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Address correspondence to Evanston Northwestern Healthcare Research Institute, 1001 University Pl., Evanston, IL 60201. Tel.: (847) 570-7660. Fax: (847) 570-8025. E-mail: c-froelich{at}northwestern.edu
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Abstract |
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Key Words: granzyme B; apoptosis; caspase-3; mitochondria; mechanism
* Abbreviations used in this paper: AD, adenovirus; cyt c, cytochrome c; GrB, granzyme B; ICAD, inhibitor of caspase-activated deoxyribonuclease; MEF, murine embryonic fibroblast; NK, natural killer; PFN, perforin; PFU, plaque-forming unit; SG, serglycin; WT, wild type.
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Introduction |
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Among the granule proteases, granzyme B (GrB) serves as a model to understand how intracellular delivery of a protease causes cell death (Barry and Bleackley, 2002). GrB shares substrate specificity with caspases for cleavage after aspartate residues (Poe et al., 1991) and has been reported to process numerous caspases in vitro including caspase-3, -6, -7, -8 and -10 (Darmon et al., 1995; Chinnaiyan et al., 1996; Duan et al., 1996; Talanian et al., 1997). The results have lead to the notion that the protease initiates death by processing any number of caspases in vivo (Medema et al., 1997; Barry et al., 2000). We have learned, however, that GrB, due to the constraints of accessibility and rates of proteolysis proceeds efficiently in vivo to first process caspase-3, which along with GrB then matures caspase-7 (Yang et al., 1998). On this basis, we have come to view GrB as apical caspase-like in function. We have nonetheless speculated that GrB might also have the capacity to initiate alternate death pathways if the caspase cascade is paralyzed, for example, by viral inhibitors (Talanian et al., 1997). Supporting this concept, GrB appears to process certain caspase substrates including PARP, NuMA, DNA-PK (Andrade et al., 1998), and DFF45/inhibitor of caspase-activated deoxyribonuclease (ICAD) (Thomas et al., 2000; Sharif-Askari et al., 2001) and thus might cause cell death independently of the caspases. In contradistinction to the activation of a proteolytic cascade by an intracellularly delivered protease, GrB has been reported to induce death through a mitochondria-centered pathway by cleaving the BH3-only proapoptotic Bcl-2 family member, Bid. Three groups have reported data linking rapid Bid proteolysis with mitochondrial permeabilization (MacDonald et al., 1999; Heibein et al., 2000; Sutton et al., 2000; Alimonti et al., 2001; Pinkoski et al., 2001), suggesting the granzyme induces death primarily through this pathway.
Mitochondrial membrane permeabilization may be mediated by cytosolic factors such as Bax/Bak which insert in the outer membrane. Alternately, changes in the mitochondrial permeability transition pore complex (Bernardi, 1999; Crompton, 2000b) may allow the release of intramembranous proteins and/or loss of membrane potential across the inner membrane (m) (Bernardi, 1999; Crompton, 2000a; Loeffler and Kroemer, 2000). The outer mitochondrial membrane, responding to proapoptotic signals, becomes permeabilized and releases factors such as cytochrome c (cyt c), apoptosis-inducing factor (Joza et al., 2001; Ye et al., 2002), and most recently the serine protease HtrA2/Omi (Suzuki et al., 2001) and endonuclease G (Hengartner, 2001; Li et al., 2001). Among these paradigms, GrB-generated truncated Bid is predicted to oligomerize with proapoptotic family members, Bax and/or Bak, forming large ion channels in the outer mitochondrial membranes. The released cyt c then coalesces into an apoptosome with Apaf-1 and dATP facilitating activation of caspase-9, which in turn processes caspase-3 (Li et al., 1997; Zou et al., 1997). GrB also has been predicted to induce apoptosis by directly perturbing mitochondrial integrity through a Bid-independent pathway (Alimonti et al., 2001). Finally, using lines deficient in Bid, Bax, and/or Bak, investigators have shown GrB readily induces mitochondrial depolarization independently of cyt c release, permeability transition, and caspase activation (Thomas et al., 2001). Together the results imply the granzyme has a multifaceted potential to ensure target cell death by initiating several pathways: (a) caspase driven, (b) BH3-only protein driven, (c) directly "damaging" mitochondria, and (d) cleavage of crucial structural and regulatory proteins.
We have reported that GrB is exclusively secreted from cytotoxic cells as a macromolecular complex bound to chondroitin sulfate proteoglycan, serglycin (SG) (Metkar et al., 2002), and have provided the biophysical basis for this observation (Raja et al., 2002). The use of free GrB could lead potentially to anomalous binding of the granzyme to anionic membranes on the target cell surface and cytosolic proteins. As a consequence, we thought it would be instructive to reassess how the granzyme initiates death after intracellular delivery of the macromolecular complexes. Using techniques that examine apoptotic events in situ, we report here that GrBSG initiates death predominantly through caspase-3, and mitochondria secondarily amplify this process.
