Adenosine stimulates DNA fragmentation in human thymocytes by Ca2+-mediated mechanisms

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Abstract

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/107 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 ≤ cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 μM) 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 Ca2+-dependent DNA fragmentation and cell killing in human thymocytes 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.

Original languageEnglish
Pages (from-to)877-885
Number of pages9
JournalBiochemical Journal
Volume304
Issue number3
DOIs
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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