thapsigargin and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacA

Topics: Activating Transcription Factor 4; Adenine; Amino Acid Chloromethyl Ketones; Animals; Autophagy; Bacterial Proteins; Cell Cycle Proteins; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Epithelial Cells; Eukaryotic Initiation Factor-2; Gene Expression Regulation, Neoplastic; Helicobacter pylori; Humans; Macrolides; Mice; Poly (ADP-Ribose) Polymerase-1; Protein Serine-Threonine Kinases; Repressor Proteins; RNA, Small Interfering; Signal Transduction; Thapsigargin; Transcription Factor CHOP; Vacuoles

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagy; Autophagy-Related Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Docetaxel; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fungal Proteins; Ganoderma; Humans; Lung Neoplasms; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Taxoids; Thapsigargin; TOR Serine-Threonine Kinases; Tunicamycin; Vincristine; Xenograft Model Antitumor Assays

The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4.

Topics: Adenocarcinoma, Clear Cell; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Division; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Recombinant Fusion Proteins; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Transfection; X-Linked Inhibitor of Apoptosis Protein

Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells.. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.. Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis.. Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.

Topics: Amino Acid Chloromethyl Ketones; Animals; Aorta; Apoptosis; Blotting, Western; Bradykinin; Calcium; Calcium-Transporting ATPases; Calpain; Caspase 12; Caspase Inhibitors; Caspases; Cells, Cultured; Chelating Agents; Cytosol; Dipeptides; DNA Fragmentation; Egtazic Acid; Endoplasmic Reticulum; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Signal Transduction; Swine; Thapsigargin

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Calcimycin; Calcium; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dantrolene; DNA, Antisense; Endoplasmic Reticulum; Gene Expression; Histocytochemistry; Homeostasis; HSP70 Heat-Shock Proteins; Humans; In Situ Nick-End Labeling; Lac Operon; Membrane Proteins; Neurons; Thapsigargin; Transfection

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Dominant; Humans; In Situ Nick-End Labeling; MAP Kinase Kinase 1; Microscopy, Fluorescence; Mitochondria; Mitogen-Activated Protein Kinase 3; Models, Biological; Phosphatidylinositol 3-Kinases; Plasmids; Protein Denaturation; Protein Folding; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Subcellular Fractions; Tetrazolium Salts; Thapsigargin; Thiazoles; Time Factors; TOR Serine-Threonine Kinases; Transcription, Genetic; Tunicamycin; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

Mature mouse oligodendrocytes (OLs) are susceptible to death in demyelinating diseases such as multiple sclerosis and in brain injury following neurotrauma, ischemia, or stroke. To understand mechanisms leading to death of mature OLs and develop strategies for protection, we utilized cultures of mature mouse OLs to investigate the role of caspases and calpains in OL cell death mediated by different mechanisms. The agents used were (i) staurosporine, which induces apoptotic death via inhibition of protein kinases; (ii) kainate, which activates non-NMDA glutamate receptors; (iii) thapsigargin, which releases intracellular calcium stores; and (iv) SNAP, which releases active NO species and causes necrotic cell death. Inhibitors blocking primary effector caspases (including caspase 3), the FAS (death receptor)-mediated initiator caspases (including caspase 8), and stress-induced caspases (including caspase 9), were tested for their protective effects. Inhibition of caspases 3, 8, and 9 each robustly protected OLs following insult with staurosporine, thapsigargin, or kainate when added at optimal times. The time of addition of the inhibitors for maximal protection varied with the agent, from 1 h of preincubation before addition of staurosporine to 6 h after addition of kainate. Much less protection was seen for the NO generator SNAP under any condition. The role of calcium in OL death in each model was investigated by chelating extracellular Ca++ with EGTA, and by inhibiting the Ca++-activated calpain proteases. Calcium chelation did not protect against staurosporine, but decreased OL death initiated by kainate, thapsigargin, or NO. The calpain inhibitors PD150606 and calpain inhibitor I protected from cell death initiated by staurosporine, kainate, and thapsigargin, but not from cell death initiated by the NO donor SNAP.

Topics: Amino Acid Chloromethyl Ketones; Animals; Calcium; Calpain; Caspase Inhibitors; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Kainic Acid; Mice; Mice, Inbred BALB C; Oligodendroglia; Penicillamine; Staurosporine; Thapsigargin

In recent years, several inhibitors that prevent caspase activation and apoptosis have emerged. At high doses, however, these inhibitors can have nonspecific effects and/or become cytotoxic. In this study, we determined the effectiveness of broad spectrum caspase inhibitors to prevent apoptosis. A carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate (Q-VD-OPh) potently inhibited apoptosis. Q-VD-OPh was significantly more effective in preventing apoptosis than the widely used inhibitors, ZVAD-fmk and Boc-D-fmk, and was also equally effective in preventing apoptosis mediated by the three major apoptotic pathways, caspase 9/3, caspase 8/10, and caspase 12. In addition to the increased effectiveness, Q-VD-OPh was not toxic to cells even at extremely high concentrations. Our data indicate that the specificity, effectiveness, and reduced toxicity of caspase inhibitors can be significantly enhanced using carboxyterminal o-phenoxy groups and may have important uses in vivo.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzyl Compounds; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Dactinomycin; DNA Fragmentation; Humans; Hydrocarbons, Fluorinated; Jurkat Cells; Mice; Nucleic Acid Synthesis Inhibitors; Quinolines; Rats; Thapsigargin; Transforming Growth Factor beta