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Results |
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Observing prominent adventitious processing in GrB-treated cells after solubilization, we speculated that a similar outcome might occur when lysates are generated after whole cell cytotoxicity assays. Using the NK-lymphoma cell line YT, which lacks detectable Bid as a surrogate effector and Jurkat cells as target, the cells were mixed at a 1:1 ratio and solubilized in the presence and absence of IETD-cho. After incubation for various times (060 min), the lysates were blotted and probed with anti-Bid antisera. In the absence of the inhibitor, there was complete processing of Bid at the earliest measurable time (Fig. 1 E). In presence of IETD-cho, disappearance of the 22-kD Bid was observed but only after 60 min, a time when the effect of the inhibitor likely is exhausted. Together the results indicate that precautions are necessary to distinguish intracellular and postlytic proteolysis (i.e., adventitious processing) during GrB-induced apoptosis assays if treated targets require solubilization. In addition, the reported cleavage of Bid during GrB-mediated cell death appears to be due to adventitious proteolysis, suggesting the mechanism(s) responsible for granzyme-mediated mitochondrial disruption remains largely unexplained.
Bax/Bak (-/-) MEFs undergo less mitochondrial potential loss after GrBSG delivery than WT MEFs
Although we failed to identify processed Bid in MCF-7(vec/casp3) cells, mitochondrial disruption via a GrB-initiated mechanism could still occur through a Bax/Bak-dependent pathway (Wei et al., 2001) or independently of BH3 domain proteins (Alimonti et al., 2001; Thomas et al., 2001). To investigate whether Bax and Bak contributed to a granzyme-initiated apoptotic cascade, double negative (DKO) Bax x Bak (-/-) murine embryonic fibroblasts (MEFs) were studied for their sensitivity to granzyme-mediated mitochondrial disruption and cell death.
To avoid adventitious processing and the difficulties inherent to interpreting the significance of cleaved proteins observed by immunoblot, we limited our examination for signs of cell death to intact cells. Furthermore, we observed that the granzyme variably detached the MEFs and thereby might obscure the results by causing anoikis. To minimize this confounding variable, MEFs were proteolytically detached for the described treatment periods. Focusing first on the nuclear aspect of the apoptotic response, the Bax/Bak (-/-) MEFs showed a reduced level of DNA fragmentation by TUNEL staining at 2 h which was then comparable to wild type (WT) at 4 h (Fig. 2 A). After determining that Bax/Bak (-/-) and WT MEFs contained comparable levels of activatable procaspase-3 (unpublished data), kinetics of intracellular active caspase-3 formation and mitochondrial potential loss (m) were investigated. The Bax/Bak (-/-) MEFs showed lower levels of active caspase-3 throughout the kinetic analysis (Fig. 2 B). Finally, the rate and level of mitochondrial potential loss was less in DKO than WT cells being imperceptible in one experiment (Fig. 2 C).
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Caspase-3 is essential for mitochondrial potential loss and DNA fragmentation
The preceding results suggest that prevention of GrB-mediated caspase-3 activation protects against mitochondrial disruption and nuclear signs of apoptosis. However, due to the conflicting data surrounding this observation the results warranted verification in cells that absolutely lacked procaspase-3. To directly examine the role of caspase-3 in GrB-mediated mitochondrial dysfunction, we evaluated alterations in MCF-7 cells lacking procaspase-3 (MCF-7vec) and a stable transfectant expressing the zymogen (MCF-7casp-3) (Yang et al., 1998). Despite the apparent absence of Bid cleavage in MCFcasp-3 cells (Fig. 1 A), mitochondrial potential loss was clearly observed at 15 min in these cells, whereas m remained intact in MCF-7vec cells lacking procaspase-3 (Fig. 3 A). The MCF-7vec cells also failed to develop TUNEL reactivity for upwards to 20 h after GrB delivery, whereas MCF-7casp-3 cells displayed substantial DNA fragmentation at 4 h (Fig. 3 B). As expected, intracellular active caspase-3 was discernible from analysis of lysates within 15 min of GrB delivery in MCFcasp-3 (unpublished data), whereas processed procaspase-3 was observed later by immunoblotting (60 min) (see above and Fig. 1 B).
