Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death
Mathieu Vinken, Elke Decrock, Elke De Vuyst, Luc Leybaert, Tamara Vanhaecke and Vera Rogiers
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.