8-Chloroadenosine Mediates 8-Chloro-Cyclic AMP-induced Down-Regulation of Cyclic AMP-dependent Protein Kinase in Normal and Neoplastic Mouse Lung Epithelial Cells by a Cyclic AMP-independent Mechanism

Abstract

The 8-chloro analogue of the regulatory molecule, cyclic AMP (cAMP), modulates the intracellular concentrations of cAMP-dependent protein kinases (PKA) and inhibits both in vitro and in vivo growth of several neoplastic cell types. Because 8-chloro-cyclic AMP (8-Cl-cAMP) can be converted to 8-chloroadenosine (8-Cl-adenosine) by serum enzymes contained in cell growth media, we tested whether 8-Cl-cAMP effects were mediated by its adenosine metabolite in normal and neoplastic cell lines of mouse lung epithelial origin. 8-Cl-adenosine, directly added to cells or derived from exogenously applied 8-Cl-cAMP, specifically decreased the intracellular concentration of the type I isozyme of cAMP-dependent protein kinase (PKA I). 8-Cl-adenosine and 8-Cl-cAMP were equipotent at inhibiting cell growth, and elicited similar changes in the proportion of cells in the G₁, S, and G₂-M phases of the cell cycle. The presence of adenosine deaminase, which converts 8-Cl-adenosine to 8-chloroinosine, completely prevented growth inhibition by 8-Cl-cAMP and the concomitant diminution of PKA I. 8-Cl-cAMP had no discernible effect on cells when its conversion into 8-Cl-adenosine was prevented by 3-isobutyl-1-methyl-xanthene, an inhibitor of phosphodiesterase, 6-(p-Nitrobenzyl)-thioinosine, an inhibitor of adenosine uptake, protected cells from cytostasis, indicating that 8-Cl-adenosine acts intracellularly. 8-Cl-adenosine greatly decreased RI (regulatory subunit of PKA I) and PKA catalytic (C) subunit protein concentrations without affecting RII (regulatory subunit of the PKA type II isozyme) or intracellular cAMP levels. Northern blot analysis of PKA subunit mRNAs following treatment of each cell line with 8-Cl-adenosine demonstrated decreased Cα mRNA expression, increased RIIα mRNA, and no change in RIα mRNA abundance. Our results indicate that 8-Cl-adenosine inhibits lung cell growth and induces PKA I down-regulation via a cAMP-independent mechanism.