Abstract
The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration.
Highlights
Isolation of hepatocytes from normal liver and establishment of in vitro culture systems have provided powerful experimental in vitro models to identify extracellular signals and to study intracellular signalling pathways regulating differentiation and controlling the ratio between proliferation and apoptosis in liver [1]
Expression of liver specific functions progressively decreases and apoptosis eventually occurs within a week through the activation of caspases 3, 8, and 9 in hepatocytes [3, 4]
We demonstrate that liver slicing procedure induces proliferation signalling pathways, which trigger entry into and progression through G1 phase of the cell cycle similar to that observed in isolated hepatocytes after liver dissociation
Summary
Isolation of hepatocytes from normal liver and establishment of in vitro culture systems have provided powerful experimental in vitro models to identify extracellular signals and to study intracellular signalling pathways regulating differentiation and controlling the ratio between proliferation and apoptosis in liver [1]. Expression of liver specific functions progressively decreases and apoptosis eventually occurs within a week through the activation of caspases 3, 8, and 9 in hepatocytes [3, 4]. This in vitro culture model has been very useful to identify apoptotic inducers, survival factors and mitogens based on their ability to increase or reduce apoptosis and induce DNA replication, respectively. In Me2SO-treated cultures [9], coculture [10] and monolayers or sandwich of collagen I [11, 12], hepatocytes are arrested in G1 phase of the cell cycle and do not replicate DNA upon stimulation by growth factors
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