Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological change s, and increases in caspase-like activities. In this study, we have found t hat intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cell s and that this decrease is followed by the ricin-mediated protein synthesi s inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the de pletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3- ABA is due to the inhibition of PARP and does not result from its other pha rmacological side effects. Moreover, the enzymatic activity of PARP gradual ly increases and reaches a maximum level after ricin exposure for 3 h, wher eas no significant change in activity was observed in untreated cells, Howe ver, 3-ABA has no effect on ricin-mediated DNA fragmentation. In. addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum l evel within 6 h of incubation. Thus, in the case of ricin-induced apoptosis , it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis, Our results suggest that multiple apoptotic si gnaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independ ent of the pathway leading to DNA fragmentation in which caspases may be pr ofoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA frag mentation and cytolysis, even at concentrations that cause significant inhi bition of protein synthesis, Thus, a specific ricin action mechanism throug h which ribosomes are inactivated may be responsible for the apoptotic even ts induced by ricin.