SEQUENTIAL ACQUISITION OF MITOCHONDRIAL AND PLASMA-MEMBRANE ALTERATIONS DURING EARLY LYMPHOCYTE APOPTOSIS

When cells undergo nuclear apoptosis (chromatin condensation, DNA frag mentation), 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 increa sed production of superoxide anions, and 3) the aberrant exposure of p hosphatidylserine (PS) residues on the outer plasma membrane leaflet. This latter alteration allows for the phagocytic recognition/eliminati on of apoptotic cells. In this work, we show that cells first undergo the Delta Psi(m) disruption and that PS exposure only affects cells th at already have a low Delta Psi(m). Pharmacologic modulation of apopto sis with inhibitors of macromolecule synthesis or proteases, as well a s with drugs stabilizing the Delta Psi(m), indicates that Delta Psi(m) disruption and PS exposure are coregulated. Interventions on apoptosi s-regulatory genes (p53, bcl-2) confirm the coregulation of Delta Psi( m) disruption, PS exposure, and nuclear signs of apoptosis. In all con ditions in which apoptosis is prevented, the Delta Psi(m) remains stab le and PS cannot be detected on the cell surface. Reactive oxygen spec ies do not contribute to PS exposure, based on two lines of evidence. First, among thymocytes undergoing apoptosis in response to dexamethas one, Delta Psi(m)(low) cells first expose PS and then hyperproduce sup eroxide 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 inhibit ors of the respiratory chain or the F-1 ATP synthase cause PS exposure in cells subsequent to Delta Psi(m) disruption. This effect is also o btained in anucleate cells, indicating that the nucleus does not inter vene in the sequence of events coupling mitochondrial dysfunction to P S exposure. Altogether, these data underline the functional impact of mitochondrial alterations on the apoptotic process.