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,

SummaryIn a number of experimental systems in which lymphocyte depletion was induced by apoptosisinducing 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 (A~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 A~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 A~m l~ splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither A~m l~ nor AlI;mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37~ 1 h) A~m l~ cells show endonucleolysis, followed by cytolysis several hours later. Incubation of AlICml~ cells in the presence of excess amount of the GC receptor antagonist RU-38486 (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 AlICml~ to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in A~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.

SummaryAnucleate 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 (A~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 the disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkrekic 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 in 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. Since it has been shown that anucleate cells (cytoblasts) can be induced to undergo programmed cell death (PCD) 1 (1-3), it has become clear that a cytoplasmic PCD pathway must function independently from the nucleus. Both mitochondria (4) and specific ced-3-1ike proteases 1Abbreviations used in this paper: