Formation of caspase-3 complexes and fragmentation of caspase-12 during anisomycin-induced apoptosis in AKR-2B cells without aggregation of Apaf-1

Treatment of AKR-2B fibroblasts with anisomycin (10 microM) led to a rapid disintegration of the cells (t1/2 = 5 h) which was complete after 24 h. Cell death was associated with typical hallmarks of apoptosis like membrane blebbing, exposure of phophatidylserine on the cell surface, nuclear condensation and specific cleavage of rRNA. However, there was no dissipation of the mitochondrial potential and no intranucleosomal fragmentation. By affinity labeling with YVK(-bio)D.aomk in combination with immunostaining against activated caspase-3 analyzed by 2-D gel electrophoresis it was shown that caspase-3 is the dominant executioner caspase. Gel filtration experiments of cytosolic extract analyzed by Western blotting revealed the formation of high-molecular-weight complexes of caspase-3 (600 kDa and 250 kDa, respectively), but there was no complex formation of Apaf-1. Anisomycin treatment led to a strong activation of the stress kinases p38 kinases and the jun kinases, that was not sufficient for the activation of caspase-3 which required much higher concentrations. By using the selective inhibitors SB 203580 for p38 kinases and SP 600125 for c-jun kinases, respectively, it is shown that activation of these kinases is not necessary for cell death induced by anisomycin in AKR-2B cells. Furthermore, we disclose the activation of caspase-12 in AKR-2B cells following the addition of anisomycin. Caspase-12 zymogen present as a cytosolic complex (> 600 kDa) is activated by anisomycin leading to an uncomplexed cleaved enzyme. Since anisomycin treatment did neither lead to stress of the endoplasmic reticulum nor to a breakdown of intracellular Ca(2+)-stores, alternative pathways involved in the activation of caspases are discussed.     

Formation of high molecular weight caspase-3 complex in neonatal rat brain

Caspase-3 plays an essential role in normal brain development. Recently, a large protein complex known as apoptosome, which catalyzes the activation of caspase-3, has been reported. To investigate structural characteristics of caspase-3 in the developing brain, rat neonatal cortex extract was analysed by gel filtration chromatography. We show here the formation of high molecular complex including procaspase-3 in the extract. When the extract was activated by cytochrome c, caspase-3 recruitment to the apoptosome was not observed, although apoptotic protease activating factor-1 (Apaf-1), caspase-9, and X-linked inhibitor of apoptosis protein (XIAP) existed in the apoptosome. These results indicate that procaspase-3 exists as a high molecular weight complex during brain development.     

High molecular weight water-soluble chitosan protects against apoptosis induced by serum starvation in human astrocytes

The effect of high molecular weight water-soluble chitosan (WSC) on serum starvation-induced apoptosis in human astrocytes (CCF-STTG1 Cells) was investigated. WSC, having an average molecular weight of 300 kDa and a degree of deacetylation over 90%, can be produced using a simple multi-step membrane separation process. Serum starvation led to growth arrest, rounding up of cells and appearance of p53 bands. Prolonged (48 h) incubation in serum starved medium led to cell detachment and death. WSC significantly protected the serum starvation-induced cellular rounding up and protected the serum starvation-induced cell death as tested by flow cytometry. WSC also protected serum starvation-induced p53 activation as determined by Western blot. These results suggest that WSC may prevent serum starvation-induced apoptosis of CCF-STTG1 cells via p53 inactivation.     

Selective degradation of the PKC-epsilon isoform during cell death in AKR-2B fibroblasts

The protein kinase C (PKC) family of serine/threonine protein kinases is involved in intracellular signals that regulate growth, differentiation, and apoptosis. AKR-2B cells express the PKC isoforms alpha, gamma, epsilon, lambda, mu, und zeta (J. Hoppe, R. Sch?fer, V. Hoppe, and A. Sachinidis, Cell Death Differ. 6, 546-556). Here we show that during serum starvation only PKC-epsilon was cleaved. An N-terminal fragment of 42 kDa remained associated with subcellular components, presumably the Golgi apparatus. The C-terminal part (catalytic domain) was further degraded and was no longer detectable in vivo. As published before, the activation of the DEVDase in AKR-2B cells is prevented by numerous agents like PDGF, TPA, and DEVD.cmk (R. Sch?fer, D. Karbach, and J. Hoppe, Exp. Cell Res. 240, 28--39). All these agents completely prevented PKC-epsilon cleavage, indicating a tight correlation between DEVDase activity and PKC-epsilon cleavage. By using recombinant caspase-3 or highly purified DEVDase from cytosolic extracts we localized by Edman degradation the cleavage site in recombinant PKC-epsilon to asp383 in the hinge region between regulatory and catalytic domains. The corresponding tetrapeptide sequences SSPD and SATD for human and mouse PKC-epsilon, respectively, are unusual for caspase-3. Expression of the catalytic domain or of the cleavage-resistant mutant D383A had no effect on cell death in AKR-2B cells.     

