thapsigargin and Necrosis

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium; Cytoplasm; Dantrolene; Degenerin Sodium Channels; Endoplasmic Reticulum; Enzyme Inhibitors; Membrane Proteins; Muscle Relaxants, Central; Necrosis; Neurons; Phosphatidylserines; Phospholipid Transfer Proteins; Sodium Channels; Thapsigargin; TRPC Cation Channels

The mechanistic link between ER stress, autophagy, and resultant cell death was investigated by the use of drugs Thapsigargin (Tg) and Chloroquine (CQ) with prior induction and or blockade of autophagy and apoptosis which modulated the ER stress response and resultant form of cell death. All these biological processes can be measured flow cytometrically allowing the determination of the type of cell death, G

Topics: Apoptosis; Autophagy; Cell Survival; Chloroquine; eIF-2 Kinase; Endoplasmic Reticulum Stress; Flow Cytometry; Fluorescent Antibody Technique; G1 Phase Cell Cycle Checkpoints; Humans; Jurkat Cells; Microtubule-Associated Proteins; Necrosis; Oligopeptides; Sirolimus; Thapsigargin; Unfolded Protein Response

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Carrier Proteins; Caspases; Cell Line, Tumor; Chromatin; Colchicine; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Microfilament Proteins; Necrosis; Neurons; Nocodazole; Peptidomimetics; Quinolines; Rotenone; Signal Transduction; Staurosporine; Thapsigargin

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.

Topics: Animals; Arachidonic Acid; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Transformed; Chelating Agents; Cyclosporine; Diglycerides; Egtazic Acid; Fibroblasts; Group IV Phospholipases A2; Isoenzymes; L-Lactate Dehydrogenase; Mice; Mice, Knockout; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Phospholipase A2 Inhibitors; Pyrrolidines; Thapsigargin

The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its microtubule interaction and trafficking (MIT) domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2. NRBF2-deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, although the mice show no increased mortality. Our data reveal a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Beclin-1; Blotting, Western; Class III Phosphatidylinositol 3-Kinases; Dimethyl Sulfoxide; Endoplasmic Reticulum Stress; Gene Knockdown Techniques; Liver; Mice, Knockout; Models, Biological; Necrosis; NF-E2-Related Factor 2; Phagosomes; Protein Binding; Protein Stability; Protein Structure, Tertiary; Proteolysis; Thapsigargin; Trans-Activators; Transcription Factors; Vesicular Transport Proteins

Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca(2+)-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca(2+) concentration ([Ca(2+)]i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca(2+)]i level. Gyp-increased [Ca(2+)]i level was partly inhibited by removal of extracellular Ca(2+) by Ca(2+) chelator EGTA, store-operated Ca(2+) channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca(2+)-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca(2+) response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca(2+)]i increase and apoptosis in HepG2 cells. Exposure to 300 μg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca(2+)]i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca(2+) release from ER stores and increased store-operated Ca(2+) entry.

Topics: Antineoplastic Agents; Apoptosis; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Survival; Chelating Agents; Down-Regulation; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Gallic Acid; Gynostemma; Hep G2 Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Necrosis; Plant Extracts; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Up-Regulation

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Calcium; Cyclosporine; Fluoresceins; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Reactive Oxygen Species; Ruthenium Compounds; Ryanodine; Thapsigargin; Tiopronin

