Bernard Mignotte

The biochemistry of programmed cell death.

Programmed cell death (PCD) is involved in the removal of superfluous and damaged cells in most organ systems. The induction phase of PCD or apoptosis is characterized by an extreme heterogeneity of potential PCD‐triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD‐indueing stimuli converge into a few stereotypical pathways and cells pass a point of no return, thus becoming irreversibly committed to death. It is only during the successive degradation phase that vital structures and functions are destroyed, giving rise to the full‐blown phenotype of PCD. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations commonly considered to define PCD (apoptotic morphology of the nucleus and regular, oligonucleosomal chromatin fragmentation) have to be ascribed to the late degradation phase. The decision as to whether a cell will undergo PCD or not may be expected to be regulated by “switches” that, once activated, trigger self‐am‐ plificatory metabolic pathways. One of these switches may reside in a perturbation of mitochondrial function. Thus, a decrease in mitochondrial transmem‐ brane potential, followed by mitochondrial uncoupling and generation of reactive oxygen species, precedes nuclear alterations. It appears that molecules that participate in apoptotic decisionmaking also exert functions that are vital for normal cell proliferation and intermediate metabolism.—Kroemer, G., Petit, P., Zamzami, N., Vayssière, J.‐L., Mignotte, B. The biochemistry of programmed cell death. FASEB J. 9, 1277‐1287 (1995)

Mitochondrial reactive oxygen species in cell death signaling.

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.

Mitochondria and programmed cell death: back to the future

Programmed cell death, or apoptosis, has in the past few years undoubtedly become one of the most intensively investigated biological processes. However, fundamental questions concerning the molecular and biochemical mechanisms remain to be elucidated. The central question concerns the biochemical steps shared by the numerous death induction pathways elicited by different stimuli. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of ‘no return’ and are engaged in a degradation phase where they acquire the typical onset of late apoptosis. Alterations in mitochondrial permeability transition linked to membrane potential disruption precede nuclear and plasma membrane changes. In vitro induction of permeability transition in isolated mitochondria provokes the release of a protein factor capable of inducing nuclear chromatin condensation and fragmentation. This permeability transition is regulated by multiple endogenous effectors, including members of the bcl‐2 gene family. Inhibition of these effects prevents apoptosis.

Implication of mitochondria in apoptosis.

The induction phase of programmed cell death (PCD) or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD-inducing stimuli converge into a few stereotypical pathways and cells pass a ‘point of no return’, thus becoming irreversibly committed to death. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations usually considered to define apoptosis, as nuclear chromatolysis and cytolysis, have to be ascribed to the late degradation phase. We and others have recently shown that nuclear features of apoptosis are preceded by alterations in mitochondrial function and structure. The importance of these alterations for the apoptotic process and also the possible link between, these observations, the permeability transition pore and the programmed cell death, are discussed. (Mol Cell Biochem 174: 185–188, 1997)

Study of PTPC Composition during Apoptosis for Identification of Viral Protein Target

Abstract: The permeability transition pore complex (PTPC), a mitochondrial polyprotein complex, has been previously described to be involved in the control of mitochondrial membrane permeabilization (MMP) during chemotherapy‐induced apoptosis. PTPC may contain proteins from both mitochondrial membranes [e.g., voltage‐dependent anion channel (VDAC), PRAX‐1, peripheral benzodiazepine receptor (PBR), adenine nucleotide translocator (ANT)], from cytosol (e.g., hexokinase II, glycerol kinase), from matrix [e.g., cyclophilin D (CypD)], and from intermembrane space (e.g., creatine kinase). PTPC may also interact with tumor suppressor proteins (i.e., Bax and Bid), oncoprotein homologues of Bcl‐2 and some viral proteins, which can regulate apoptosis induced by pore opening. ANT and VDAC are the target of numerous pro‐apoptotic MMP inducers. However, the precise composition of PTPC as well as the respective role of each PTPC component represent major issues in the understanding MMP process. Using several experimental strategiesthat combine co‐immunoprecipitation, proteomics, and functional tests with proteoliposomes, we and others have been able to characterize some of the intra/inter‐PTPC protein interactions leading to a better understanding of the process of MMP. In addition, this approach could identify new putative members and regulators of PTPC pro‐apoptotic function and new targets of viral protein involved in the modulation of apoptosis during infection.

