By
From the * BASF Bioresearch Corporation, Worcester, Massachusetts 01605; Department of Medicine,
Evanston Hospital, Northwestern University, Evanston, Illinois 60201; § W.M. Keck Autoimmune
Disease Center, Scripps Research Institute, La Jolla, California 92037;
Biological Chemistry,
University of Michigan Medical School, Ann Arbor, Michigan 48109; and the ¶ Medicine Branch,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.
Lymphocyte granule-mediated cytotoxicity is designed
to protect the host from invasion by intracellular pathogens, tumor, and nonself cells. Two distinct mechanisms encompass this phenomenon: (a) perforin (PFN)1-mediated
necrosis of the target and (b) PFN/granzyme-induced apoptosis in which granzyme B (GrB) plays a pivotal role. Unlike PFN-induced target cell necrosis, the mechanism of
lymphocyte granule-mediated apoptosis has only recently
become apparent (1). An important clue to its function is
the preference of GrB for cleavage of peptide bonds after
Asp residues. The caspases, which are expressed as zymogens, are activated by proteolytic cleavage at specific
Asp residues, and act by cleaving substrates at Asp residues
as well. Except for caspase-1 (2), all caspases that have been
tested as GrB substrates can be processed and activated by
GrB in vitro: caspase-3 (3), caspase-6 (6, 7), caspase-7 (8), caspase-8 (11), caspase-9 (14), and caspase-10
(15, 16). The relative rates of processing of these enzymes
by GrB is not known, nor is it clear whether GrB can access and cleave these substrates in vivo.
We have proposed that GrB is delivered to target cells by
a mechanism unique to mammalian cells (17). Secreted
PFN and GrB are cointernalized into endosomes of the target cell during granule-mediated cytotoxicity. PFN then
permeabilizes the vesicles, delivering GrB to the cytosol.
Subsequently, GrB induces cell death by activating the
caspases. Caspase-1, -2, -3, -6, and -7 have been reported to undergo processing in target cells during GrB-mediated
apoptosis (3, 8, 17, 18). Because many caspases can auto- or
cross-activate one another, and because the relative contribution of caspase activation by caspases and by GrB is unknown, it remains unclear whether a subset of caspase(s) are
directly cleaved by GrB to initiate the death pathway. Although the apparent polyspecificity of GrB toward multiple
caspases complicates dissection of the pathway(s) activated
by GrB, this attribute may be crucial under conditions
where full activation of the caspases is hampered by the absence of a specific caspase (19) or by the presence of an inhibitor that inactivates one or more members of the pathway (20, 21). Knowledge of the preferences of GrB toward
the caspases would enable predictions of the caspase(s) first
processed by GrB to initiate apoptosis under physiologic
conditions and of downstream caspases that may be activated by the granzyme when some enzymes are disrupted.
Although it is commonly accepted that GrB induces cell
death only through activation of the caspases, this notion must
be reconciled with the evidence that GrB rapidly translocates
to the nucleus in targets treated with a combination of GrB
and PFN or replication-deficient adenovirus type 2 (AD; reference 22) (Pinkoski, M., A. Caputo, P. Seth, C.J. Froelich, and R.C. Bleackley, manuscript submitted for publication);
and (Trapani, J., P. Jans, M. Smyth, C.J. Froelich, V. Sutton, and D. Jans, manuscript submitted for publication).
These results argue that GrB induces cell death by an intranuclear process separate from or in addition to the caspases.
We report experiments designed to define the mechanism(s) of GrB-induced apoptosis. The catalytic efficiencies
of GrB against all 10 known caspases was measured. We
developed a protocol for fully blocking caspase activity in
whole cells. This system was used to demonstrate the kinetics of caspase protein cleavage due only to GrB activity
(and not to caspase auto- or cross-activation), and to reveal
that GrB can induce both growth arrest and cell death in
target cells in the absence of caspase activity.
Cell lines.
