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1
Slee, Elizabeth A., Mary T. Harte, Ruth M. Kluck, Beni B. Wolf, Carlos A. Casiano, Donald D. Newmeyer, Hong-Gang Wang, et al. "Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner." Journal of Cell Biology 144, no. 2 (January 25, 1999): 281–92. http://dx.doi.org/10.1083/jcb.144.2.281.
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Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.
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Zhuang, Shougang, and Gabriel Simon. "Peroxynitrite-induced apoptosis involves activation of multiple caspases in HL-60 cells." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C341—C351. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c341.
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In this study, we show that caspases 2, 3, 6, and 7 were activated during peroxynitrite-induced apoptosis in human leukemia HL-60 cells and that processing of these caspases was accompanied by cleavage of poly(ADP-ribose) polymerase and lamin B. Treatment of cells with DEVD-fluoromethyl ketone (FMK), a selective inhibitor for caspase 3-like proteases, resulted in a marked diminution of apoptotic cells. VAVAD-FMK, an inhibitor of caspase 2, partially inhibited the apoptotic response to peroxynitrite. However, selective inactivation of caspase 6 by VEID-FMK did not affect apoptosis rates. These data suggest that caspase 3-like proteases and caspase 2, but not caspase 6, are required for peroxynitrite-induced apoptosis in this cell type. Moreover, we demonstrate that peroxynitrite treatment stimulated activation of caspases 8 and 9, two initial caspases in the apoptotic signaling pathway, and preincubation of cells with their inhibitor, IETD-FMK, inhibited activation of caspase 3-like proteases and caspase 2 at the concentration that prevents the apoptosis. These observations, together, suggest that caspase 8 and/or caspase 9 mediates activation of caspase 3-like proteases and caspase 2 during the apoptosis induced by peroxynitrite in HL-60 cells.
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Qin, Yimin, Terry L. Vanden Hoek, Kim Wojcik, Travis Anderson, Chang-Qing Li, Zuo-Hui Shao, Lance B. Becker, and Kimm J. Hamann. "Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion." American Journal of Physiology-Heart and Circulatory Physiology 286, no. 6 (June 2004): H2280—H2286. http://dx.doi.org/10.1152/ajpheart.01063.2003.
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We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 ± 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH2F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.
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MEERGANS, Thomas, Ann-Kristin HILDEBRANDT, Daniel HORAK, Christina HAENISCH, and Albrecht WENDEL. "The short prodomain influences caspase-3 activation in HeLa cells." Biochemical Journal 349, no. 1 (June 26, 2000): 135–40. http://dx.doi.org/10.1042/bj3490135.
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Proteolytic activation of caspases is a key step in the process of apoptosis. According to their primary structure, caspases can be divided into a group with a long prodomain and a group with a short prodomain. Whereas long prodomains play a role in autocatalytic processing, little is known about the function of the short prodomain, for example the prodomain of caspase-3. We constructed caspase-3 variants lacking the prodomain and overexpressed these in HeLa and yeast cells. We found that removal of the caspase-3 prodomain resulted in spontaneous proteolytic activation of the protein when expressed in HeLa cells. This processing was only partially autocatalytic, as demonstrated by a catalytically inactive caspase-3 mutant. Co-expression of the anti-apoptotic protein XIAP (X-chromosome-linked inhibitor of apoptosis protein) completely blocked the observed spontaneous activation, which excluded a direct involvement of caspase-8. Our findings indicate that the short prodomain of caspase-3 serves as a silencing component in mammalian cells by retaining this executioner caspase in an inactive state.
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Rathbun, R. Keaney, Tracy A. Christianson, Gregory R. Faulkner, Gary Jones, Winifred Keeble, Michael O'Dwyer, and Grover C. Bagby. "Interferon-γ–induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8–dependent activation of caspase 3 family members." Blood 96, no. 13 (December 15, 2000): 4204–11. http://dx.doi.org/10.1182/blood.v96.13.4204.h8004204_4204_4211.
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Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-γ) and tumor necrosis factor-α. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-γ contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-γ-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-γ upstream of caspase 3 activation.
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CAPANO, Michela, Sukaina VIRJI, and Martin CROMPTON. "Cyclophilin-A is involved in excitotoxin-induced caspase activation in rat neuronal B50 cells." Biochemical Journal 363, no. 1 (March 22, 2002): 29–36. http://dx.doi.org/10.1042/bj3630029.
