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

A series of promoting and non-promoting barbiturates and hydantoins wereexamined for their ability to sustain the growth of a phenobarbital(PB)-dependent hepatocyte line in cell culture. The effective liver tumorpromoters, pentobarbital, allobarbital and 5- ethyl-5-phenylhydantoin,replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at52-87% of the level induced by PB. The weak promoters secobarbital andamobarbital supported colony formation at only 11-19% of the PB control. Asignificant correlation was observed for in vivo and in vitro promotionactivities of barbiturates and hydantoins, indicating that clonal expansionby 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared toinfluence hepatocyte growth in vitro. Hepatocyte colonies acquired theability to grow in the absence of PB, such that after 10 days incubationwith PB, approximately 50% of colonies continued to grow in the absence ofpromoter. This phenomenon of clone-size-dependent hepatocyte growthsuggested the operation of an autocrine growth factor pathway. Addition ofthe hepatocyte mitogen and autocrine growth factor, transforming growthfactor-alpha (TGF-alpha), to culture medium lacking PB induced adose-dependent increase in 6/27C1 hepatocyte colony formation. At theoptimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonalexpansion at 84% of the level induced by 2 mM PB. Individual 6/27C1colonies that grew from single cells in the presence of TGF-alpha weretested for promoter-dependent colony formation. Either PB or TGF-alphasupported colony formation by these cells at similar levels and whencombined 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 theparental 6/27C1 line. The ability of TGF-alpha to replace PB and sustainhepatocyte clonal expansion was confirmed with the tumorigenic 6/15hepatocyte line. These results suggest that TGF- alpha and PB may promotehepatocarcinogenesis by stimulating a common signal transduction pathway.