TGF-ALPHA SUSTAINS CLONAL EXPANSION BY PROMOTER-DEPENDENT, CHEMICALLY-INITIATED RAT HEPATOCYTES

A series of promoting and non-promoting barbiturates and hydantoins we re examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture, The effective liver t umor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydant oin, replaced PB and supported 6/27C1 hepatocyte colony formation in v itro at 52-87% of the level induced by PB, The weak promoters secobarb ital 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. Ce ll density also appeared to influence hepatocyte growth in vitro, Hepa tocyte colonies acquired the ability to grow in the absence of PB, suc h that after 10 days incubation with PB, similar to 50% of colonies co ntinued to grow in the absence of promoter, This phenomenon of clone-s ize-dependent hepatocyte growth suggested the operation of an autocrin e growth factor pathway, Addition of the hepatocyte mitogen and autocr ine growth factor, transforming growth factor-alpha (TGF-alpha), to cu lture medium lacking PB induced a dose-dependent increase in 6/27C1 he patocyte colony formation, At the optimal concentration of 3 ng/ml, TG F-alpha sustained hepatocyte clonal expansion at 84% of the level indu ced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colon y formation, Either PB or TGF-alpha supported colony formation by thes e 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 a dditive for supporting colony formation by the parental 6/27C1 line, T he ability of TGF-alpha to replace PB and sustain hepatocyte clonal ex pansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesi s by stimulating a common signal transduction pathway.