8-Chloroadenosine 3′,5′-monophosphate (8-Cl-cAMP) selectively eliminates protein kinase A type I to induce growth inhibition in c-ras-transformed fibroblasts

Abstract 8-Chloroadenosine 3′,5′-monophosphate (8-Cl-cAMP), a site-selective cyclic adenosine 3′,5′-monophosphate (cAMP) analogue, exhibits growth inhibition in a broad spectrum of cancer cell lines. We investigated the effect of 8-Cl-cAMP on c- ras -transformed mouse fibroblasts (MP3/3T3) which were established by transfection of Balb3T3 cells (Balb3T3) with the point-mutated c- ras gene [G12V12]. 8-Cl-cAMP (2–5 μM) exerted over 80% growth inhibition by day 4 on MP3/3T3, while inhibiting parental Balb3T3 cell growth less than 40%. In order to distinguish the effect of 8-Cl-cAMP from that of 8-chloroadenosine (8-Cl-adenosine), we examined the effect of 8-Cl-cAMP in serum-free medium. 8-Cl-cAMP demonstrated a potent growth inhibition of MP3/3T3 cells cultured in serum-free medium, suggesting that the growth inhibitory effect of 8-Cl-cAMP was not due to its hydrolysed product, 8-Cl-adenosine. In addition, both Balb3T3 and MP3/3T3 contained cAMP phosphodiesterases mainly composed of isozyme IV which has previously been reported to be insensitive towards the hydrolysis of 8-Cl-cAMP. Non-transformed Balb3T3 cells contained only type II cAMP-dependent protein kinase (PKA), whereas transformed MP3/3T3 exhibited a marked increase in type I PKA. The growth inhibition of MP3/3T3 by 8-Cl-cAMP accompanied almost complete elimination of type I PKA without affecting type II PKA. Moreover, 8-Cl-cAMP induced an arrest in the G 0 /G 1 -phase of the cell cycle in MP3/3T3. 8-Cl-adenosine had little or no effect on the cell cycle kinetics of MP3/3T3 cells. These results show that 8-Cl-cAMP is a novel cAMP analogue which selectively eliminates type I PKA to induce growth inhibition in transformed fibroblasts.