['Ceramide is an important messenger of TNF- and lipid-induced apoptosis. We previously demonstrated the adverse effect of TNF in the process of reendothelialization as well as the dependence of its effect on cell-cycle regulation. The current study was designed to investigate the linkage between ceramide induced toxicity and growth arrest in human endothelial cells.', 'Cultured human arterial endothelial cells (HAEC) served as an in-vitro model to test the cellular effects of C2-ceramide (C2). C2-induced cell death in HAECs occurred time- and dose-dependently. The LD(50) in subconfluent cells was three times lower than in confluent cell layers (25 vs. 75 microM). C2 caused up to 70% inhibition of BrdU and [3H]thymidine incorporation at non-toxic concentrations as a result of G1 cell-cycle arrest. Downregulation of cyclin A and p21(Cip1/Waf1) protein expression was observed independently of C2-toxicity, while expression of other cell-cycle regulatory genes was not affected. Inhibition of cyclin A protein expression by sequence-specific antisense-oligonucleotides was paralleled by significant growth-inhibition. The protein phosphatase inhibitor okadaic acid induced endothelial cell proliferation, which was completely abrogated by C2. In contrast, aphidicolin-synchronized endothelial cells demonstrated elevated cyclin A levels along with 30% higher BrdU-incorporation and 70% less C2-toxicity. G1-arrested cells, however, showed significantly enhanced C2-toxicity, lack of cyclin A expression and induction of uncleaved caspase-3 (CPP32).'] Are loss of cyclin A and G1-cell cycle arrest a prerequisite of ceramide-induced toxicity in human arterial endothelial cells?