PROTECTION BY ASCORBATE AGAINST APOPTOSIS OF THYMOCYTES - IMPLICATIONS OF ASCORBATE-INDUCED NONLETHAL OXIDATIVE STRESS AND POLY(ADP-RIBOSYL)ATION

Apoptosis can be triggered in thymocytes with stimuli (6 alpha-methylp rednisolone, thapsigargin, and etoposide) acting by different mechanis ms. In each of these instances cell death is extensively prevented unt il 5 h of incubation when cells are preincubated with 250 mu M ascorbi c acid (AA) for 1 h, then washed, and incubated in fresh medium contai ning the above mentioned apoptotic stimuli, In addition, the degree of spontaneous apoptosis of untreated thymocytes is somewhat lower in th e AA-preincubated cells. The protection against apoptosis does not see m to be dependent on the intracellular enrichment of AA, as measured a t the end of the preincubation period. On the contrary, such a protect ion is strictly related to a partial loss of ascorbate in the medium ( possibly due to its autooxidation), is catalase-inhibitable, and is re produced by a preincubation of the cells with nontoxic concentrations of hydrogen peroxide. The AA-supplemented cells show a remarkable decr ease in NAD(+) levels and a significant increase of poly(ADP-ribose) p olymerase (PARP) activity, Consistently with these results, the additi on of PARP inhibitors, such as thymidine and S-aminobenzamide, during the preincubation with AA, prevents NAD(+) depletion and abolishes the protective effect of AA against apoptosis, The possibility is discuss ed that an early activation of PARP by stimuli which are nontoxic per se makes the cells able to withstand subsequent apoptotic stimuli whic h are otherwise lethal. (C) 1996 Academic Press, Inc.