Adenosine stimulates DNA fragmentation in human thymocytes by Ca(2+)-mediated mechanisms.
Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine-triggered signal transduction cascade and the activation state of protein kinase C in regulating apoptosis of human thymocytes.
Szondy
Z
Z
Department of Biochemistry, University Medical School of Debrecen, Hungary.
D015762:2-Chloroadenosine / Q000633:toxicity; D000241:Adenosine / Q000031:analogs & derivatives / Q000633:toxicity*; D000262:Adenylyl Cyclases / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D017252:CD3 Complex / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002675:Child, Preschool; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000187:drug effects* / Q000378:metabolism*; D004249:DNA Damage; D004789:Enzyme Activation; D006801:Humans; D007223:Infant; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D018655:Lymphocyte Count / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000502:physiology; D013268:Stimulation, Chemical; D016176:T-Lymphocyte Subsets / Q000187:drug effects; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*
D017252:CD3 Complex; D007295:Inositol Phosphates; D058906:Purinergic P1 Receptor Agonists; D011948:Receptors, Antigen, T-Cell; D018047:Receptors, Purinergic P1; D015762:2-Chloroadenosine; D004247:DNA; D000242:Cyclic AMP; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D000241:Adenosine; D002118:Calcium