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Glutamate and the NO donor, nitroprusside, synergistically induced the death of B50 cells from a rat CNS-derived neuroblastoma cell line. With low [nitroprusside] (10μM) both nitroprusside and glutamate were required. Under these conditions, nuclei became pyknotic and caspases were activated. The activities of caspase-3 and caspase-6 (effector caspases) were higher than those of caspase-8 and caspase-9 (initiator caspases). The activation of all four caspases was inhibited by cyclosporin A, with the order of susceptibility caspase-8=caspase-9=caspase-6>caspase-3. To identify the possible locus of cyclosporin A action, we used an antisense oligodeoxynucleotide to suppress the level of cyclophilin-A to < 5% of its control value. Cyclophilin-A suppression largely reproduced the inhibitory effects of cyclosporin A. These results provide the first indication that cyclophilin-A participates in the activation of the caspase cascade in neuronal cells, in particular in the form of cascade elicited by excitotoxic stimuli. It is concluded that neuroprotection by cyclosporin A against excitotoxin-induced apoptosis is, at least partly, due to inhibition of cyclophilin-A.
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Boatright, Kelly M., and Guy S. Salvesen. "Caspase activation." Biochemical Society Symposia 70 (September 1, 2003): 233–42. http://dx.doi.org/10.1042/bss0700233.
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Caspase activation is the 'point of no return' commitment to cell death. Synthesized as inactive zymogens, it is essential that the caspases remain inactive until the death signal is received. It is known for the downstream executioner caspases-3 and -7 that the activation event is proteolytic cleavage, and this had been assumed to apply to the initiator caspases as well. However, recent studies conducted on caspases-2, -8 and -9 have challenged this tenet of caspase activation. In this review we focus on the molecular details of caspase activation, with emphasis on recent work that provides a pleasing explanation for the differential requirements for the activation of executioner and initiator caspases.
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Fahy, Ruairi J., Andrea I. Doseff, and Mark D. Wewers. "Spontaneous Human Monocyte Apoptosis Utilizes a Caspase-3-Dependent Pathway That Is Blocked by Endotoxin and Is Independent of Caspase-1." Journal of Immunology 163, no. 4 (August 15, 1999): 1755–62. http://dx.doi.org/10.4049/jimmunol.163.4.1755.
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Abstract Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 μg/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.
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Rathbun, R. Keaney, Tracy A. Christianson, Gregory R. Faulkner, Gary Jones, Winifred Keeble, Michael O'Dwyer, and Grover C. Bagby. "Interferon-γ–induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8–dependent activation of caspase 3 family members." Blood 96, no. 13 (December 15, 2000): 4204–11. http://dx.doi.org/10.1182/blood.v96.13.4204.
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Abstract Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-γ) and tumor necrosis factor-α. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-γ contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-γ-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-γ upstream of caspase 3 activation.
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Pu, Xuan, Sarah J. Storr, Yimin Zhang, Emad A. Rakha, Andrew R. Green, Ian O. Ellis, and Stewart G. Martin. "Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival." Apoptosis 22, no. 3 (October 31, 2016): 357–68. http://dx.doi.org/10.1007/s10495-016-1323-5.
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Demirci, Umut, Melek Yaman, and Umit E. Bagriacik. "Effects of acute doxorubicin exposure on caspase-mediated apoptosis in cardiomyocytes." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e12036-e12036. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e12036.
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e12036 Background: Doxorubicin binds to DNA-associated enzymes, intercalates the base pairs of the DNA and induces apoptosis in cancerous and healthy tissues especially in cardiomyocytes. Caspase mediated apoptosis in cardiomyocytes remains largely unknown. We investigated the role of doxorubicin via caspase system on apoptosis of cardiomyocytes. Methods: H9C2ratcardiomyocytes were incubated with doxorubicin a concentration of 10-6 Mfor 4 or 24 hours to perform gene expression and activity of caspases, respectively. Total RNA isolated and expression analysis were performed by real time PCR assay. Caspase activity was determined by colorimetric caspase assay kit. At least two fold increases in gene expression were accepted statistically significant. Results: In comparison to controls, bothof caspase expression and activity increased in doxorubicin-treated samples. Increase in expression of Caspase 9 and 2 as initiator caspases and caspases 7 and 3 as effector caspases were detected statistically higher than control values. In contrast, expression of caspase 1, 4, 6, 8 and 12 were detected lower than controls. Activity of both caspase 3 and 9 increased significantly. Conclusions: Acute doxorubicin administration caused a significant caspase activation on apoptosis of cardiomyocytes.
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Heilig, Rosalie, Marisa Dilucca, Dave Boucher, Kaiwen W. Chen, Dora Hancz, Benjamin Demarco, Kateryna Shkarina, and Petr Broz. "Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD." Life Science Alliance 3, no. 6 (April 28, 2020): e202000735. http://dx.doi.org/10.26508/lsa.202000735.
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Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). Gsdmd deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1–driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd-deficient cells is caused by a synergistic effect of rapid caspase-1–driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1–dependent inflammatory disease.
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Campbell, Douglas S., and Hitoshi Okamoto. "Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization." Journal of Cell Biology 203, no. 4 (November 25, 2013): 657–72. http://dx.doi.org/10.1083/jcb.201303072.
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In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. We show that caspases were locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system.