Jurkat cells were maintained in RPMI-1640, 10%
heat-inactivated FCS supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin. MCF7w and
MCF7i, breast cancer cells reported to be deficient in capases -3 and -10 (16, 19), were provided by Drs. D. Boothman (University of Wisconsin, Madison, WI) and K. Tomaselli (IDUN, Inc.
San Diego, CA), respectively.
Reagents.
Human GrB was purified to homogeneity from a
human NK cell line (YT cells; reference 23). Titration with the
GrB-specific protease inhibitor, anti-GraB, an antichymotrypsin
engineered to react specifically with GrB (17), showed that
~80% of the serine protease is present in its active form (data not
shown). A nonreplicating strain of AD was cultured and isolated
as described (24). The peptidic inhibitors and substrates z-DEVD-fmk, z-VAD-fmk, and Ac-DEVD-afc were supplied by Kamiya
Biomedical (Spokane, WA). Human sera containing highly specific autoantibodies to poly-(ADP-ribose) polymerase (PARP),
small nuclear ribonucleoprotein (snRNP) and lamin B were from
the serum bank of the W.M. Keck Autoimmune Disease Center
of The Scripps Research Institute (La Jolla, CA; reference 25).
Caspase Cleavage by GrB.
Caspases-1-9 and 10/b were encoded on vectors under the control of a T7 RNA polymerase
promoter (12, 16, 26). [35S]Methionine-labeled proteins were
prepared from these vectors using a T7-coupled reticulocyte lysate transcription translation (TnT) system (Promega, Madison,
WI). Cleavage assayed consisted of 75 µl of TnT reaction mix
and 75 µl of reaction buffer (100 mM Hepes, pH 7.5, 20% glycerol, 0.5 mM EDTA, 5 mM DTT) containing purified GrB at a
final concentration of 5.2 nM. Incubations were at room temperature, and 10 µl aliquots were removed at various times between
0 and 30 min, and stopped by diluting with 75 µl of a buffer containing SDS and heating to 90°C for 5 min. Aliquots (7.5 µl)
were separated by SDS-PAGE using 10-20% Tris-tricine gels
(Integrated Separation Systems, Natick, MA). Dried gels were imaged, and bands corresponding to caspases were quantitated using a GS-250 Molecular Imager and Molecular Imaging Screen
CS (BioRad, Hercules, CA). Apparent Vmax/Km values were obtained by plotting substrate band intensity versus time and fitting
to an exponential decay curve (where kobs = Vmax/Km) as described (29). Reported kcat/Km values were obtained by dividing
kobs by enzyme concentration corrected for fractional activity as
described above, and are means of assays performed in triplicate.
Target Cells.
Jurkat cells were treated with GrB and AD as
described (17); unless indicated, cells (106/ml) were mixed with
GrB (1 µg/ml; 30 nM) and AD (100 PFU) in 1-ml microfuge
tubes containing RPMI supplemented with 0.5% BSA. Target
cells were pretreated with z-DEVD-fmk and/or z-VAD-fmk (100 µM) except as indicated. The peptides were dissolved in
Me2SO and used at <0.5% (vol/vol). Cell number and viability
were determined by Trypan Blue dye exclusion and conventional
light microscopy. To minimize in vitro caspase processing in cell
lysates for immunoblotting, 5 min before the end of the assay target cells were washed with PBS to remove excess GrB, transferred to a fresh tube, and lysed in buffer containing the GrB-specific antiprotease, anti-GraB (17).
Terminal Deoxyribonucleotidyl Transferase Labeling of DNA Strand
Breaks with FITC-dUTP, Propidium Iodide Reactivity, and Hoescht
33342 Staining.
Cell death was measured by terminal deoxyribonucleotidyl transferase catalyzed labeling of DNA strand breaks
with FITC-dUTP (FITC-TUNEL; reference 30) and/or propidium iodide (PI) staining followed by flow cytometry. Data acquisition consisted of 5,000 events/analysis on a Coulter Epic V. For Hoescht staining, cells were fixed with 0.5% paraformaldehyde for 15 min, cytospun to microscope slides, and stained with
Hoescht 33342 (1 µg/ml). Cells were visualized with a Zeiss Fluorescent microscope.
