The hepatocellular binucleation rate, measured as the percentage of binuclear cells amongst newly formed bromodeoxyuridine-labelled and immunostained collagenase-isolated rat hepatocytes, decreased from 12% to 4% between days 30 and 40 after birth, rose to 20% between days 50 and 60, and then declined again to the adult rate of about 10% at day 80. During regenerative growth following a two-thirds partial hepatectomy, the rate of binucleation declined to about 3%, causing the fraction of binuclear cells to fall from 27% (before hepactectomy) to 5% (at 45 h after hepactectomy) as pre-existing binuclear cells replicated and formed mononuclear daughter cells. Essentially all (97%) hepatocytes replicated at least once, starting their DNA synthesis at around 13 h and reaching a peak at 30 h, irrespective of ploidy and nuclearity. At later time points, the diploid hepatocytes had a higher labelling index than the polyploid cells, suggesting a greater tendency to go through several cell cycles.

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The question of a possible precursor--product relationship between oval cells and hepatocytes was examined in rats treated for 2 weeks with 2-acetylaminofluorene (2-AAF) with a two-thirds partial hepatectomy (PH) performed after the first week of 2-AAF treatment (modified Solt-Farber model). Liver cells were pulse-chase labelled with bromodeoxyuridine (BrdU) on day 6 post PH. On day 7 post PH the nonparenchymal (NPC) fraction, which contains the oval cells, exhibited a labelling index (LI) approximately 10 times higher than that of the hepatocytes as analysed by flow cytometry (FCM), the majority of the proliferating cells being oval cells. At later time points, there was no significant increase in the LI of diploid hepatocytes, and no detectable shift of BrdU-labelled cells from the NPC fraction to the hepatocyte fraction, suggesting that no extensive conversion of BrdU-labelled oval cells to hepatocytes was taking place. Throughout the experimental period there was a significant increase in the diploid hepatocyte cell fraction, from 12% on day 7 to 25% on day 13 post PH. Diploid hepatocytes pulse-labelled on days 7 or 9 post PH had a high LI (7-8%), in contrast to the low LI (1%) of tetra- and octoploid cells. Proliferation of diploid hepatocytes may thus explain the large increase in the diploid hepatocyte fraction observed from days 9 through 15 post PH. Our results, therefore, provide no reason to invoke oval cells as precursors of hepatocytes in the modified Solt-Farber carcinogenesis model.

The complete liver carcinogen 2-acetylaminofluorene (2-AAF) promoted the outgrowth of large neoplastic liver nodules and hepatocellular carcinomas in diethylnitrosamine-treated rats. 2-AAF did not alter the overall proliferative activity of normal hepatocytes, but suppressed binucleation and induced, on a long-term basis, an increase in proliferative activity and in the fraction of diploid hepatocytes relative to control animals. The analogue 4-acetylaminofluorene (4-AAF) was much less effective than 2-AAF as a promoter of large nodules and carcinomas, but promoted the outgrowth of medium-sized nodules (1 < 2.5 mm). While 2-AAF specifically stimulated the growth of cells in enzyme-altered foci, the cells responding to 4-AAF were more randomly distributed throughout the liver tissue. In contrast to 2-AAF, 4-AAF strongly stimulated the growth (DNA synthesis) of normal hepatocytes, but like 2-AAF it suppressed binucleation and caused a long-term increase in the proliferative activity and in the fraction of diploid hepatocytes. Other liver tumour promoters (cyproterone acetate, alpha-hexachlorocyclohexane, methylclofenapate) likewise stimulated the growth and suppressed the binucleation of normal hepatocytes. All hepatocellular ploidy classes were affected virtually equally by mitogenic stimulation, but at low proliferation rates the mononuclear cells were more proliferative than the binuclear cells. Since this difference could be eliminated by increasing the mitogen dose, it would seem that mononuclear cells may be somewhat more sensitive towards mitogens than binuclear cells. In contrast to previously reported results [Styles et al. (1990) Carcinogenesis, 11, 1149-1152], methylclofenapate was not found to specifically stimulate binuclear hepatocytes. Our results indicate that liver tumour promoters in general tend to induce a non-binucleating, non-polyploidizing hepatocellular growth pattern, similar to that observed during liver regeneration. 4-AAF is confirmed to be, at best, a very weak promoter of liver carcinogenesis, but appears to be an effective promoter of benign tumours.

The proliferative activity of tumor cells differing in DNA content (ploidy) and nuclearity was investigated in primary hepatocellular carcinomas of the rat by flow cytometric analysis of collagenase-isolated cells immunostained after labelling with bromodeoxyuridine (BrdU) in vivo. The diploid cell fraction in these euploid tumours was higher than in normal liver, and the rate of binucleation as well as the proliferative activity of the binuclear cells was very low. The highest proliferative activity (BrdU labelling index) was found among the diploid tumour cells. The activity in mononuclear tetraploid and octoploid cells was reduced in inverse proportion to their increasing DNA content, possibly suggesting a loss of proliferative potential associated with polyploidization. There was a significant correlation between the proliferative activity of hepatocellular carcinoma cells and nonparenchymal liver cells in the different tumours, indicating that different cell types within a tumour may respond to common growth stimuli. Treatment of tumour-bearing rats with a promoting carcinogen (2-acetylaminofluorene) resulted in significant stimulation of tumour cell proliferation (all ploidy classes), whereas the proliferation of non-parenchymal (stromal) cells in the tumour was slightly inhibited.