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Wolf, Beni B., Joshua C. Goldstein, Henning R. Stennicke, Helen Beere, Gustavo P. Amarante-Mendes, Guy S. Salvesen, and Douglas R. Green. "Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation." Blood 94, no. 5 (September 1, 1999): 1683–92. http://dx.doi.org/10.1182/blood.v94.5.1683.
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Abstract Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.
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Wolf, Beni B., Joshua C. Goldstein, Henning R. Stennicke, Helen Beere, Gustavo P. Amarante-Mendes, Guy S. Salvesen, and Douglas R. Green. "Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation." Blood 94, no. 5 (September 1, 1999): 1683–92. http://dx.doi.org/10.1182/blood.v94.5.1683.417k37_1683_1692.
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Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.
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Fennell, Myles, Helen Chan, and Andrew Wood. "Multiparameter Measurement of Caspase 3 Activation and Apoptotic Cell Death in NT2 Neuronal Precursor Cells Using High-Content Analysis." Journal of Biomolecular Screening 11, no. 3 (February 20, 2006): 296–302. http://dx.doi.org/10.1177/1087057105284618.
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Caspase activation is a component of a number of neurodegenerative disorders, including stroke. In this study, the authors describe a multiplexed assay for caspase 3 activation, nuclear condensation, and cell viability in a neuronal precursor cell line Ntera-2, injuredwith staurosporine and etoposide. Using a high-content screening approach, cells were identified by staining with the nuclear stain Hoechst 33342; cell viability wasmeasured by staining cells with YoPro-1, which is taken up by damaged cells but excluded from healthy cells; and caspase 3/7 activation was detected using the cell-permeable probe PhiPhi-Lux, which becomes fluorescentwhen cleaved by active caspase 3 or 7. These 3 dyeswere detected simultaneously using a 4-band pass filter set on a Cellomics Arrayscan. The authors used peptide-fmk inhibitors selective for a variety of caspases, demonstrating that the injury is mediated primarily through caspase 3 or 7, although other caspases or related proteases may play aminor role. The general caspase inhibitor zVAD-fmkwas able to block cell death and caspase activationwith the highest potency. The caspase 3 selective inhibitor DEVD-fmkwas almost as potent as zVAD-fmk; other peptide caspase inhibitors displayed onlymodest inhibition of cell death. This assay was also used as a high-content screening tool for the evaluation of novel caspase 3 inhibitors for the potential treatment of degenerative disorders.
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Zheng, Timothy S., Stéphane Hunot, Keisuke Kuida, Takashi Momoi, Anu Srinivasan, Donald W. Nicholson, Yuri Lazebnik, and Richard A. Flavell. "Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation." Nature Medicine 6, no. 11 (November 2000): 1241–47. http://dx.doi.org/10.1038/81343.
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Kang, Shin-Jung, Suyue Wang, Hideaki Hara, Erin P. Peterson, Shobu Namura, Sepideh Amin-Hanjani, Zhihong Huang, et al. "Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions." Journal of Cell Biology 149, no. 3 (May 1, 2000): 613–22. http://dx.doi.org/10.1083/jcb.149.3.613.
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Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.
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MacFarlane, Marion, Wendy Merrison, David Dinsdale, and Gerald M. Cohen. "Active Caspases and Cleaved Cytokeratins Are Sequestered into Cytoplasmic Inclusions in Trail-Induced Apoptosis." Journal of Cell Biology 148, no. 6 (March 20, 2000): 1239–54. http://dx.doi.org/10.1083/jcb.148.6.1239.
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Tumor necrosis factor–related apoptosis- inducing ligand (TRAIL) –induced apoptosis, in transformed human breast epithelial MCF-7 cells, resulted in a time-dependent activation of the initiator caspases-8 and -9 and the effector caspase-7. Cleavage of caspase-8 and its preferred substrate, Bid, preceded processing of caspases-7 and -9, indicating that caspase-8 is the apical initiator caspase in TRAIL-induced apoptosis. Using transient transfection of COOH-terminal–tagged green fluorescent protein fusion constructs, caspases-3, -7, and -8 were localized throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in activation of caspases-3 and -7, and the redistribution of most of their detectable catalytically active small subunits into large spheroidal cytoplasmic inclusions, which lacked a limiting membrane. These inclusions, which were also induced in untransfected cells, contained cytokeratins 8, 18, and 19, together with both a phosphorylated form and a caspase-cleavage fragment of cytokeratin 18. Similarly, in untransfected breast HBL100 and lung A549 epithelial cells, TRAIL induced the formation of cytoplasmic inclusions that contained cleaved cytokeratin 18 and colocalized with active endogenous caspase-3. We propose that effector caspase-mediated cleavage of cytokeratins, resulting in disassembly of the cytoskeleton and formation of cytoplasmic inclusions, may be a characteristic feature of epithelial cell apoptosis.