Calcium signaling, as a key to early step of the elementary intracellular events, has been implicated in controlling the development of atherosclerosis. We have shown previously that oxidized low density lipoprotein OxLDL-induced spatiotemporal increases of intracellular free calcium ([Ca(2+)](i)) in the early formation of macrophage foam cells. Here, we evaluated how spatiotemporal redistribution of intracellular calcium occurs and would affect OxLDL-induced apoptosis. Confocal laser scanning microscopy and flow cytometry showed the time-dependent increase of mitochondrial Ca(2+) ([Ca(2+)](m)) in acute and chronic exposure of U937-derived macrophages to OxLDL (100 microg/ml). Independent of the presence or absence of external Ca(2+), OxLDL-induced a peak of [Ca(2+)](m) in acute exposure, whose amplitude in the absence of extracellular Ca(2+) was obviously lower than the presence of extracellular Ca(2+). In addition, the thapsigargin-mediated increase of [Ca(2+)](i), through endoplasmic reticulum (ER) Ca(2+) pump depletion, was obviously reduced by 1-h pretreatment of OxLDL. OxLDL also caused a time-dependent opening of mitochondrial permeability transition pores (PTPs). EGTA/AM, an intracellular Ca(2+) chelator, significantly reduced OxLDL-induced apoptosis and failed to prevent OxLDL-induced necrosis at 6h. In contrast to control cells, chelation of cytosolic Ca(2+) by EGTA/AM at 6h did not completely reverse OxLDL-induced apoptosis. OxLDL stimulated depolarization of mitochondrial membrane potential (Deltapsi) in time-dependent manner. Our data demonstrated that OxLDL-induced spatiotemporal Ca(2+) redistribution in appropriate organelles and mediated Ca(2+)-dependent apoptosis in relation to depolarization of Deltapsi. These findings suggested that manipulation of the intracellular calcium balance may be a useful strategy to limit the loss of macrophages in early atherosclerosis.

Topics: Apoptosis; Calcium Signaling; Cell Differentiation; Culture Media, Serum-Free; Egtazic Acid; Humans; Lipoproteins, LDL; Macrophages; Membrane Potentials; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Necrosis; Tetradecanoylphorbol Acetate; Thapsigargin; U937 Cells

Tumor cells deficient in the proapoptotic proteins Bak and Bax are resistant to chemotherapeutic drugs. Here, we demonstrate that murine embryonic fibroblasts deficient for both Bak and Bax are, however, efficiently killed by thapsigargin, a specific inhibitor of ER Ca(2+) pumps that induces ER stress by depleting ER Ca(2+) stores. In the presence of Bak and Bax, thapsigargin eliminates cells by release of mitochondrial cytochrome c and subsequent caspase activation, which leads to the proteolytic inactivation of the molecular necrosis switch PARP-1 and results in apoptosis. By contrast, in the absence of Bak and Bax, a failure to activate caspases results in PARP-1-mediated ATP depletion. The subsequent necrosis is not prevented by autophagy as an alternative energy source. Moreover, in cells deficient for both Bak and Bax, thapsigargin induces permanent mitochondrial damage by Ca(2+) overload, permeability transition and membrane rupture. Thus, even though deficiency in Bak and Bax protects these cells against apoptosis, it does not compromise necrosis induced by SERCA inhibitors. Importantly, thapsigargin induces caspase-independent cell death also in colon and prostate carcinoma cells deficient in Bak and Bax expression. Therefore, targeted application of ER stressors such as thapsigargin might be a promising approach for the treatment of Bak- and Bax-deficient, drug-resistant tumors.

Topics: Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biological Transport; Calcium; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Humans; Mice; Mice, Knockout; Mitochondria; Necrosis; Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell type-dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control.

Topics: Activating Transcription Factor 4; Antineoplastic Agents; Apoptosis; Autophagy; Boronic Acids; Bortezomib; Cell Hypoxia; Cell Line, Tumor; DNA-Binding Proteins; Endoplasmic Reticulum; Humans; Microtubule-Associated Proteins; Necrosis; Protease Inhibitors; Protein Folding; Pyrazines; Regulatory Factor X Transcription Factors; Signal Transduction; Stress, Physiological; Thapsigargin; Transcription Factors

The signaling pathways governing pathophysiologically important autophagic (ACD) and necrotic (NCD) cell death are not entirely known. In the Dictyostelium eukaryote model, which benefits from both unique analytical and genetic advantages and absence of potentially interfering apoptotic machinery, the differentiation factor DIF leads from starvation-induced autophagy to ACD, or, if atg1 is inactivated, to NCD. Here, through random insertional mutagenesis, we found that inactivation of the iplA gene, the only gene encoding an inositol 1,4,5-trisphosphate receptor (IP3R) in this organism, prevented ACD. The IP3R is a ligand-gated channel governing Ca(2+) efflux from endoplasmic reticulum stores to the cytosol. Accordingly, Ca(2+)-related drugs also affected DIF signaling leading to ACD. Thus, in this system, a main pathway signaling ACD requires IP3R and further Ca(2+)-dependent steps. This is one of the first insights in the molecular understanding of a signaling pathway leading to autophagic cell death.