Western Blotting of Caspases.
Processing of caspase-3, -6, and -7 was measured as described (4, 6, 17, 31). Treated cells (106/ml)
were lysed, resolved by SDS-PAGE (10%), and transferred to nitrocellulose. Anti-caspase-3, -6, and -7 rabbit antisera were used
at dilutions of 1:1,000 followed by incubation with anti-rabbit Ig-horseradish peroxidase (Amersham, Arlington Heights, IL) at a
dilution of 1:10,000. The signal was visualized with the ECL kit
(Amersham).
Western Blotting for PARP, snRNP, and Lamin B.
Harvested
target cells were resuspended at 107 cells/ml in lysis buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1%
Protease Assay.
Proteolytic activity in cell lysates was measured using the fluorogenic substrate Ac-DEVD-afc as described (32).
Caspase-1, -2, -3, -6, and -7 have been reported to undergo processing in
target cells during GrB-mediated apoptosis (3, 8, 17, 18). It
is not known whether GrB processes each of these proteases directly or whether some are matured by a subset of
the caspases that are the direct GrB substrates. To compare the caspases as GrB substrates, all 10 known family members were expressed in an in vitro transcription-translation
system, and tested as GrB substrates. By monitoring the decrease in caspase substrate parent band intensity, a single
value potentially including GrB cleavage at more than one
site, plus autoactivation by matured caspase, was obtained.
Caspase-7 and -10 were clearly preferred GrB substrates,
displaying observed kcat/Km values of 533,000 and 325,000 M GrB displays a preference for peptide substrates in vitro
consistent with cleavage of caspase-7 and -10 at IxxD motifs (IQAD198-S and IEAD372-A, respectively), which separate the pSmall and pLarge subunits (Talanian, R.V., unpublished results). Caspase-3 contains a similar sequence in the
analogous position (IETD174-S), so it seemed likely that
this enzyme would be cleaved by GrB preferentially with
respect to the other caspases. When sixfold higher concentrations of GrB was added to in vitro translated caspase-3,
processing could be detected at 30 min (Fig. 1 a). In contrast, virtually all caspase-7 and most of caspase-10 were processed within 5 min. Cleavage of another nonpreferred
GrB substrate, caspase-6, was not observed until 120 min
(Fig. 1 d). Although caspase-7 and -10 are clearly preferred
GrB substrates, the results suggest that caspase-3 and others
can be matured directly by GrB under circumstances where
caspase-7 and/or -10 are absent.
To allow the assessment of caspase activation by
GrB in cells independently of caspase auto- and trans-activation, we explored the use of irreversible peptidic ligands
for full caspase inhibition. Caspase activation in GrB/AD-treated cells was measured by cleavage of the peptidic substrate Ac-DEVD-afc, a readout of caspase-3-like proteolytic activity that is typical of lysates of cells undergoing apoptosis. On addition of GrB/AD, we observed a transient
induction of caspase-3-like activity that was fully inhibited
by z-DEVD-fmk at 100 µM (Fig. 2 a). To test more stringently for full inhibition of all caspases, we evaluated cell
lysates for GrB/AD-induced cleavage of PARP, snRNP,
and lamin B to the signature apoptotic fragments of 85, 40, and 45 kD, respectively. Because z-DEVD-fmk at 100 µM
failed to block PARP cleavage completely (data not shown), we used a combination of z-DEVD-fmk and z-VAD-fmk
each at 100 µM. The two inhibitors completely prevented
cleavage of all three proteins up to 18 h after GrB/AD application (Fig. 2 b). We conclude that these inhibitors at
100 µM each give full caspase inhibition.
