Room temperature-induced apoptosis of Jurkat cells sensitive to both caspase-1 and caspase-3 inhibitors

1998 ◽  
Vol 132 (1-2) ◽  
pp. 7-16 ◽  
Author(s):  
Mari Shimura ◽  
Emiko Okuma ◽  
Akira Yuo ◽  
Takehito Sasaki ◽  
Chiaki Mukai ◽  
...  
Keyword(s):  
Room Temperature ◽  
Caspase 3 ◽  
Jurkat Cells ◽  
Caspase 1 ◽  
Induced Apoptosis

scholarly journals Salmonella-Induced Caspase-2 Activation in Macrophages

2000 ◽  
Vol 192 (7) ◽  
pp. 1035-1046 ◽  
Author(s):  
Veronika Jesenberger ◽  
Katarzyna J. Procyk ◽  
Junying Yuan ◽  
Siegfried Reipert ◽  
Manuela Baccarini
Keyword(s):  
Type Iii Secretion ◽  
Caspase 3 ◽  
Secretion System ◽  
Caspase 1 ◽  
Chemical Inhibition ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

scholarly journals Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

1999 ◽  
Vol 19 (8) ◽  
pp. 5659-5674 ◽  
Author(s):  
Rakesh K. Srivastava ◽  
Steven J. Sollott ◽  
Leila Khan ◽  
Richard Hansford ◽  
Edward G. Lakatta ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Caspase 3 ◽  
No Synthase ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
No Production ◽  
Acetoxymethyl Ester ◽  
Jnk Pathway ◽  
Induced Apoptosis ◽  
Jnk Activation

ABSTRACT The proteins Bcl-2 and Bcl-XL prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-XL in the regulation of cytosolic Ca2+, nitric oxide production (NO), c-Jun NH2-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca2+ATPase, was used to disrupt Ca2+ homeostasis. TG acutely elevated intracellular free Ca2+ and mitochondrial Ca2+ levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca2+ response with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N G-nitro-l-arginine methyl ester hydrochloride (l-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca2+ level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-XL (JT/Bcl-2 or JT/Bcl-XL), NO production, late (36-h) Ca2+ accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-XL cells to the NO donor,S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by l-NAME. Transient expression of a dominant negative mutant SEK1 (Lys→Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-XL inhibited TG-induced loss in mitochondrial membrane potential, release of cytochromec, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca2+ release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca2+-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-XL cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-XL protect the cells against TG-induced apoptosis by negatively regulating Ca2+-sensitive NO synthase activity or expression.

SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2177-2177
Author(s):  
Duncan H Mak ◽  
Christa Manton ◽  
Michael Andreeff ◽  
Bing Z Carter
Keyword(s):  
Cell Death ◽  
Vector Control ◽  
Caspase 3 ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Smac Mimetic ◽  
P53 Activation ◽  
Induced Apoptosis

Abstract Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.

Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation

2001 ◽  
Vol 353 (2) ◽  
pp. 357-367 ◽  
Author(s):  
Patrice Xavier PETIT ◽  
Marie-Claude GENDRON ◽  
Nicolas SCHRANTZ ◽  
Didier MÉTIVIER ◽  
Guido KROEMER ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Nuclear Dna ◽  
Mitochondrial Permeability Transition ◽  
Kinetic Studies ◽  
Permeability Transition ◽  
Jurkat Cells ◽  
Glutathione Depletion ◽  
Late Apoptosis ◽  
Induced Apoptosis ◽  
Nuclear Dna Fragmentation

Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (∆Ψm). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K+, decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the ∆Ψm disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.

scholarly journals Caspases Are Activated in a Branched Protease Cascade and Control Distinct Downstream Processes in Fas-induced Apoptosis

1998 ◽  
Vol 187 (4) ◽  
pp. 587-600 ◽  
Author(s):  
Hirokazu Hirata ◽  
Atsushi Takahashi ◽  
Susumu Kobayashi ◽  
Shin Yonehara ◽  
Hirofumi Sawai ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Chromatin Condensation ◽  
Permeability Transition ◽  
Jurkat Cells ◽  
Mitotic Apparatus ◽  
Caspase 6 ◽  
Induced Apoptosis ◽  
Nuclear Apoptosis ◽  
Downstream Processes ◽  
And Control

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.

scholarly journals Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells

2013 ◽  
Vol 8 ◽  
pp. 2 ◽  
Author(s):  
Faisal Thayyullathil ◽  
Siraj Pallichankandy ◽  
Anees Rahman ◽  
Jaleel Kizhakkayil ◽  
Shahanas Chathoth ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Jurkat Cells ◽  
Induced Apoptosis

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

2002 ◽  
Vol 282 (5) ◽  
pp. G825-G834 ◽  
Author(s):  
Guoping Feng ◽  
Neil Kaplowitz
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Jurkat Cells ◽  
Primary Hepatocytes ◽  
Iκb Kinase ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-α-induced apoptosis. STS activated IκB kinase and nuclear factor-κB (NF-κB) nuclear translocation and DNA binding but inhibited transactivation of IκB-α, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-κB reporter in transfected Huh-7 cells.

scholarly journals Cif (Cytochrome c Efflux-Inducing Factor) Activity Is Regulated by Bcl-2 and Caspases and Correlates with the Activation of Bid

1999 ◽  
Vol 19 (2) ◽  
pp. 1381-1389 ◽  
Author(s):  
Zhiyong Han ◽  
Kapil Bhalla ◽  
Panayotis Pantazis ◽  
Eric A. Hendrickson ◽  
James H. Wyche
Keyword(s):  
Caspase 3 ◽  
Signal Transduction Pathway ◽  
Jurkat Cells ◽  
Caspase 8 ◽  
Caspase 7 ◽  
Cytosolic Extract ◽  
Induced Apoptosis ◽  
Cytosolic Factor ◽  
Apoptotic Signal Transduction ◽  
Apoptotic Signal

ABSTRACT The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochromec from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.

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