8-Chloro-cAMP Serves as a Prodrug for the RNA Directed Nucleoside Analog, 8-Chloro-Adenosine

Previous preclinical and clinical work with 8chloro-cAMP (8-Cl-cAMP) has raised questions whether it works as a cAMP analog or as a nucleoside analog, 8-Cl-Adenosine (Ado). Although degradation of 8-Cl-cAMP to 8-ClAdo in culture medium or plasma has been shown, cellular pharmacology is missing. Additionally, conversion of 8-Cl-Ado to 8-ClcAMP in the cell and its effects via a Protein Kinase A (PKA) dependent pathway is possible. However, there was no measurable increase in PKA activity by 8-Cl-Ado. The phorbol ester PMA, which blocks both glucocorticoid and cAMP mediated cell death, was not able to block 8-Cl-Ado mediated apoptosis. These data suggested that the mechanism of action of 8-Cl-Ado is via a separate pathway than either glucocorticoid or PKA signaling. In multiple myeloma (MM) and leukemia cells, both 8-Cl-cAMP and 8-Cl-Ado incubation resulted in the accumulation of 8Cl-Ado mono-, di-, and tri-phosphate (8-ClATP). Accumulation of 8-Cl-ATP was dependent on both the exogenous concentration of 8-Cl-Ado and incubation time. At the 10 μM level of 8-Cl-Ado, more than 400 pM 8-ClATP accumulated in MM cells after 12 hours. Similar incubation with 8-Cl-cAMP also resulted in accumulation of 8-Cl-ATP, albeit at a lower level. The formation of 8-Cl-ATP from 8-Cl-cAMP was inhibited by more that 80% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in the medium, suggesting extracellular conversion of 8-Cl-cAMP to 8-Cl-Ado. Parallel to accumulation of 8-Cl-ATP, there was a decline in the endogenous level of cellular ATP pool. This was associated with inhibition of RNA synthesis but did not affect DNA synthesis. Among the RNA species, there was maximum inhibition of mRNA, which was consistent with highest incorporation of 8-Cl-Ado resi­dues. Taken together, these data demonstrate that 8-Cl-cAMP serves as a prodrug for 8-Cl­Ado. Because 8-Cl-Ado induces apoptosis in MM and leukemia cells resistant to traditio­nal chemotherapeutic drugs, this analog may be a potential new agent for these diseases.