Showing papers by "Guido Kroemer published in 1998"

The mitochondrial death/life regulator in apoptosis and necrosis

Abstract: Both physiological cell death (apoptosis) and, in some cases, accidental cell death (necrosis) involve a two-step process. At a first level, numerous physiological and some pathological stimuli trigger an increase in mitochondrial membrane permeability. The mitochondria release apoptogenic factors through the outer membrane and dissipate the electrochemical gradient of the inner membrane. Mitochondrial permeability transition (PT) involves a dynamic multiprotein complex formed in the contact site between the inner and outer mitochondrial membranes. The PT complex can function as a sensor for stress and damage, as well as for certain signals connected to receptors. Inhibition of PT by pharmacological intervention on mitochondrial structures or mitochondrial expression of the apoptosis-inhibitory oncoprotein Bcl-2 prevents cell death, suggesting that PT is a rate-limiting event of the death process. At a second level, the consequences of mitochondrial dysfunction (collapse of the mitochondrial inner transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins) entails a bioenergetic catastrophe culminating in the disruption of plasma membrane integrity (necrosis) and/or the activation of specific apoptogenic proteases (caspases) by mitochondrial proteins that leak into the cytosol (cytochrome c, apoptosis-inducing factor) with secondary endonuclease activation (apoptosis). The relative rate of these two processes (bioenergetic catastrophe versus protease and endonuclease activation) determines whether a cell will undergo primary necrosis or apoptosis. The acquisition of the biochemical and ultrastructural features of apoptosis critically relies on the liberation of apoptogenic proteases or protease activators from mitochondria. The fact that mitochondrial events control cell death has major implications for the development of cytoprotective and cytotoxic drugs.

Bax and Adenine Nucleotide Translocator Cooperate in the Mitochondrial Control of Apoptosis

Abstract: The proapoptotic Bax protein induces cell death by acting on mitochondria. Bax binds to the permeability transition pore complex (PTPC), a composite proteaceous channel that is involved in the regulation of mitochondrial membrane permeability. Immunodepletion of Bax from PTPC or purification of PTPC from Bax-deficient mice yielded a PTPC that could not permeabilize membranes in response to atractyloside, a proapoptotic ligand of the adenine nucleotide translocator (ANT). Bax and ANT coimmunoprecipitated and interacted in the yeast two-hybrid system. Ectopic expression of Bax induced cell death in wild-type but not in ANT-deficient yeast. Recombinant Bax and purified ANT, but neither of them alone, efficiently formed atractyloside-responsive channels in artificial membranes. Hence, the proapoptotic molecule Bax and the constitutive mitochondrial protein ANT cooperate within the PTPC to increase mitochondrial membrane permeability and to trigger cell death.

The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins.

Abstract: Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2–like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.

Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins.

Abstract: 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, deltapsim, 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 principal (but not exclusive) mechanism of Bcl-2-mediated cytoprotection.

Molecular Ordering of Apoptosis Induced by Anticancer Drugs in Neuroblastoma Cells

Abstract: Apoptosis mediated by anticancer drugs may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas), cleavage of caspases, and perturbance of mitochondrial functions. We investigated the sequence of these events in SHEP neuroblastoma cells transfected with Bcl-2 or Bcl-X(L) using two different drugs, namely, doxorubicin (Doxo), which activates the CD95/CD95 ligand (CD95-L) system, and betulinic acid (Bet A), which does not enhance the expression of CD95 or CD95-L and which, as shown here, directly targets mitochondria. Apoptosis induced by both drugs was inhibited by Bcl-2 or Bcl-X(L) overexpression or by bongkrekic acid, an agent that stabilizes mitochondrial membrane barrier function, suggesting a critical role for mitochondria. After Doxo treatment, enhanced CD95/CD95-L expression and caspase-8 activation were not blocked by Bcl-2 or Bcl-X(L) and were found in cells with a mitochondrial transmembrane potential (delta psi(m)) that was still normal (delta psi(m)high cells). In marked contrast, after Bet A treatment, caspase-8 activation occurred in a Bcl-2- or Bcl-X(L)-inhibitable fashion and was confined to cells that had lost their delta psi(m) (delta psi(m)low cells). Mitochondria from cells treated with either Doxo or Bet A induced cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Thus, caspase-8 activation may occur upstream or downstream of mitochondria, depending on the apoptosis-initiating stimulus. In contrast to caspase-8, cleavage of caspase-3 or poly(ADP-ribose)polymerase was always restricted to delta psi(m)low cells, downstream of the Bcl-2- or Bcl-X(L)-controlled checkpoint of apoptosis. Cytochrome c, released from mitochondria undergoing permeability transition, activated caspase-3 but not caspase-8 in a cell-free system. However, both caspases were activated by apoptosis-inducing factor, indicating that the mechanism of caspase-8 activation differed from that of caspase-3 activation. Taken together, our findings demonstrate that perturbance of mitochondrial function constitutes a central coordinating event in drug-induced cell death.

