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 define d by alterations in nuclear morphology (apoptosis) and/or characterist ic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondr ial reductase activity or cytolysis can be induced in enucleated cells , indicating cytoplasmic PCD control. Here we report a sequential disr egulation of mitochondrial function that precedes cell shrinkage and n uclear fragmentation. A first cyclosporin A-inhibitable step of ongoin g PCD is characterized by a reduction of mitochondrial transmembrane p otential, as determined by specific fluorochromes hloro-1,1',3,3'-tetr aethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodi de). Cytofluorometrically purified cells with reduced mitochondrial tr ansmembrane potential are initially incapable of oxidizing hydroethidi ne (HE) into ethidium. Upon short-term in vitro culture, such cells ac quire the capacity of HE oxidation, thus revealing a second step of PC D 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 volu me, a step that is delayed by radical scavengers, indicating the impli cation of ROS in the apoptotic process. This sequence of alterations a ccompanying early PCD is found in very different models of apoptosis i nduction: glucocorticoid-induced death of lymphocytes, activation-indu ced 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 fluoroch romes such as hloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodid e; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection o f cells that are programmed for death but still lack nuclear DNA fragm entation. In particular, assessment of mitochondrial ROS generation pr ovides an accurate picture of PCD-mediated lymphocyte depletion. In co nclusion, alterations of mitochondrial function constitute an importan t feature of early PCD.