ADENOSINE STIMULATES DNA FRAGMENTATION IN HUMAN THYMOCYTES BY CA2-MEDIATED MECHANISMS()

Incubation of human thymocytes with an optimum concentration of adenos ine and its receptor site agonist, 2-chloroadenosine, induced increase s 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 internucl eosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being oxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adeno sine less than or equal to cyclopentylad enosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum co ncentration of 40 mu M) 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 positi ve immature thymocyte population. DNA fragmentation was prevented by t he addition of actinomycin D or cycloheximide to the thymocyte suspens ion, indicating that this process required both mRNA and protein synth esis. Endonuclease activation and cell killing were dependent on an ea rly, 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 intracel lular 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. Phor bol dibutyrate, a protein kinase C activator, previously shown to inhi bit Ca2+-dependent DNA fragmentation and cell killing in human thymocy tes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264 , 13399-13402], at 60 ng/ml concentration also prevented adenosine-ind uced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine-triggered signal transductio n cascade and the activation state of protein kinase C in regulating a poptosis of human thymocytes.