Potassium Leakage During the Apoptotic Degradation Phase

Abstract: The subcellular compartmentalization of ions is perturbed during the process of apoptosis. In this work, we investigated the impact of K+ on the apoptotic process in thymocytes and T cell hybridoma cells. Irrespective of the death-inducing stimulus (glucocorticoids, topoisomerase inhibition, or Fas-crosslinking), a significant K+ outflow was observed during apoptosis, as determined on the single-cell level by means of the K+-sensitive fluorochrome, benzofuran isophtalate. This loss of cytosolic K+ only occurs in cells that have completely disrupted their inner mitochondrial transmembrane potential. Inhibition of this mitochondrial transmembrane potential loss by Bcl-2 or by specific inhibitors acting on the mitochondrial permeability transition pore (bongkrekic acid, cyclosporin A) prevents K+ leakage. K+ drops at the same stage at which cells expose phosphatidylserine residues on the outer leaflet of the membrane and reduce the levels of nonoxidized glutathione, but before they hyperproduce reactive oxygen species, undergo massive Ca2+ influx, shrink, and lyse. In a cell-free system of apoptosis, isolated nuclei exposed to the supernatant of mitochondria that have undergone permeability transition only manifest chromatinolysis when the K+ concentration is lowered from physiologic to apoptotic levels. Accordingly, massive DNA fragmentation causing subdiploidy is confined to cells that have undergone K+ leakage. Together, these data point to the step-wise acquisition of membrane dysfunction in apoptosis and indicate an important role for the disruption of normal K+ homeostasis in apoptotic degradation. Derepression of endonucleases due to low K+ concentrations may be a decisive prerequisite for end-stage DNA fragmentation.

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition.

Abstract: 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 his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) 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 delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) 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 delta psi(m) 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-X(L) 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 protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.

Mitochondria in chemotherapy-induced apoptosis: a prospective novel target of cancer therapy (review).

Abstract: Resistance to apoptosis is a frequent characteristic of cancer cells and participates both in the initial phase of carcinogenesis and in the development of chemotherapy resistance. Recently, it has become clear that a disruption in mitochondrial membrane function is a decisive event of the apoptotic process leading to the disposal of chemotherapy-treated cells. Opening of the mitochondrial megachannel (also called permeability transition pore) is at least in part responsible for the disruption of mitochondrial membrane integrity in apoptosis. The megachannel is regulated by numerous endogenous effectors including members of the Bcl-2/Bax family, the redox status of the cell, cytosolic Ca2+ levels, ceramide, and amphipathic peptides. Chemotherapeutic agents may induce opening of the megachannel by modulating some of these endogenous effectors. The disruption of mitochondrial membrane integrity involves a loss of metabolic functions and the liberation of intermembrane proteins into the cytosol. Such proteins, which normally are well secluded in mitochondria, include cytochrome c and AIF (apoptosis inducing factor), which both activate caspases and endonucleases upon release into the cytosol. Strategies for the development of chemotherapeutic agents acting on mitochondria are discussed.

Mitochondrial permeability transition in apoptosis and necrosis.

