Hepatocyte DNA Replication Research Articles

Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S‐phase: With a note on the use of nucleolar and extranucleolar [3H]‐thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication

Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1- to S-phase, the duration of G2- and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1- to S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.

Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities.

Dexamethasone inhibited the basal and EGF-stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid-specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU-38486 in a concentration-dependent manner. Dexamethasone acted by decreasing the rate of entry into S-phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half-maximal effect occurring 11 hr after the addition of dexamethasone. Densely populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densely populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.

The kinetics of DNA alkylation, repair and replication in hepatocytes, Kupffer cells, and sinusoidal endothelial cells in rat liver during continuous exposure to 1,2-dimethylhydrazine.

We have previously shown that when rats are continually exposed to 1,2-dimethylhydrazine (SDMH) in a manner that induces malignant hemangioendotheliomas of the liver there was both accumulation of promutagenic DNA damage, as measured by O6-methylguanine (O6MG) and a selective increase in cell replication in the target cell population, the nonparenchymal cells (NPC). Hepatocytes rapidly repaired O6MG and had only modest increases in cell replication. In this study, we have further separated the NPC into Kupffer cells (KC) and sinusoidal endothelial cells (SEC) and have determined the kinetics of O6MG and 7-methylguanine (7MG) formation and repair, and cell replication in each individual cell type. O6MG concentrations were highest over the entire exposure period in the SEC, lowest in the hepatocytes, with intermediate concentrations occurring in KC. 7MG concentrations were lower in SEC than in KC or hepatocytes resulting in greatly increased O6/7MG ratios in SEC suggesting a lower capacity for SDMH metabolism and O6MG repair. Moreover, the concentrations of 7MG and the pattern of accumulation were sufficiently similar in KC and hepatocytes to suggest similar capacities of these two cell types to activate SDMH. Rates of cell replication were greatly elevated in both KC and SEC by SDMH, while hepatocyte replication was barely affected. These data suggest that SEC and to a lesser extent KC have greater potential for initiation due to mispairing of O6MG than do hepatocytes. The data further suggest that alkylation in SEC may arise from metabolites generated by neighboring hepatocytes or KC.