Phenobarbital-dependent proliferation of putative initiated rat hepatocytes

The mitogenic effects of phenobarbital (PB) were examined using cultures of putative initiated hepatocytes that proliferate and form colonies under conditions in which normal hepatocytes senesce and die. The frequencies of colony-forming hepatocytes in primary cultures isolated 2 weeks after initiation with methyl(acetoxymethyl)nitrosamine or benzo[a]pyrene-7,8-diol-9,10-epoxide(anti) were in the range of 2-38 per million in the presence of PB. Colony-formation frequencies were 0.1 per million in the absence of PB. Proliferative hepatocyte colonies were not observed in cultures grown in serum-free medium containing PB, epidermal growth factor, nor-epinephrine and insulin. The requirement for PB was characterized further using secondary cultures of hepatocytes that had been isolated from a liver 5 weeks after initiation. The colony-forming efficiency of these hepatocytes was about 10% in the presence of 2 mM PB and less than 0.2% in its absence. Colony formation displayed a linear response to concentrations of PB in the range of 0.5-2 mM and a decline above the optimal 2 mM concentration. Autoradiography was used to determine the percentages of hepatocytes in secondary cultures that synthesized DNA in the presence or absence of PB. By the third day after seeding as single cells, hepatocytes exhibited a labeling index of about 50% and this level of labeling was preserved for up to 2 weeks after seeding. Very few hepatocytes were found to synthesize DNA in the absence of PB and most senesced. A small fraction of the colony-forming hepatocytes continued to proliferate in the absence of PB and formed colonies with a high labeling index. These results suggest that the proliferation of initiated hepatocytes in vivo may be conditional upon the presence of the hepatic tumor promoter, PB.