Growth factor dependence of progression through G(1) and S phases of adult rat hepatocytes in vitro - Evidence of a mitogen restriction point in mid-late G(1)

Several hepatocyte mitogens have been identified, but the signals triggering the G(0)/G(1) 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 G(1) phase and analyzed the expression of cell cycle markers. we show that the G(0)/G(1) 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 G(1) regardless of growth factor stimulation until a restriction point (R point) in mid-late G(1) beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell. cycle gene expression were associated with progression in G(1); the cyclin E mRNA level is low early in G(1) but increases at the G(1)/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G(1) after the It 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 It point. In contrast, cyclin D3 mRNA appears early in G(1), 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 G(1) phase in hepatocytes.