APOPTOSIS INDUCED BY MICROINJECTION OF CYTOCHROME-C IS CASPASE-DEPENDENT AND IS INHIBITED BY BCL-2

Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was ch aracterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastr uctural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of t he cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl- 2 and these all showed a markedly decreased sensitivity towards inject ed cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. Th ey also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, bu t can also interfere with caspase activation induced by cytochrome c m icroinjected directly into the cytoplasm.