Philippe Marchetti

Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis

In a number of experimental systems, the early stage of the apoptotic process, i.e. the stage which precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (ΔΨ m). Here we address the question as to whether mitochondrial permeability transition (PT) pores may account for the ΔΨ m dissipation in lymphocyte apoptosis. Drugs known for their PT‐inhibitory potential (bongkrekic acid, cyclosporin A, and the non‐immunosuppressive cyclosporin A analogue N‐methyl‐Val‐4‐cyclosporin A) are capable of preventing the apoptotic ΔΨ m disruption. Moreover, pharmacological modulation of PT‐mediated ΔΨ m dissipation can prevent apoptosis. Thus, while suppressing the ΔΨ m disruption, bongkrekic acid also inhibits the apoptotic chromatinolysis. In conclusion, these data are compatible with the hypothesis that apoptotic ΔΨ m disruption is mediated by the formation of PT pores and that PT‐mediated ΔΨ m disruption is a critical event of the apoptotic cascade.

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

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 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.

Apoptosis of cells lacking mitochondrial DNA

The mitochondrial genome of animals encodes a few subcomponents of the respiratory chain complexes I, III and IV, whereas nuclear DNA encodes the overwhelming majority, both in quantitative and qualitative terms, of mitochondrial proteins. Complete depletion of mitochondrial DNA (mtDNA) can be achieved by culturing cells in the presence of inhibitors of mtDNA replication or mitochondrial protein synthesis, giving rise to mutant cells (ϱ∘ cells) which carry morphological near-to-intact mitochondria with respiratory defects. Such cells can be used to study the impact of mitochondrial respiration on apoptosis. ϱ∘ cells do not undergo cell death in response to determined stimuli, yet they conserve their potential to undergo full-blown apoptosis in many experimental systems. This indicates that mtDNA and associated functions (in particular mitochondrial respiration) are irrelevant to apoptosis execution. However, the finding that mtDNA-deficient mitochondria can undergo apoptosis does not argue against the involvement of mitochondria in the apoptotic process, since mitochondria from ϱ∘ cells conserve most of their functions including those involved in the execution of the death programme: permeability transition and release of one or several intermembrane proteins causing nuclear apoptosis.

Maintenance of the T Lymphocyte Pool by Inhibition of Apoptosis: A Novel Strategy of Immunostimulation?

Throughout development T lymphocytes are constantly confronted with a series of vital options. First, T cells can decide to either ignore an antigen or to become activated upon antigenic stimulation. Second, T cell activation may have rather disparate consequences, T lymphocytes may become productively activated to proliferate, differentiate or exert an effector function. Alternatively, they can become activated in an abortive or aberrant fashion, leading to anergy or apoptosis. The option that the T cell will choose among these possibilities is not only dictated by the conformation and concentration of the antigenic peptide/MHC complex, but depends also on a series of further circumstances: the particular context of cosignais perceived via receptors interacting with the antigen-presenting cell (APC), bystander cells (Ding and Shevach 1994), cell matrix proteins and soluble factors (cytokines and hormones), and the particular subpopulation to which the responding cells belong and their differentiation stage and (pre-) activation state. In this sense, T cells function as semiotic entities; they integrate signals from a complex universe as a function of their previous experiences to act in a quantitatively and qualitatively graded, rather than all-or-nothing, fashion.