TGF-alpha sustains clonal expansion by promoter-dependent, chemically initiated rat hepatocytes

Abstract

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.

Greater Expression of Transforming Growth Factor α and Proliferating Cell Nuclear Antigen Staining in Mouse Hepatoblastomas Than Hepatocellular Carcinomas Induced by a Diethylnitrosamine-Sodium Phenobarbital Regimen

“…It shares about 30% structural similarity with EGF and can bind to the EGF receptor (3). Several reports have suggested TGF-a to be a potent stimulator of normal hepatocyte proliferation and its overexpression may play an important role in hepatocarcinogenesis (13,22,23). Thus, TGF-a has been detected in many malignant cell lines including human and rodent hepatocellular tumors (4,19,20), and overexpression in the liver results in hepatocarcinogenesis in transgenic mice (18,26).…”

Effect of phenobarbital on hepatic cell proliferation and apoptosis in mice deficient in the p50 subunit of NF-κB

“…It has been postulated that PB acts through the same autocrine growth pathway as TGF-α by inhibiting apoptosis (Sandgren et al, 1995;Smith et al, 1995;Kaufmann et al, 1997;SantoniRugiu et al, 1998), which is thought to be caused by activation of reactive oxygen species (ROS). ROS may also be involved in the intracellular regulation of transcription factors like NF-κB and AP-1 through protein kinase-C activation (Feig et al, 1994;Brawn et al, 1995;Storz and Polla, 1996;Klaunig et al, 1998).…”