2'-Deoxyadenosine Induces Apoptosis in Rat Chromaffin Cells

Abstract: We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3–4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 µ M 2'-dAdo plus 3 µ M deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 µ M dilazep or 1 µ M nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 µ M uridine or 100 µ M 2'-deoxycytidine), or 5 m M nicotinamide. Cells incubated with 2'-dAdo (100 and 300 µ M ) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.