MODULATION OF MITOGEN-INDEPENDENT HEPATOCYTE PROLIFERATION DURING THEPERINATAL-PERIOD IN THE RAT

Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capac ity declines with advancing gestational age of tile fetus from which t he hepatocytes are derived. The present studies were undertaken to inv estigate this change in fetal hepatocyte growth regulation. Examinatio n of E19 fetal hepatocyte primary cultures using immunocytochemistry f or 5-bromo-2'-deoxyuridine (BrdU) incorporation showed that approximat ely 80% of these cells traverse S-phase of the cell cycle over the fir st 48 h in culture. Similarly, 65% of EIS hepatocytes maintained in cu lture under defined mitogen-free conditions for 24 h showed nuclear ex pression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreacting PCNA in immuno fluorescent analyses of E19 liver. In contrast, E21 (term) liver showe d little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative s hortly after isolation. However, within 12 h of plating, E21 hepatocyt es showed cytoplasmic staining for PCNA. Although maintained under mit ogen-free conditions, PCNA expression progressed synchronously to a nu cleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This appa rently synchronous cell cycle progression was confirmed by studies sho wing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transform ing growth factor alpha (TGF alpha), considerable mitogen-independent DWA synthesis was seen in hepatocytes from both gestational ages. Thes e results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultu red.