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 (35), caspase-6 (6, 7), caspase-7 (810), caspase-8 (1113), 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.

Materials And Methods

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, 2628). [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% β-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).

Protease Assay.

Proteolytic activity in cell lysates was measured using the fluorogenic substrate Ac-DEVD-afc as described (32).

Results

Comparison of Caspases as GrB Substrates.

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−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.

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.

Inhibition of Caspase Activation during GrB-mediated Apoptosis.

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.

Caspase-3 and -7 Are Processed Directly by GrB in Cells with Inactivated Caspases.

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.

GrB Activates the Caspases through Caspase-10.

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.

Caspase Inhibitors Do Not Prevent GrB-mediated Cell Death.

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).

Discussion

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.

Acknowledgments

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.

References

References
1
Atkinson
EA
,
Bleackley
RC
Mechanisms of lysis by cytotoxic T cells
Crit Rev Immunol
1995
15
359
384
[PubMed]
2
Darmon
AJ
,
Ehrman
N
,
Caputo
A
,
Fujinaga
J
,
Bleackley
RC
The cytotoxic T cell proteinase granzyme B does not activate interleukin-1β–converting enzyme
J Biol Chem
1994
269
32043
32046
[PubMed]
3
Darmon
AJ
,
Nicholson
DW
,
Bleackley
RC
Activation of the apoptotic protease CPP32 by cytotoxic T-cell–derived granzyme B
Nature (Lond)
1995
377
446
448
[PubMed]
4
Quan
LT
,
Tewari
M
,
O'Rourke
K
,
Dixit
VM
,
Snipas
SJ
,
Poirier
GG
,
Ray
C
,
Pickup
DJ
,
Salvesen
GS
Proteolytic activation of the cell death protease Yama/ CPP32 by granzyme B
Proc Natl Acad Sci USA
1996
93
1972
1976
[PubMed]
5
Martin
SJ
,
Amarante-Mendes
GP
,
Shi
LF
,
Chuang
TH
,
Casiano
CA
,
O'Brien
GA
,
Fitzgerald
P
,
Tan
EM
,
Bokoch
GM
,
Greenberg
AH
,
Green
DR
The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/ CED-3 family protease, CPP32, via a novel two-step mechanism
EMBO (Eur Mol Biol Organ) J
1996
15
2407
2416
[PubMed]
6
Orth
K
,
Chinnaiyan
AM
,
Garg
M
,
Froelich
CJ
,
Dixit
VM
The CED-3/ICE–like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A
J Biol Chem
1996
271
16443
16446
[PubMed]
7
Fernandes-Alnemri
T
,
Litwack
G
,
Alnemri
ES
Mch2, a new member of the apoptotic Ced-3/Icecysteine protease gene family
Cancer Res
1995
55
2737
2742
[PubMed]
8
Chinnaiyan
AM
,
Orth
K
,
Hanna
WL
,
Duan
HJ
,
Poirier
GG
,
Froelich
CJ
,
Dixit
VM
Cytotoxic T cell–derived granzyme B activates the apoptotic protease ICE–LAP3
Curr Biol
1996
6
897
899
[PubMed]
9
Gu
Y
,
Sarnecki
C
,
Fleming
MA
,
Lippke
JA
,
Bleackley
RC
,
Su
MSS
Processing and activation of CMH-1 by granzyme B
J Biol Chem
1996
271
10816
10820
[PubMed]
10
Fernandes-Alnemri
T
,
Takahashi
A
,
Armstrong
R
,
Krebs
J
,
Fritz
L
,
Tomaselli
KJ
,
Wang
L
,
Yu
Z
,
Croce
CM
,
Salveson
G
et al
