Effect of inhibition of poly (AOP-ribose) synthetase on peroxynitrrte-induced thymocyte apoptosis

Introduction: Negative selection of thymocytes, i.e. elimination of potentially autoreactive T cells in the thymus, plays a crucial role in the formation of the T cell repertoire and in maintaining autotolerance. The mechanisms by which immature thymocytes are induced 10 undergo apoptosis during negative selection are poorly defined. Prior reports demonstrated that crosslinking of T cell receptor in vivo leads to siromal cell activation, expression of the inducible nitric oxide synthase (iNOS) and. subsequently, to ihymocyte apoptosis. Here we examined, therefore, whether NO directly, or indirectly through the formation of peroxynitrite, may be responsible for ihymocyte apoptosis. Furthermore, since we has demonstrated the role of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) in peroxynitrite-relaied cellular injury, we also investigated the effect of PARS inhibition on thymocyte apoptosis. Methods and résolu : To study the effect of peroxynitrite on thymocytes, thymocytes were isolated and exposed to peroxynitrite in vitro. Apoptotic and necrobc cell death were characterized by flow cytometnc analysis. Peroxynitrite treatment (20μM) resulted in an increase of the number of apoptotic cells. Higher concentrations (lOOuM), however, caused a marked necrosis. Treatment with 3-aminobenzarrude (3-AB), an inhibitor of PARS produced a dramatic decrease in the number of necrotk cells, with a concomitant increase of apoptotic cells. PARS deficient thymocytes behaved similarly to the 3-AB treated wild type cells. 3-AB had no effect on the apoptosis of PARS deficient thymocytes. During apoptosis DNA is cleaved by endonucleases into oligonucleosomal fragments, the detection of which in the cytoplasmic fraction of cell lysates is commonly used to verify apoptotic cell death. In line with the flow cytometnc data, agarose gel electrophoresis of cellular DNA showed that 20 uM peroxynitrile, but not higher concentrations of the oxidant, induced DNA fragmentation. At higher concentrations of the oxidant, a marked inhibition of the apoptotic process was found, which was reversed by 3-aminobenzamide. PARS-deficient thymocytes exposed to peroxynitrite demonstrated a dose-dependent increase of DNA fragmentation, similar to the results of the wild-type cells treated with 3-aminobenzamide. We also investigated whether PARS is activated in thymocytes in response to peroxyniirite treatment and whether PARS activation-related energy depletion. A significant decrease in the intracellular ATP level was found Ih after peroxynitrite pretreatment of thymocytes, which was prevented by inhibition of PARS. Conclusions: Based on the present results, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. We propose that the physiological role of PARS cleavage during apoptosis may serve as an energy conserving step, enabling (he cell to complete the process of apoptosis.