Patterns of alpha-1-adrenergic receptor expression in regenerating and neoplastic hepatic tissue.

As norepinephrine is a potent hepatocyte comitogen through binding to the alpha 1-adrenergic receptor, we have examined mRNA levels of the alpha 1a- and alpha 1b-adrenergic receptor subtypes in normal and regenerating rat hepatocytes as well as in several different rat hepatoma cell lines. All rat hepatomas examined lacked both alpha 1a- and alpha 1b-receptor message and receptor binding in radioligand binding experiments, suggesting that the growth of dedifferentiated neoplastic rat hepatocytes is not regulated by the alpha 1-adrenergic receptor. Interestingly, unlike the rat hepatomas analyzed, the human hepatocellular carcinoma cell line, HepG2, was positive for both alpha 1a and alpha 1b message at 4.5 kb, yet this cell line lacked receptor binding in radioligand binding assays. While normal and regenerating liver is negative for alpha 1a-receptor expression, it is positive for alpha 1b expression and is characterized by the presence of two bands at approximately 4.0 and 3.2 kb which peaked between 20 and 48 h after partial hepatectomy. A dramatic decrease in message level of the lower band and the continued presence of the upper band between 6 and 12 h after partial hepatectomy, and before the peak in DNA synthesis in regenerating rat liver, may correspond with observed differences in alpha 1-receptor function during liver regeneration.

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.

Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture.

Two percent dimethyl sulfoxide (DMSO) reversibly inhibited DNA synthesis in primary rat hepatocyte cultures maintained with epidermal growth factor (EGF) or hepatocyte growth factor (HGF). These data suggest that, in vitro, DMSO is a non-specific inhibitor of hepatocyte proliferation, regardless of the stimulating mitogen. In addition, removal of DMSO from mitogen-free cultures resulted in an increase in DNA synthesis. Protein synthesis gradually but irreversibly declined in all cultures after DMSO removal. The relevance of these findings to regulation of hepatocyte growth is discussed.

Effect of epidermal growth factor on the expression of protooncogenes c-myc and c-Ha-ras in short-term primary hepatocyte culture.

We have characterized the effect of the hepatomitogen epidermal growth factor (EGF) on the expression of the cellular protooncogenes c-Ha-ras and c-myc in short-term (48 hours) primary hepatocyte culture. mRNA concentrations of both protooncogenes increased dramatically in nonproliferating cultures and in the absence of EGF, suggesting that the isolation procedure or the culture conditions may trigger expression of these genes or potentially increase the lifetime of transcripts in vitro, regardless of the presence of a mitogen. In cells treated with EGF, a distinct peak in c-Ha-ras expression was seen 24 hours after EGF treatment. This coincided with the onset of DNA synthesis. No such peak was seen in cultures not treated with EGF. The c-myc mRNA concentrations were increased relatively equally in all cultures with or without the addition of EGF. These data show a differential response of these two cell-cycle-associated genes to the culture conditions and EGF stimulation. It also demonstrated that enhanced gene expression for Ha-ras and myc in hepatocytes can occur in the absence of cell proliferation.

Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO-EGF on/off protocol.

Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self-limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.