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Bcl-2 suppresses GrBSG-mediated caspase-3 activation followed by mitochondrial membrane potential loss and DNA fragmentation
Among the findings reported in cells undergoing apoptosis in the presence of purified GrB, perhaps the most consistent observation is the capacity of Bcl-2 to prevent cell death. The results here show that GrB first activates caspase-3 and this or a related family member then contributes to the loss in mitochondrial membrane potential and DNA fragmentation. Since Bcl-2 has been reported to minimize activation of caspase-3 (Krebs et al., 1999), we asked whether Bcl-2 might inhibit GrB-associated mitochondrial depolarization by preventing the activation of caspase-3. Using a Jurkat cell line as a model, we examined how overexpression of Bcl-2 influenced mitochondrial membrane potential, intracellular caspase activation, and DNA fragmentation after granzyme delivery. As a prelude to these studies, we documented that recombinant Bcl-2 does not directly inhibit the enzymatic activity of either soluble GrB or active caspase-3 (unpublished data). Bcl-2transfected Jurkat cells (JurkatBcl-2) were then found to show a substantial inhibition of caspase-3 activity compared with the control (Fig. 4 A); a result paralleled by the caspase-3 fluorogenic assay performed on cell lysates (unpublished data). In comparison, mitochondrial potential loss and DNA fragmentation were also markedly reduced in JurkatBcl-2 cells (Fig. 4, B and C). Finally, despite the virtual absence of activated caspase-3 in Bcl-2transfected cells, processed procaspase-3 could be detected by immunoblotting (Fig. 4 D). Together the results reinforce the concept that mitochondria are undisturbed after granzyme delivery if the level of active caspase-3 is minimized and, Bcl-2, at least during GrB-induced killing, acts by preventing the activation of caspase-3.
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Discussion |
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Several laboratories have reported that GrB apparently cleaves Bid in situ; an event associated with the reduction in m and cyt C release. When performed with targets treated with caspase inhibitors, the combined result of Bid cleavage and persistent mitochondrial disruption has been interpreted to indicate the granzyme initiates death through a mitochondria-centered, caspase-independent pathway. We emphasize that the reliability of these interpretations are hampered by the lack of safeguards to block GrB-associated adventitious proteolysis which could yield spurious immunoblot and cyt c release data and to ensure caspases are inactivated in GrB-treated cells.
Solubilization of targets exposed to GrB release sequestered granzyme allowing cleavage of susceptible substrates. Among the proteins processed by GrB perhaps the two most readily cleaved are Bid and procaspase-7. We first recognized this problem of adventitious proteolysis during studies performed to delineate the sequence of caspases processed after delivery of free GrB (Yang et al., 1998). In the absence of controls exposed to GrB alone and a strategy to inactivating the granzyme immediately before and during solubilization, immunoblot data that compared rates of processing of caspase-3 and -7 would be interpreted to indicate that caspase-7 is processed first after GrB delivery. Therefore, it is not surprising that Bid has been considered a key substrate through which GrB apparently activates a mitochondrial death pathway and that homogenates from GrB-treated cells contain released cyt c. Notably, a similar outcome may occur during whole cell cytotoxicity assays (Thomas et al., 2001) (Fig. 1 E), and the use of PFN at permeabilizing concentrations also could produce anomalous proteolysis due to diffusion of the granzyme into necrotic but otherwise intact cells.
Despite the lack of proof that Bid is a dominant initiator of a mitochondrial death pathway, available data indicate that mitochondria play a crucial role in GrB-mediated apoptosis. Unlike cytochrome release assays, which are hampered by the lack of safeguards against adventitious proteolysis, support for GrB-dependent disruption of mitochondrial integrity is based more reliably on studies which examine changes in mitochondrial membrane potential and the detection of released cyt c in whole cells by imaging techniques (Pinkoski et al., 2001; Thomas et al., 2001). What has been uncertain is whether mitochondrial depolarization occurs primarily via BH3-only/Bax/Bak pathway and whether the granzyme directly mediates these effects or participates indirectly via one or more of the caspases.
Studies in Bax/Bak MEFs have shown that almost every described form of cell death requires the presence of these proteins (Wei et al., 2001). Therefore, existence of Bax/Bak-dependent and -independent pathways during GrB-mediated apoptosis is most reliably assessed with Bax/Bak (-/-) MEFs. In comparison to a previous report (Thomas et al., 2001), we observed that the predominate reduction of m occurred in WT, and only a minor alteration was observed in Bax/Bak (-/-) MEFs. The disparate observations may be due to differences in the techniques used to deliver the granzyme and the confounding effect GrB-mediated detachment may have on mitochondrial depolarization.
