DIFFERENTIAL-EFFECTS OF 2 STRUCTURALLY RELATED N-6-SUBSTITUTED CAMP ANALOGS ON C6 GLIOMA-CELLS

Membrane permeable derivatives of cAMP are widely used to investigate the role of cAMP in the regulation of cell growth and differentiation. To further investigate the molecular mechanisms, underlying the effec ts of cAMP analogues on growth control and differentiation, the concen tration-dependent action of four structurally related cAMP analogues w ith substitutions at the N6-position in the adenine moiety, namely N6- benzyl-cAMP (Bn-cAMP), N6-benzoyl-cAMP (Bz-cAMP), N6-butyryl-cAMP (Bt- cAMP) and N 6, O2'-cAMP (Bt2-cAMP), on C6 rat glioma cell proliferatio n was determined. The four analogues tested showed different specifici ties, and the order of growth inhibitory potency was: Bn-cAMP much gre ater than Bt-cAMP=Bt2-cAMP much greater than Bz-cAMP. Thus, although b oth derivatives have been described to equally bind and activate cAMP- dependent protein kinase (cAK) isozymes, Bn-cAMP most effectively inhi bited C6 glioma cell proliferation with an IC50 of 25 muM, while Bz-cA MP was almost ineffective in C6 cells (IC50 much greater than 1000 muM ). In vivo and in vitro studies using HPLC analysis, revealed that Bn- cAMP was subject to enzymatic degradation and that the metabolite Bn-a denosine (Bn-Ado) exerted growth inhibitory effects at a concentration even below 10 muM. Additionally, C6 glioma cells morphologically diff erentiated in the presence of Bn-cAMP (100 muM) and of Bn-Ado (10 muM) , by extending long cellular processes. The growth inhibitory activity of Bn-Ado was not influenced, when dipyridamole, an inhibitor of aden osine uptake, was added to the incubation medium, indicating that aden osine action was mediated through a receptor-mediated mechanism. These data presented here show that the fate of externally applied nucleoti des in cell culture systems has to be thoroughly investigated, before conclusions about specific effects of cAMP and its activation of cAKs may be drawn.