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Suresh, Karthik, Kathleen Carino, Laura Johnston, Laura Servinsky, Carolyn E. Machamer, Todd M. Kolb, Hong Lam, et al. "A nonapoptotic endothelial barrier-protective role for caspase-3." American Journal of Physiology-Lung Cellular and Molecular Physiology 316, no. 6 (June 1, 2019): L1118—L1126. http://dx.doi.org/10.1152/ajplung.00487.2018.
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Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.
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Wang, J., and M. J. Lenardo. "Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies." Journal of Cell Science 113, no. 5 (March 1, 2000): 753–57. http://dx.doi.org/10.1242/jcs.113.5.753.
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Caspases are a group of cysteine proteases critical for apoptosis of eukaryotic cells. Deletion of genes that encode murine caspases suggests that caspases are involved not only in apoptosis but also in cytokine maturation and cell growth and differentiation. Among them, caspase-1 and caspase-11 are primarily involved in the processing of pro-inflammatory cytokines. Caspase-3 and caspase-9 are essential for apoptosis during brain development. Caspase-8 is required for the development of heart muscle, cell proliferation in the hematopoietic lineage and death-receptor-mediated apoptosis. These studies suggest that caspases function in cell signaling events including apoptosis, cell growth and differentiation.
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Hirata, Hirokazu, Atsushi Takahashi, Susumu Kobayashi, Shin Yonehara, Hirofumi Sawai, Toshiro Okazaki, Kokichi Yamamoto, and Masataka Sasada. "Caspases Are Activated in a Branched Protease Cascade and Control Distinct Downstream Processes in Fas-induced Apoptosis." Journal of Experimental Medicine 187, no. 4 (February 16, 1998): 587–600. http://dx.doi.org/10.1084/jem.187.4.587.
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Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.
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Takemoto, Kiwamu, Takeharu Nagai, Atsushi Miyawaki, and Masayuki Miura. "Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects." Journal of Cell Biology 160, no. 2 (January 13, 2003): 235–43. http://dx.doi.org/10.1083/jcb.200207111.
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Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton (H+) and chloride ion (Cl−) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an H+- and Cl−-insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.
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Mannick, Joan B., Christopher Schonhoff, Natalia Papeta, Pedram Ghafourifar, Marten Szibor, Kezhong Fang, and Benjamin Gaston. "S-Nitrosylation of mitochondrial caspases." Journal of Cell Biology 154, no. 6 (September 10, 2001): 1111–16. http://dx.doi.org/10.1083/jcb.200104008.
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Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.
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Duval, R., V. Bellet, S. Delebassée, and C. Bosgiraud. "Implication of caspases during maedi–visna virus-induced apoptosis." Journal of General Virology 83, no. 12 (December 1, 2002): 3153–61. http://dx.doi.org/10.1099/0022-1317-83-12-3153.
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Maedi–visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep. In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.
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Sgorbissa, A., R. Benetti, S. Marzinotto, C. Schneider, and C. Brancolini. "Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis." Journal of Cell Science 112, no. 23 (December 1, 1999): 4475–82. http://dx.doi.org/10.1242/jcs.112.23.4475.
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Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. We now demonstrate that Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage was only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 was processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 was detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it was coupled to the specific rearrangement of the microfilament characterizing cell death. Finally we also demonstrated that Gas2 in vitro binds to F-actin, but this interaction was unaffected by the caspase-3 dependent proteolytic processing.
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McComb, Scott, Pik Ki Chan, Anna Guinot, Holmfridur Hartmannsdottir, Silvia Jenni, Maria Pamela Dobay, Jean-Pierre Bourquin, and Beat C. Bornhauser. "Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7." Science Advances 5, no. 7 (July 2019): eaau9433. http://dx.doi.org/10.1126/sciadv.aau9433.
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Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.
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Faleiro, Lavina, and Yuri Lazebnik. "Caspases Disrupt the Nuclear-Cytoplasmic Barrier." Journal of Cell Biology 151, no. 5 (November 27, 2000): 951–60. http://dx.doi.org/10.1083/jcb.151.5.951.
Full textAbstract:
During apoptosis, caspases, a family of proteases, disassemble a cell by cleaving a set of proteins. Caspase-3 plays a major role in the disassembly of the nucleus by processing several nuclear substrates. The question is how caspase-3, which is usually cytoplasmic, gains access to its nuclear targets. It was suggested that caspase-3 is actively transported to the nucleus through the nuclear pores. We found that caspase-9, which is activated earlier than caspase-3, directly or indirectly inactivates nuclear transport and increases the diffusion limit of the nuclear pores. This increase allows caspase-3 and other molecules that could not pass through the nuclear pores in living cells to enter or leave the nucleus during apoptosis by diffusion. Hence, caspase-9 contributes to cell disassembly by disrupting the nuclear-cytoplasmic barrier.
