AIM: To investigate the effect of stable c-Fos over-expression on immortalized human hepatocyte (IHH) proliferation. METHODS: IHHs Stably transfected with c-Fos (IHH-Fos) or an empty "vector (IHH-C) were grown in me-dium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, to-tal and phosphorylated GSK-3?and epidermal growth factor receptor (EGF-R) were assayed by Western blot-ting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments. RESULTS: Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorpora-tion following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpress-ing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3? A kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein com-pared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyro-sine kinase inhibitor, prevented the phosphorylation of GSK-3?induced by serum stimulation and decreased Cyclin D1 stability in the nucleus. CONCLUSION: Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis throughstabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular car-cinoma.