Until now, caspases have not been considered crucial for GrB-mediated mitochondrial disruption in a variety of cell lines (Heibein et al., 2000; Sutton et al., 2000; Alimonti et al., 2001; Thomas et al., 2001). We suggest the major reason for this discrepancy rests on whether caspases were completely inhibited in the respective studies. Unlike the forms of cells death where caspases undergo controlled autoactivation via proteinprotein interactions, the cytosolic delivery of GrB presents the pool of procaspases with a renewable activator that is only exhausted once the granzyme is blocked by cognate inhibitors (e.g., PI-9). This, we suspect, is the basis for the greater difficulty encountered in blocking the caspase cascade in GrB-treated cells (Talanian et al., 1997). Although the study of cells deficient in key caspases (see below) offers a more satisfactory approach, we believed it was crucial to examine cells which lacked Bax/Bak and active caspase-3 to determine whether the granzyme directly disrupts mitochondrial integrity. We had relied previously on measuring caspase activity colorimetrically in lysates after preincubation with the designated inhibitors (Talanian et al., 1997). However, the level of activated caspase-3 in solubilized cells is reduced in the presence of cell lysates (unpublished data), resulting in an underestimation of the active protease intracellularly. To circumvent this problem, we verified the effectiveness of the caspase inhibitors in whole cells using the PhiPhiLux assay (Komoriya et al., 2000). Relying on the combination of DEVD-fmk and VAD-fmk, mitochondrial depolarization was blocked in WT cells, suggesting caspases rather than GrB were responsible.
To decisively establish that caspases play a central role in mitochondrial depolarization during GrB-mediated apoptosis, we then showed that m only occurred in targets containing procaspase-3 (e.g., MCF-7casp3 cells). Notably, similar results were obtained with procaspase-3 (-/-) MEFs (unpublished data), indicating the observation is not cell type specific. Coupled with the observation that
m was more reduced in WT than DKO MEFs, the results indicate that the predominant GrB death pathway starts with intracellular delivery of the granzyme, continues with activation of procaspase-3, and is followed by caspase-mediated engagement of a BH3-only/Bax/Bak pathway. The BH3-only protein that is cleaved by caspase-3 is unclear. In other systems, caspase-3 has been reported to process BID, amplifying the death process (Slee et al., 2000). As observed for caspase-7 where the active protease was undetected by blotting but seemed apparent by PARP cleavage, more sensitive assays may reveal that BID is processed to amplify GrB-mediated apoptosis. However, at present the mechanism underlying GrB-initiated, caspase-3mediated engagement of the Bax/Bak pathway may be implemented by BID or other BH3-only proteins. Consistent with data presented here that Bid may not be critical for GrB induced apoptosis, it has been shown recently that the GrB cleavage site in Bid is not conserved among different species which are known to have granzyme-laden cytotoxic T lymphocytes (Coultas et al., 2002).
Since caspase-3 and -7 are rapidly processed during GrB-induced apoptosis (Yang et al., 1998), it was unclear whether both proteases contribute to the observed reduction in m. Despite our inability to detect processed caspase-7 by immunoblotting in MCF-7vec cells during a 4-h assay (Yang et al., 1998), the results suggests GrB can directly activate this caspase after intracellular delivery. This interpretation is supported by evidence that PARP is similarly cut by both caspase-3 and -7 (Germain et al., 1999) and by data here, which indicates PARP cleavage in MCF-7vec cells is inhibited by DEVD-fmk (Fig. 3 E). Therefore, although processed caspase-7 was not apparent in procaspase-3deficient cells by immunoblotting, GrB appears to access and activate this procaspase. Furthermore, sufficient active caspase-7 is generated to cleave PARP and yet a reduction in
m does not occur. Together, the results suggest that caspase-7 does not disrupt mitochondrial function. Furthermore, MCF-7vec cells do not show signs of DNA fragmentation by TUNEL upwards to 20 h after GrB delivery (unpublished data), but Hoechst staining reveals that most MCF-7vec cells contained condensed nonfragmented nuclei (unpublished data). Consistent with a recent observation (Marsden et al., 2002), caspase-7 directly activated by GrB may be sufficient to induce apoptosis and thus represent a unique death pathway which is entirely independent of mitochondria.
The WT MEFs clearly produced higher levels of active caspase-3 than DKO MEFs. This observation may provide clues as to how mitochondria amplify GrB-mediated apoptosis. Since the cell-associated procaspase-3 does not appear to be completely activated in DKO cells, a portion of the zymogen may be sequestered from the granzyme. Procaspase-3 has been reported to reside in mitochondrial (Mancini et al., 1998) and cytosolic compartments. Therefore, with mitochondrial permeabilization the zymogen leaves the mitochondria and becomes accessible to processing by the granzyme. However, recent evidence cast doubts on the localization of procaspase-3 to mitochondria (van Loo et al., 2002). Therefore, coupled with the observation that cytosolic procaspase-3 is compartmentalized to free and membrane-bound fractions (Krebs et al., 1999), a plausible alternative entails the generation of an amplification loop where caspase-9 rather than GrB activates the membrane-associated procaspase-3 zymogen.
