FL-1 than cells in
which the mitochondria had released their cytochrome c-GFP.
PS exposure was detected by staining the cells with 10 µl/ml v/v
annexin V-Alexa 568 (red) (CLONTECH). Cells with PS exposed on the outside were two logs brighter than control cells when
analyzed by FACS using the FL-1 (green) detector.
Confocal Microscopy and Supplemental Material--
Real time
apoptosis assays were performed on 2H18 and 2H18-Bcl-2 cells with a
Bio-Rad MRC1024ES confocal laser scanning microscope. During the
killing assays, cells were cultured in phenol red-free medium under a
layer of mineral oil to prevent evaporation. Individual frames and
QuicktimeTM movies were analyzed for cytochrome
c release according to Goldstein et al. (27).
zVAD-fmk, when used, was added to a final
concentration of 100 µM 15 min prior to the addition of
granzyme B and maintained in the culture for the duration of the
killing assays.
The Primary Granzyme B-mediated Death Pathway Involves
Mitochondrial Cytochrome c Release--
To study the molecular events
during target cell apoptosis, we utilized an experimental system of
granule-mediated killing wherein purified granzyme B is added directly
to the medium of target cells in the presence of a
replication-deficient adenovirus (28, 33, 34). Although granzyme B is
capable of entering the target autonomously, apoptosis does not occur
in the absence of virus. The granzyme B/adenovirus system has been
shown to trigger all biochemical and cellular manifestations of target
cell apoptosis observed when target cells are treated with granzyme B
and perforin and those treated with whole CTL (9, 28, 29, 33-36).
Using 2H18 and 2H18-Bcl-2, novel cell lines stably expressing
cytochrome c-GFP ± Bcl-2, we examined molecular events
upstream and downstream of mitochondrial cytochrome c
release in granzyme-mediated killing. Using this system we sought to
establish a preference, if any, given to Bcl-2-inhibitable events by
delivering granzyme B at limiting doses. 2H18 and 2H18-Bcl-2 cells were
treated with decreasing amounts of granzyme B along with a constant
amount of adenovirus. After 2 and 4 h, cells were harvested and
quantitatively assayed by flow cytometry for apoptosis by annexin V
binding. At concentrations of granzyme B as low as 10 ng/ml we observed
rapid release of cytochrome c which corresponded with PS
externalization (Fig. 1). Bcl-2 was
capable of inhibiting death at doses of granzyme B as high as 1 µg/ml. As observed earlier, the presence of
zVAD-fmk, which afforded no protection from
cytochrome c release, inhibited PS externalization, which
presumably requires caspase activity in short term assays.
To determine if granzyme B can overcome the Bcl-2 block at longer time
points, we treated 2H18-Bcl-2 cells with granzyme and adenovirus and
followed the target cells in culture by time lapse video confocal
microscopy. As shown in Fig. 2, when
2H18-Bcl-2 cells were incubated in the presence of granzyme B and
adenovirus, there was no release of cytochrome c. After an
extensive incubation (24 h), we observed only a small number of
apoptotic cells, and we did not observe cytochrome c release
in the apoptotic cells. This suggests that granzyme B treatment cannot
override the Bcl-2 blockade of cytochrome c release and is
consistent with the model in which granzyme B is capable of activating
effector caspases directly (10, 21, 22). However, it is clear from our
observations that the most efficient pathway to death in targets of
granzyme B involves includes release of cytochrome c from
the mitochondria.
Cytochrome c Release by Granzyme Is Caspase-independent--
To
characterize further the events leading to cytochrome c
release during target cell apoptosis, we utilized target cells expressing cytochrome c-GFP. HeLa has proven to be an
excellent target for killing by granzyme B in combination with
adenovirus (28, 29), and therefore we utilized 2H18, the HeLa sublines expressing cytochrome c-GFP described above to study
cytochrome c release during granzyme B-mediated apoptosis.
The top series of panels in Fig.
3 shows a representative field of 2H18
cells incubated in the presence of granzyme B and adenovirus in which cytochrome c release occurs within 60 min as indicated by
the diffuse cytoplasmic pattern of cytochrome c-GFP. As
shown in the bottom series of panels, the
addition of the broad spectrum caspase inhibitor
zVAD-fmk did not diminish the time required to
achieve comparable levels of cytochrome c release.
