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N Zamzami

Publications and source records attributed to N Zamzami.

At least 73 records · Page 4Linked to original sources
PubMed1996

Mitochondrial permeability transition triggers lymphocyte apoptosis.

In a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (delta psi(m)). This delta psi(m) disruption may involve mitochondrial permeability transition (PT). Here, we address the question of whether PT transition would suffice to cause apoptosis or, rather, it would constitute a secondary event not causally involved in the apoptotic cascade. Protoporphyrin IX (PPIX), a ligand of the mitochondrial benzodiazepin receptor that is well known for its PT-triggering capacity, induces delta psi(m) disruption, enhanced generation of superoxide anions, as well as signs of nuclear apoptosis in thymocytes and in T cells. The sequence of events triggered by PPIX mimics that observed in natural apoptosis. The PT inhibitory compound bongkrekic acid, a specific ligand of the mitochondrial adenine nucleotide translocator, is an efficient inhibitor of protoporphyrin IX-induced delta psi(m) disruption. Bongkrekic acid prevents all PPIX-induced phenomena, including superoxide anion generation, chromatinolysis, and oligonucleosomal DNA fragmentation. In contrast, inhibitors of mRNA or protein synthesis fail to suppress PPIX-induced delta psi(m) disruption and apoptosis. Transfection-enforced hyperexpression of the apoptosis-inhibitory proto-oncogene bcl-2 also inhibits PPIX-induced delta psi(m) disruption, hyperproduction of reactive oxygen species, and nuclear DNA loss. The delta psi(m)-stabilizing effect of Bcl-2 is observed both in cells and in isolated mitochondria. In conclusion, these data are compatible with the hypotheses that mitochondrial PT is self-sufficient to trigger apoptosis and that Bcl-2 may directly regulate PT.

Amino Acid Chloromethyl Ketones↗
PubMed1996

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.

Animals↗
PubMed1996

Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.

Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.

Amino Acid Chloromethyl Ketones↗
PubMed1996

CD69-induced monocyte apoptosis involves multiple nonredundant signaling pathways.

Simultaneous stimulation of human monocytes/macrophages or THP1 cells with LPS and an antibody specific for the activation marker CD69 induces apoptosis. Here we demonstrate the involvement of multiple independent signals that are necessary for apoptosis induction. Thus, inhibitors of phospholipase A2 and lipoxygenase prevent apoptosis induction. Similarly, the ADP-ribosylating G-protein-reactive pertussis toxin (PTX) but not a mutant toxin lacking the ADP-ribosylating moiety (mPTX) prevents apoptosis induction. Furthermore, inhibition of NO generation abrogates completely the induction of apoptosis by LPS/CD69 ligation. These three pathways can be dissociated from each other in the sense that interventions on the arachidonic acid metabolism or G proteins do not inhibit the generation of NO and that exogenous NO cannot reverse the inhibition of cell death by inhibitors of phospholipase A2 or PTX. In addition, both PTX and mPTX affect arachidonic acid mobilization only partially, indicating that the apoptosis-inhibitory effect of PTX (which is not shared by mPTX) cannot be explained by its effect on phospholipase A2 activation. Both LPS and anti-CD69 are sufficient on their own to activate cells, as determined by TNF production, NO generation, or arachidonic acid metabolism, but neither LPS nor anti-CD69 can induce apoptosis on their own. Thus, apoptosis induction in this system involves at least three independent signal transduction systems--(i) arachidonic acid metabolism, (ii) NO, and (iii) PTX-sensitive events--each of which is necessary but insufficient to induce monocyte/macrophage apoptosis. These findings underline the complex control of activation-induced apoptosis in cells of the myelomonocytic lineage.

Adult↗
PubMed1996

Mitochondrial permeability transition is a central coordinating event of apoptosis.

In a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (delta psi m). This delta psi m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system of apoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic delta psi m disruption, BA prevents a number of phenomena linked to apoptosis: depletion of nonoxidized glutathione, generation of reactive oxygen species, translocation of NF kappa B, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic delta psi m disruption is secondary to transcriptional events. These data connect the PT control point to the p53- and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.

Animals↗
PubMed1996

Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis.

