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

doi:10.1074/jbc.271.19.11484

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

EVIDENCE OF A MITOGEN RESTRICTION POINT IN MID-LATE G(*)

From the From INSERM U49, Unité de Recherches Hépatologiques, Hôpital Pontchaillou, 35033 Rennes Cedex, France

^¶ To whom correspondence should be addressed: Tel.: 33-99-54-37-37; Fax: 33-99-54-01-37.

Abstract

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

Footnotes

↵^§ Recipient of a fellowship from l'Association pour la Recherche Contre le Cancer et le Ministère de la Recherche et de l'Espace (MRE), respectively.
↵* This work was supported by l'Institut National de la Santé et de la Recherche Médicale (INSERM) and the European Community (BIOT-CT 900189). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviationa used are:

PHT

partial hepatectomy

HGF

hepatocyte growth factor

TGF-α

transforming growth factor-α

pyr

pyruvate

EGF

epidermal growth factor

R point

restriction point

cdk

cyclin-dependent kinase

TBS

Tris-buffered saline

cdc2

cell division cycle mutant 2

FCS

fetal calf serum.
- Received October 11, 1995.
- Revision received February 15, 1996.