Abstract: Apoptosis has classically been viewed as a process not involving mitochondria, whereas the implication of mitochondrial dysfunction in necrosis has been recognized for several decades. Recently, it has become clear that apoptosis implies a disruption of mitochondrial membrane intregrity that is decisive for the cell death process. Cytofluorometric methods assessing the mitochondrial membrane function and structure can be employed to demonstrate that, at least in most models of apoptosis, mitochondrial changes precede caspase and nuclease activation. Moreover, pharmacological and genetic experiments suggest that the loss of mitochondrial membrane integrity is a critical event of the apoptotic process, beyond or at the point of no return of programmed cell death. Inhibitors of the mitochondrial megachannel (= permeability transition pore) can prevent both the mitochondrial and the post-mitochondrial manifestations of apoptosis.

The mitochondrion as an integrator/coordinator of cell death pathways.

Abstract: Although it has been a widely accepted dogma thatmitochondria are not involved in the process of apoptosis,recent evidence indicates thatmitochondria do playa decisiverole in both apoptosis and necrosis. Indeed, mitochondria canfunction as integrators of different pro-apoptotic signalingpathways and, simultaneously, constitute the target of anumber of apoptosis-inhibitory molecules. It appears that themitochondrial megachannel also called ‘multiple conductancechannel’ or ‘permeability transition pore’) is activated inresponse to different pro-apoptotic signal transductionmolecules. The megachannel is a composite channel formedby apposition of several proteins in the contact site betweenthe inner and the outer mitochondrial membranes, and it isdirectly regulated by the Bcl-2/Bax complex. It participates inthe regulation of matrix Ca

Proteasome Activation Occurs at an Early, Premitochondrial Step of Thymocyte Apoptosis

Abstract: Proteasomes and mitochondrial membrane changes are involved in thymocyte apoptosis. The hierarchical relationship between protease activation and mitochondrial alterations has been elusive. Here we show that inhibition of proteasomes by two specific agents, lactacystin or MG132, prevents all manifestations of thymocyte apoptosis induced by the glucocorticoid receptor agonist dexamethasone or by the topoisomerase II inhibitor etoposide. Lactacystin and MG132 prevent the early disruption of the mitochondrial transmembrane potential (ΔΨ m ), which precedes caspase activation, exposure of phosphatidylserine, and nuclear DNA fragmentation. In contrast, stabilization of the ΔΨ m using the permeability transition pore inhibitor bongkrekic acid or inhibition of caspases by N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone does not prevent the activation of proteasomes, as determined with the fluorogenic substrate N -succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin. Thus, proteasome activation occurs upstream from mitochondrial changes and caspase activation. Whereas the proteasome-specific agents lactacystin and MG132 truly maintain thymocyte viability, a number of protease inhibitors that inhibit nuclear DNA fragmentation (acetyl-Asp-Glu-Val-Asp-fluoromethylketone; N- Boc-Asp(OMe)-fluoromethylketone; N- tosyl-l-Phe-chloromethylketone) do not prevent the cytolysis induced by DEX or etoposide. These latter agents fail to interfere with the preapoptotic ΔΨ m disruption. Altogether, our data indicate that different proteases may be involved in the pre- or postmitochondrial phase of apoptosis. Only those protease inhibitors that interrupt the apoptotic process at the premitochondrial stage can actually preserve cell viability.

Molecular and Cellular Mechanisms of T Lymphocyte Apoptosis

Abstract: Publisher Summary This chapter focuses on the genetic and molecular mechanisms of T lymphocyte apoptosis and also deals with the various phases of apoptosis initiation, execution, and degradation of lymphocytes. Apoptosis is a regulated (programmed) device for the removal of superfluous, aged, or damaged cells within the immune system, as well as in all other organ systems. It is fundamental for development, throughout embryogenesis, organ metamorphosis and organogenesis, including synaptic interactions of neurons, and repertoire selection of T lymphocytes. The activation of T cells generates lymphocytes that are prone to apoptosis, unless these cells are rescued by IL-2 or reside in a specialized environment. Thus, activated lymphocytes undergo programmed cell death by default in the absence of rescue signals mediated by cytokines or cell-cell contacts. Although in determined circumstances T cell death can be induced by starvation from IL-2, this lymphokine can also facilitate T cell apoptosis. Thus, pretreatment with IL-2 renders T cells susceptible to T cell receptor-induced apoptosis, perhaps by dysregulated activation of the cell cycle or by influencing Fas/Fas-L signaling.