Therefore, caspase-8 is not an obligate requirement for cytochrome
c release in granzyme B-treated target cells. These data are
consistent with the observation that granzyme B/adenovirus induces a
drop in the mitochondrial transmembrane potential which does not
require active caspases (36).
In a similar set of experiments we treated Jurkat targets with granzyme
B and adenovirus in the presence and absence of
zVAD-fmk. Fig.
4A shows an immunoblot time
course of cytosol isolated from treated Jurkat. In granzyme
B/adenovirus-treated cells cytosolic cytochrome c appeared
within 15-30 min, whereas no cytosolic cytochrome c was
observed in cells treated with adenovirus alone. In Jurkat incubated in
100 µM zVAD-fmk prior to the
addition of granzyme and adenovirus, there was no reduction in the
appearance of cytosolic cytochrome c, thus demonstrating
further that caspases are not required for this phenomenon to occur.
Similarly, experiments in Jurkat-Bcl-2 targets showed that expression
of Bcl-2 completely prevented granzyme B-mediated cytochrome
c release as noted by the absence of cytosolic cytochrome
c. Analysis of the mitochondrial pellets (Fig.
4A, bottom panel) showed that cytochrome
c was maintained in its original cellular fraction.
Cytochrome c Release by Granzyme B-activated Bid--
Because
granzyme B directly cleaves Bid and cytochrome c release can
be achieved without caspase activation, we reasoned that granzyme B
might mediate cellular cytochrome c release in a
caspase-independent manner through Bid. To test this model, we treated
isolated mitochondria with purified Bid processed by granzyme B. Fig.
4B shows an immunoblot of supernatants from mitochondria
treated with increasing concentrations of granzyme B-cleaved and
uncleaved Bid. At the highest concentrations of untreated Bid tested
(100 ng/ml) we observed some cytochrome c release, but Bid
that had been activated by prior treatment with granzyme B was
significantly more potent with respect to its cytochrome c
releasing activity. Full-length GST-Bid, which possesses no cytochrome
c releasing activity, became active after treatment with
granzyme B. When GST-Bid was processed by granzyme B, the cytochrome
c releasing activity was comparable to that observed with
processed wild type Bid. It is important to note that mitochondria
treated with granzyme B alone did not release cytochrome c,
indicating that granzyme B does not directly cleave any
mitochondria-associated proteins that result in cytochrome c
release. Although granzyme B cleaves Bid at only one of the caspase-8
sites, Bid processed by granzyme B is active in releasing mitochondrial
cytochrome c.
Granzyme B cleaves and activates Bid in vitro, so we next
set out to ascertain the pattern and requirements for Bid cleavage in
target cells treated with granzyme B. Jurkat targets were treated with
granzyme B and adenovirus for 3 h at 37 °C. Whole cell lysates from these cells were analyzed for the presence and cleavage of Bid
protein. As shown in Fig. 4C, there was significant cleavage of Bid in granzyme B/adenovirus-treated cells, which corresponds with
cytosolic cytochrome c release (Fig. 4A). The
reduction in full-length Bid protein in granzyme-treated targets was
not inhibited in the presence of the broad spectrum caspase inhibitor
zVAD-fmk. Whereas granzyme-mediated Bid cleavage
was independent of caspase activation, Jurkat treated with anti-Fas
required active caspases for Bid cleavage to occur because cleavage of
Bid in anti-Fas-treated Jurkat, which proceeds via caspase-8
activation, was inhibited by zVAD-fmk. These data
are in agreement with our previous findings that cleavage of Bid in
target cells still occurs in the presence of the viral serpin caspase-8
inhibitor, SPI-2 (9). Similar treatment of targets overexpressing Bcl-2
did not block cleavage of Bid by death stimulus, granzyme B/adenovirus,
or anti-Fas, suggesting that Bid processing occurs upstream of the
apoptotic inhibition imparted by Bcl-2.
Kinetics of Bid Cleavage by Granzyme B--
Granzyme B cleaves and
activates Bid, thus providing a mechanism by which granzyme B can cause
caspase-independent cytochrome c release. In light of these
observations we analyzed Bid as a substrate for granzyme B. To
facilitate purification of the C-terminal cleavage product away from
the similar sized N-terminal fragment, we used purified GST-Bid fusion
protein. As shown in Fig. 5A, granzyme B cleaves GST-Bid in a dose-dependent manner.