Mch3, a novel human apoptotic cysteine protease highly related to CPP32
Cancer Res
1995
55
6045
6052
[PubMed]
11
Boldin
MP
,
Goncharov
TM
,
Goltsev
YV
,
Wallach
D
Involvement of MACH, a novel MORT1/ FADD–interacting protease, in Fas/APO-1– and TNF receptor–induced cell death
Cell
1996
85
803
815
[PubMed]
12
Muzio
M
,
Chinnaiyan
AM
,
Kischkel
FC
,
O'Rourke
K
,
Shevchenko
A
,
Scaffid
C
,
Bretz
JD
,
Zhang
M
,
Ni
J
,
Gentz
R
et al
FLICE, a novel FADD-homologous ICE/ CED-3–like protease, is recruited to the CD-95 (FAS/Apo-1) death-inducing signaling complex (DISC)
Cell
1996
86
817
821
[PubMed]
13
Srinivasula
SM
,
Ahmad
M
,
Fernandes-Alnemri
T
,
Litwack
G
,
Alnemri
ES
Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ ICE–like cysteine proteases
Proc Natl Acad Sci USA
1996
93
14486
14491
[PubMed]
14
Duan
HJ
,
Orth
K
,
Chinnaiyan
AM
,
Poirier
GG
,
Froelich
CJ
,
He
W-W
,
Dixit
VM
ICE–LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B
J Biol Chem
1996
171
16720
16724
15
Fernandes-Alnemri
T
,
Armstrong
RC
,
Krebs
J
,
Srinivasula
SM
,
Wang
L
,
Bullrich
F
,
Fritz
LC
,
Trapani
JA
,
Tomaselli
KJ
,
Litwack
G
,
Alnemri
ES
In vitroactivation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains
Proc Natl Acad Sci USA
1996
93
7464
7469
[PubMed]
16
Vincenz
C
,
Dixit
VM
Fas-associated–death domain protein interleukin-1β–converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling
J Biol Chem
1997
272
6578
6583
[PubMed]
17
Froelich
CJ
,
Orth
K
,
Turbov
J
,
Seth
P
,
Babior
BM
,
Gottlieb
RA
,
Shah
GM
,
Bleackley
RC
,
Dixit
VM
,
Hanna
WL
New paradigm for lymphocyte granule mediated cytotoxicity: targets bind and internalize granzyme B but a endosomolytic agent is necessary for cytosolic delivery and apoptosis
J Biol Chem
1996
271
29073
29081
[PubMed]
18
Shi
LF
,
Chen
G
,
MacDonald
G
,
Bergeron
L
,
Li
HL
,
Miura
M
,
Rotello
RJ
,
Miller
DK
,
Li
P
,
Seshadri
T
,
Yuan
JY
,
Greenberg
AH
Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B
Proc Natl Acad Sci USA
1996
93
11002
11007
[PubMed]
19
Krajewska
M
,
Wang
HG
,
Krajewski
S
,
Zapata
JM
,
Shabaik
A
,
Gascoyne
R
,
Reed
JC
Immunohistochemical analysis of in vivopatterns of expression of CPP32 (Caspase-3), a cell death protease
Cancer Res
1997
57
1605
1613
[PubMed]
20
Komiyama
T
,
Ray
CA
,
Pickup
DJ
,
Howard
AD
,
Thornberry
NA
,
Peterson
EP
,
Salvesen
G
Inhibition of interleukin-1β converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition
J Biol Chem
1994
269
19331
19337
[PubMed]
21
Thome
M
,
Schneider
P
,
Hofmann
K
,
Fickenscher
H
,
Meinl
E
,
Neipel
F
,
Mattmann
C
,
Burns
K
,
Bodmer
JL
,
Schröter
M
et al
Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors
Nature (Lond)
1997
386
517
521
[PubMed]
22
Shi
L
,
Mai
S
,
Israels
S
,
Browne
KA
,
Trapani
JA
,
Greenberg
AH
Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization
J Exp Med
1997
185
855
866
[PubMed]
23
Hanna
WL
,
Zhang
X
,
Turbov
J
,
Winkler
U
,
Hudig
D
,
Froelich
CJ
Rapid purification of cationic granule proteases: application to human granzymes
Protein Purif Exp
1993
4
398
402
[PubMed]
24
Seth
P
A simple and efficient method of protein delivery into cells using adenovirus
Biochem Biophys Res Commun
1994
203
582
587
[PubMed]
25
Casiano
CA
,
Martin
SJ
,
Green
DR
,
Tan
