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1.
Cell Death Differ ; 10(10): 1188-203, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14502242

ABSTRACT

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.


Subject(s)
Apoptosis/physiology , Caspase Inhibitors , Phagocytosis/physiology , Serine Endopeptidases/metabolism , Amino Acid Chloromethyl Ketones/chemistry , Amino Acid Chloromethyl Ketones/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Blotting, Western , Brefeldin A/pharmacology , Caspase 3 , Caspases/chemistry , Caspases/metabolism , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Line, Tumor , Cycloheximide/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Flow Cytometry , Gene Expression Regulation , HeLa Cells , Humans , Interleukin-3/deficiency , Interleukin-3/pharmacology , Mice , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Models, Biological , Phagocytosis/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , Rats , Sulfones/pharmacology , Thapsigargin/pharmacology , Tunicamycin/pharmacology , U937 Cells , fas Receptor/immunology
2.
Oncogene ; 19(19): 2286-95, 2000 May 04.
Article in English | MEDLINE | ID: mdl-10822379

ABSTRACT

Apoptosis involves mitochondrial steps such as the release of the apoptogenic factor cytochrome c which are effectively blocked by Bcl-2. Although Bcl-2 may have a direct action on the mitochondrial membrane, it also resides and functions on the endoplasmic reticulum (ER), and there is increasing evidence for a role of the ER in apoptosis regulation as well. Here we uncover a hitherto unrecognized, apoptotic crosstalk between the ER and mitochondria that is controlled by Bcl-2. After triggering massive ER dilation due to an inhibition of secretion, the drug brefeldin A (BFA) induces the release of cytochrome c from mitochondria in a caspase-8- and Bid-independent manner. This is followed by caspase-3 activation and DNA/nuclear fragmentation. Surprisingly, cytochrome c release by BFA is not only blocked by wild-type Bcl-2 but also by a Bcl-2 variant that is exclusively targeted to the ER (Bcl-2/cb5). Similar findings were obtained with tunicamycin, an agent interfering with N-linked glycosylations in the secretory system. Thus, apoptotic agents perturbing ER functions induce a novel crosstalk between the ER and mitochondria that can be interrupted by ER-based Bcl-2.


Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Biological Transport/drug effects , Brefeldin A/metabolism , Brefeldin A/pharmacology , Caspase 3 , Caspase 8 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cell Nucleus/metabolism , Coumarins/metabolism , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome c Group/drug effects , Cytochrome c Group/metabolism , Endoplasmic Reticulum/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Humans , Mitochondria/drug effects , Mitochondria/ultrastructure , Oligopeptides/metabolism , Protein Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Rats , Tunicamycin/pharmacology
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