Phenobarbital-dependent proliferation of putative initiated rat hepatocytes

The mitogenic effects of phenobarbital (PB) were examined usingcultures of putative initiated hepatocytes that proliferateand form colonies under conditions in which normal hepatocytessenesce and die. The frequencies of colony-forming hepatocytesin primary cultures isolated 2 weeks after initiation with methyl(acetoxymethyl)nitrosamineor benzo-[a]pyrene-7, 8-diol-9, 10-epoxide(anti) were in therange of 2–38 per million in the presence of PB. Colony-formationfrequencies were 0.1 per million in the absence of PB. Proliferativehepatocyte colonies were not observed in cultures grown in serum-freemedium containing PB, epidermal growth factor, nor-epinephrineand insulin. The requirement for PB was characterized furtherusing secondary cultures of hepatocytes that had been isolatedfrom a liver 5 weeks after initiation. The colony-fonning efficiencyof these hepatocytes was about 10% in the presence of 2 mM PBand less than 0.2% in its absence. Colony formation displayeda linear response to concentrations of PB in the range of 0.5–2mM and a decline above the optimal 2 mM concentration. Autoradiographywas used to determine the percentages of hepatocytes in secondarycultures that synthesized DNA in the presence or absence ofPB. By the third day after seeding as single cells, hepatocytesexhibited a labeling index of about 50% and this level of labelingwas preserved for up to 2 weeks after seeding. Very few hepatocyteswere found to synthesize DNA In the absence of PB and most senesced.A small fraction of the colony-forming hepatocytes continuedto proliferate in the absence of PB and formed colonies witha high labeling index. These results suggest that the proliferationof initiated hepatocytes in vivo may be conditional upon thepresence of the hepatic tumor promoter, PB.