After resolution by SDS-PAGE and transfer to polyvinylidene difluoride,
the C-terminal 12-kDa cleavage fragment was subjected to N-terminal
sequencing, and the scissile bond in Bid was identified as C-terminal
to aspartate 75 (Fig. 5A). Asp-75 corresponds to the
IEAD75SESQED sequence that can also be recognized by
caspase-8. Even at the highest doses of granzyme B there were no
detectable fragments corresponding to cleavage at Asp-59, the preferred
caspase-8 cleavage site. This confirms the earlier observation of Li
et al. (37) using Bid mutants, suggesting that granzyme B
cleaves at Asp-75, whereas caspase-8 prefers Asp-59 and is consistent
with the pattern of Bid cleavage products observed during granzyme
B-mediated events reported recently (9). It is important to note that
although the granzyme B and caspase-8 cleavage sites differ within Bid, both cleavage events are capable of activating Bid.
In Fas-mediated apoptosis the apical death-inducing signaling
complex-associated caspase, caspase-8, has been shown to
activate Bid to effect cytochrome c release (37, 38). We
recently demonstrated that granzyme B can bypass caspase-8 activation
(9). We sought to clarify the relevance of granzyme
B-dependent Bid cleavage by comparing the preference of
granzyme B for Bid and caspase-8 as substrates. We performed in
vitro cleavage assays to compare the rates of cleavage of Bid and
caspases. Equimolar amounts of radiolabeled in vitro
transcribed and translated substrate were digested with increasing
concentrations of granzyme B. As shown in Fig. 5, B and
C, granzyme B cleaved Bid more efficiently than caspase-8 or
caspase-3. Granzyme B at concentrations as low as 3.75 nM
cleaved Bid, whereas these concentrations induced little or no
caspase-3 or caspase-8 cleavage. Based on the published kcat/Km value for cleavage of
caspase-3 by granzyme B (3.6 × 104
M Granzyme B is a critical mediator of target cell death. This is
demonstrated by the inability of CTL and natural killer cells from
granzyme B Granzyme B cleaves and activates Bid, which provides a
caspase-independent means of releasing cytochrome c during
CTL granule-mediated apoptosis. In physiological settings it is
possible that granzyme B also utilizes caspase-8 activation as a means
to bring about the rapid destruction of its targets. However, direct
activation of Bid by granzyme B provides a caspase-independent pathway
to mitochondria. Thus, caspase-8 may contribute but is not required for
cytochrome c release in granule-mediated apoptosis. It is also clear from our data and others (45) that regardless of the
availability of caspase-8, the most efficient pathway to target cell
apoptosis is via the mitochondria. As one might expect, redundance is
built into the CTL arsenal, and granzyme B is capable of initiating apoptotic events through a mitochondrial bypass route by direct activation of downstream caspases, but with greatly reduced efficiency.
It was shown previously that Bid is cleaved by granzyme B (37),
but it remained to be demonstrated that this cleavage event resulted in
active Bid with respect to its cytochrome c releasing activity. However, it is abundantly evident from our observations that
although the preferred caspase-8 and granzyme B cleavage sites in Bid
differ, cleavage of Bid by granzyme B results in productive activation
and potent cytochrome c releasing activity. At first glance
this may appear to be a subtle distinction, but one must consider that
cleavage by granzymes or caspases does not always result in activation.
For example, granzyme B cleaves ICAD (inhibitor of CAD, the
caspase-activated DNase) (46, 47) at a site different from the scissile
bond for caspase-3. However, the primary cleavage by granzyme does not
result in productive activation of CAD and nucleolytic activity as a
result of ICAD cleavage (46). A second cleavage event occurs at
significantly elevated granzyme concentrations and even then in a much
less efficient manner than that observed in caspase-3-treated
ICAD·CAD (47). Whereas a concentration of 4.7 µM
granzyme B was required to activate the ICAD·CAD complex (47), as
little as 3.75 nM (0.1 ng/µl) granzyme B was sufficient
to cleave enough Bid to trigger the cytochrome c releasing
activity of Bid (see Figs. 4 and 5). This 3,000-fold difference in
sensitivity strongly implicates the
mitochondria/caspase-dependent pathway as the principal
mechanism for granzyme B-mediated activation of CAD, with resultant DNA fragmentation.