EM
Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1)–mediated T cell apoptosis
J Exp Med
1996
184
765
770
[PubMed]
26
Hugunin
M
,
Quintal
LJ
,
Mankovich
JA
,
Ghayur
T
Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3)product
J Biol Chem
1996
271
3517
3522
[PubMed]
27
Ghayur
T
,
Hugunin
M
,
Talanian
RV
,
Ratnofsky
S
,
Quinlan
C
,
Emoto
Y
,
Pandey
P
,
Datta
R
,
Huang
YY
,
Kharbanda
S
et al
Proteolytic activation of protein kinase C d by an ICE/CED 3–like protease induces characteristics of apoptosis
J Exp Med
1996
184
2399
2404
[PubMed]
28
Orth
K
,
O'Rourke
K
,
Salvesen
GS
,
Dixit
VM
Molecular ordering of apoptotic mammalian Ced-3/ ICE proteases
J Biol Chem
1996
171
16443
16446
[PubMed]
29
Talanian
RV
,
Quinlan
C
,
Trautz
S
,
Hackett
MC
,
Mankovich
JA
,
Banach
D
,
Ghayur
T
,
Brady
KD
,
Wong
WW
Substrate specificities of caspase family proteases
J Biol Chem
1997
272
9677
9682
[PubMed]
30
Gorczyca
W
,
Gong
J
,
Darzynkiewicz
Z
Detection of DNA strand breaks in individual apoptotic cells by the in situterminal deoxynucleotidyl transferase and nick translation assays
Cancer Res
1993
53
1945
1951
[PubMed]
31
Duan
HJ
,
Chinnaiyan
AM
,
Hudson
PL
,
Wing
JP
,
He
WW
,
Dixit
VM
ICE–LAP3, a novel mammalian homologue of the Caenorhabditis eleganscell death protein ced-3 is activated during fas- and tumor necrosis factor– induced apoptosis
J Biol Chem
1996
271
1621
1625
[PubMed]
32
Enari
M
,
Talanian
RV
,
Wong
WW
,
Nagata
S
Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis
Nature (Lond)
1996
380
723
726
[PubMed]
33
Fernandes-Alnemri
T
,
Litwack
G
,
Alnemri
ES
CPP32, a novel human apoptotic protein with homology to Caenorhabditis eleganscell death protein Ced-3 and mammalian interleukin-1b–converting enzyme
J Biol Chem
1994
269
30761
30764
[PubMed]
34
Müller
C
,
Tschopp
J
Resistance of CTL to perforin-mediated lysis: evidence for a lymphocyte membrane protein interacting with perforin
J Immunol
1994
153
2470
2478
[PubMed]
35
Sarin
A
,
Williams
MS
,
Alexander-Miller
MA
,
Berzofsky
JA
,
Zacharchuk
C M
,
Henkart
PA
Target cell lysis by CTL granule exocytosis is independent of ICE/ Ced-3 family proteases
Immunity
1997
6
209
215
[PubMed]
36
Shi
LF
,
Chen
G
,
He
DL
,
Bosc
DG
,
Litchfield
DW
,
Greenberg
AH
Granzyme B induces apoptosis and cyclin A–associated cyclin-dependent kinase activity in all stages of the cell cycle
J Immunol
1996
157
2381
2385
[PubMed]
37
Ongkeko
W
,
Ferguson
DJP
,
Harris
AL
,
Norbury
C
Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage
J Cell Sci
1995
108
2897
2904
[PubMed]
38
Schröter
M
,
Peitsch
MC
,
Tschopp
J
Increased p34cdc2-dependent kinase activity during apoptosis: a possible activation mechanism of DNase I leading to DNA breakdown
Eur J Cell Biol
1996
69
143
150
[PubMed]
39
Chen
G
,
Shi
L
,
Litchfield
DW
,
Greenberg
AH
Rescue from granzyme B–induced apoptosis by Wee1kinase
J Exp Med
1995
181
2295
2300
[PubMed]
40
Zhou
Q
,
Snipas
S
,
Orth
K
,
Muzio
M
,
Dixit
VM
,
Salvesen
GS
Target protease specificity of the viral serpin CrmA: analysis of five caspases
J Biol Chem
1997
272
7797
7800
[PubMed]
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.

Author notes

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@merle.acns.nwu.edu