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EDELSTEIN, CHARLES L., YUEXIAN SHI, and ROBERT W. SCHRIER. "Role of Caspases in Hypoxia-Induced Necrosis of Rat Renal Proximal Tubules." Journal of the American Society of Nephrology 10, no. 9 (September 1999): 1940–49. http://dx.doi.org/10.1681/asn.v1091940.
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Abstract. The role of the caspases, a newly discovered group of cysteine proteases, was investigated in a model of hypoxia-induced necrotic injury of rat renal proximal tubules. An assay for caspases in freshly isolated rat proximal tubules was developed. There was a 40% increase in tubular caspase activity after 15 min of hypoxia in association with increased cell membrane damage as indicated by a threefold increase in lactate dehydrogenase release. The specific caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB) attenuated the increase in caspase activity during 15 min of hypoxia and markedly decreased lactate dehydrogenase release in a dose-dependent manner. In the proximal tubules, Z-D-DCB also inhibited the hypoxia-induced increase in calpain activity, another cysteine protease. In contrast, when Z-D-DCB was added to purified calpainin vitro, there was no inhibition of calpain activity. The calpain inhibitor (2)-3-(4-iodophenyl)-2-mercapto-2-propenoic acid (PD150606) also inhibited the hypoxia-induced increase in caspase activity in proximal tubules, but did not inhibit the activity of purified caspase 1in vitro. In these experiments, caspase activity was detected with the fluorescence substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), which is preferentially cleaved by caspase 1. However, minimal caspase activity was detected with the fluorescence substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC), which is cleaved by caspases 2, 3, and 7. The present study in proximal tubules demonstrates that (1) caspase inhibition protects against necrotic injury by inhibition of hypoxia-induced caspase activity; and (2) caspase 1 may be the caspase involved. Thus, although the role of caspases in apoptotic cell death is well established, this study provides new evidence that caspases contribute to necrotic cell death as well.
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Zeng, Lizhi, and Larry D. Smith. "Caspase-3 Colorimetric Assay." BioTechniques 33, no. 6 (December 2002): 1196–97. http://dx.doi.org/10.2144/02336bm01.
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Komoriya, Akira, Beverly Z. Packard, Martin J. Brown, Ming-Lei Wu, and Pierre A. Henkart. "Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates." Journal of Experimental Medicine 191, no. 11 (May 30, 2000): 1819–28. http://dx.doi.org/10.1084/jem.191.11.1819.
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To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.
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Dursun, Belda, Zhibin He, Hilary Somerset, Dong-Jin Oh, Sarah Faubel, and Charles L. Edelstein. "Caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis." American Journal of Physiology-Renal Physiology 291, no. 3 (September 2006): F578—F587. http://dx.doi.org/10.1152/ajprenal.00455.2005.
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The role of caspases and calpain in cisplatin-induced endothelial cell death is unknown. Thus we investigated whether caspases and calpain are mediators of cisplatin-induced apoptosis and necrosis in endothelial cells. Cultured pancreatic microvascular endothelial (MS1) cells were exposed to 10 and 50 μM cisplatin. Apoptosis or necrosis was determined by Hoechst 33342 and propidium iodide (PI) nuclear staining. Cells treated with 10 μM cisplatin had normal ATP levels, increased caspase-3-like activity, excluded PI and demonstrated morphological characteristics of apoptosis at 24 h. Cells treated with 50 μM cisplatin had severe ATP depletion, increased caspase-3-like activity, and displayed extensive PI staining indicative of necrosis at 24 h. There was a dose-dependent increase in caspase-2-like activity and Smac/DIABLO protein. Calpain activity increased significantly with 50 μM, but not 10 μM cisplatin at 24 h. With 50 μM cisplatin, ATP levels were significantly reduced starting at 18 h, caspase-2- and caspase-3-like activities were significantly increased starting at 18 h, and LDH release started at 8 h with maximum increase at 18–24 h. Calpain activity was not increased before 24 h. The increase in LDH release and the nuclear PI staining with 50 μM cisplatin at 24 h was reduced by either the pancaspase inhibitor, Q-VD-OPH, or the calpain inhibitor, PD-150606. Calpain inhibitor had no effect on caspase-3-like activity. In conclusion, in cisplatin-treated endothelial cells, caspases, the major mediators of apoptosis, can also cause necrosis. A calpain inhibitor protects against necrosis without affecting caspase-3-like activity suggesting that calpain-mediated necrosis is independent of caspase-3.
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Nowak, Grażyna, Peter M. Price, and Rick G. Schnellmann. "Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells." American Journal of Physiology-Renal Physiology 285, no. 3 (September 2003): F440—F450. http://dx.doi.org/10.1152/ajprenal.00233.2002.