It has been observed that in some cells, Bcl-2 gives only partial or
temporary protection from apoptosis and that effector caspases may lead
to proteolytic inactivation of Bcl-2 (48, 49). In that model, removal
of Bcl-2 then allows for cytochrome c release and a
full-blown apoptotic response. However, our experiments show that a
small percentage of granzyme B-treated Bcl-2-expressing target cells
eventually undergo apoptosis, but they do so without the coordinate
release of cytochrome c. This suggests that in this system
Bcl-2 retains its activity to block cytochrome c release but
does not prevent the end result of apoptosis. This is believed to be
the result of engaging the apoptotic machinery downstream of the
mitochondria, which is consistent with the ability of granzyme B to
activate caspases directly, albeit less efficiently than Bid.
Therefore, granzyme B can circumvent the necessity, although inefficiently, for mitochondrial cytochrome c release, but
it does not override the protection afforded by Bcl-2 to inhibit cytochrome c release.
Although a physiological relevance has yet to be established for the
nuclear translocation of granzyme B in target cell apoptosis (29,
50-52), it has been shown that movement of granzyme B into the nucleus
is inhibited by Bcl-2 (51). However, if cytochrome c release
is a key event controlling all of the downstream events during target
cell apoptosis, then it is likely that the Bcl-2 block of nuclear
granzyme is due to the fact that release of mitochondrial proteins,
such as cytochrome c, is the switch necessary for all downstream effects. This situation may parallel that reported earlier
in which it was found that the mitochondrial components of the death
machinery were necessary for caspase-8-activated cell free extracts to
elicit all of the downstream apoptotic events, notably nuclear membrane
changes (53).
As demonstrated for death receptor-mediated apoptosis, some cell types
are prone to death by a type I or type II pathway, characterized by the
independence or dependence, respectively, of mitochondrial events (20).
For example, hepatocytes from Bid In our studies we have demonstrated that although granzyme B can
activate caspases directly, the preferred route to apoptosis under
limiting conditions is via Bid cleavage, mitochondrial outer membrane
permeabilization, and cytochrome c release. It is also likely that these events are accompanied by the release of Smac/Diablo from the mitochondrial intermembrane space, which functions to interfere with the inhibitory activity of inhibitor of apoptosis proteins. The latter may block caspases activated by granzyme B, and
therefore the involvement of mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis might be through this
molecule (or in conjunction with cytochrome c). In any case, the involvement of the mitochondria in granzyme B-induced apoptosis provides an explanation for the ability of Bcl-2 to block CTL-induced apoptosis in many cases, depending on the cells being targeted and the
amount of granzyme B that enters those cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Quantitation of PS flip and cytochrome
c release in 2H18 ± Bcl-2. Rapid cell death
is associated with cytochrome c release. 2H18 and 2H18-Bcl-2
targets were incubated in the presence of 10 plaque-forming units/ml
granzyme B and adenovirus (Ad). The broad spectrum caspase
inhibitor zVAD-fmk was added to a final
concentration of 100 µM 15-30 min prior to the addition
of granzyme and adenovirus. Cells were harvested at 2 and 4 h to
measure cytoplasmic cytochrome c and annexin V binding. In
the bottom panel, 2H18-Bcl-2 were incubated at 37 °C for
12 h in 0.5 µg/ml granzyme B and 10 plaque-forming units/ml
adenovirus. PS externalization was assayed by annexin V-alexa 568 binding as a measure of apoptosis. Cytochrome c release was
quantitated by measurement of loss of cytochrome c-GFP from
the mitochondria by flow cytometry as described under "Experimental
Procedures."
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Fig. 2.
Bcl-2 inhibits cytochrome c
release and rapid apoptosis in target cells. 2H18-Bcl-2
cells were treated with 1 µg/ml granzyme B and 10 plaque-forming
units/ml adenovirus in the presence of annexin V-alexa 568 (red). Confocal images were captured at 2-min intervals to
assess cytochrome c release and PS externalization.
