Cyclic AMP-Dependent Protein Kinase Activation and the Induction of Ornithine Decarboxylase during Lymphocyte Mitogenesis

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Abstract
Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase (ODC) were studied along with RNA and DNA synthesis in human peripheral blood lymphocytes following in vitro culture with either mitogens, nonmitogenic agglutinins, or dibutyryl cyclic AMP (DBcAMP). The mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) all resulted in activation of soluble cyclic AMP-dependent protein kinase, the induction of increased levels of ODC activity, and increases in the rate of incorporation of (3H)-thymidine (DNA synthesis) and (3H)-uridine (RNA synthesis) into acid-insoluble material. High concentrations of PHA or Con A, which resulted in maximal cyclic AMP-dependent protein kinase activation, were only slightly mitogenic. The time at which maximum activation of protein kinase occurred differed for each mitogen but, in each case, always preceded ODC induction and increased RNA and DNA synthesis. In Con A-stimulated cultures, cyclic AMP-dependent protein kinase activation was maximal at 4 hr of culture and remained elevated at 24 and 48 hr. In these cultures increased ODC activity was first observed at 6 to 8 hr of culture and preceded increased RNA synthesis which was detectable at 12 hr of culture. DNA synthesis in all mitogen-stimulated cultures was first observed at 48 hr with maximum (3H)-thymidine incorporation being at 72 hr of culture. Cyclic AMP-dependent protein kinase activation, induction of ODC, and increased RNA and DNA synthesis were all shown to be dependent upon lymphocyte-Con A binding and were blocked by early addition of α-methyl mannoside. After 12 hr of culture, α-methyl mannoside addition had little effect on Con A induction of ODC or on RNA and DNA synthesis measured at 24 to 72 hr. In contrast, cyclic AMP-dependent protein kinase activation, measured at 24 hr, could be completely inhibited when α-methyl mannoside was added as late as 12 or 20 hr to Con A-stimulated cultures. The nonmitogenic agglutinins, wheat germ agglutinin (WGA) and Agaricus bisporus lectin (ABL), failed to increase any of the parameters tested. DBcAMP also did not induce ODC or cause any increased RNA or DNA synthesis. However, DBcAMP (10-3 M) did cause activation of cyclic AMP-dependent protein kinase as early as 15 min of culture. DBcAMP activation of protein kinase was concentration-dependent, maximal at 12 hr, and remained elevated through 48 hr of culture. The addition of DBcAMP (10-3 M) to Con A-stimulated cultures caused a 90 to 95% inhibition of ODC activity and RNA and DNA synthesis. This inhibition was concentration-dependent and paralleled the degree of activation of cyclic AMP-dependent protein kinase elicited by each concentration. These results suggest that the selective activation of cyclic AMP-dependent protein kinase and induction of increased ODC activity are early integral events linked to and regulating lymphocyte mitogenesis.