Bcl-2 can promote p53-dependent senescence versus apoptosis without affecting the G1/S transition

With the aim to identify events involved in the determination of p53-dependent apoptosis versus growth arrest, we used rat embryo fibroblasts expressing a temperature-sensitive mutant (tsA58) of the SV40 large tumour antigen (LT). Heat-inactivation of LT leads to p53 activation and commitment to a senescent-like state (REtsA15 cell line) or apoptosis (REtsAF cell line). We report that senescence is associated with high levels of the anti-apoptotic Bcl-2 protein and a cell cycle arrest in G1 phase, whereas apoptosis is associated with low levels of Bcl-2 and a cell cycle arrest in G2 phase. Here we show that Bcl-2, which can inhibit apoptosis and proliferation, turns the apoptotic phenotype into a senescent-like phenotype in G2 phase. This result suggests that Bcl-2-dependent inhibition of apoptosis could be crucial for the commitment to replicative senescence, whereas its ability to inhibit G1 progression would not be required.

Yeast as a model to study apoptosis

Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms.The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, . . .) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis.Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics).This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.

The mitochondrial TOM complex modulates bax-induced apoptosis in Drosophila.

Bax is a pro-apoptotic member of the Bcl-2 family proteins involved in the release of apoptogenic factors from mitochondria to the cytosol. Recently, it has been shown both in mammals and yeast that Bax insertion in the mitochondrial outer membrane involves at least two distinct mechanisms, one of which uses the TOM complex. Here, we show that in Drosophila, heterozygous loss of function mutations of Tom22 or Tom70, two receptors of the TOM complex, attenuates bax-induced phenotypes in vivo. These results argue that the TOM complex may be used as a mitochondrial Bax receptor in Drosophila.

Evidence for a mitochondrial localization of the retinoblastoma protein

BackgroundThe retinoblastoma protein (Rb) plays a central role in the regulation of cell cycle, differentiation and apoptosis. In cancer cells, ablation of Rb function or its pathway is a consequence of genetic inactivation, viral oncoprotein binding or deregulated hyperphosphorylation. Some recent data suggest that Rb relocation could also account for the regulation of its tumor suppressor activity, as is the case for other tumor suppressor proteins, such as p53.ResultsIn this reported study, we present evidence that a fraction of the total amount of Rb protein can localize to the mitochondria in proliferative cells taken from both rodent and human cells. This result is also supported by the use of Rb siRNAs, which substantially reduced the amount of mitochondrial Rb, and by acellular assays, in which [35S]-Methionine-labeled Rb proteins bind strongly to mitochondria isolated from rat liver. Moreover, endogenous Rb is found in an internal compartment of the mitochondria, within the inner-membrane. This is consistent with the protection of Rb from alkaline treatment, which destroys any interaction of proteins that are weakly bound to mitochondria.ConclusionAlthough a few data regarding an unspecific cytosolic localization of Rb protein have been reported for some tumor cells, our results are the first evidence of a mitochondrial localization of Rb. The mitochondrial localization of Rb is observed in parallel with its classic nuclear location and paves the way for the study of potential as-yet-unknown roles of Rb at this site.

p53 and Retinoblastoma protein (pRb): a complex network of interactions.

p53 and the Retinoblastoma protein (pRb) are two oncosuppressive proteins whose loss of function is linked to the development of many cancers. Both of them play a central role in the regulation of both cell cycle and apoptosis. Different observations have led to a paradigm in which p53 and pRb can regulate each other, pRb playing an important role in the outcome—growth arrest versus apoptosis—of p53 activation.