A CYTOFLUOROMETRIC ASSAY OF NUCLEAR APOPTOSIS INDUCED IN A CELL-FREE SYSTEM - APPLICATION TO CERAMIDE-INDUCED APOPTOSIS

Purified nuclei exposed to apoptogenic factors in vitro undergo morpho logical and biochemical changes in chromatin organization. Most cell-f ree models of nuclear apoptosis are based on the quantitation of endon uclease-mediated DNA fragmentation on agarose gels or on the changes o f nuclear morphology revealed by the DNA-intercalating fluorochrome 4' -6-diamidino-2-phenylindole dihydrochloride. In this work we develop a cytofluorometric system for the accurate quantitation of nuclear DNA loss. This system has been used to determine the conditions of nuclear apoptosis induced by apoptosis-inducing factor (AIF) contained in the supernatant of mitochondria induced to undergo permeability transitio n. AIF can provoke significant nuclear DNA loss in less than or equal to 5 min, acts over a wide pH range (pH 6 to 9), and resists cysteine protease inhibitors such as iodoacetamide and N-ethylmaleimide. Moreov er, we applied this system to the question of how the proapoptotic sec ond messenger ceramide would induce apoptosis in vitro: via a direct e ffect on nuclei, a direct effect on mitochondria, or via indirect mech anisms? Our data indicate that ceramide has to activate yet unknown cy tosolic effecters that, in the presence of mitochondria, can induce nu clear apoptosis in vitro. (C) 1997 Academic Press.