Using Bcl-2transfected cells, we verify previous studies which showed that the antiapoptotic protein protects targets from GrB-mediated death (Sutton et al., 1997; Pinkoski et al., 2001). However, the results here establish that Bcl-2 acts at the most proximal aspect of the granzyme-mediated death pathway by inhibiting the activation of caspase-3. Despite the marked reduction in active caspase-3 within cells, the processed subunits were identified by immunoblot (Fig. 4 D), an observation we made previously in targets undergoing GrB-induced apoptosis in the presence of DEVD-fmk (Talanian et al., 1997). However, isolated Bcl-2 does not directly inhibit the proteolytic activity of caspase-3 (unpublished data). Together, the results suggest that Bcl-2 interferes with oligomerization of the caspase into active heterodimers. We thus identify a novel mechanism through which the potent antiapoptotic protein prevents cell death (Cory and Adams, 2002) akin to the described Bcl-2associated sequestration of BH3-only domain proteins (Cheng et al., 2001).
Combining three separate strategies to ablate procaspase-3 function with three assays that focus on intracellular events in whole cells, the results indicate that GrB initiates death by activating procaspase-3 rather than by modifying BH3-only proteins (Heibein et al., 2000; Sutton et al., 2000; Wang et al., 2001). Thereafter, caspase-3 permeabilizes mitochondria primarily in a BH3-dependent fashion. The release of mitochondrial products then amplify the death process, ensuring efficient execution of the cell. This pathway appears to represent the optimal sequence for cell death. However, the results clearly indicate that mitochondria are not essential for the granzyme-mediated killing. Independently of BH3 proteins, caspase-3 induces DNA fragmentation presumably through cleavage of ICAD (Wolf et al., 1999). If caspase-3 is lacking, DNA fragmentation is conspicuously absent, suggesting a pathway where the granzyme directly cleaves ICAD is not operative in situ (Thomas et al., 2000). Finally, in the absence of procaspase-3, activated caspase-7 produces mitochondrial independent nuclear condensation, emphasizing the crucial role the caspases play in GrB-mediated apoptosis (Fig. 5).
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Materials and methods |
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Reagents
L-glutamine, EDTA, glycerol, PFA, 3-aminopropyltrietoxy-silane, and propidium iodide were purchased from Sigma-Aldrich. RPMI 1640 was from GIBCO BRL. GELCODE blue stain reagent and Hoechst 33342 were from Pierce Chemical Co. and Molecular Probes, respectively. HRP rat antimouse kappa mAb was from Accurate Chemical, and ready gels, silver stain kit, Immunoblot and Sequiblot-PVDF membranes were purchased from Bio-Rad Laboratories. ECL Western blot system and Hyperfilm ECL and HRP-tagged antimouse and rabbit secondary antibodies were from Amersham Biosciences. Human GrB and PFN were isolated as described (Hanna et al., 1993; Froelich et al., 1996b). Human serglycin (SG) was isolated from supernatants of YT cells (Raja et al., 2002). The GrB inhibitor Ac-IETD-cho was from Alexis Biochemicals. Z-DEVD-fmk and Z-VAD-fmk were from Calbiochem. Anticaspase-3 and -7, anti-PARP, and anticyt c were from BD PharMingen, and anti-Bid was either provided by Dr. Xiaodong Wang (University of Texas Southwestern Medical Center, Dallas, TX) or obtained from Cell Signaling Technology. Antiactin antibody is from ICN. Recombinant TRAIL was expressed as the cDNA fragment (aa 114281) in pET-23d plasmid.
Generation and analysis of GrBSG complexes
GrBSG complexes were prepared as reported previously (Metkar et al., 2002). The concentrations of GrB retentates were assessed by ELISA (Spaeny-Dekking et al., 1998). GrB enzymatic activity was measured using Boc-Ala-Asp-thiobenzyl ester (BAADT) or Ac-Ile-Glu-Thr-Asp-pNA (IETD-paranitroanilide; Calbiochem). Cleavage was monitored at 405 nm.
Protein delivery for induction of cell death
AD was employed at a plaque-forming unit (PFU) ranging from 5001,000 per cell. The endosomolytic activity of the AD against the various target cells was determined as follows: AD, containing the ß-galactosidase construct, was added to the designated target cell at increasing PFU/cell. The cells were stained 18 h later for ß-galactosidase expression. The PFU that resulted in >80% positive target cells was used. GrB and GrBSG were used at 1 and 2 µg/ml, respectively, for the experiments. Cells (106) were treated for times indicated in the presence and absence of designated inhibitors and then processed for assays described below. Viability of treated cells were comparable to media controls based on similar light scatter characteristic and acquisition rates by flow cytometry.
