SummaryProgrammed 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-l,l',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-l,l',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. p rogrammed cell death (PCD) 1 is likely to occur during the whole lifetime of higher organisms, including early embryogenesis, to affect any cell type, and to constitute the normal fate of cells (1-3). In spite of its physiological and pathological importance, at least two major problems concerning PCD remain still elusive: (a) an operative definition of PCD, and (b) an efficient system to detect PCD in vivo.PCD is commonly defined by characteristic morpholog-N. Zamzami and P. Marchetti contributed equally to this work.1 Abbreviations used in this paper: CHX,

SummaryBcl-2 belongs to a family ofapoptosis-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 (AXltm), suggesting the involvement ofmitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed ~50-kD protein which is released upon A~ m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and intemucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1]3-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 A~ 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. Before cells manifest nuclear signs ofapoptosis, they disrupt the mitochondrial transmembrane potential (ARtm) 1. This pre-apoptotic AXI*,,~ collapse occurs in nu~Abbreviations used in this paper: Ac-DEVD.CHO, acetyl-Asp-Glu-ValAsp-aldehyde; Ac-YVAD.CHO; acetyl-Tyr-Val-Ala-Asp-aldehyde; Ac-YVAD.cmk, aceyl-Tyr-Val-Ala-Asp-chloromethylketone; AIF, apoptosisinducing factor; Atr, atractyloside; CMXP,.os, chloromethyl-X-rosamine; Axlt,,,, mitochondrial transmembrane potential; mC1CCP, carbonylcyanide m-chlorophenylhydrazone; Neo, neomycin resistance gene; MDH, malate dehydrogenase; MAO, monoamine oxidase; PI, propidium iodine; PT, pemleability transition; P, OS, reactive oxygen species; SDH, succihate dehydrogenase; t-BHP, ter-butylhydroperoxide; TLCK, N-tosylt-lysyl chloromethylketone; TPCK, N-tosyl-l-Phe-chloromethylketone; PARP, poly (ADP-ribose) polymerase; Z-D.CH2-dcb, N-benzyloxycarbonyl-Asp-CH20C(O)-2-6,-dichlorobenzene; Z-FA.fmk, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone; Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. S.A. Susin and N. Zamnzami contributed equally to this paper. merous cell types (neurons, fibroblasts, myelomonocytic cells, lymphocytes, hepatocytes)...

SummaryIn 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 (Aqtm). This A~ 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 ofapoptosis. 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 A~,n disruption, BA prevents a number of phenomena linked to apoptosis: depletion ofnonoxidized glutathione, generation of reactive oxygen species, translocation of NFKB, 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 Aqr,, 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.

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.