Differential effects of two structurally related N6-substituted cAMP analogues 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 effects of cAMP analogues on growth control and differentiation, the concentration-dependent action of four structurally related cAMP analogues with 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 N6, O2'-cAMP (Bt2-cAMP), on C6 rat glioma cell proliferation was determined. The four analogues tested showed different specificities, and the order of growth inhibitory potency was: Bn-cAMP >> Bt-cAMP = Bt2-cAMP >> Bz-cAMP. Thus, although both derivatives have been described to equally bind and activate cAMP-dependent protein kinase (cAK) isozymes, Bn-cAMP most effectively inhibited C6 glioma cell proliferation with an IC50 of 25 microM, while Bz-cAMP was almost ineffective in C6 cells (IC50 >> 1000 microM). In vivo and in vitro studies using HPLC analysis, revealed that Bn-cAMP was subject to enzymatic degradation and that the metabolite Bn-adenosine (Bn-Ado) exerted growth inhibitory effects at a concentration even below 10 microM. Additionally, C6 glioma cells morphologically differentiated in the presence of Bn-cAMP (100 microM) and of Bn-Ado (10 microM), by extending long cellular processes. The growth inhibitory activity of Bn-Ado was not influenced, when dipyridamole, an inhibitor of adenosine uptake, was added to the incubation medium, indicating that adenosine action was mediated through a receptor-mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)