When cells undergo nuclear apoptosis (chromatin condensation, DNA fragmentation), they already manifest at least three alterations that can be quantified cytofluorometrically at the single-cell level: 1) a loss of mitochondrial transmembrane potential (delta psi m), 2) an increased production of superoxide anions, and 3) the aberrant exposure of phosphatidylserine (PS) residues on the outer plasma membrane leaflet. This latter alteration allows for the phagocytic recognition/elimination of apoptotic cells. In this work, we show that cells first undergo the delta psi m disruption and that PS exposure only affects cells that already have a low delta psi m. Pharmacologic modulation of apoptosis with inhibitors of macromolecule synthesis or proteases, as well as with drugs stabilizing the delta psi m, indicates that delta psi m disruption and PS exposure are coregulated. Interventions on apoptosis-regulatory genes (p53, bcl-2) confirm the coregulation of delta-psi-m disruption, PS exposure, and nuclear signs of apoptosis. In all conditions in which apoptosis is prevented, the delta psi m remains stable and PS cannot be detected on the cell surface. Reactive oxygen species do not contribute to PS exposure, based on two lines of evidence. First, among thymocytes undergoing apoptosis in response to dexamethasone, delta psi mlow cells first expose PS and then hyperproduce superoxide anion. Second, exogenous sources of reactive oxygen species or the superoxide anion-generating drug menadione fail to cause rapid PS exposure. Instead, direct interventions on mitochondria using inhibitors of the respiratory chain or the F1 ATP synthase cause PS exposure in cells subsequent to delta psi m disruption. This effect is also obtained in anucleate cells, indicating that the nucleus does not intervene in the sequence of events coupling mitochondrial dysfunction to PS exposure. Altogether, these data underline the functional impact of mitochondrial alterations on the apoptotic process.

Animals↗
PubMed1996

Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA.

U937 cells lacking mitochondrial DNA (rho [symbol: see text] cells) are auxotrophic for uridine and pyruvate, hypersensitive to hypoglycemic conditions, and resistant to antimycin A-induced apoptosis. In spite of their obvious metabolic defects, rho [symbol: see text] cells possess a normal mitochondrial transmembrane potential, as well as near-normal capacity to generate superoxide anion after menadione treatment. Similarly to rho + controls, rho [symbol: see text] cells undergo apoptosis in response to tumor necrosis factor-alpha plus cycloheximide. Detailed comparison of the apoptotic process in rho + and rho [symbol: see text] cells reveals essentially the same sequence of events. In response to tumor necrosis factor/cycloheximide, cells first lose their mitochondrial transmembrane potential (delta psi m) and then manifest late apoptotic alterations, such as generation of reactive oxygen species and DNA fragmentation. Experiments involving isolated mitochondria from rho + and rho [symbol: see text] cells confirm that rho [symbol: see text] mitochondria can be induced to undergo permeability transition, a process thought to account for the pre-apoptotic delta psi m disruption in cells. Like rho + mitochondria, rho [symbol: see text] mitochondria contain a pre-formed soluble factor that is capable of inducing chromatin condensation in isolated nuclei in vitro. This factor is released from mitochondria upon induction of permeability transition by calcium or the specific ligand of the adenine nucleotide translocator atractyloside. In conclusion, it appears that all structures involved in the maintenance and pre-apoptotic disruption of the delta psi m, as well as a mitochondrial apoptotic factor(s), are present in rho [symbol: see text] cells and thus are controlled by the nuclear rather than by the mitochondrial genome. These findings underline the contribution of mitochondria to the apoptotic process.

Apoptosis↗
PubMed1996

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 (delta psi m). Here we address the question as to whether mitochondrial permeability transition (PT) pores may account for the delta psi 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 delta psi m disruption. Moreover, pharmacological modulation of PT-mediated delta psi m dissipation can prevent apoptosis. Thus, while suppressing the delta psi m disruption, bongkrekic acid also inhibits the apoptotic chromatinolysis. In conclusion, these data are compatible with the hypothesis that apoptotic delta psi m disruption is mediated by the formation of PT pores and that PT-mediated delta psi m disruption is a critical event of the apoptotic cascade.

Animals↗
PubMed1996

Mitochondrial control of nuclear apoptosis.

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.

Animals↗
PubMed1996

Polyethylene glycol-modified IL-2 abrogates superantigen-induced anergy without affecting peripheral clonal deletion in vivo.

