A [Lys49]phospholipase A2 from Protobothrops flavoviridis Venom Induces Caspase-Independent Apoptotic Cell Death Accompanied by Rapid Plasma-Membrane Rupture in Human Leukemia Cells

Protobothrops flavoviridis venom contains plural phospholipase A₂ (PLA₂) isozymes. A [Lys⁴⁹]PLA₂ called BPII induced cell death in human leukemia cells. PLA2, an [Asp⁴⁹]PLA₂ that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA₂ lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeast Saccharomyces cerevisiae

Ciclopirox olamine (CPO), a fungicidal agent widely used in clinical practice, induced in Saccharomyces cerevisiae an active cell death (ACD) process characterized by changes in nuclear morphology and chromatin condensation associated with the appearance of a population in the sub-G(0)/G(1) cell cycle phase and an arrest delay in the G(2)/M phases. This ACD was associated neither with intracellular reactive oxygen species (ROS) signaling, as revealed by the use of different classes of ROS scavengers, nor with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive phenotype. Furthermore, CPO-induced cell death seems to be dependent on unknown protease activity but independent of the apoptotic regulators Aif1p and Yca1p and of autophagic pathways involving Apg5p, Apg8p and Uth1p. Our results show that CPO triggers in S. cerevisiae an atypical nonapoptotic, nonautophagic ACD with as yet unknown regulators.     

A novel paraptosis pathway involving LEI/L-DNaseII for EGF-induced cell death in somato-lactotrope pituitary cells

We have recently reported that EGF triggers an original form of cell death in pituitary cell line (GH4C1) with a phenotype sharing some characteristics of both apoptosis (internucleosomal DNA fragmentation) and paraptosis (caspase-independence and cytoplasmic vacuolization). However, the endonuclease involved in EGF-induced DNA fragmentation has not been assessed so far. In the present work we therefore further explored the putative paraptosis involvement in EGF-induced cell death and asked whether L-DNaseII might be involved. Indeed, this endonuclease is known to mediate internucleosomal DNA fragmentation in caspase independent manner. Our Western blot, immunocytochemistry and enzymatic measurement assays show that EGF triggers a cleavage of Leukocyte Elastase Inhibitor (LEI) precursor into L-DNaseII, its subsequent enzymatic activation and nuclear translocation thus pointing to the involvement of this endonuclease pathway in caspase-independent DNA fragmentation. In addition, EGF-induced cell death can be blocked by paraptosis inhibitor AIP-1/Alix, but not with its anti-apoptotic C-terminal fragment (Alix-CT). Altogether these data suggest that EGF-induced cell death defines a novel, L-DNaseII-mediated form of paraptosis. This work was supported by fellowships from Fondation pour la Recherche medicale (FRM: FDT20030627283), from Association pour la Recherche contre le Cancer (ARC: ML/MLD/CM-A04/4) to JF and Retina France to AT and LP. These two authors equally contributed to this work.     

A cellular mechanism that reversibly inactivates pancaspase inhibitor zAsp-CH(2)-DCB: a potential pitfall causing discrepancy between in vitro and in vivo caspase assays

Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.     

Ceramide Induces Non-Apoptotic Cell Death in Human Glioma Cells

Ceramide causes either apoptosis or non-apoptotic cell death depending on model system and experimental conditions. The present study was undertaken to examine the effect of ceramide on cell viability and its molecular events leading to cell death in A172 human glioma cells. Ceramide induced cell death in a dose-dependent manner and the cell death was dependent on generation of reactive oxygen species and lipid peroxidation. TUNEL assay, Hoechst 33258 staining, and flow cytometric analysis did not show typical apoptotic morphological features. Ceramide caused phosphorylation of extracellular signal-regulated kinase (ERK) and p38, but the cell death was not affected by inhibitors of MAPK subfamilies. Ceramide caused ATP depletion without loss of mitochondrial membrane potential. Ceramide did not induce caspase activation and ceramide-induced cell death was also not altered by inhibitors of caspase activation. Transfection of dominant inhibitory mutant of IκBα (S32A/36A) and pretreatment of pyrrolidinedithiocarbamate, an inhibitor of NF-κB, enhanced ceramide-induced cell death. These results indicate that ceramide causes non-apoptotic, caspase-independent cell death by inducing reactive oxygen species generation in A172 human glioma cells. NF-κB is involved in the regulation of ceramide-induced cell death in human glioma cells.     

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS

Abstract

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.     

Caspase-independent cell death by Fas ligation in human thymus-derived T cell line, HPB-ALL cells

In HPB-ALL cells, a human thymus-derived T-cell line, Fas (CD95)-mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-CH(2)F (zVAD-fmk) sufficient to block the caspase activity. Fas-mediated caspase-independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB-ALL cells. This suggests that Fas-mediated caspase-independent cell death is a common feature in thymocytes. The signaling pathway of caspase-independent cell death has not yet been fully elucidated. In HPB-ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase-independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB-ALL cells have a caspase-independent RIP kinasedependent pathway for Fas ligation.     

Caspase-independent apoptotic pathways in T lymphocytes: a minireview

Cell death by apoptosis is involved in the maintenance of T cell receptor diversity, self tolerance, and T-cell number homeostasis. Until recently, apoptosis was thought to require caspase activation. Evidence is now accumulating that a caspase-independent pathway exists, shown by in vitro experiments with broad-range caspase inhibitors. Mature T lymphocytes readily undergo caspase-independent apoptosis in vitro, and recent data suggest that this type of apoptosis may be involved in the negative selection of thymocytes. Mitochondria likely release death triggers specific for both caspase-dependent and caspase-independent apoptotic pathways (cytochrome c and AIF respectively) in response to apoptotic stimuli. A caspase-independent pathway is triggered first in activated T lymphocytes subjected to apoptotic stimuli that do not rely on receptors with death domains. In this pathway, the early commitment phase to apoptosis involves cell shrinkage, peripheral DNA condensation and the translocation of mitochondrial AIF to the cytosol and nucleus. This process is reversible until mitochondrial cytochrome c is released and m dissipated. Only at this stage are caspases activated.