Cytochrome release was not observed at any point over the duration of
these assays, but some cells eventually became annexin V-positive after
>6 h, blebbed, and rounded up (see Fig. 2 movie in Supplemental
Material). The time for induction of apoptosis was significantly
longer than in cells not expressing Bcl-2 (see Fig. 3).
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Fig. 3.
Cytochrome c release is not
dependent on active caspases in granzyme B-treated cells. 2H18
targets were incubated with 0.5 µg/ml granzyme B and adenovirus and
observed by confocal video microscopy. The top series of
panels shows the progression of apoptosis as indicated by
cytochrome c release. As cytochrome c is released
during apoptosis the fluorescent label changes from a punctate pattern
of healthy mitochondria to a diffuse cytoplasmic pattern. As cells
condense, bleb, and round up, cytochrome c-GFP resumes a
granular pattern in late stage apoptotic cells. The bottom
series of panels shows 2H18 treated with 100 µM zVAD-fmk prior to the addition
of granzyme and adenovirus. These panels demonstrate that
cytochrome c release proceeds in a caspase-independent
manner in these cells but that other indices of apoptosis, such as
rounding up and blebbing, require active caspases (see Fig. 3 movies in
Supplemental Material).
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Fig. 4.
Bid cleavage and cytochrome c
release are caspase-independent in granzyme B-treated cells.
Panel A, cytochrome c release in Jurkat by
granzyme B/adenovirus (Ad) is caspase-independent but is
inhibited by Bcl-2. Jurkat were treated with granzyme B and adenovirus
for the times indicated in the presence or absence of the caspase
inhibitor zVAD-fmk or Bcl-2. Cytosol was isolated
away from mitochondria and lysates analyzed by immunoblot. Bands
corresponding to cytochrome c are marked by
asterisks for each treatment. Cytochrome c
release was detected in the presence of zVAD-fmk
but was inhibited by Bcl-2. Controls in which cells incubated for 120 min with granzyme B or adenovirus alone were also included. Panel
B, granzyme B-"activated" Bid causes cytochrome c
release in isolated mitochondria. Isolated Xenopus
mitochondria were treated with full-length Bid or Bid that had been
digested with 17.5 ng/µl granzyme B prior to the addition to
mitochondria. After incubation of mitochondria with 1, 10, or 100 ng/ml
purified Bid or equimolar amounts of GST-Bid at 30 °C for 1 h,
mitochondrial pellets were removed by centrifugation, and the
supernatants were analyzed for the presence of cytochrome c
by immunoblot analysis. Untreated mitochondria incubated at 30 °C
and 4 °C were included as negative controls, and the supernatant
from a Nonidet P-40-treated mitochondria sample was included as a
positive control for cytochrome c release. Panel
C, cleavage of Bid is caspase-independent and upstream of Bcl-2.
Jurkat and Jurkat stably expressing Bcl-2 were treated with 1 µg/ml
granzyme and adenovirus for 3 h in the absence or presence of 100 µM zVAD-fmk. Cell lysates were collected,
analyzed by immunoblot for disappearance of the full-length Bid, and
quantitated with NIH-Image software. The percentage of Bid cleavage was
normalized according to the amount of full-length Bid in untreated
cells.
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[in a new window]
Fig. 5.
Kinetics of Bid cleavage by granzyme B. Purified GST-Bid was incubated in increasing doses of granzyme B (0.1, 0.25, 1, 2.5, 10, and 25 ng/µl; 3.75 nM-0.94
µM), resolved on standard SDS-PAGE, transferred to
sequencing polyvinylidene difluoride, and stained in Coomassie
Brilliant Blue. To determine the site at which granzyme B cleaves Bid,
we subjected GST-Bid to complete digestion by granzyme B, and the band
corresponding to the new C-terminal fragment(s) was excised and
subjected to N-terminal sequencing. No other fragments were observed,
even at high concentrations of granzyme B. For comparison of Bid,
caspase-3, and caspase-8 as substrates for granzyme B, equimolar
amounts of in vitro transcribed and translated proteins were
subjected to digestion by increasing amounts of granzyme B for 30 min
at 30 °C. The amount of granzyme B (0.1, 0.25, 1, 2.5, 10, and 25 ng/µl; 3.75 nM-0.94 µM) was kept
consistent between substrates. The cleavage products were analyzed by
SDS-PAGE and autoradiography. Concentrations of granzyme B required to
cleave detectable amounts of caspase-3 and caspase-8 (lower
panels) were capable of cleaving Bid to completion, suggesting
that Bid is a better substrate than either of these caspases for
granzyme B.
