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.

中文翻译：

---

人外周血单核细胞中 2-氯-2'-脱氧腺苷和 2-氯-腺苷引起的细胞凋亡。


腺苷对免疫细胞具有深远的影响，并与发育过程中 T 细胞的胸腺内凋亡缺失有关。为了表征腺苷对静止外周血单核细胞 (PBMC) 的作用，我们评估了先前表征的腺苷受体激动剂 2-氯-腺苷 (2CA; Ceruti, Barbieri 等人，1997) 和抗肿瘤药物的能力2-氯-2'-脱氧腺苷（2CdA，克拉屈滨）触发 PBMC 凋亡。通过形态学变化、通过琼脂糖凝胶电泳的DNA片段化和通过流式细胞术的亚二倍体DNA峰的出现来评估细胞凋亡。 2CA (10 microM) 和 2CdA (1 microM) 诱导人 PBMC 凋亡，而 PBMC 对细胞凋亡相对不敏感。对于这两种药物，其效果均依赖于浓度和时间，尽管 2CdA 比 2CA 更有效地诱导细胞凋亡。使用线粒体膜电位特异性染料 JC-1 对平行样品中的线粒体功能进行评估表明，线粒体损伤遵循与细胞凋亡相同的动力学，因此线粒体的早期损伤可能不是腺苷诱导的 PBMC 死亡的原因。添加双嘧达莫 (DP)（一种核苷转运抑制剂）可部分阻止 2CA 的作用，因此该核苷的部分凋亡作用至少部分是由于细胞内作用所致。或者，DP 不影响 2CdA 诱导的细胞凋亡，表明 2CdA 可能通过 DP 不敏感转运蛋白进入细胞。 5-碘结核菌素 (5-Itu) 是一种核苷激酶抑制剂，也能够部分阻止 2CA 的作用，但不能影响 2CdA 诱导的细胞凋亡，这表明磷酸化在 2CA 与 2CdA 诱导的细胞凋亡中的作用不同。 为了测试 P1 受体的作用，使用了针对各种 P1 受体亚型的选择性激动剂和拮抗剂。数据表明，对于 2CA，细胞凋亡部分是通过 A2A 受体亚型的激活来维持的，而 P1 受体在 2CdA 依赖性细胞凋亡中不起任何作用。此外，在这些细胞中，通过选择性激动剂如 2-氯-N6-(3-碘苄基)腺苷-5'-N-甲基脲酰胺 (Cl-IB-MECA) 强烈激活 A3 受体也可以引发细胞凋亡。但这种机制在 2CA 或 2CdA 诱导的细胞凋亡中不起作用。总的来说，我们的结果表明2CA和2CdA在诱导免疫细胞凋亡方面遵循不同的途径。此外，我们的数据还表明，腺苷衍生物至少可以通过三种不同的方式诱导人PBMC凋亡：（i）通过A2A样细胞外膜受体； (ii) 通过核苷进入细胞并直接激活细胞凋亡过程中涉及的细胞内事件；或 (iii) 通过激活 A3 受体。