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The lack of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivity to acute renal failure produced by the chemotherapeutic agent cisplatin. We used primary cultures of mouse renal proximal tubular cells (MPTC) grown in optimized culture conditions to investigate the cellular basis for increased apoptosis in p21 knockout mice. Cisplatin (15 μM) activated caspase-3 but not caspase-8 or caspase-9 and produced phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation in wild-type [p21(+/+)] MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treated MPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21(+/+) MPTC, cisplatin activated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3 activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis in p21(+/+) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activity and decreased chromatin condensation by 51% in p21(-/-) but not in p21(+/+) MPTC. However, cisplatin-induced phosphatidylserine externalization was not inhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1) in the presence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4) MPTC apoptosis is caspase independent in the presence of p21 but partially dependent on caspases in the absence of p21.
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Talanian, Robert V., XiaoHe Yang, Jane Turbov, Prem Seth, Tariq Ghayur, Carlos A. Casiano, Kim Orth, and Christopher J. Froelich. "Granule-mediated Killing: Pathways for Granzyme B–initiated Apoptosis." Journal of Experimental Medicine 186, no. 8 (October 20, 1997): 1323–31. http://dx.doi.org/10.1084/jem.186.8.1323.
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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.
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ANNAND, Robert R., Jeffrey R. DAHLEN, Cindy A. SPRECHER, Piet DE DREU, Donald C. FOSTER, John A. MANKOVICH, Robert V. TALANIAN, Walter KISIEL, and David A. GIEGEL. "Caspase-1 (interleukin-1β-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9." Biochemical Journal 342, no. 3 (September 5, 1999): 655–65. http://dx.doi.org/10.1042/bj3420655.
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The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1β-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7×102 M-1˙s-1. The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.
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Kamada, Shinji, Ushio Kikkawa, Yoshihide Tsujimoto, and Tony Hunter. "A-Kinase-Anchoring Protein 95 Functions as a Potential Carrier for the Nuclear Translocation of Active Caspase 3 through an Enzyme-Substrate-Like Association." Molecular and Cellular Biology 25, no. 21 (November 1, 2005): 9469–77. http://dx.doi.org/10.1128/mcb.25.21.9469-9477.2005.
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ABSTRACT Caspase-mediated proteolysis is a critical and central element of the apoptotic process, and caspase 3, one of the effector caspases, is proposed to play essential roles in the nuclear morphological changes of apoptotic cells. Although many substrates for caspase 3 localize in the nucleus and caspase 3 translocates from the cytoplasm to the nuclei after activation in apoptotic cells, the molecular mechanisms of nuclear translocation of active caspase 3 have been unclear. Recently, we suggested that a substrate-like protein(s) served as a carrier to transport caspase 3 from the cytoplasm into the nucleus. In the present study, we identified A-kinase-anchoring protein 95 (AKAP95) as a caspase 3-binding protein. Small interfering RNA-mediated depletion of AKAP95 reduced apoptotic nuclear morphological changes, suggesting that AKAP95 is involved in the process of apoptotic nuclear morphological changes. The association of AKAP95 with active caspase 3 was analogous to an enzyme-substrate interaction. Furthermore, overexpression of AKAP95 with nuclear localization sequence mutations inhibited nuclear morphological changes in apoptotic cells. These results indicate that AKAP95 is a potential carrier protein for active caspase 3 from the cytoplasm into the nuclei in apoptotic cells.
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Hernandez, Ambrosio, Xiang Y. Xue, Alex Westerband, Lois A. Killewich, and Glenn C. Hunter. "TRAIL Mediated Apoptosis Increases Carotid Plaque Vulnerability." Stroke 32, suppl_1 (January 2001): 360. http://dx.doi.org/10.1161/str.32.suppl_1.360-a.
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P116 Background: TRAIL, a TNF family protein, and its receptors DR 4 and 5 (TNF-R) are known to induce apoptosis in tumor cells via caspase activation. In this study, we examined the relationship between TRAIL, its effector caspases and apoptosis in carotid plaque (CP). Materials and Methods: Forty CPs were examined by immunohistochemistry, Western blotting and RNA protection assay for evidence of apoptosis (Tunel), TRAIL, caspase 3, 8 and PARP. We compared the expression of these pro-apoptotic proteins within plaque (P), and in areas of intimal thickening (IT) adjacent to the plaque to that of normal intima (N). Results: Apoptosis was detected in 10–25% of ECs, SMCs, macrophages and T lymphocytes in complex CP, but only in 1–3% SMCs in fibrous plaques. TRAIL and TNF-R, as well as caspases 3, 8, and PARP were present in all complex lesions. In N, there were focal areas of caspase 3 expression localized to ECs, whereas both caspase 3 and TRAIL were detected in all 3 regions of complex CP. There was a significant increase in the expression of TRAIL, TNF-R and caspase 8 in areas of IT compared to N (Fig. 1). Conclusions: The increased expression of TRAIL, TNF-R and caspases 3 and 8 in areas of IT and caspase 3 in ECs from N suggest that the mechanisms for ongoing EC injury and TRAIL-mediated cell death are present in complex CP. Modulation of these pro-apoptotic proteins and/or their inhibitors could potentially result in the introduction of therapeutic agents to attenuate plaque vulnerability.
