Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNAreplication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes

In this study, we investigated the combined effects of EGF and collagen I g el on the phenotype of cultured rat hepatocytes and we focussed our investi gations on the regulation of xenobiotic-mediated induction of CYP, cell cyc le progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provok ed a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitor?; effect on 3-methylcholantrene (5 muM) and beta -n aphtoflavone (25 muM) induction of CYP1A1 activities. In collagen gel sandw ich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cyc le, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In coll agen gel sandwich cultures. we found that neither collagen I nor EGF preven ted activation of caspase 8 while collagen I gel inhibited activation of ca spase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In c ontrast, EGF transiently increased caspase 3 activity at day 1 after platin g. Altogether, our data demonstrate that collagen I gel triggers intracellu lar signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibit ion of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures. (C) 2001 Elsevier Science Inc. All rights reserved.