Search

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspaseindependent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of ∼ 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Focuses on the key role mitochondria play in the regulation of apoptosis. The identification and cloning of an apoptosis-inducing factor, which is sufficient to induce apoptosis of isolate nuclei.

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid bis 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential ΔΨm that is not inhibited by the broad spectrum caspase inhibitor Z-VAD.fmk, although Z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the ΔΨm of t-BHP-treated cells but has no inhibitory effect on the ΔΨm collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of ΔΨm disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered ΔΨm collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-XL proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer... [ABSTRACT FROM AUTHOR]

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12 – 24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice... [ABSTRACT FROM AUTHOR]

Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-XL only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells. Oncogene (2001) 20, 1063–1075. [ABSTRACT FROM AUTHOR]

The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore. [ABSTRACT FROM AUTHOR]

Bcl-2 is the prototype of a class of oncogenes which regulates apoptosis. Bcl-2-related gene products with either death-promoting and death-inhibitory activity are critically involved in numerous disease states and thus constitute prime targets for therapeutic interventions. The relative amount of death agonists and antagonists from the Bcl-2 family constitutes a regulatory rheostat whose function is determined, at least in part, by selective protein-protein interactions. Bcl-2 and its homologs insert into intracellular membranes including mitochondria, the endoplasmatic reticulum and the nuclear envelope. Many of the molecular genetic, ultrastructural, crystallographic and functional studies suggest that Bcl-2-related molecules exert their apoptosis-regulatory effects via regulating mitochondrial alterations preceding the activation of apoptogenic proteases and nucleases. Via a direct effect on mitochondrial membranes, Bcl-2 prevents all hallmarks of the early stage of apoptosis including disruption of the inner mitochondrial transmembrane potential and the release of apoptogenic protease activators from mitochondria. The mitochondrial permeability transition (PT) pore, also called mitochondrial megachannel or multiple conductance channel, is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly at the same localization at which Bax, Bcl-2, and Bcl-XL are particularly abundant. The PT pore participates in the regulation of matrix Ca2+, pH, ΔΨm, and volume and functions as a Ca2+-, voltage-, pH-, and redox-gated channel with several levels of conductance and little if any ion selectivity. Experiments involving the purified PT pore complex indicate that Bax, Bcl-2, and Bcl-XL exert at least part of their apoptosis-regulatory function by facilitating (Bax) or inhibiting (Bcl-2, Bcl-XL) PT pore opening. These findings clarify the... [ABSTRACT FROM AUTHOR]