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Lu, Ying, and Guo-Qiang Chen. "Effector Caspases and Leukemia." International Journal of Cell Biology 2011 (2011): 1–8. http://dx.doi.org/10.1155/2011/738301.
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Caspases, a family of aspartate-specific cysteine proteases, play a major role in apoptosis and a variety of physiological and pathological processes. Fourteen mammalian caspases have been identified and can be divided into two groups: inflammatory caspases and apoptotic caspases. Based on the structure and function, the apoptotic caspases are further grouped into initiator/apical caspases (caspase-2, -8, -9, and -10) and effector/executioner caspases (caspase-3, -6, and -7). In this paper, we discuss what we have learned about the role of individual effector caspase in mediating both apoptotic and nonapoptotic events, with special emphasis on leukemia-specific oncoproteins in relation to effector caspases.
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Denault, Jean-Bernard, Brendan P. Eckelman, Hwain Shin, Cristina Pop, and Guy S. Salvesen. "Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)–mediated inhibition of caspase 9." Biochemical Journal 405, no. 1 (June 13, 2007): 11–19. http://dx.doi.org/10.1042/bj20070288.
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During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp330, and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
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Ekert, Paul G., John Silke, Christine J. Hawkins, Anne M. Verhagen, and David L. Vaux. "Diablo Promotes Apoptosis by Removing Miha/Xiap from Processed Caspase 9." Journal of Cell Biology 152, no. 3 (January 29, 2001): 483–90. http://dx.doi.org/10.1083/jcb.152.3.483.
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MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.
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COHEN, Gerald M. "Caspases: the executioners of apoptosis." Biochemical Journal 326, no. 1 (August 15, 1997): 1–16. http://dx.doi.org/10.1042/bj3260001.
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Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1β-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Caspases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.
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Renema, Phoibe, Natalya Kozhukhar, Viktoriya Pastukh, Domenico Spadafora, Sunita Subedi Paudel, Dhananjay T. Tambe, Mikhail Alexeyev, Dara W. Frank, and Troy Stevens. "Exoenzyme Y induces extracellular active caspase-7 accumulation independent from apoptosis: modulation of transmissible cytotoxicity." American Journal of Physiology-Lung Cellular and Molecular Physiology 319, no. 2 (August 1, 2020): L380—L390. http://dx.doi.org/10.1152/ajplung.00508.2019.
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Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY+ (PA103 Δ exoUexoT::Tc pUCP exoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY+ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY+ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY+ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.
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Shahzad, Khurrum, Fabian Bock, Moh’d Mohanad Al-Dabet, Ihsan Gadi, Shrey Kohli, Sumra Nazir, Sanchita Ghosh, et al. "Caspase-1, but Not Caspase-3, Promotes Diabetic Nephropathy." Journal of the American Society of Nephrology 27, no. 8 (February 1, 2016): 2270–75. http://dx.doi.org/10.1681/asn.2015060676.
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Ge, Y., Y.-M. Cai, L. Bonneau, V. Rotari, A. Danon, E. A. McKenzie, H. McLellan, L. Mach, and P. Gallois. "Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis." Cell Death & Differentiation 23, no. 9 (April 8, 2016): 1493–501. http://dx.doi.org/10.1038/cdd.2016.34.
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Abstract Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.
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Wilkins, Nikhil, Timothy Stephens, and Laura Felix. "EXTH-49. BXQ-350 TARGETS TO THE LYSOSOME AND KILLS GLIOBLASTOMA (GBM) CELLS VIA ACTIVATION OF APOPTOTIC CASPASES IN VITRO." Neuro-Oncology 22, Supplement_2 (November 2020): ii97—ii98. http://dx.doi.org/10.1093/neuonc/noaa215.403.
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Abstract BACKGROUND Apoptosis is a programmed cell death mechanism where cells respond to internal or external stimuli by initiating a cascade of events and enzymes leading to cell death. One of the hallmarks of cancer is the ability to resist apoptotic stimuli. Removing resistances to apoptosis can result in the death of these tumor cells. METHOD The GBM cell line Gli36ΔEGFR was used to determine Caspase activity and BXQ-350 cytotoxicity. Cells were treated with 9uM to 30uM BXQ-350 in triplicate and incubated for 24 hours at 37oC. Promega’s Caspase-Glo 9 or Caspase-Glo 3/7 reagent was added to each well of a plate and was incubated at room temperature in the dark for 3 hours then luminescence was read. The parallel cytotoxic assay was run under the same conditions except Roche’s MTT labeling reagent was added to each well after 24 hours and the plate was incubated at 370C for 4 hours. Solubilization solution was added to each well, the plate was incubated overnight then absorbance was read. The GBM cell line U87 MG was used to determine lysosomal targeting by treating with 10uM BXQ-350 and incubated at 37oC overnight. They were stained with anti-SapC (RFP) and anti-LAMP1 (GFP) antibodies and images were taken. RESULT BXQ-350 mediated cell death is correlated with a rise in Caspase 3, Caspase 7 and Caspase 9 activity. The caspase activity levels did not rise until after BXQ-350 passed its IC50 and stayed elevated. Caspases 3/7 levels showed higher activity compared to untreated than Caspase 9. BXQ-350 was seen to colocalize to LAMP1, a lysosomal membrane protein. CONCLUSION BXQ-350 tracks to the lysosomal membrane where it initiates a cascade of enzymes necessary to cause apoptosis. Caspases 3/7 are the effector caspases that complete the apoptotic process removing a major barrier to fight cancer.
