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.

Bacteria-induced phagocyte secondary necrosis as a pathogenicity mechanism

“…As expected, cultured metazoan cells apoptosing in vitro express eat-me signals [62,74], and consequently, when coincubated with phagocytes, they are ingested [78 -80].…”

“…1). In the first (pre-necrotic) phase, molecular alterations produce the classical apoptotic morphotype that allows the identification of this mode of cell death [36,60] and the expression of eat-me signals [62,74]. The terminal phase is secondary necrosis with rupture of the cytoplasmic membrane and autolytic cell disintegration into cell debris [32,46,50].…”

“…1), including oxidative burst, mitochondrial membrane hyperpolarization, fall in the intracellular ATP pool, lysosomal membrane permeabilization, and most importantly, terminal cytoplasmic membrane permeabilization, the necrotic hallmark of this apoptotic outcome. These events lead to cytoplasmic swelling, rupture of the cytoplasmic membrane, and terminal cell dismantling into cell debris with spilling of the intracellular content [59,62,63], the crucial event that confers to secondary necrosis its pathological character in multicellular animals. This terminal cytolysis is autolytic, as it is carried out from within by the ongoing autonomous, proteolytic process, mainly operated by released lysosomal enzymes [81] and thus, contrasts with the heterolytic disintegration of apoptosing cells occurring within the phagolysosomes of the scavenger cell when clearance occurs [33].…”

Experimental models of hepatotoxicity related to acute liver failure

“…Primary hepatocytes may offer a better alternative, as they display in vivo -like sensitivity to apoptosis, at least during short-term cultivation regimes (Schulze-Bergkamen et al 2003). Among the numerous experimental strategies that have been followed to provoke apoptotic cell death in primary hepatocyte cultures, the use of Fas triggers is a most reasonable approach, as it directly activates the physiological pathway (Vinken et al 2009). Monoclonal agonistic antibodies directed against the Fas receptor are frequently used in primary hepatocyte cultures.…”

“…For this reason, Fas antibodies are often combined with inhibitors of protein production or gene expression, such as cycloheximide and actinomycin D, respectively (Nagata 1999; Ni et al 1994; Rouquet et al 1996). A more rationalized strategy that resembles the natural Fas pathway is the use of FasL as such (Fu et al 2004; Reinehr et al 2002; Vinken et al 2009). FasL can also be presented to hepatocytes by cultivation partners.…”