J. Exp. Med., Volume 189, Number 1, January 4, 1999 131-144

Mitochondria-dependent and -independent Regulation of Granzyme B-induced Apoptosis

By Glen MacDonald,^* Lianfa Shi,^* Christine Vande Velde,^* Judy Lieberman, and Arnold H. Greenberg^*

From the ^* Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9; and the  Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115

Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death.

Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-X_L suppressed caspase activation, mitochondrial cytochrome c release, suppression, and apoptosis and cell death induced by GraB, GraK, or perforin.

In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Harari, A., Enders, F. B., Cellerai, C., Bart, P.-A., Pantaleo, G. (2009). Distinct Profiles of Cytotoxic Granules in Memory CD8 T Cells Correlate with Function, Differentiation Stage, and Antigen Exposure. J. Virol. 83: 2862-2871 [Abstract] [Full Text]  
• Hua, G., Wang, S., Zhong, C., Xue, P., Fan, Z. (2009). Ignition of p53 Bomb Sensitizes Tumor Cells to Granzyme K-Mediated Cytolysis. J. Immunol. 182: 2152-2159 [Abstract] [Full Text]  
• Bovenschen, N., Quadir, R., van den Berg, A. L., Brenkman, A. B., Vandenberghe, I., Devreese, B., Joore, J., Kummer, J. A. (2009). Granzyme K Displays Highly Restricted Substrate Specificity That Only Partially Overlaps with Granzyme A. J. Biol. Chem. 284: 3504-3512 [Abstract] [Full Text]  
• Goping, I. S., Sawchuk, T., Rieger, A., Shostak, I., Bleackley, R. C. (2008). Cytotoxic T lymphocytes overcome Bcl-2 inhibition: target cells contribute to their own demise. Blood 111: 2142-2151 [Abstract] [Full Text]  
• de Vries, J. F., von dem Borne, P. A., van Luxemburg-Heijs, S. A.P., Heemskerk, M. H.M., Willemze, R., Falkenburg, J.H. F., Barge, R. M.Y. (2007). Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing. haematol 92: 1671-1678 [Abstract] [Full Text]  
• Hua, G., Zhang, Q., Fan, Z. (2007). Heat Shock Protein 75 (TRAP1) Antagonizes Reactive Oxygen Species Generation and Protects Cells from Granzyme M-mediated Apoptosis. J. Biol. Chem. 282: 20553-20560 [Abstract] [Full Text]  
• Zhao, T., Zhang, H., Guo, Y., Fan, Z. (2007). Granzyme K Directly Processes Bid to Release Cytochrome c and Endonuclease G Leading to Mitochondria-dependent Cell Death. J. Biol. Chem. 282: 12104-12111 [Abstract] [Full Text]  
• Bots, M., Medema, J. P. (2006). Granzymes at a glance. J. Cell Sci. 119: 5011-5014 [Full Text]  
• Waterhouse, N. J., Sutton, V. R., Sedelies, K. A, Ciccone, A., Jenkins, M., Turner, S. J., Bird, P. I., Trapani, J. A. (2006). Cytotoxic T lymphocyte-induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis.. JCB 173: 133-144 [Abstract] [Full Text]  
• Ravi, R., Fuchs, E. J., Jain, A., Pham, V., Yoshimura, K., Prouser, T., Jalla, S., Zhou, X., Garrett-Mayer, E., Kaufmann, S. H., Schulick, R. D., Pardoll, D. M., Bedi, A. (2006). Resistance of Cancers to Immunologic Cytotoxicity and Adoptive Immunotherapy via X-Linked Inhibitor of Apoptosis Protein Expression and Coexisting Defects in Mitochondrial Death Signaling. Cancer Res. 66: 1730-1739 [Abstract] [Full Text]  
• Bade, B., Boettcher, H. E., Lohrmann, J., Hink-Schauer, C., Bratke, K., Jenne, D. E., Virchow, J. C. Jr, Luttmann, W. (2005). Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes. Int Immunol 17: 1419-1428 [Abstract] [Full Text]  
• Han, J., Goldstein, L. A., Gastman, B. R., Rabinovitz, A., Rabinowich, H. (2005). Disruption of Mcl-1{middle dot}Bim Complex in Granzyme B-mediated Mitochondrial Apoptosis. J. Biol. Chem. 280: 16383-16392 [Abstract] [Full Text]  
• Parker, G. A., Picut, C. A. (2005). Liver Immunobiology. Toxicol Pathol 33: 52-62 [Abstract] [Full Text]  
• Rode, M., Balkow, S., Sobek, V., Brehm, R., Martin, P., Kersten, A., Dumrese, T., Stehle, T., Mullbacher, A., Wallich, R., Simon, M. M. (2004). Perforin and Fas Act Together in the Induction of Apoptosis, and Both Are Critical in the Clearance of Lymphocytic Choriomeningitis Virus Infection. J. Virol. 78: 12395-12405 [Abstract] [Full Text]  
