Growth Factor Dependence of Progression through G1 and S Phases of Adult Rat Hepatocytes in Vitro

Abstract

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.

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…In marked contrast to our observations in PC12 cells, NGF treatment of hepatocytes stimulated the ability of these cells to synthesize DNA, which was also blocked by either PD98059 treatment or by expression of dominant negative MEK1 (Table 1). In a parallel experiment, EGF treatment also stimulated the ability of primary cultures of hepatocytes to synthesize DNA, in agreement with data of others [3].…”

“…Hepatocytes 20-24 h after partial hepatectomy in i o are at the end of G1 and most cells are in S phase, whereas hepatocytes cultured in itro for 20-24 h are mostly in G1 [3]. Based on the studies Ethanol treatment (A) temporally extends the ability of NGF to activate the MAP kinase cascade and (B) increases expression of the cdk inhibitor proteins p16 INK4a and p21 Cip-1 .…”

“…Partial hepatectomy or acute dissociation by collagenase treatment and primary culture of hepatocytes triggers their entry into the cell cycle [1][2][3][4][5][6][7][8][9][10]. Maximal DNA synthesis in i o occurs 12-36 h post partial hepatectomy and within 5-7 days the liver has regained its original mass [1][2][3].…”

“…Maximal DNA synthesis in i o occurs 12-36 h post partial hepatectomy and within 5-7 days the liver has regained its original mass [1][2][3]. Primary culture in itro in the presence of growth factors such as insulin or epidermal growth factor (EGF) stimulates hepatocyte DNA synthesis which is maximally observed 40-70 h post isolation [1][2][3][4]. Treatment of hepatocytes, isolated from either proliferating or nonproliferating livers, with either insulin or EGF has also been shown to cause an acute phasic increase of mitogen activated protein (MAP) kinase activity with a subsequent stimulation of DNA synthesis [3].…”

“…Primary culture in itro in the presence of growth factors such as insulin or epidermal growth factor (EGF) stimulates hepatocyte DNA synthesis which is maximally observed 40-70 h post isolation [1][2][3][4]. Treatment of hepatocytes, isolated from either proliferating or nonproliferating livers, with either insulin or EGF has also been shown to cause an acute phasic increase of mitogen activated protein (MAP) kinase activity with a subsequent stimulation of DNA synthesis [3]. However, little is understood about the activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21 Cip-"/WAF" and p16 INK%a cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased.…”