Intracellular phosphorylation of benzyladenosine is related to apoptosis induction in tobacco BY-2 cells

Treatment of tobacco BY-2 cells with micromolar concentration of benzyladenosine ([9R]BA) resulted in the loss of cell viability in a time- and concentration-dependent manner. Cell death induced by [9R]BA exhibited typical apoptotic hallmarks including cell shrinkage, chromatin condensation and degradation of nuclear DNA to characteristic high molecular weight (HMW) as well as nucleosomal size fragments. Externally added [9R]BA was very rapidly and almost quantitatively phosphorylated within BY-2 cells. Accumulation of [9R]BA-monophosphate was accompanied by massive production of endogenous reactive oxygen species (ROS), intracellular ATP depletion, and these events were followed by the loss of cell viability. Inhibition of intracellular phosphorylation of [9R]BA by adenosin kinase inhibitor, 5'-amino-5'-deoxyadenosine (AdAs), diminished ROS production, ATP depletion, and consequently prevented cells from death. Selective inhibition of ROS production without restoring ATP production, however, did not provide any protection to cells. In contrast, even enhanced phosphorylation of [9R]BA caused by adenosine that simultaneously revived ATP synthesis reduced the number of dying cells. This is the first evidence of a direct relationship between intracellular phosphorylation of [9R]BA and apoptosis induction in BY-2 cells. ATP depletion but not ROS production is the key secondary event that determines the cellular decision between life and death.