A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: application to ceramide-induced apoptosis.

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.

Statistics

050100150'99'01'03'05'07'09'11'13'15'17
Citations per Year

349 Citations

Semantic Scholar estimates that this publication has 349 citations based on the available data.

See our FAQ for additional information.

Cite this paper

@article{Susin1997ACA, title={A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: application to ceramide-induced apoptosis.}, author={Santos A . Susin and Naoufal Zamzami and Nathanael Larochette and Bruno Dallaporta and Isabel Marzo and Catherine Brenner and Tamara Hirsch and P X Petit and Maurice Geuskens and Guido Kroemer}, journal={Experimental cell research}, year={1997}, volume={236 2}, pages={397-403} }