Stimulation of enzyme synthesis and inhibition of DNA synthesis and growth rate by cyclic AMP derivatives in cultured hepatoma cells

Abstract

The pattern of regulation of the activities of PEP carboxykinase and tyrosine transaminase by glucocorticoids, insulin and dibutyryl cyclic AMP in cultured H35 hepatoma cells has been found to be remarkably similar to that in rat liver. These results coupled with the obvious advantages of cell culture make this an attractive model system for the study of enzyme regulation. The increase in the activity of these two enzymes in rat liver and H35 cells is due to a selective increase in the rate of de novo enzyme synthesis. Hormones which induce the transaminase and carboxykinase in rat liver by stimulating cyclic AMP production are not effective in H35 cells. The adenylate cyclase in these cells appears to possess a functional catalytic component but the normal transduction of the hormonal input signal is aberrant. A variety of experimental approaches all support the conclusion that dibutyryl cyclic AMP stimulates specific enzyme synthesis by facilitating the translation of pre-existing templates. The rapid increase and decay in enzyme activities after addition and removal of dibutyryl cyclic AMP and the resistance to actinomycin D of the early response of both enzymes to dibutyryl cyclic AMP were consistent with this hypothesis. Similarly, preincubation of H35 cells with dibutyryl cyclic AMP and cycloheximide followed by transfer to medium devoid of additions produced little subsequent increase in the activity of either enzymes in contrast to similar experiments with dexamethasone. The pattern of regulation of these two enzymes in two other hepatoma and one normal liver cell culture was found to vary. Thus, glucocorticoid, insulin and dibutyryl cyclic AMP produced the same effects in MH 1C 1 hepatoma cells, except that tyrosine transaminase exhibited little increase after addition of dibutyryl cyclic AMP. In contrast, in HTC and RLC cells only glucocorticoids produced a consistently significant effect and then only on the transaminase. Dibutyryl cyclic AMP produces a rapid, reversible and marked inhibition of the rate of growth of H35 cells. Among other compounds tested only those capable of inducing tyrosine transaminase and PEP carboxykinase had any effect on growth. This effect has been traced to an inhibition of overall DNA synthesis. Although the exact mechanism by which DNA synthesis is inhibited is not known, it was observed that deoxycytidine, uridine, deoxyuridine and low concentrations of thymidine reversed the effects of dibutyryl cyclic AMP. These results suggest that the activity of CDP ribonucleotide reductase might be responsible for the observed inhibition. Dibutyryl cyclic AMP also inhibited the growth rate and DNA synthesis in MH 1C 1 cells which could also be reversed by addition of deoxyribopyrimidines. In contrast, HTC and RLC cells proved to be resistant to the action of the cyclic nucleotide analog.