As compared with the native molecule, recombinant human interleukin-2 that is modified by covalently attached polyethylene glycol residues (IL-2-PEG) exhibits a markedly enhanced half-life in vivo, thus facilitating its biological evaluation. We have characterized the effect of IL-2-PEG on the Staphylococcus aureus enterotoxin B (SEB)-induced tolerance of peripheral SEB-reactive (V beta 8+) T cells. Treatment with sublethal doses of IL-2-PEG does not modulate (inhibit or enhance) the SEB-triggered apoptosis and deletion of V beta 8+ T cells. In contrast, in vivo treatment with IL-2-PEG partially abolishes the SEB-triggered anergy of V beta 8+ T cells, i.e., the failure to proliferate in response to SEB in vitro. To abolish SEB-triggered anergy, IL-2-PEG must act for an extended period in vivo; short term treatment in vivo (2 days) or exposure of anergic T cells to IL-2 in vitro fails to reconstitute proliferative responses. Moreover, the effect of in vivo treatment with IL-2-PEG on lymphokine production by anergic T cells is partial. IL-2-PEG restores IL-4-dependent autocrine proliferation in response to SEB but does not reestablish defective IL-2 production. These data are compatible with the notion that IL-2 is a regulator of postdeletional rather than deletional T cell tolerance.

Animals↗
PubMed1995

Mitochondrial dysfunctions in circulating T lymphocytes from human immunodeficiency virus-1 carriers.

In several models of lymphocyte apoptosis, two alterations of mitochondrial function precede advanced DNA fragmentation: (1) a reduction of mitochondrial transmembrane potential (delta psi m) and (2) an increase in mitochondrial generation of superoxide anion. Here we show that two fluorochromes allow for the identification of analogous mitochondrial perturbations in circulating T lymphocytes from human immunodeficiency virus (HIV)-1+ donors. The first among these fluorochromes, the cationic lipophilic dye DiOC6(3), measures delta psi m; the second marker, hydroethidine (HE), is nonfluorescent, unless it is oxidized by superoxide anions to the product ethidium (Eth). CD4+ or CD8+ cells from clinically asymptomatic HIV-1 carriers contain a significantly elevated percentage of cells endowed with enhanced HE --> Eth conversion and/or reduced DiOC6(3) uptake as compared with normal controls. Phenotypic characterization of (HE --> Eth)high cells from HIV+ donors shows that these cells possess a low delta psi m, thus demonstrating a functional alteration of mitochondria. In addition, (HE --> Eth)high cells display a reduced incorporation of the cardiolipin-specific dye nonyl-acridine orange (NAO), showing a structural defect of the cardiolipin-containing inner mitochondrial membrane. Control experiments involving rotenone, an inhibitor of the respiratory chain complex I, indicate that the reactive oxygen species responsible for HE --> Eth conversion is generated during mitochondrial electron transport. In synthesis, it appears that mitochondrial alterations occur in a significant percentage of circulating T lymphocytes from HIV-1 carriers. The extent of delta psi m reduction, as determined ex vivo, correlates with the frequency of cells undergoing DNA fragmentation after overnight in vitro culture. These observations may be important for the understanding and for the direct ex vivo quantitation of HIV-triggered lymphocyte destruction.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.

Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.

Animals↗
PubMed1995

Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.

In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.

Animals↗
PubMed1995

Chlorpromazine amplifies macrophage-dependent IL-10 production in vivo.

