Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-c yclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 tr eatment, chromatin condensation and fragmentation, and the number of termin al deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and A nnexin V-positive cells increased dose dependently without any observable l actate dehydrogenase leakage. The activity of the cysteine protease, caspas e-3, was increased, but not that of caspase-1 and -6. The specific caspase- 3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation an d attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakag e. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were f urther increased. Decreased mitochondrial membrane potential (measured by r hodamine 123 uptake) and cytochrome c release went in parallel with ultrast ructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cyto plasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transm ission electron microscopy, treatment with SDZ IMM 125 resulted in an incre ase in the number of necrotic cells after 20 h, but not after 4 h. Our find ings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific ind ucer of apoptosis after short-term incubation, and this overlaps with necro sis after longer treatment periods. it is very likely that the necrosis occ urring later is the result of the early apoptotic events.