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Apoptosis; Blotting, Western; Brefeldin A; Caspase 3; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Line, Tumor; Cycloheximide; fas Receptor; Fibroblasts; Flow Cytometry; Gene Expression Regulation; HeLa Cells; Humans; Interleukin-3; Mice; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Models, Biological; Phagocytosis; Proto-Oncogene Proteins c-bcl-2; Rats; Serine Endopeptidases; Sulfones; Thapsigargin; Tunicamycin; U937 Cells

In early, androgen dependent stages of prostate cancer, androgen withdrawal, the major course of therapy in prostate cancer, leads to a rapid regression of the tumor as a result of apoptosis. However, prostate cancer invariably progresses to an androgen independent and apoptosis resistant stage for which no curative treatment is available. The molecular details of regression upon androgen withdrawal and progression to a resistant state are largely unknown. Here we show that c-Jun N-terminal Kinase (JNK) is activated strongly and in a sustained fashion by 12-O-tetradecanoylphorbol 13-acetate (TPA) and thapsigargin (TG), two agents which were previously shown to lead to apoptosis in the androgen responsive prostate cancer cell line LNCaP. The time course of JNK induction by both compounds correlated very well with the onset and progression of apoptosis in LNCaP cells. Inhibition of either ERK or p38 pathways did not affect TPA-induced cell death. In the androgen-independent prostate cancer cell lines DU-145 and PC-3, and in the cervical carcinoma cell line HeLaS3, TPA did not lead to apoptosis and there were no significant changes in JNK activity upon TPA treatment. The failure of TPA to induce JNK activity in PC-3, DU-145, and HelaS3 cells was not due to a general defect in JNK signaling since ultraviolet (UV) irradiation dramatically increased JNK activity in all four cell lines. Specific inhibition of JNK by expression of the JNK Inhibitory Protein (JIP) dramatically inhibited both TPA- and TG-induced apoptosis. Furthermore, apoptosis induced by both agents was completely blocked by ectopic expression of the baculovirus caspase-inhibitor P35. Surprisingly, ZVAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases, had no effect on TPA-induced apoptosis, whereas it completely inhibited TG-induced cell death; JNK activity was not affected in either case. This indicates that ZVAD-fmk does not inhibit some of the caspases involved in TPA-induced apoptosis, and that despite the common requirement of JNK activation, TPA- and TG-induced cell death are mechanistically different. Furthermore, it also suggests that JNK is either upstream or independent of caspases in LNCaP cells. Collectively, these results indicate that apoptosis in LNCaP cells requires a sustained increase in JNK activity and caspase activation; components of these signaling pathways may be defective in the androgen independent prostate cancer cell lines.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Androgens; Apoptosis; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; Male; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Tetradecanoylphorbol Acetate; Thapsigargin; Transfection; Tumor Cells, Cultured; Viral Proteins

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

Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells.. Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure.. During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU.. Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Survival; Clone Cells; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Floxuridine; Humans; Male; Oligopeptides; Prostatic Neoplasms; Rats; Retinoblastoma Protein; Signal Transduction; Thapsigargin; Tumor Cells, Cultured

We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Aspartic Acid; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Caspase 3; Caspases; Chlorobenzoates; Cysteine Endopeptidases; Enzyme Inhibitors; Humans; Mice; Neuroblastoma; Peptide Fragments; Recombinant Proteins; Staurosporine; Thapsigargin; Tumor Cells, Cultured

Programmed cell death, or apoptosis, is inhibited by the antiapoptotic oncogene, Bcl-2, and is mediated by a cascade of aspartate-specific cysteine proteases, or caspases, related to interleukin 1-beta converting enzyme. Depending on cell type, apoptosis can be induced by treatment with thapsigargin (TG); a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase. The role of caspases in mediating TG-induced apoptosis was investigated in the Bcl-2-negative human breast cancer cell line, MDA-MB-468. Apoptosis developed in MDA-MB-468 cells over a period of 24-72 h following treatment with 100 nM TG, and was prevented by Bcl-2 overexpression. TG-induced apoptosis was associated with activation of caspase-3 and was inhibited by stable expression of the baculovirus p35 protein, an inhibitor of caspase activity. Also, TG-induced apoptosis was inhibited by treating cells with Z-VAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases. These findings indicate that TG-induced apoptosis of MDA-MB-468 breast cancer cells is subject to inhibition by Bcl-2 and is mediated by caspase activity. This model system should be useful for further investigation directed toward understanding the role of calcium in signaling apoptosis, and its relationship to Bcl-2 and the caspase proteolytic cascade.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Breast Neoplasms; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Viral Proteins

Apoptosis was induced in thymocytes using diverse stimuli in order to identify events within a common apoptotic pathway. Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone (Z-VAD.FMK), an interleukin-1 beta-converting enzyme (ICE)-like protease inhibitor, inhibited apoptosis assessed by flow cytometry, proteolysis of poly (ADP)-ribose polymerase of DNA to both large kilobase pair fragments (30-50 and 200-300 kbp) and to nucleosomal fragments. Z-VAD.FMK also blocked all the classical ultrastructural features of apoptosis including chromatin condensation to one pole of the nucleus, nucleolar disintegration and cytoplasmic vacuolation. These results suggest the involvement of an ICE-like protease as a common mediator of apoptosis in thymocytes.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 1; Cells, Cultured; Cysteine Endopeptidases; Dexamethasone; DNA; Etoposide; Flow Cytometry; Kinetics; Male; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Rats; Rats, Inbred F344; Terpenes; Thapsigargin; Thymus Gland