Chlorpromazine (CPZ) has potent immunomodulatory effects in vivo; it induces humoral autoimmunity in up to 50% of patients, inhibits delayed-type hypersensitivity reactions, and suppresses lethal immune hyperactivation in animal models of septic shock. Here, we show that in an in vivo model of acute superantigen-driven immune activation, CPZ independently down-regulates the production of various T cell-derived lymphokines (IL-2, IFN-gamma, IL-4, TNF, and GM-CSF) and up-regulates the secretion of IL-10. Whereas only low, if any, serum IL-10 levels are detectable by ELISA after injection of CPZ, bacterial LPS, or staphylococcal enterotoxin B (SEB) alone, simultaneous administration of CPZ + LPS or CPZ + SEB causes a significant increase in IL-10 production in vivo. CPZ-mediated amplification of the SEB-driven CPZ secretion is accompanied by an enhanced IL-10 mRNA accumulation, as shown by PCR analysis and in situ hybridization. Determination of IL-10 production in mice lacking T cells, B cells, or phagocytes revealed that SEB + CPZ-induced IL-10 was produced by phagocytic cells, but not by lymphocytes, a finding that is in accord with the distribution of splenic cells transcribing the IL-10 gene in response to SEB + CPZ. Moreover, these data indicate that bacterial superantigen can directly stimulate tissue phagocytes, even in the virtual absence of T lymphocytes. The blockade of dopamine type 1 (D1) but not type 2 (D2) receptors abolishes the CPZ effect on IL-10 production. Inhibition of Th1 and Th2 lymphokine production by CPZ is not mediated by dopamine receptors and is independent of IL-10 up-regulation. These findings may explain the mechanism by which CPZ and related drugs enhance humoral autoimmune reactions, block cellular immune responses, and prevent lethal septic shock in vivo.

Animals↗
PubMed1995

Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo.

We have recently shown that lymphocyte apoptosis induced by dexamethasone or superantigens is accompanied by reduction of mitochondrial transmembrane potential (delta psi m) which precedes nuclear DNA fragmentation. Here, we demonstrate that fluorochromes such as 3,3' dihexyloxacarbocyanine iodide [DiOC6(3)] which measure delta psi m, or fluorochromes such as hydroethidine (HE) which measure mitochondrial superoxide anion production allow the identification of thymocytes or peripheral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic alpha/beta T cell receptor (TCR) specific for a class I-restricted male-specific peptide, the superoxide-mediated oxidation of HE into ethidium (Eth) is enhanced among thymocytes which are being deleted due to negative selection (CD4+ CD8+ cells expressing the transgenic TCR in male mice) or lack of positive selection (CD4+ CD8- thymocytes from female mice). delta psi m reduction and/or enhanced HE oxidation are also found when apoptosis is induced by a series of pathogenic agents. Thus, lethal irradiation provokes mitochondrial and nuclear signs of apoptosis, and both these alterations are absent in mice bearing a p53 null mutation, underlying the correlation between mitochondrial perturbation and nuclear apoptosis. Similarly, superantigen-triggered deletion of peripheral T cells in vivo is accompanied by enhanced HE-->Eth conversion and reduced DiOC6(3) uptake. More importantly, as compared to normal controls, CD4+ or CD8+ cells from clinically asymptomatic human immunodeficiency virus-1 (HIV-1) carriers also contain a significantly elevated percentage of cells endowed with reduced DiOC6(3) uptake. In superantigen- and HIV-induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC6(3) uptake is more important than that of cells marked by enhanced HE-->Eth conversion. In conclusion, mitochondrial alterations precede and/or accompany nuclear signs of apoptosis induced by physiological and a variety of different pathogenic agents in vivo.

Animals↗
PubMed1995

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-inducing 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-amplificatory metabolic pathways. One of these switches may reside in a perturbation of mitochondrial function. Thus, a decrease in mitochondrial transmembrane potential, followed by mitochondrial uncoupling and generation of reactive oxygen species, precedes nuclear alterations. It appears that molecules that participate in apoptotic decision-making also exert functions that are vital for normal cell proliferation and intermediate metabolism.

Apoptosis↗
PubMed1994

Pertussis toxin inhibits activation-induced cell death of human thymocytes, pre-B leukemia cells and monocytes.

Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. Similarly, cross-linking of the activation marker CD69, which belongs to the natural killer complex, causes apoptosis of lipopolysaccharide-preactivated monocytes. Here we show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal, because thymocytes exposed to PTX are still capable of mobilizing Ca2+ after TCR-alpha/beta cross-linking and proliferate in response to costimulation with PTX and CD3/TCR ligation. The apoptosis-inhibitory effect of PTX depends on the presence of an intact adenosine diphosphate (ADP)-ribosylating moiety, since a mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity, fails to interfere with activation-induced cell death. A toxin that induces a different spectrum of ADP ribosylation than PTX, cholera toxin, fails to inhibit apoptosis. To suppress apoptosis, the intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis. These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activation-induced cell death of immune cells.

Apoptosis↗