Estrogen receptor, epidermal growth factor receptor and cellular ploidy in elutriated subpopulations of hepatocytes during liver tumor promotion by 17 alpha-ethinylestradiol in rats.

Abstract

17 alpha-Ethinylestradiol (EE2)-mediated promotion of diethylnitrosamine (DEN)-initiated liver tumors was evaluated in distinct hepatocyte subpopulations. Our initiation-promotion regime consisted of a single dose of DEN (200 mg/kg) to ovariectomized rats, followed by chronic exposure to EE2 (90 micrograms/kg/day for 30 weeks). We observed significant increases in liver and uterine organ wts which were associated with liver tumor formation. Isolated hepatocytes were separated by elutriation into seven subpopulations. The early eluting subpopulations consisted of a greater proportion of diploid cells and they exhibited a preferential uptake of acridine orange, which is characteristic of periportal cells. With the increasing order of elutriated fractions, hepatocyte subpopulations of tetraploid and octaploid cells were obtained. Elutriation revealed that EE2 promotion enhanced nuclear estrogen receptor levels (3-fold) and gamma-glutamyltranspeptidase activity (5-fold) to a greater extent in the early eluting diploid subpopulations (1 and 2), even though total estrogen receptor (ER) levels were higher in the later eluting subpopulations. The stimulatory effect of EE2 on ER levels was associated with an increased ER occupancy in all subpopulations, although the effect was greatest in the later eluting fractions. Chronic EE2 exposure induced the emergence of new hepatocyte populations within fractions 6 and 7. Enhanced cell growth was observed in the DEN/EE2-derived hepatocytes by flow cytometric measurements of DNA synthesis. The new populations of altered cells expressed high levels of epidermal growth factor receptor (EGFR), with 90% of the cells positive for EGFR-antibody. In summary, our data demonstrate that many effects of EE2 on hepatocyte pathways involved in growth control occur in nearly all populations of cells, derived by elutriation although some effects such as the emergence of an EGFR-enriched population of hepatocytes are localized in specific populations.