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.
Loyer
P
P
INSERM U49, Hôpital Pontchaillou, Rennes, France.
Cariou
S
S
Glaise
D
D
Bilodeau
M
M
Baffet
G
G
Guguen-Guillouzo
C
C
D000818:Animals; D015415:Biomarkers; D002453:Cell Cycle; D002478:Cells, Cultured; D019938:Cyclin D1; D056742:Cyclin D2; D056743:Cyclin D3; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis; D016213:Cyclins / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D016193:G1 Phase; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008297:Male; D015513:Oncogene Proteins / Q000096:biosynthesis; D006656:Protamine Kinase / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011519:Proto-Oncogenes; D011773:Pyruvates / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase
D015415:Biomarkers; C486197:Ccnd2 protein, rat; C534698:Ccnd3 protein, rat; D056742:Cyclin D2; D056743:Cyclin D3; D016213:Cyclins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011773:Pyruvates; D012333:RNA, Messenger; D019938:Cyclin D1; D004815:Epidermal Growth Factor; D004247:DNA; D006656:Protamine Kinase; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases