A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: application to ceramide-induced apoptosis.
Exp Cell Res
; 236(2): 397-403, 1997 Nov 01.
Article
en En
| MEDLINE
| ID: mdl-9367623
ABSTRACT
Purified nuclei exposed to apoptogenic factors in vitro undergo morphological and biochemical changes in chromatin organization. Most cell-free models of nuclear apoptosis are based on the quantitation of endonuclease-mediated DNA fragmentation on agarose gels or on the changes of 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 transition. AIF can provoke significant nuclear DNA loss in < or = 5 min, acts over a wide pH range (pH 6 to 9), and resists cysteine protease inhibitors such as iodoacetamide and N-ethylmaleimide. Moreover, we applied this system to the question of how the proapoptotic second messenger ceramide would induce apoptosis in vitro via a direct effect on nuclei, a direct effect on mitochondria, or via indirect mechanisms? Our data indicate that ceramide has to activate yet unknown cytosolic effectors that, in the presence of mitochondria, can induce nuclear apoptosis in vitro.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Núcleo Celular
/
Ceramidas
/
Apoptosis
/
Citometría de Flujo
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Exp Cell Res
Año:
1997
Tipo del documento:
Article
País de afiliación:
Francia