APOPTOSIS BY 2-CHLORO-2'-DEOXY-ADENOSINE AND 2-CHLORO-ADENOSINE IN HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS

Adenosine has profound effects on immune cells and has been implicated in the intrathymic apoptotic deletion of T-cells during development. In order to characterize adenosine effects on quiescent peripheral blo od mononuclear cells (PBMC), we have evaluated the ability of the prev iously characterized adenosine receptor agonist 2-chloro-adenosine (2C A; Ceruti, Barbieri et al., 1997) and of the antineoplastic drug 2-chl oro-2'-deoxy-adenosine (2CdA, cladribine) to trigger apoptosis of PBMC . Apoptosis was assessed by morphological changes, DNA fragmentation b y agarose gel electrophoresis and appearance of hypodiploid DNA peak b y flow cytometry. 2CA (10 mu M) and 2CdA (1 mu M) induced apoptosis in human PBMC, which are relatively insensitive to apoptosis. For both a gents, the effect was concentration- and time-dependent, although 2CdA induced apoptosis more potently than 2CA. Evaluation of mitochondrial function in parallel samples using the mitochondrial membrane-potenti al-specific dye JC-1 showed that mitochondrial damage followed the sam e kinetics as apoptosis, hence an early damage of mitochondria is like ly not responsible for adenosine-induced death of PBMC. The effect of 2CA was partially prevented by addition of dipyridamole (DP), a nucleo side transport inhibitor, hence some of the apoptotic effect of this n ucleoside is, at least in part, due to intracellular action. Alternati vely, DP did not affect 2CdA-induced apoptosis, suggesting that 2CdA m ay enter cells via a DP-insensitive transporter. 5-Iodotubercidin (5-I tu), a nucleoside kinase inhibitor, was also able to partially prevent the action of 2CA and was not able to affect 2CdA-induced apoptosis, suggesting a different role for phosphorylation in 2CA- vs 2CdA-induce d apoptosis. To test the role of P1 receptors, agonists and antagonist s selective at various P1 receptor subtypes were used. Data suggest th at, for 2CA, apoptosis is partially sustained by activation of the A,, receptor subtype, whereas no role is exerted by P1 receptors in 2CdA- dependent apoptosis. Moreover, in these cells, apoptosis could also be triggered through intense activation of the A(3) receptor via selecti ve agonists such as o-N-6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA), but this mechanism plays no role in either 2CA- or 2CdA -induced apoptosis. On the whole, our results suggest that 2CA and 2Cd A follow different pathways in inducing apoptosis of immune cells. Mor eover, our data also suggest that there are at least three different w ays by which adenosine derivatives may induce apoptosis of human PBMC: (i) through an A(2A)-like extracellular membrane receptor; (ii) throu gh entry of nucleosides into cells and direct activation of intracellu lar events involved in the apoptotic process; or (iii) through activat ion of the A(3) receptor. (C) 1998 Elsevier Science Ltd. All rights re served.