2'-DEOXYADENOSINE INDUCES APOPTOSIS IN RAT CHROMAFFIN CELLS

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was to xic 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 mu M 2'-dAdo plus 3 mu M deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and te rminal deoxynucleotidyl transferase-mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, a nd DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibi ts adenosine deaminase, 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 mu M dilazep or 1 mu M nitr obenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 mu M uridine or 100 mu M 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 mu M) showed a three- and n inefold, 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 iodotuber cidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP b y nucleoside kinase. It is interesting that nanomolar concentrations o f ITu also completely protected chromaffin cells from 2'-dAdo lethalit y. Our study demonstrates for the first time that mammalian adrenal ch romaffin cells undergo apoptotic cell death by a natural nucleoside an d suggests that this model could be used to study apoptosis and cell f unction.