Mitochondrial Regulation of Apoptosis

Abstract: Physiological cell death (PCD) constitutes a strictly regulated process which is responsible for the removal of superfluous, aged, or damaged cells. An abnormal resistance to PCD entails malformations, autoimmune disease, or cancer due to the persistence of superfluous, self-specific, or mutated cells, respectively. In contrast, enhanced removal of cells by PCD participates in acute diseases (intoxications, septic shock, anoxia), as well as in chronic pathologies (neurodegenerative and neuromuscular diseases, AIDS). PCD, whose morphological and biochemical phenotype is referred to as “apoptosis”, is characterized by the action of catabolic enzymes, mostly proteases and nucleases, within the limits of a near-to-intact plasma membrane. Thus, the cell actively contributes to its removal and undergoes a series of stereotyped biochemical and ultrastructural alterations (Table 8.1). Apoptosis is the final outcome of multiple different death-inducing pathways. In mammalian cells, such apoptosis-triggering stimuli include interventions on second messenger systems, ligation of certain receptors (Fas/APO-1/CD95, TGF-R, TNF-R, etc.) or, in the case of obligate growth factor receptors, the absence of receptor occupancy. In addition, suboptimal culture conditions (lack of essential compounds, shortage of nutrients, deficiency of oxygen), mild physical damage (radiotherapy), and numerous toxins (chemotherapy and toxins stricto sensu) can provoke apoptosis.1–5 Thus, apoptosis can result both from physiological and from pathological triggers. In vivo, cells undergoing apoptosis are recognized and removed by phagocytes before they undergo lysis. Phagocytic recognition of apoptotic cells is facilitated by characteristic changes in plasma membrane structure, namely the loss of plasma membrane asymmetry with a consequent aberrant exposure of phosphatidylserine (PS) residues (normally only located in the inner membrane leaflet) on the cell surface.

Contrôle mitochondrial de l'apoptose : la mort cellulaire programmée est-elle apparue à la suite de l'événement endosymbiotique à l'origine des mitochondries ?

Abstract: La plupart des cellules animales peuvent mourir par un processus de mort cellulaire que l'on applelle apoptose. Les proteines cellulaires impliquees dans ce processus d'autodestruction ont relativement peu change depuis la dichotomie nematodes/vertebres. Il apparait maintenant que les mitochondries occupent une place determinante dans le controle de l'apoptose. Chez les mammiferes, comme chez les nematodes, les proteines de la famille Bcl-2/Bcl-X/Ced-9 inhibent l'apoptose au moins a deux niveaux : regulation du passage d'ions ou de molecules pro-apoptotiques au travers de pores transmembranaires, et ancrage, au niveau des mitochondries, des proteines impliquees dans la transduction de signaux apoptotiques. Par ailleurs, il est maintenat admis que les mitochondries sont des endosymbiontes ayant pour origine une bacterie aerobie qui aurait ete absorbee par l'ancetre des cellules eucaryotes. Une partie de la machinerie apoptotique existerait chez les eucaryotes unicellulaires et certains des composants controlant l'apoptose pourraient meme etre presents chez les bacteries. Il est donc possible que le mecanisme a l'origine du maintien de la symbiose entre la bacterie ancetre des mitochondries et la cellule hote a l'origine des eucaryotes ait fourni les bases d'un controle de la survie cellulaire. Les metazoaires auraient exploite cette possibilite en connectant les effecteurs mitochondriaux de la mort cellulaire aux voies de transduction de signaux.

Methode de detection in vitro du potentiel transmembranaire mitochondrial

Abstract: L'invention a pour objet l'utilisation de composes lipophiles, cationiques et comportant au moins un groupe halogenoalkyle, notamment un groupe chloromethyle, ces composes etant marques ou etant par eux-memes des marqueurs susceptibles de pouvoir etre detectes, pour la mise en oeuvre d'une methode de detection, et/ou de mesure in vitro du potentiel transmembranaire mitochondrial. L'invention a egalement pour objet l'utilisation de ces composes pour la mise en oeuvre d'une methode de diagnostic in vitro de modifications du processus d'apoptose, ainsi que de pathologies associees a de telles modifications.