Apoptosis induced by nitric oxide is associated with nuclear p53 protein expression in cultured osteoarthritic synoviocytes

Objective. Nitric oxide (NO) is a free radical molecule endogenously produc ed by NO synthases that may play a critical role in inflammation. It inhibi ts cell proliferation and may be involved in the induction of apoptosis in various cellular models. Recently, the presence of apoptotic cells was repo rted in the synovium of osteoarthritic (OA) patients. The aim of this study was to determine whether synovial fibroblasts are target cells for NO-indu ced apoptosis. The expression of p53 protein was also studied to evaluate t he ability of synovial cells to repair DNA fragmentation. Methods. Synoviocytes from OA patients were treated with two NO donors: sod ium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Apopto sis was analysed by transmission electron microscopy. DNA content was evalu ated by flow cytometric analysis after propidium iodide staining and recogn ition of DNA strand break determined by the TUNEL (TdT-mediated dUTP nick e nd labeling) method. P53 protein expression was studied by immunofluorescen ce using a monoclonal antibody. Results. After 6 hours, cells treated with NO donors (1.25 mM) showed a cyt oplasmic condensation and vacuolization. DNA strand break analysis by the T UNEL method confirmed the presence of a DNA fragmentation after 24 hours of NO treatment. There was also a progressive decrease in the DNA diploid pea k in response to NO donors. In parallel, p53 protein, constitutively expres sed in cytoplasmic synovial cells, showed markedly increased expression aft er a 6-hour NO exposure and displayed prominent nuclear staining after 12 h ours. Conclusions. This study demonstrates the potential role of NO for the induc tion of synoviocyte apoptosis in OA. The increased expression of p53 in the nucleus may play a protective role in the control of apoptosis.