6th ESACP Congress, Heidelberg, April 7-11, 1999

A114
OXIDATION OF PYRIMIDINE NUCLEOTIDES DURING APOPTOSIS: CLOSE CORRELATION WITH MITOCHONDRIAL ALTERATIONS, GLUTATHIONE DEPLETION, AND INTRACELLULAR ACIDIFICATION
Petit PX 1, Métivier D 2, Gendron MC 3, Sureau F 3, Macioroska Z 4, Kroemer G 2, Koester S 5

1) INSERM U129, ICGM, Faculté de Médecine Cochin Port-Royal, Paris, France 2) IRC Villejuif, 3) Université Pierre et Marie Curie, 4) Institut Curie, 5) Coulter/Beckman, Paris, France

In response to Fas/Apo-1/CD95 crosslinking or stimumation with cell-permeant ceramide, Jurkat T cells exhibit a major, early decrease in autofluorescence at 424±40 nm indicative of the oxidation/depletion of NADH or NADPH. Based on kinetic studies, cytofluorometric multiparameter analyses and cell sorting experiments, the NAD(P)H depletion was found to occur simultaneously with the dissipation of the mitochondrial inner transmembrane potential (delta-Phi-m), depletion/oxidation of GSH, and intracellular acidification. In contrast, NAD(P)H depletion becomes detectable well before a number of additional changes associated with late apoptosis occur: enhanced superoxide generation, phosphatidyl serine exposure on the cell surface, cytosolic K+ loss, nuclear DNA fragmentation, cellular shrinkage, loss of viability, and formation of the mitochondrial neo-antigen APO2.7. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which is an inhibitor of the mitochondrial permeability transition (PT) pore, retarded both the delta-Phi-m disruption and the depletion of NAD(P)H. Similar effects were observed for the pharmacological PT pore inhibitors bongkrekic acid or cyclosporin A. Altogether, these data point to a close functional relationship between mitochondrial and redox changes during early apoptosis.