thioinosine and 2--deoxyadenosine

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; Thioinosine

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Apoptosis; Chromaffin Cells; Deoxyadenosines; Dilazep; Enzyme Inhibitors; Epinephrine; Mutagens; Norepinephrine; Phosphorylation; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Thioinosine; Tubercidin; Vasodilator Agents