Epidermal Growth Factor Induction of Cellular Proliferation and Protooncogene Expression in Growth-Arrested Rat H4IIE Hepatoma Cells: Role of Cyclic Adenosine Monophosphate

Abstract

The precise molecular events involved in growth factor-mediated cell proliferation in eukaryotes have not been entirely elucidated. Identification and characterization of the itnracellular molecular signaling systems linking growth factor function with nuclear events would provide insight into the regulatory mechanisms governing eukaryotic cell growth. In this report, we demonstrate that serum-deprived rat H4IIE hepatoma cells enter a quiescent state and remain viable in the absence of serum for up to 7 days. These cells can be stimulated to transverse the cell cycle and proliferate in response to epidermal growth factor (EGF) after a 24-h lag phase. We were able to completely mimic the mitogenic effects of EGF with 8-p-chlorophenylthio-cAMP (8-CPT-cAMP) but only partially with N6-(Bu)2-cAMP. EGF and 8-CPT-cAMP together induce a synergistic increase in H4IIE hepatoma cell proliferation. The calcium ionophore A23187 and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate had little effect on H4IIE cell proliferation. EGF treatment led to a rapid and transient increase of intracellular cAMP concentration. Both 8-CPT-cAMP and EGF were also equally effective in causing a rapid and transient induction of c-fos and c-myc protooncogene mRNA levels when added to growth-arrested H4IIE cells while A23187, N-(Bu)2-cAMP, and 4 beta-phorbol 12-myristate 13-acetate were significantly less effective. Both EGF and 8-CPT-cAMP affect protooncogene expression in growth-arrested rat H4IIE hepatoma cells primarily at the transcriptional level. Localization and semi-quantification of nuclear pp55c-fos and 63 (kilodalton)-myc protooncoproteins by immunocolloidal gold electron microscopy revealed that EGF and/or 8-CPT-cAMP treatment of quiescent H4IIE hepatoma cells led to a marked and rapid nuclear accumulation of these proteins in discrete nuclear substructures. Cummulatively, these results suggest that cAMP participates in the intracellular signaling system mediating the mitogenic and protooncogene inducing effects of EGF on growth-arrested rat H4IIE hepatoma cells.