Immunoblotting
Detection of processed caspase-3 and -7 and Bid was performed as reported previously (Froelich et al., 1996a) with modifications described here. 15 min before the completion of the assay, IETD-cho (250500 µM) was added to targets. The cells were then incubated in solubilization buffer (1% NP-40, 1 mM EDTA, 50 mM Tris-HCl, 165 mM NaCl) containing additional IETD-cho. Thereafter, debris was cleared by centrifugation, and the postnuclear lysate was immediately frozen (-80°C) with aliquots held to measure protein content. Each lot of IETD-CHO was evaluated for the capacity to completely inactivate GrB in the lysates by measuring residual GrB activity with the chromogenic substrate, IETD-pNA. For immunoblotting, postnuclear lysates of varying protein content were thawed, mixed with Laemelli buffer, heated, resolved by SDS-PAGE (1015%), and transferred to Immunoblot PVDF membranes (Bio-Rad Laboratories). Signal was visualized with the ECL kit (Amersham Biosciences).
Apoptosis assays
Hoechst assay.
Cells were fixed in 4% PFA and dried on microscope slides. For analysis, cells were hydrated, stained with Hoechst 33342 (10 µg/ml), and mounted with a drop of 50% glycerol in PBS. For each sample, 200 cells were counted to determine the percentage of cells with condensed and/or fragmented nuclei.
FITC-TUNEL.
Target cells (106/ml) were treated with the appropriate reagents in microfuge tubes for the required time and death was measured by terminal deoxyribonucleotidyl transferasecatalyzed labeling of DNA strand breaks with FITC-dUTP followed by flow cytometry (TUNEL).
PhiPhiLux caspase-3 assay.
Cells were stained with the cell permeable fluorogenic caspase 3 substrate PhiPhiLux (G1-D2; OncoImmunin, Inc.) as per the manufacturer's instructions followed by flow cytometry (Komoriya et al., 2000).
FAM-DEVD-FMK caspase-3 assay.
Cells were stained with the cell permeable fluorogenic caspase-3 inhibitor FAM-DEVD-FMK (APOLOGIX, Inc.) as per the manufacturer's instructions followed by flow cytometry.
Mitochondrial potential loss (m)
After treatment, cells were stained with MitoTracker red CMXRos (Molecular Probes) as described (Metkar et al., 2000).
Imaging, computers, and software
Images of immunoblots were captured either with a Eastman Kodak digital camera or Saphir Ultra 2 flatbed scanner, exported to Adobe Photoshop® 7.0, after which the Tiff images were placed for final presentation in Adobe Illustrator® 10.0 using a Macintosh Power G4 computer (OS X).
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Acknowledgments |
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This work supported in part by 1 RO1 AI/GM 44941 (to C.J. Froelich), a National Cancer Institute grant CA48000 (to Y.J. Lee), and the Elsa U. Pardee Foundation (to Y.J. Lee).
Submitted: 29 October 2002
Revised: 24 January 2003
Accepted: 27 January 2003
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References |
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Alimonti, J.B., L. Shi, P.K. Baijal, and A.H. Greenberg. 2001. Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition. J. Biol. Chem. 276:69746982.
Andrade, F., S. Roy, D. Nicholson, N. Thornberry, A. Rosen, and L. Casciola-Rosen. 1998. Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity. 8:451460.[Medline]
Barry, M., and R.C. Bleackley. 2002. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2:401409.[Medline]
Barry, M., J.A. Heibein, M.J. Pinkoski, S.F. Lee, R.W. Moyer, D.R. Green, and R.C. Bleackley. 2000. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol. Cell. Biol. 20:37813794.
Bernardi, P. 1999. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol. Rev. 79:11271155.
Cheng, E.H., M.C. Wei, S. Weiler, R.A. Flavell, T.W. Mak, T. Lindsten, and S.J. Korsmeyer. 2001. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell. 8:705711.[CrossRef][Medline]
Chinnaiyan, A.M., K. Orth, W.L. Hanna, H.J. Duan, G.G. Poirier, C.J. Froelich, and V.M. Dixit. 1996. Cytotoxic T cell-derived granzyme B activates the apoptotic protease ICE-LAP3. Curr. Biol. 6:897899.[Medline]
Cory, S., and J.M. Adams. 2002. The bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer. 2:647665.[CrossRef][Medline]
Coultas, L., D.C. Huang, J.M. Adams, and A. Strasser. 2002. Pro-apoptotic BH3-only Bcl-2 family members in vertebrate model organisms suitable for genetic experimentation. Cell Death Differ. 9:11631166.[CrossRef][Medline]
Crompton, M. 2000a. Bax, Bid and the permeabilization of the mitochondrial outer membrane in apoptosis. Curr. Opin. Cell Biol. 12:414419.[CrossRef][Medline]
Crompton, M. 2000b. Mitochondrial intermembrane junctional complexes and their role in cell death. J. Physiol. 529:1121.
Darmon, A.J., D.W. Nicholson, and R.C. Bleackley. 1995. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 377:446448.[CrossRef][Medline]
Duan, H.J., K. Orth, A.M. Chinnaiyan, G.G. Poirier, C.J. Froelich, W.-W. He, and V.M. Dixit. 1996. 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. 271:1672016724.