To confirm direct caspase inhibition by the peptidic
ligands in cells, caspase-3 and -7 were examined by immunoblot analysis of cells treated with z-DEVD-fmk and GrB
(Fig. 3). Inhibitor treatment resulted in a shift of the pLarge
subunit of each enzyme to higher molecular weight, consistent with the location of the putative catalytic nucleophiles (Cys163 and Cys186 for caspase-3 and -7, respectively) on the pLarge subunits (10, 33). Caspase-3 also displayed a shifted pro/pLarge fragment (Fig. 3 b), suggesting that cleavage at that site (ESMD28-S) is autocatalytic. The
results demonstrate that z-DEVD-fmk inactivates caspase-3
and -7 in cells directly, and suggest that the combination of
z-DEVD-fmk and z-VAD-fmk gives similar inactivation of
the other caspases as well.
Using z-DEVD-fmk and z-VAD-fmk to block auto- and trans-caspase activation, we used
immunoblotting to examine caspase processing directly by
GrB in Jurkat cells. Owing to the lack of a suitable antibody to detect processed forms of caspase-10, our efforts focused on caspase-3 and -7. In the absence of inhibitors,
GrB/AD resulted in rapid maturation of caspase-7 characterized by removal of the propeptide by cleavage at
DSVD23-A and apparently slower cleavage between the
pLarge and pSmall subunits at IQAD198-S (Fig. 4 a). With
addition of the inhibitors, caspase-7 was cleaved only between the pLarge and pSmall subunits (Fig. 4 a). The results show that GrB initiates caspase-7 activation by cleavage between the pLarge and pSmall subunits. Pro-region
removal, which is rapid compared with GrB-mediated
cleavage between the pLarge and pSmall subunits, is conducted by and requires active caspases.
Caspase-3 processing in Jurkat cells by GrB/AD can be
detected in 15-30 min (Fig. 4 b; reference 17). The onset
of proteolysis (60 min) was slower and the quantity matured was reduced compared with caspase-7 (Fig. 4 b). Like
caspase-7, the inhibitors prevented removal of the pro-
region, showing that cleavage at this site is also caspase dependent and that GrB initiates caspase-3 maturation by
cleavage at the IxxD motif between its large and small subunits. The results also provide evidence that GrB can process a less preferred substrate such as caspase-3 directly in
cells, at correspondingly lower efficiency.
The processing pattern of caspase-7 in cells suggests that its pro-region is cleaved either by another caspase or autocatalytically. Because caspase-10 is also a preferred substrate for
GrB, it is a likely candidate for the completion of caspase-7
maturation. We find that the breast carcinoma cell lines
MCF7w and MCF7i, which are deficient in caspase-3 and
-10 (16, 19), are also deficient in GrB/AD-induced caspase-7
pro-region removal (Fig. 5). In these cells, GrB cleaves
caspase-7 between the pLarge and pSmall subunits as expected. The resulting species cannot remove its pro-region
(in cis or trans), demonstrating that caspase-7 pro-region
cleavage requires the action of other caspase(s). We propose
that this occurs through the preferred GrB substrate
caspase-10, and thus that caspase activation initiated by GrB
occurs primarily through caspase-10.
We have observed extremely rapid nuclear translocation of endocytosed GrB into the majority of target
cells after treatment with PFN or AD. We reasoned that
GrB might catalyze intranuclear proteolysis directly and
cause cell death independently of caspase action. Using the
irreversible caspase inhibitors, we examined the cells for
morphologic evidence of cell death. Similar to target cells
treated only with z-DEVD-fmk (17), the two caspase inhibitors blocked DNA fragmentation (TUNEL) but only a
minor portion of the cells expressed condensed nuclei and
PI staining at 4 h (<15%; data not shown). Longer-term
effects of intranuclear GrB was examined by PI and Hoescht
over a 4-d period. In these experiments, fresh inhibitors
were added to the target cells at 24 h. Compared with controls, cells treated with the caspase inhibitors and GrB/AD were present in reduced numbers and completely failed to
proliferate throughout a 96-h period (Fig. 6 a). Morphologic analysis by PI and Hoescht stain showed two populations: an increased percentage of dying cells (Fig. 6, b and c)
whose nuclei became progressively more condensed throughout the culture period (40% at 96 h), and a second group
with normal sized nuclei and intact plasma membrane (60%)
(Fig. 7 c). These results suggest that GrB induces dimorphic changes in the targets in which one subset has died and the
other is in growth arrest. The mechanisms that result in
these outcomes do not involve proteolysis of substrates
usually associated with apoptotic cell death mediated by activated caspases (see Fig. 2 b).