• Pardo, J., Bosque, A., Brehm, R., Wallich, R., Naval, J., Mullbacher, A., Anel, A., Simon, M. M. (2004). Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis. JCB 167: 457-468 [Abstract] [Full Text]  
• Seye, C. I., Knaapen, M. W.M., Daret, D., Desgranges, C., Herman, A. G., Kockx, M. M., Bult, H. (2004). 7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling. Cardiovasc Res 64: 144-153 [Abstract] [Full Text]  
• Veugelers, K., Motyka, B., Frantz, C., Shostak, I., Sawchuk, T., Bleackley, R. C. (2004). The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis. Blood 103: 3845-3853 [Abstract] [Full Text]  
• Reed, J. C., Wilson, D. B. (2003). Cancer Immunotherapy Targeting Survivin: Commentary re: V. Pisarev et al., Full-Length Dominant-Negative Survivin for Cancer Immunotherapy. Clin. Cancer Res., 9:6523-6533, 2003.. Clin. Cancer Res. 9: 6310-6315 [Full Text]  
• Andrade, F., Casciola-Rosen, L. A., Rosen, A. (2003). A Novel Domain in Adenovirus L4-100K Is Required for Stable Binding and Efficient Inhibition of Human Granzyme B: Possible Interaction with a Species-Specific Exosite. Mol. Cell. Biol. 23: 6315-6326 [Abstract] [Full Text]  
• Mackewicz, C. E., Wang, B., Metkar, S., Richey, M., Froelich, C. J., Levy, J. A. (2003). Lack of the CD8+ cell anti-HIV factor in CD8+ cell granules. Blood 102: 180-183 [Abstract] [Full Text]  
• Kasibhatla, S., Tseng, B. (2003). Why Target Apoptosis in Cancer Treatment?. Molecular Cancer Therapeutics 2: 573-580 [Abstract] [Full Text]  
• Metkar, S. S., Wang, B., Ebbs, M. L., Kim, J. H., Lee, Y. J., Raja, S. M., Froelich, C. J. (2003). Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. JCB 160: 875-885 [Abstract] [Full Text]  
• Vermijlen, D., Luo, D., Froelich, C. J, Medema, J. P., Kummer, J. A., Willems, E., Braet, F., Wisse, E. (2002). Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J. Leukoc. Biol. 72: 668-676 [Abstract] [Full Text]  
• Thomas, D. A., Scorrano, L., Putcha, G. V., Korsmeyer, S. J., Ley, T. J. (2001). Granzyme B can cause mitochondrial depolarization and cell death in the absence of BID, BAX, and BAK. Proc. Natl. Acad. Sci. USA 98: 14985-14990 [Abstract] [Full Text]  
• Heibein, J. A., Goping, I. S., Barry, M., Pinkoski, M. J., Shore, G. C., Green, D. R., Bleackley, R. C. (2000). Granzyme B-mediated Cytochrome c Release Is Regulated by the Bcl-2 Family Members Bid and Bax. JEM 192: 1391-1402 [Abstract] [Full Text]  
• Sutton, V. R., Davis, J. E., Cancilla, M., Johnstone, R. W., Ruefli, A. A., Sedelies, K., Browne, K. A., Trapani, J. A. (2000). Initiation of Apoptosis by Granzyme B Requires Direct Cleavage of Bid, but Not Direct Granzyme B-mediated Caspase Activation. JEM 192: 1403-1414 [Abstract] [Full Text]  
• Barry, M., Heibein, J. A., Pinkoski, M. J., Lee, S.-F., Moyer, R. W., Green, D. R., Bleackley, R. C. (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: 3781-3794 [Abstract] [Full Text]  
• Ruefli, A. A., Smyth, M. J., Johnstone, R. W. (2000). HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance. Blood 95: 2378-2385 [Abstract] [Full Text]  
• Heibein, J. A., Barry, M., Motyka, B., Bleackley, R. C. (1999). Granzyme B-Induced Loss of Mitochondrial Inner Membrane Potential ({Delta}{Psi}m) and Cytochrome c Release Are Caspase Independent. J. Immunol. 163: 4683-4693 [Abstract] [Full Text]  
• Casciola-Rosen, L., Andrade, F., Ulanet, D., Wong, W. B., Rosen, A. (1999). Cleavage by Granzyme B Is Strongly Predictive of Autoantigen Status: Implications for Initiation of Autoimmunity. JEM 190: 815-826 [Abstract] [Full Text]  
• Alimonti, J. B., Shi, L., Baijal, P. K., Greenberg, A. H. (2001). Granzyme B Induces BID-mediated Cytochrome c Release and Mitochondrial Permeability Transition. J. Biol. Chem. 276: 6974-6982 [Abstract] [Full Text]  
• Pinkoski, M. J., Waterhouse, N. J., Heibein, J. A., Wolf, B. B., Kuwana, T., Goldstein, J. C., Newmeyer, D. D., Bleackley, R. C., Green, D. R. (2001). Granzyme B-mediated Apoptosis Proceeds Predominantly through a Bcl-2-inhibitable Mitochondrial Pathway. J. Biol. Chem. 276: 12060-12067 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search

TABLE OF CONTENTS