Froelich, C.J., K. Orth, J. Turbov, P. Seth, B.M. Babior, R.A. Gottlieb, G.M. Shah, R.C. Bleackley, V.M. Dixit, and W.L. Hanna. 1996a. 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. 271:2907329079.
Froelich, C.J., J. Turbov, and W. Hanna. 1996b. Human perforin: rapid enrichment by immobilized metal affinity chromatography (IMAC). Biochem. Biophys. Res. Commun. 229:4449.[CrossRef][Medline]
Germain, M., E.B. Affar, D. D'Amours, V.M. Dixit, G.S. Salvesen, and G.G. Poirier. 1999. Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J. Biol. Chem. 274:2837928384.
Guo, Y., S.M. Srinivasula, A. Druilhe, T. Fernandes-Alnemri, and E.S. Alnemri. 2002. Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J. Biol. Chem. 277:1343013437.
Hanna, W.L., X. Zhang, J. Turbov, U. Winkler, D. Hudig, and C.J. Froelich. 1993. Rapid purification of cationic granule proteases: application to human granzymes. Protein Expr. Purif. 4:398402.[CrossRef][Medline]
Heibein, J.A., I.S. Goping, M. Barry, M.J. Pinkoski, G.C. Shore, D.R. Green, and R.C. Bleackley. 2000. Granzyme Bmediated cytochrome c release is regulated by the Bcl-2 family members Bid and Bax. J. Exp. Med. 192:13911402.
Hengartner, M.O. 2001. Apoptosis. DNA destroyers. Nature. 412:2729.[CrossRef][Medline]
Joza, N., S.A. Susin, E. Daugas, W.L. Stanford, S.K. Cho, C.Y. Li, T. Sasaki, A.J. Elia, H.Y. Cheng, L. Ravagnan, et al. 2001. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 410:549554.[CrossRef][Medline]
Komoriya, A., B.Z. Packard, M.J. Brown, M.L. Wu, and P.A. Henkart. 2000. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med. 191:18191828.
Krebs, J.F., R.C. Armstrong, A. Srinivasan, T. Aja, A.M. Wong, A. Aboy, R. Sayers, B. Pham, T. Vu, K. Hoang, et al. 1999. Activation of membrane-associated procaspase-3 is regulated by Bcl-2. J. Cell Biol. 144:915926.
Lassus, P., X. Opitz-Araya, and Y. Lazebnik. 2002. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science. 297:13521354.
Li, L.Y., X. Luo, and X. Wang. 2001. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 412:9599.[CrossRef][Medline]
Li, P., D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, and X.D. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479489.[Medline]
Loeffler, M., and G. Kroemer. 2000. The mitochondrion in cell death control: certainties and incognita. Exp. Cell Res. 256:1926.[CrossRef][Medline]
MacDonald, G., L. Shi, C. Vande Velde, J. Lieberman, and A.H. Greenberg. 1999. Mitochondria-dependent and -independent regulation of granzyme Binduced apoptosis. J. Exp. Med. 189:131144.
Mancini, M., D.W. Nicholson, S. Roy, N.A. Thornberry, E.P. Peterson, L.A. Casciola-Rosen, and A. Rosen. 1998. The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J. Cell Biol. 140:14851495.
Marsden, V.S., L. O'Connor, L.A. O'Reilly, J. Silke, D. Metcalf, P.G. Ekert, D.C. Huang, F. Cecconi, K. Kuida, K.J. Tomaselli, et al. 2002. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature. 419:634637.[CrossRef][Medline]
Medema, J.P., R.E.M. Toes, C. Scaffidi, T.S. Zheng, R.A. Flavell, C.J.M. Melief, M.E. Peter, R. Offringa, and P.H. Krammer. 1997. Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis. Eur. J. Immunol. 27:34923498.[Medline]
Metkar, S., B. Wang, M. Aguilar-Santelises, S.M. Raja, L. Uhlin-Hansen, E. Podack, J.A. Trapani, and C.J. Froelich. 2002. Cytotoxic cell granule-mediated apoptosis: a multimeric delivery system where perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity. 16:417428.[CrossRef][Medline]
Metkar, S.S., M. Anand, P.P. Manna, K.N. Naresh, and J.J. Nadkarni. 2000. Ceramide-induced apoptosis in fas-resistant Hodgkin's disease cell lines is caspase independent. Exp. Cell Res. 255:1829.[CrossRef][Medline]
Pinkoski, M.J., N.J. Waterhouse, J.A. Heibein, B.B. Wolf, T. Kuwana, J.C. Goldstein, D.D. Newmeyer, R.C. Bleackley, and D.R. Green. 2001. Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway. J. Biol. Chem. 276:1206012067.