1 s
1)
using similar measurements (22) we calculated relative
kcat/Km values for granzyme B
cleavage of Bid (6.0 × 105
M
1 s
1)
and caspase-8 (2.4 × 104
M
1 s
1).
These data suggest that Bid is a better substrate for granzyme B than
caspase-8 and caspase-3 by more than 10-fold and support the notion
that Bid represents a target protein sensitive to activation by
granzyme B. They also support our contention that the Bcl-2-nhibitable mitochondrial pathway is utilized more efficiently by granzyme B than
that involving direct activation of caspases.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice to induce rapid DNA fragmentation and subsequent
apoptosis in allogeneic target cells (39, 40). These knockout mice also
have severely depressed ability to overcome infection by
cytomegalovirus (39, 41) and ectromelia (42). There is some low
efficiency residual killing activity in the granzyme B
/
mice which
is attributed to another granule protease, granzyme A, in a mechanism
that is thought to provide a backup to the principal mechanism
involving granzyme B (43). Double knockout of granzymes B and A results
in a phenotype similar to that observed in the perforin knockout mouse
(42, 44). The mechanism by which granzyme B activate apoptosis is
therefore of significant interest.
/
mice are resistant to killing by
anti-Fas, suggesting that the preferred apoptotic pathway in these
cells involves the Bid-mediated, Bcl-2-inhibitable release of proteins
from the mitochondria (54). The preference for the Bcl-2-inhibitable
mitochondrial pathway as the primary apoptotic pathway utilized by
granzyme B provides an explanation for the earlier observations that
Bcl-2 offers protection from rapid apoptosis induced by granzyme B and
perforin (24-26, 55). Our model also accounts for the reduced levels
of apoptosis observed in early studies, by a type I-like mitochondrial bypass pathway (20) engaged directly by granzyme B, which we found to
be less efficient in our experimental system. In the studies of Sutton
et al. (25), the Bcl-2 block of apoptosis could be overcome
at higher effector to target ratios and increased doses of granzyme B
in CTL-free killing (25), which is consistent with the notion that
granzyme B, like caspase-8, acts most efficiently via the release of
cytochrome c (53). It is possible that there are some cell
types that are more sensitive to direct activation of caspase-3 by
granzyme B, and in such cells Bcl-2 would not be expected to interfere
with this form of death. In cells lacking Bid, for example, granzyme B
may not be able to induce release of proteins from the mitochondria;
however, higher levels of granzyme would be necessary to induce
apoptosis. It remains to be determined what effect the newly identified
Smac/Diablo protein plays in granzyme B-mediated apoptosis. It is
possible that release of mitochondrial Smac/Diablo (56, 57) also
contributes toward the rapid destruction of target cells through
repression of the inhibitor XIAP in target cells, making caspase-3 more
readily activated directly by granzyme B. If the release of Smac/Diablo from the mitochondria is inhibited by Bcl-2, then it is reasonable to
propose that there are important mitochondrial events in
granzyme-induced apoptosis in addition to the release of cytochrome
c.
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FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health Grants GM52735 and AI40646 and a Canadian Institute for Health Research (CIHR) grant (to D. R. G.) and by CIHR and National Cancer Institute of Canada grants (to R. C. B.). This is La Jolla Institute for Allergy and Immunology Paper 373.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.
The on-line version of this article (available at
http://www.jbc.org) contains Quicktime movies for Figs. 2 and 3.
§ Supported by a postdoctoral fellowship from the CIHR.
Supported by a studentship from the CIHR.
** Medical scientist of the Alberta Heritage Foundation for Medical Research, CIHR distinguished scientist, and an international scholar of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Division of
Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA, 92121. E-mail:
dgreen5240@aol.com.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M009038200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CTL, cytotoxic T lymphocytes; GFP, green fluorescent protein; PS, phosphatidylserine; FACS, fluorescence-activated cell sorter; CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase.
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