DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion

Carcinogenesis was initiated in female rat liver by a single dose of N-nitrosomorpholine; subsequently phenobarbital (PB) was administered via the diet at a daily dose of 50 mg/kg body weight for up to 49 weeks. Subgroups of rats were left untreated after 10 or 28 weeks on PB. PB produced the following changes: (a) accelerated appearance of neoplastic nodules and hepatocellular carcinoma (from 28 weeks onwards); (b) phenotypic changes in altered foci such as a shift from clear to eosinophilic appearance, enhanced expression of gamma-glutamyltranspeptidase and other markers, and more distinct borders from surrounding liver; (c) an increase in foci number; and (d) accelerated foci enlargement. The increase in foci number was found to be due to increased phenotypic expression of foci. DNA synthesis was measured by [3H]thymidine labeling at multiple time points. The rate of DNA synthesis was always approximately 10-fold higher in foci than in surrounding liver tissue. Despite this, after N-nitrosomorpholine alone foci grew little before 18-24 weeks. Continuous treatment with PB did not produce a persistent further increase of DNA synthesis in foci, although it accelerated foci growth. Furthermore, at early stages small and larger foci showed similar DNA synthesis activity. These findings indicate that the rate of cell replication as measured by DNA synthesis is not the only determinant of the growth rate of foci. Further studies showed that foci with indistinct borders (reflecting weak expression of the altered phenotype) grew much less than foci with distinct borders; this was at least in part due to an increased rate of cell death by apoptosis found in foci with indistinct borders. In conclusion, besides cell replication, apoptosis and the extent of phenotypic expression (remodeling) determine the growth rate of foci. Foci with weak phenotypic expression predominated after N-nitrosomorpholine alone; in these, a high incidence of apoptosis counterbalanced cell replication. In contrast, during PB treatment foci with strong phenotypic expression predominated; in these, apoptotic activity was lower and the high replicative activity could manifest itself. Finally, all effects of PB on foci were largely although not completely reversible upon cessation of treatment; as a result phenotypic expression declined, and "remodeling" foci with high apoptotic activity predominated again.