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A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochrome c, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochrome c plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL.

In multicellular organisms, the total number of cells is a balance between the cell-generating effects of mitosis and cell death that is induced through apoptosis. A disruption of this delicate balance can lead to the development of cancer. This Timeline article focuses on how the field of apoptosis biology has developed in the context of its contribution to our understanding of cell death, or lack of it, in the development of malignant disease. It traces the course of research from key discoveries in fundamental biology to potential therapeutic applications.

Reactive oxygen species (ROS) are a group of molecules produced in the cell through metabolism of oxygen. Endogenous ROS such as hydrogen peroxide (H2O2) have long been recognised as destructive molecules. The well-established roles they have in the phagosome and genomic instability has led to the characterisation of these molecules as non-specific agents of destruction. Interestingly, there is a growing body of literature suggesting a less sinister role for this Jekyll and Hyde molecule. It is now evident that at lower physiological levels, H2O2 can act as a classical intracellular signalling molecule regulating kinase-driven pathways. The newly discovered biological functions attributed to ROS include proliferation, migration, anoikis, survival and autophagy. Furthermore, recent advances in detection and quantification of ROS-family members have revealed that the diverse functions of ROS can be determined by the subcellular source, location and duration of these molecules within the cell. In light of this confounding paradox, we will examine the factors and circumstances that determine whether H2O2 acts in a pro-survival or deleterious manner.

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Cell death may occur by either of two mechanisms: apoptosis or necrosis. Necrosis, the first type of cell death to be recognized, is an uncontrolled degenerative phenomenon invariably caused by noxious stimuli and is the result of irreversible failure of membrane function. Apoptosis, on the other hand, is a death process which involves a series of well-organized events which require active cell participation, and is primarily caused by physiological stimuli. In the present study we show that cell death induced by a range of varied agents may take the form of either apoptosis or necrosis. Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis occurred. Hence, cells which are not killed directly, but merely injured by these agents, have the capacity to activate an internally programmed suicide death mechanism, whereas cells receiving greater injuries apparently do not. In addition, the presence of extracellular calcium was found to be necessary for the induction of apoptosis with all agents tested.

Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

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