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Peluffo, Marina C., Richard L. Stouffer, and Marta Tesone. "Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum." American Journal of Physiology-Endocrinology and Metabolism 293, no. 5 (November 2007): E1215—E1223. http://dx.doi.org/10.1152/ajpendo.00261.2007.
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Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy ( days 7, 17, 19, and 21 of gestation), postpartum ( days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2α. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2α injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2α. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2α may regulate members of the caspase family.
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Malloy, Peter J., and David Feldman. "Inactivation of the Human Vitamin D Receptor by Caspase-3." Endocrinology 150, no. 2 (February 1, 2009): 679–86. http://dx.doi.org/10.1210/en.2008-1217.
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Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [3H]1,25-dihydroxy vitamin D3 binding and VDR protein, suggesting that the VDR may be targeted for inactivation by caspases during apoptosis. A potential caspase-3 site (D195MMD198S) was identified in the human VDR ligand-binding domain. Mutations D195A, D198A, and S199A were generated in the putative capase-3 cleavage site. In transfected COS-7 cells, STS treatment resulted in the cleavage of the wild-type (WT) VDR and S199A mutant VDR but not the D195A or D198A mutants. In in vitro assays, the WT VDR and S199A mutant VDR were cleaved by caspase-3, although the D195A and D198A mutants were resistant to caspase-3. In vitro, the WT VDR was also cleaved by caspase-6 and caspase-7 and in extracts of STS-treated LNCaP cells. In STS-treated LNCaP cells and human skin fibroblasts, the proteasome inhibitor MG-132 protected the VDR caspase cleavage fragment from further degradation by the 26S proteasome. The rat VDR that does not contain the caspase-3 cleavage site was not cleaved in STS-treated COS-7 cells. In conclusion, our results demonstrate that the human VDR is a target of caspase-3 and suggest that activation of caspase-3 may limit VDR activity. The vitamin D receptor contains a caspase-3 cleavage site in the ligand-binding domain that can be cleaved by caspase-3 in vitro and in intact cells.
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Brentnall, Matthew, Luis Rodriguez-Menocal, Rebeka De Guevara, Enrique Cepero, and Lawrence H. Boise. "Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis." BMC Cell Biology 14, no. 1 (2013): 32. http://dx.doi.org/10.1186/1471-2121-14-32.
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Linder, Manuel, and Thomas Tschernig. "Vasculogenic mimicry: Possible role of effector caspase-3, caspase-6 and caspase-7." Annals of Anatomy - Anatomischer Anzeiger 204 (March 2016): 114–17. http://dx.doi.org/10.1016/j.aanat.2015.11.007.
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Brad, Amber M., Katherine E. M. Hendricks, and Peter J. Hansen. "The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage." Reproduction 134, no. 6 (December 2007): 789–97. http://dx.doi.org/10.1530/rep-07-0146.
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The capacity of the preimplantation embryo to undergo apoptosis in response to external stimuli is developmentally regulated. Acquisition of apoptosis does not occur in the cow embryo until between the 8- and 16-cell stages. The purpose of the present experiments was to determine the mechanism by which apoptosis is blocked in the bovine two-cell embryo. Heat shock (41 °C for 15 h) did not increase activity of caspase-9 or group II caspases (caspase-2, -3, and -7) in two-cell embryos but did in day 5 embryos. Exposure of embryos to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to depolarize mitochondria resulted in activation of caspase-9 and group II caspases at both stages of development. For day 5 embryos, CCCP also increased the proportion of blastomeres that underwent DNA fragmentation as determined by the TUNEL assay. In contrast, CCCP did not increase TUNEL labeling when applied at the two-cell stage. In conclusion, failure of heat shock to increase caspase-9 and group II caspase activity in the two-cell embryo indicates that the signaling pathway leading to mitochondrial depolarization and caspase activation is inhibited at this stage of development. The fact that CCCP treatment of two-cell embryos induced caspase-9 and group II-caspase activity indicates that caspase activation is possible following mitochondrial depolarization. However, since CCCP did not increase TUNEL labeling of two-cell embryos, actions of group II-caspases to activate DNases is inhibited.