We recently proposed that granule-mediated apoptosis
mimics a pathway used by viruses to enter nucleated cells
(17). In this model, apoptosis is induced in target cells
through the delivery of GrB by PFN. GrB and PFN are internalized into a coated vesicle, and during fusion with an
early endosome, PFN, by an endosomolytic mechanism,
releases GrB to the cytosol. Consistent with this model, the
replication-deficient type 2 AD can substitute for PFN
(17). Cytosolic delivery of GrB by AD offers two advantages to study the processing of caspases in target cells undergoing apoptosis. First, target cells vary markedly in their
susceptibility to the apoptotic action of PFN and granzymes.
Although this is often attributed to variable effects of the
granzymes, the membranolytic activity of PFN is also highly
variable (34). AD, on the other hand, when properly engineered, can be used to deliver GrB in a highly reproducible
fashion. Second, unlike AD, PFN may lyse a proportion of
the target cells, exposing cytosol components to the proteolytic action of the granzymes. Consequently, target cells
treated with GrB/PFN contain a mixture of products that reflect GrB-mediated cleavage of proteins in whole cells as
well as cellular extracts. In the present study, we took advantage of the GrB/AD model to determine the mechanism(s) by which GrB initiates apoptosis in target cells, asking
which caspase(s) are activated directly by GrB, and whether
GrB can effect apoptosis independently of the caspases.
Measuring the catalytic efficiencies expressed by GrB
against the known caspases showed that GrB has a substantial preference for caspase-7 and -10. Therefore, either caspase
may be cleaved by GrB to initiate cell death. We find that
caspase-7 pro-region removal after GrB cleavage between
the pLarge and pSmall subunits is deficient in cell lines
lacking caspase-3 and -10, suggesting that full caspase activation requires one or both of those enzymes. Caspase-10
is probably situated near the cytoplasmic aspect of the plasma
membrane (12), making it accessible to GrB released from
the early endosomal compartment. Thus, caspase-10 may
be the more important GrB target for initiation of apoptosis.
This represents a proposed functional role for caspase-10,
and suggests a novel model for GrB-initiated apoptosis in
cells (Fig. 8).
The subsequent sequence by which caspases are activated
is suggested by the pattern in which caspase-6 and -7 are
cleaved in whole cells. Both are first cleaved free of the
propeptide, represented by sequences TETD23-A and
DSVD23-A, respectively (17). The former matches the substrate specificity of caspase-6 itself (29), and might be
largely autocatalytic. The latter matches well the preferences of caspase-3 and -7 (29). Thus, in vivo, caspase-10
may activate caspase-3 by cleaving the latter between the
pLarge and pSmall subunits (IETD174-S). The activation of
caspase-7 plus presumably other caspases is then followed
by rapid auto- and cross-activation in an explosive process
resulting in full activation of the caspases (Fig. 8). We note
that the participation of undiscovered caspases or other proteases in this process is neither ruled out by our data nor particularly unlikely.
Although GrB most efficiently elicits apoptosis in target
cells by initiating caspase activation through caspase-10,
there is sufficient evidence to propose also that GrB can still
activate caspases through caspase-7 or through less preferred substrates such as caspase-3 if the target cells lack
functional caspase-10 (see Fig. 8, secondary pathway). Owing
to mutation, inhibitory viral serpins, or lack of expression
in a given tissue or stage of differentiation, any particular
caspase may be nonfunctional; therefore, consistent with its
dominant role in tumor surveillance and viral clearance,
GrB has the redundant capacity to initiate caspase activation despite the absence of specific caspases.