Topics: Animals; Apoptosis; Autophagy; Cyclosporine; Dictyostelium; Egtazic Acid; Genes, Protozoan; Inositol 1,4,5-Trisphosphate Receptors; Models, Biological; Mutagenesis, Insertional; Mutation; Necrosis; Protozoan Proteins; Signal Transduction; Thapsigargin

Little is known about the effect of microglial activation on cell death. This study examines the effects of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), triggers of microglial activation, on cell death induced by several agents in cultured rat microglia. For comparison, the effect of LPS on cell death is also examined in cultured astrocytes. LPS or IFN-gamma enhanced cell death induced by thapsigargin or ionomycin, an agent that increases intracellular Ca2+ concentration, although LPS or IFN-gamma alone did not affect cell viability. Thapsigargin or ionomycin induced apoptosis in LPS-untreated microglia, while they induced necrosis in LPS-treated microglia, which were partially reversed by O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, an intracellular Ca2+ chelator). In contrast, LPS treatment did not affect tunicamycin- or staurosporine-induced apoptosis, while it inhibited S-nitroso-N-acetylpenicillamine-induced apoptosis. The effect of LPS on thapsigargin or ionomycin-induced apoptosis was not observed in astrocytes. These results indicate that microglial activation sensitizes the cells toward cell death induced by the change in intracellular Ca2+ concentration and shifts the mode of cell death from apoptosis to necrosis.

Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; Calcium; Calcium Signaling; Cells, Cultured; Drug Interactions; Egtazic Acid; Encephalitis; Gliosis; Inflammation Mediators; Interferon-gamma; Intracellular Fluid; Ionomycin; Lipopolysaccharides; Microglia; Necrosis; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Thapsigargin

The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines.. Glycosaminoglycan synthesis was analyzed by metabolic labeling with (35)S-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry.. The main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of (35)S-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased (35)S-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium.. Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.

Topics: Animals; Calcium; Calcium Oxalate; Cell Death; Cell Survival; Crystallization; Dogs; Durapatite; Endocytosis; Formates; Glycosaminoglycans; Ionophores; Ions; Kidney Tubules, Distal; Kidney Tubules, Proximal; LLC-PK1 Cells; Necrosis; Oxalates; Sulfates; Sulfur Radioisotopes; Swine; Thapsigargin; Urinary Calculi

Prostate cancer is the second leading cause of cancer death in men. The most common treatment of prostate cancer is androgen ablation therapy which leads to regression of the tumor due to increased cell death. However, at later stages, the tumor becomes resistant to androgen ablation. Ceramide is a lipid second messenger that mediates cell death in prostate cancer cells. Previous studies suggested that ceramide may cause either apoptosis or growth arrest in the androgen-responsive prostate cancer cell line LNCaP. However, the molecular details of ceramide-induced cell death in LNCaP cells remain to be elucidated.. To investigate the mechanisms of cell death in LNCaP cells, we used various methods, including cell viability assays, fluorescence image analysis, internucleosomal DNA fragmentation analysis, Western blotting, and protein kinase assays.. Ceramide caused LNCaP cell death without exhibiting typical signs of apoptosis, such as internucleosomal DNA fragmentation and poly(ADP)-ribose-polymerase (PARP) proteolysis. In addition, the general caspase inhibitor z-VAD-fmk did not alter ceramide-induced cell death in LNCaP cells, whereas it efficiently inhibited thapsigargin-induced apoptosis under similar conditions. However, ceramide treatment of LNCaP cells resulted in nuclear fragmentation, which is characteristic of apoptosis. Ceramide induced a strong and prolonged activation of c-Jun N-terminal Kinase (JNK) that correlated very well with the time course of cell death. Whereas the PKC inhibitor bisindolylmaleimide prevented phorbol ester-induced apoptosis in LNCaP cells, it did not affect ceramide-induced cell death. These results suggest that LNCaP cell death induced by ceramide progresses through a novel pathway that is more necrotic than apoptotic.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Death; Ceramides; DNA, Neoplasm; Enzyme Inhibitors; Fluorescence; Humans; Indoles; Male; Maleimides; Necrosis; Prostatic Neoplasms; Protein Kinase C; Thapsigargin; Time Factors; Tumor Cells, Cultured

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.

Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium Channels; Calcium Signaling; Calcium-Binding Proteins; Calnexin; Calreticulin; Cell Size; Chromosome Mapping; Endoplasmic Reticulum; Helminth Proteins; Heterotrimeric GTP-Binding Proteins; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate Receptors; Ion Transport; Larva; Membrane Proteins; Molecular Sequence Data; Mutation; Necrosis; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Ribonucleoproteins; Ryanodine Receptor Calcium Release Channel; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thapsigargin; Touch

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A(2). The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43 degrees C). The appearance of membrane susceptibility to secretory phospholipase A(2) was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A(2), live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA(2) was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA(2) is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.

Topics: Animals; Apoptosis; Bucladesine; Cell Membrane; Cell Membrane Permeability; Dexamethasone; Hot Temperature; Ionophores; Necrosis; Phospholipases A; Staining and Labeling; T-Lymphocytes; Thapsigargin; Time Factors; Tumor Cells, Cultured

The proinflammatory protein endothelial monocyte-activating polypeptide II (EMAP-II) was first detected in supernatants of murine tumor cells by virtue of its ability to stimulate endothelial-dependent coagulation in vitro. The purified protein has pleiotropic effects on endothelial cells, monocytes, and neutrophils; however, its function in vivo is unknown, and the mechanism whereby it is released from cells is poorly understood. We investigated the expression of EMAP-II in human prostate adenocarcinoma specimens by immunohistochemistry and in LNCaP and DU-145 human prostate adenocarcinoma cells by reverse transcription-PCR, flow cytometry, and Western blotting. We then examined the effects of chemical and physiological stress on release and processing of EMAP-II by LNCaP and DU-145 cells. These cells constitutively express a Mr 34,000 form of EMAP-II that is retained intracellularly. Exposure to agents that induce apoptosis or, in some cases, necrosis induces the release of the Mr 34,000 form and further processing to the Mr 27,000 and Mr 22,000 forms. Hypoxia, but not heat shock, is a potent inducer of release and processing of biologically active EMAP-II by LNCaP and DU-145 cells. We suggest that release of EMAP-II by prostate adenocarcinoma cells as a consequence of treatment with anticancer agents or as a result of constitutive hypoxia may potentiate the effects of those agents through the localized activation of host effector mechanisms.

Topics: Adenocarcinoma; Anti-Bacterial Agents; Antimycin A; Apoptosis; Blotting, Western; Cell Hypoxia; Cytokines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Glucose; Humans; Immunohistochemistry; Ionomycin; Ionophores; Male; Necrosis; Neoplasm Proteins; Peptides; Prostate; Prostatic Neoplasms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Stress, Physiological; Thapsigargin; Tumor Cells, Cultured

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Apoptosis; Benzodiazepines; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cell Survival; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Electric Conductivity; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Ion Channel Gating; Kainic Acid; Necrosis; Neurons; Nifedipine; Oligopeptides; Phosphodiesterase Inhibitors; Rats; Ryanodine; Sodium; Sucrose; Thapsigargin

Phosphatidylserine (PS) exposure on propidium iodide negative cells using FITC labelled annexin-V has been used to quantify apoptosis in vitro and in vivo. Detection of PS within cells undergoing necrosis is also possible if labelled annexin-V specific for PS enters the cell following early membrane damage. Necrotic or late apoptotic cells can be excluded from flow cytometric analysis using propidium iodide which enters and stains cells with compromised membrane integrity. Here we show that thymocytes undergoing death exclusively by necrosis show early exposure of PS prior to loss of membrane integrity. This early exposure of PS occurs in cells treated with agents which both raise intracellular calcium levels and are also capable of interacting with protein thiol groups. We also demonstrate that PS exposure in thymocytes induced to undergo apoptosis by three different agents does not correlate with calcium rises but correlates with and precedes DNA fragmentation.

Topics: Animals; Calcium; Cell Death; Cell Membrane; Dexamethasone; Disulfides; DNA Fragmentation; Gliotoxin; In Vitro Techniques; Mice; Mice, Inbred BALB C; Necrosis; Phosphatidylserines; Propidium; Pyridines; Sulfhydryl Reagents; T-Lymphocytes; Thapsigargin

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAM-induced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.

Topics: Alkylating Agents; Animals; Antioxidants; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Iodoacetamide; LLC-PK1 Cells; Necrosis; Protein Biosynthesis; Signal Transduction; Swine; Thapsigargin