Protective effects of vitamin K2 on 6-OHDA-induced apoptosis in PC12 cells through modulation bax and caspase-3 activation

Molecular Biology Reports - Neuroprotection using compounds with dual functions of anti-apoptotic and antioxidant effects fight against neurodegeneration. Vitamin K2 acts as a cofactor in many...     

ATP and adenosine prevent via different pathways the activation of caspases in apoptotic AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly disintegrate after serum deprivation.27 ATP or adenosine added immediately after serum removal afforded substantial protection against cell death even for a long period of 24 h. ED50 values were 14 and 110 microM for ATP and adenosine, respectively. In the presence of 5 microg/ml cycloheximide the protective effect of both substances was suppressed, indicating that protein synthesis is required. The protective effect of ATP was highly specific since among numerous tested derivatives only ATP-[gamma-S] exhibited a substantial protective effect.The ability of ATP and adenosine to modulate cell division was analyzed. Both substances did not exhibit any mitogenic effect. Adenosine completely blocked PDGF-BB induced cell division, whereas ATP had no effect. Unlike adenosine, ATP strongly stimulated Ca2+-release from intracellular stores. On the other hand, adenosine stimulated an increase in the intracellular concentration of cAMP from 0.4 - 1.5 microM, whereas ATP decreased the content below 0.1 microM. ATP stimulated the phosphorylation of MAP-kinase, RSK and p70S6-kinase; adenosine was inactive. After complexation of [Ca2+]i the protective effect of ATP was greatly lost while adenosine was still active. Surprisingly neither ATP nor adenosine caused an activation of PKC-isoforms. After incubation with pertussis toxin, the protection by ATP was reduced indicating an involvement of Gi-proteins in the signal transduction induced by ATP. Our results indicate that ATP as well as adenosine are potent inhibitors of cell death caused by serum deprivation and that this protective effect apparently occurs via distinct pathways. However, both pathways must converge at the point of caspase activation, since the stimulation of DEVDase- and VEIDase-activities, respectively, are suppressed by either ATP or adenosine.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos

BACKGROUND: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. METHODS: Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. RESULTS: Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. CONCLUSIONS: HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

Differential activation by platelet-derived growth factor-BB of mitogen activated protein kinases in starved or nonstarved AKR-2B fibroblasts

More than 90% of serum-deprived (starved) AKR-2B mouse fibroblasts are stimulated to divide by the addition of platelet-derived growth factor (PDGF)-BB. In density-arrested (nonstarved) cells, PDGF-BB affords protection from cell death without stimulation of cell division. In both cultivation conditions the cells express similar amounts of PDGF β-receptors and the receptor kinase activity was identical as judged by its autophosphorylation capacity. Three signaling pathways were studied in detail: (1) Phospholipase C-γ (PLC-γ) and [Ca²⁺]i increase, (2) activation of the phosphatidylinositol-3 kinase (PI-3 kinase), and (3) activation of mitogen activated kinases I and II (MAP kinases I and II). There was no difference in starved or nonstarved cells regarding PLC-γ activation, increase of [Ca²⁺]i, and stimulation of PL-3 kinase activity. But most remarkably the activation of MAP-I was largely suppressed in nonstarved cells. The implications of these signaling pathways in cell protection or cell division are discussed. © 1994 Wiley-Liss, Inc.     

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Background

Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, themerAgene coding for the mercuric reductase. We report on the development of a profiling method formerAand its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation.

Results

Based on an alignment of 30merAsequences from Gram negative bacteria, conserved primers were designed for amplification ofmerAfragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with differentmerAsequences. ThemerAprofiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of themerAcommunity profile was also detected in a biocatalyzer effluent isolate, which was identified asPseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing.

Conclusions

ThemerAprofiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducingPs. aeruginosastrain was identified by its unique mercuric reductase gene.     

Withdrawn: ABCA4 is ubiquitously expressed in mouse tissues and forms high molecular weight complexes

ABC transporters are the focus of extensive research attempts due to their natural ability of selective transport of huge variety of substances into and out of the cells. They are being a potential target for pharmacologists and drug designers as well as basic scientists. We were interested to study the expression patterns of mouse proteins which belong to the “A” family of ABCs as well as to analyze their protein–protein interactions. The most exciting finding came with the studies of ABCA4, which mRNA was distributed in several mouse tissues, including eyes, brain, heart, lungs, liver, and testis, and the corresponding protein was present in brain, heart, eyes, and testis. Previously, ABCA4 was described as retina‐specific transporter, therefore, we extended our research to clarify where ABCA4 is expressed on RNA level, where its protein is expressed and what are its interacting proteins, in tissues different then retina. By several techniques which utilized the protein‐specific antibody we proved that ABCA4 is not a retina‐specific ABC transporter and that we purified it from brain and testis as well as from eyes and the heart. Analysis of the co‐purifying proteins by mass‐spectrometry had shown that apart from ABCA4, ABCA1, and ABCC3 were present in cross‐linked fraction. We also identified map kinase 12 and jade1S protein as putative ABCA4 interacting proteins. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.