Apoptosis by 2-chloro-2′-deoxy-adenosine and 2-chloro-adenosine in human peripheral blood mononuclear cells

Abstract

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 blood mononuclear cells (PBMC), we have evaluated the ability of the previously characterized adenosine receptor agonist 2-chloro-adenosine (2CA; Ceruti, Barbieri et al., 1997) and of the antineoplastic drug 2-chloro-2'-deoxy-adenosine (2CdA, cladribine) to trigger apoptosis of PBMC. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and appearance of hypodiploid DNA peak by flow cytometry. 2CA (10 microM) and 2CdA (1 microM) induced apoptosis in human PBMC, which are relatively insensitive to apoptosis. For both agents, 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-potential-specific dye JC-1 showed that mitochondrial damage followed the same kinetics as apoptosis, hence an early damage of mitochondria is likely not responsible for adenosine-induced death of PBMC. The effect of 2CA was partially prevented by addition of dipyridamole (DP), a nucleoside transport inhibitor, hence some of the apoptotic effect of this nucleoside is, at least in part, due to intracellular action. Alternatively, DP did not affect 2CdA-induced apoptosis, suggesting that 2CdA may enter cells via a DP-insensitive transporter. 5-Iodotubercidin (5-Itu), 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-induced apoptosis. To test the role of P1 receptors, agonists and antagonists selective at various P1 receptor subtypes were used. Data suggest that, for 2CA, apoptosis is partially sustained by activation of the A2A 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 A3 receptor via selective agonists such as 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), but this mechanism plays no role in either 2CA- or 2CdA-induced apoptosis. On the whole, our results suggest that 2CA and 2CdA follow different pathways in inducing apoptosis of immune cells. Moreover, our data also suggest that there are at least three different ways by which adenosine derivatives may induce apoptosis of human PBMC: (i) through an A2A-like extracellular membrane receptor; (ii) through entry of nucleosides into cells and direct activation of intracellular events involved in the apoptotic process; or (iii) through activation of the A3 receptor.

A3Adenosine Receptor Activation Triggers Phosphorylation of Protein Kinase B and Protects Rat Basophilic Leukemia 2H3 Mast Cells from Apoptosis

“…In human mononuclear cells, apoptosis is stimulated by adenosine analogs through activation of A 2A or A 3 receptors or by non-receptor-mediated effects of intracellular nucleosides (Barbieri et al, 1998). In cultured newborn rat cardiomyocytes, high concentrations of IB-MECA trigger apoptosis Shneyvays et al, 1998).…”

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

“…Adenosine receptors (ARs) are also found on cells of the immune system and have been recognized to play an important role in inflammatory conditions [4][5][6][7], in which adenosine can act as an antiinflammatory and immunosuppressive mediator mainly via the activation of A 2A receptors. It inhibits TNF-α production and has been implicated as a physiological signal in the apoptotic deletion of T cells during intrathymic cell selection, a process which functions to prevent autoimmunity [8][9][10][11].…”

“…In contrast to these findings, adenosine and various related AR agonists as well as antagonists can trigger apoptosis by receptor-independent mechanisms that require the entry of the compounds into the cells. A prominent representative is 2-chloro-2′-deoxyadenosine (cladribine) which is applied as a chemotherapeutic anticancer agent for the treatment of chronic lymphoid malignancies [10,33,34]. Further examples include 2-chloroadenosine (nonselective AR agonist) [10,33], 1,3-dipropyl-8-cyclopentylxanthine (A 1 AR antagonist) [35], and 2-chloro-N 6 -(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (A 3 AR agonist) [36].…”

Expression of A3Adenosine Receptors in Human Lymphocytes: Up-Regulation in T Cell Activation

“…In contrast, its stimulation induces antiinflammatory effects by inhibiting pro-inflammatory cytokine release, formyl-Met-Leu-Phe-triggered respiratory burst, and tissue factor expression in monocytic cells (Sajjadi et al, 1996;Broussas et al, 1999Broussas et al, , 2002. Moreover, antiinflammatory effects are evoked by reduction of superoxide anion generation and degranulation in eosinophils and neutrophils (Bouma et al, 1997;Ezeamuzie and Philips, 1999;Gessi et al, 2002) and by induction of apoptosis in blood mononuclear cells (Barbieri et al, 1998). However, in the case of human lymphocytes, novel roles for the A 3 receptor must be considered with caution until the ligand binding profile of this subtype has been properly defined.…”