Reflecting another level of redundancy in its apoptogenic potential, GrB apparently has the capacity to activate
cell death independently of the caspases. Caspases cleave
substrates after Asp residues, and are also activated by cleavage after Asp. Because GrB also has a preference for cleavage of proteins after Asp residues, in principle GrB can induce apoptosis both by activating caspases and by cleaving
substrates also recognized by the caspases. We find that in
the absence of caspase activation, cleavage of cellular substrates considered critical for the induction of apoptosis (PARP, snRNP, and Lamin B) did not occur (Fig. 2 b),
but GrB still caused cell death. In our model system for
granzyme delivery, GrB enters the cytosol and nucleus of
all target cells. In the presence of the inhibitors, intranuclear delivery of GrB resulted in two distinct responses: the
target cells either die or experience growth arrest. Therefore, our data reveal an unforseen biologic role for GrB
during granule-mediated cytotoxicity. In another system
that examined the role of caspases during CTL-mediated apoptosis, the results showed caspase inhibitors blocked
DNA fragmentation but not cell death (35). These results
are consistent with our previous observation that z-DEVD-fmk (40 µM) blocked only DNA cleavage (17) and with
the data reported here. Using CTLs to identify the granule
components and the pathways that these proteins activate
to cause cell death is not possible without specific inhibitors. Our experimental system extends these studies by
clearly demonstrating that GrB alone is sufficient to induce
cell death in target cells. The substrates directly cleaved by
GrB to induce this response have not been identified, and
call into question the significance of several so-called universal markers of apoptosis such as PARP cleavage.
The finding that target cells underwent growth arrest
was unexpected. GrB is reported to rapidly induce both cyclin A/cdc 2 and cyclin A/Cdk 2 kinase activities (36). Although these activities are temporally related to the apoptotic response in GrB-treated cells, the biologic significance
of these findings has remained enigmatic. Based on evidence that cells with increased cdc2 kinase activity are
more susceptible to apoptotic stresses (37, 38) and that inhibition of cdc2 kinase activation by Wee 1 kinase inhibits
GrB-induced apoptosis (39), we predict that efficient induction of granule-mediated cell death results from the interplay of these two pathways triggered by GrB: activation of cytosolic caspases and induction of intranuclear cyclin
A-kinase complexes.
The evolution of cytopathic virus-host interactions has
led viruses to adopt strategies that prevent or delay the
death of the host until productive replication has occurred.
The cytokine response modifier A (CrmA) of the cowpox
virus (20) as well as the description of a new family of viral
inhibitors (21) exemplify this strategy. Additional viral as
well as tumor-associated inhibitors that inactivate apoptotic
proteases will undoubtedly be discovered. Furthermore,
MCF7 cells express minimal caspase-3 and -10 (16, 19).
Despite the absence of these caspases, microinjection of
GrB results in rapid apoptosis (Pinkoski, M., A. Caputo, P. Seth, C.J. Froelich, and R.C. Bleackley, manuscript submitted for publication). Cytotoxic cells have evolved a
family of serine proteases that are delivered to pathogenic
cells ensuring apoptotic cell death by activating distinct but
interwoven pathways. The ability of GrB to induce cell
death in the presence of a partially or completely inactivated caspase pathway typifies the robustness of this important host defense system.
-mercaptoethanol, and the COMPLETE protease inhibitor
cocktail (Boehringer Mannheim, Indianapolis, IN). Lysates were
heated at 100°C for 5 min, passed several times through a 27-gauge
needle to shear DNA, and stored at
70°C until use. Samples
containing total protein from ~106 cells were applied to individual lanes in 12% polyacrylamide-SDS gels. After electrophoresis
under reducing conditions, proteins were transferred to nitrocellulose at 220 mA for 4-5 h. Nitrocellulose strips corresponding to
the individual lanes of gels were blocked for 30 min in PBS containing 0.05% Tween-20 (PBST) and 5% nonfat dried milk,
probed for 45 min with the appropriate human antibody diluted
from 1:100 to 1:400 in the same buffer (25), washed for 1 h in
several changes of PBST with gentle shaking, and probed for 30 min with a peroxidase-coupled secondary antibody (Zymed Laboratories, South San Francisco, CA). Bound antibody was detected with the ECL kit (Amersham).
