title: Pharmacological screening and enzymatic assays for apoptosis.
abstract: Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.
authors.affiliation: University of Versailles/SQY, PRES UniverSud Paris, CNRS UMR 8159, Versailles, France.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Belzacq-Casagrande
authors.forename: Cecile
authors.initials: C
authors.lastname: Martel
authors.forename: Claire
authors.initials: C
authors.lastname: Pertuiset
authors.forename: Annie
authors.initials: A
authors.lastname: Borgne-Sanchez
authors.forename: Etienne
authors.initials: E
authors.lastname: Jacotot
authors.forename: Catherine
authors.initials: C
authors.lastname: Brenner
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects / Q000648:ultrastructure; D008933:Mitochondrial Swelling / Q000187:drug effects; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology
chemical_list: D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; D002118:Calcium