title: Biochemical characterisation of an in vitro model of hepatocellular apoptotic cell death.
abstract: This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.
authors.affiliation: Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium. mvinken@vub.ac.be
authors.forename: Mathieu
authors.initials: M
authors.lastname: Vinken
authors.forename: Elke
authors.initials: E
authors.lastname: Decrock
authors.forename: Elke
authors.initials: E
authors.lastname: De Vuyst
authors.forename: Luc
authors.initials: L
authors.lastname: Leybaert
authors.forename: Tamara
authors.initials: T
authors.lastname: Vanhaecke
authors.forename: Vera
authors.initials: V
authors.lastname: Rogiers
mesh_terms: D000826:Animal Testing Alternatives / Q000379:methods*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D016923:Cell Death; D006498:Hepatectomy; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D006706:Homeostasis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000166:cytology / Q000502:physiology; D009336:Necrosis; D051381:Rats; D012377:Rodentia
chemical_list: D007770:L-Lactate Dehydrogenase; D020169:Caspases