Comparison of Caspases as GrB Substrates.
1s
1, respectively. Under conditions that resulted in
~90% cleavage of caspase-10 by GrB after 30 min, processing of caspase -1, -2, -3, -4, -5, -6, -8, and -9 was not observed.
Fig. 1.
Comparison of kinetics of proteolysis in vitro that
GrB manifests against caspase-3,
-6, -7 and -10. The [35S]methionine caspases were generated and isolated as described (28). In a total volume of 320 µl,
translated caspases were mixed
with GrB (30 nM) for the times
indicated and analyzed by SDS-PAGE and autoradiography. (a)
Kinetics of [35S]-caspase-3
(~250 ng) processing; (b) kinetics of [35S]-caspase-7 (~300 ng)
processing; (c) kinetics of [35S]-
caspase-10 processing (~300 ng);
and (d) kinetics of [35S]-caspase-6
processing (~250 ng).
[View Larger Version of this Image (38K GIF file)]
Fig. 2.
Blockade of caspase proteolytic activities with oligopeptide
inhibitors during GrB-induced apoptosis: effect on Ac-DEVD-afc fluorogenic activity and cleavage of death substrates, PARP, snRNP, and lamin
B. (a) z-DEVD-fmk (100 µM) inhibits Ac-DEVD-afc cleavage during
GrB/AD-mediated apoptosis. Target cells were pretreated with z-DEVD-fmk for 15 min. After exposure to GrB/AD for the times indicated, cells
were withdrawn for measurement of fluorogenic Ac-DEVD-afc activity.
(b) Generation of the apoptotic fragments of PARP, snRNP, and lamin B
is completely inhibited by the combination of z-DEVD-fmk and z-VAD-fmk. Total Jurkat cell lysates obtained from control cells and cells treated
for 1, 4, and 18 h with either GrB alone, GrB/AD, or GrB/AD plus
z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in 12%
SDS-polyacrylamide gels and proteins were transferred to nitrocellulose.
Individual lanes (containing protein corresponding to ~106 cells) were
reacted with specific human autoantibodies to PARP, snRNP, and lamin
B. Representative blots are shown. Intact proteins are indicated by lines,
whereas proteolytic fragments are indicated by arrows. Numbers to the
right represent relative molecular weights.
[View Larger Versions of these Images (17 + 45K GIF file)]
Fig. 3.
Complex formation between large subunits of caspase-7 and -3 and z-DEVD-fmk in Jurkat cells subjected to GrB-induced apoptosis. Jurkat
cells were treated with z-DEVD-fmk
(40 µM) for 30 min followed by GrB/
AD for 1 h. Lysates were immunoblotted from 10-20% gradient gels and
reacted with the polyclonal Abs against
(a) caspase-7 and (b) caspase-3. Inhibitor treatment resulted in a shift of the large subunits to higher molecular
weight (closed circle to the right), demonstrating that the inhibitor reacted directly with the caspase large subunits.
Caspase-3 similarly displayed a shifted
pro/pLarge fragment (open circle to the
right).
[View Larger Version of this Image (40K GIF file)]
Fig. 4.
GrB can directly
process caspase-7 and -3 in Jurkat cells with inhibited caspases:
effect on pattern and rate of processing. Lysates obtained from
Jurkat cells treated for 15, 30, 60, and 120 min with either GrB/
AD or GrB/AD plus z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in
12% SDS-polyacrylamide gels and caspases were detected by
immunoblotting. Individual lanes (containing protein corresponding to ~106 cells) were reacted
with (a) anti-caspase-7 Ab or (b)
anti-caspase-3 Ab.
[View Larger Version of this Image (83K GIF file)]
Fig. 5.
