Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.