Wesley D. Wicks

Evidence for translational regulation of specific enzyme synthesis by N6, O2′-dibutyryl cyclic AMP in hepatoma cell cultures☆

Abstract N 6 ,O 2′ -dibutyryl cyclic 3′:5′ AMP (dibutyryl cAMP) produces a rapid increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK) in Reuber H35 hepatoma cell cultures. This effect is shown to result from a stimulation of enzyme synthesis. Low concentrations of actinomycin D completely block the response of PEPCK to dexamethasone but not that at early intervals after addition of dibutyryl cAMP. These results suggest that dibutyryl cAMP affects a post-transcriptional step in PEPCK synthesis. Superinduction of tyrosine transaminase after addition of high concentrations of actinomycin D is observed with both dexamethasone and dibutyryl cAMP; PEPCK does not exhibit this phenomenon with either inducer.

Induction of phosphoenolpyruvate carboxykinase by N6,O2′-dibutyryl cyclic amp in rat liver

Abstract 1. 1. Treatment of intact rats with N 6 , O 2′ -dibutyryl cyclic AMP prompts a rapid increase in the activities of hepatic PEP carboxykinase and tyrosine transaminase and an equally rapid loss of liver glycogen. The increase in PEP carboxykinase activity is shown to result from an increase in the rate of de novo synthesis by radioimmunological techniques. 2. 2. Induction of PEP carboxykinase by dibutyryl cyclic AMP is not associated with any detectable alteration in the properties of the enzyme. The affinity for substrate, nucleotide cofactors or metal ions, the stability to thermal denaturation, the relative activity at various pH values and the immunological reactivity of the enzyme were not noticeablychanged as a consequence of induction. It is concluded that the effect of dibutyryl cyclic AMP on the activity of this enzyme most likely results solely from an increase in the rate of synthesis of the basal enzyme species.

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 MH1C1 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 MH1C1 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.

Glucocorticoid requirement for tyrosine aminotransferase induction by adenosine 3′:5′-cyclic phosphate analogs in somatic cell hybrids☆

Abstract The ability of adenosine 3′:5′-cyclic phosphate (cyclic AMP) analogs to induce l -tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) in a rat hepatoma (H35)-rat liver cell (BRL) hybrid (BF5) and a subclone which has lost 29 chromosomes (BF5-1-1) has been analyzed. Cyclic AMP analogs alone were unable to increase TAT activity in either hybrid cell line or in the “normal” liver cells despite three- to fivefold induction of this enzyme in the hepatoma parental cells. In contrast, dexamethasone by itself reproducibly increased TAT activity both in BF5-1-1 cells and in the parental H35 hepatoma cells. Pretreatment of the hybrid cells with dexamethasone revealed a synergistic increase in TAT activity when a cyclic AMP analog was added. From studies of the thermal stability and immunological inhibition of TAT activity, it is concluded that the low basal activity in BRL, BF5, and BF5-1-1 cells represents tyrosine transamination catalyzed by a different aminotransferase, whereas all the induced activity does represent bona fide TAT. The results suggest that functional TAT mRNA may not be present in significant quantities in the hybrid cells in the absence of adrenal steroids and that this could account for the inability of cyclic AMP analogs to exert their presumably translational effect on TAT synthesis.