GrB directly processes caspase-7 in cells lacking
caspase-10. MCF cells (106/ml)
were allowed to adhere to 24-well culture plates overnight and
then treated with GrB/AD as
described in Materials and Methods. After 4 h, cells were harvested by trypsinization, and
lysates were subjected to immunoblotting for caspase-7. For
comparison, the processing of
caspase-7 in Jurkat cells exposed
to GrB/AD for 2 h is also
shown.
[View Larger Version of this Image (46K GIF file)]
Fig. 6.
GrB/AD treatment produces growth arrest and cell death in Jurkat cells with inhibited caspases. Target cells (106/ml) in RMPI, 0.5% BSA
were added to 24-well plates and pretreated with caspase inhibitors (100 µM) for 30 min followed by GrB/AD. At 18 h, 0.5 ml of the media was removed and replaced with RMPI, 10% FCS plus fresh inhibitors. (a) Total number of viable (i.e., PI impermeable) cells; (b) percentage of cells that developed permeability to PI staining; and (c) percentage of cells with condensed nuclei. Control cells treated with AD alone at the same multiciplicity of infection were not significantly growth impaired (data not shown).
[View Larger Versions of these Images (38 + 29 + 29K GIF file)]
Fig. 7.
Morphology during GrB-mediated death in Jurkat cells with
inhibited caspases. Target cells (106/ml) in RMPI, 0.5% BSA were added
to 24-well plates and pretreated with caspase inhibitors (100 µM) for 30 min followed by GrB/AD. At 24 h, 0.5 ml of the media was removed
and replaced with RMPI, 10% FCS plus fresh inhibitors. Photomicrograph displays cells at 96 h after Hoescht stain and imaging at 100× magnification. (a) Media; (b) GrB/AD; and (c) GrB/AD treated with caspase
inhibitors. Cells treated with AD alone at the multiplicity of infection
used here did not impair growth (data not shown).
[View Larger Version of this Image (82K GIF file)]
Fig. 8.
Model for caspase activation in whole cells undergoing GrB-mediated apoptosis. The dominant pathway (closed arrows) describes the
effect of GrB in cells that contain a full complement of known caspases. In
this case, we propose that GrB initially activates caspase-10, which then
matures caspase-3 and -7. These caspases then mature the other members
of the family. The open arrows (secondary pathway) signify how GrB can
directly activate other caspases under conditions where caspase-10 is disrupted by a mutation or a viral serpin (e.g., CrmA; reference 40). The
gray arrow suggests a pathway in which intranuclear GrB can induce cell
death independently of the caspases.
[View Larger Version of this Image (23K GIF file)]
Address correspondence to Dr. Christopher J. Froelich, Evanston Hospital, Research Department, WH Building, Rm B624, 2650 Ridge Ave., Evanston, IL 60201. Phone: 847-570-2348; FAX: 847-570-1253; E-mail: granzyme{at}merle.acns.nwu.edu
Received for publication 13 June 1997 and in revised form 6 August 1997.
1 Abbreviations used in this paper: AD, replication-deficient adenovirus type 2; GrB, granzyme B; PFN, perforin; PARP, poly-(ADP-ribose) polymerase; PI, propidium iodide; FITC-TUNEL, terminal deoxyribonucleotidyl transferase labeling of DNA strand breaks with FITC-dUTP; snRNP, small nuclear ribonucleoprotein.This work was supported by the Rice Foundation and the Arthritis Foundation, Illinois Chapter (C.J. Froelich). K. Orth is a recipient of National Institutes of Health postdoctoral fellowship CA68769.
We thank Dr. Vishva Dixit for kindly providing the anti-caspase-3, -6, and -7 antibodies plus the constructs for caspase-3, -6, -7, -8, and -9. We also thank Dr. Claudius Vincenz for providing the construct for caspase-10/b and greatly appreciate receipt of the caspase-3 and -10-deficient MCF7 lines from Drs. David Boothman and Kevin Tomaselli.
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