Poe, M., J.T. Blake, D.A. Boulton, M. Gammon, N.H. Sigal, J.K. Wu, and H.J. Zweerink. 1991. Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity. J. Biol. Chem. 266:98103.
Raja, S.M., B. Wang, M. Dantuluri, U.R. Desai, B. Demeler, K. Spiegel, S.S. Metkar, and C.J. Froelich. 2002. Cytotoxic cell granule-mediated apoptosis: characterization of the macromolecular complex of granzyme B with serglycin. J. Biol. Chem. 277:4952349530.
Sharif-Askari, E., A. Alam, E. Rheaume, P.J. Beresford, C. Scotto, K. Sharma, D. Lee, W.E. DeWolf, M.E. Nuttall, J. Lieberman, et al. 2001. Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J. 20:31013113.
Slee, E.A., S.A. Keogh, and S.J. Martin. 2000. Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ. 7:556565.[CrossRef][Medline]
Spaeny-Dekking, E.H.A., W.L. Hanna, A.M. Wolbink, P.C. Wever, A.J. Kummer, A.J.G. Swaak, J.M. Middeldorp, H.G. Huisman, C.J. Froelich, and C.E. Hack. 1998. Extracellular granzymes A and B: detection of native species during CTL responses in vitro and in vivo. J. Immunol. 160:1361013616.
Stegh, A.H., B.C. Barnhart, J. Volkland, A. Algeciras-Schimnich, N. Ke, J.C. Reed, and M.E. Peter. 2002. Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein. J. Biol. Chem. 277:43514360.
Sutton, V.R., D.L. Vaux, and J.A. Trapani. 1997. Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J. Immunol. 158:57835790.[Abstract]
Sutton, V.R., J.E. Davis, M. Cancilla, R.W. Johnstone, A.A. Ruefli, K. Sedelies, K.A. Browne, and J.A. Trapani. 2000. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme Bmediated caspase activation. J. Exp. Med. 192:14031414.
Suzuki, Y., Y. Imai, H. Nakayama, K. Takahashi, K. Takio, and R. Takahashi. 2001. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell. 8:613621.[Medline]
Talanian, R.V., X. Yang, J. Turbov, P. Seth, T. Ghayur, C.A. Casiano, and C.J. Froelich. 1997. Granule-mediated killing: pathways for granzyme Binitiated apoptosis. J. Exp. Med. 186:13231331.
Thomas, D.A., C.Y. Du, M. Xu, X.D. Wang, and T.J. Ley. 2000. DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity. 12:621632.[Medline]
Thomas, D.A., L. Scorrano, G.V. Putcha, S.J. Korsmeyer, and T.J. Ley. 2001. Granzyme B can cause mitochondrial depolarization and cell death in the absence of BID, BAX, and BAK. Proc. Natl. Acad. Sci. USA. 98:1498514990.
van Loo, G., X. Saelens, F. Matthijssens, P. Schotte, R. Beyaert, W. Declercq, and P. Vandenabeele. 2002. Caspases are not localized in mitochondria during life or death. Cell Death Differ. 9:12071211.[CrossRef][Medline]
Wang, G.Q., E. Wieckowski, L.A. Goldstein, B.R. Gastman, A. Rabinovitz, A. Gambotto, S. Li, B. Fang, X.M. Yin, and H. Rabinowich. 2001. Resistance to granzyme Bmediated cytochrome c release in Bak-deficient cells. J. Exp. Med. 194:13251337.
Wei, M.C., W.X. Zong, E.H.Y. Cheng, T. Lindsten, V. Panoutsakopoulou, A.J. Ross, K.A. Roth, G.R. MacCregor, C.B. Thompson, and S.J. Korsmeyer. 2001. Proapoptotic BAX and BAK: arequisite gateway to mitochondrial dysfunction and death. Science. 292:727730.
Wolf, B.B., M. Schuler, F. Echeverri, and D.R. Green. 1999. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J. Biol. Chem. 274:3065130656.
Yang, X., H.R. Stennicke, B. Wang, D.R. Green, R.U. Jänicke, A. Srinivasan, P. Seth, G.S. Salvesen, and C.J. Froelich. 1998. Granzyme B mimics apical caspases: description of a unified pathway for trans-activation of executioner caspases-3 and -7. J. Biol. Chem. 273:3427834283.
Ye, H., C. Cande, N.C. Stephanou, S. Jiang, S. Gurbuxani, N. Larochette, E. Daugas, C. Garrido, G. Kroemer, and H. Wu. 2002. DNA binding is required for the apoptogenic action of apoptosis inducing factor. Nat. Struct. Biol. 9:680684.[CrossRef][Medline]
Zou, H., W.J. Henzel, X.S. Liu, A. Lutschg, and X.D. Wang. 1997. Apaf-1, a human protein homologous to C-elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 90:405413.[Medline]
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