Loss of cyclin A and G1-cell cycle arrest are a prerequisite of ceramide-induced toxicity in human arterial endothelial cells.

BACKGROUND: 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. METHODS AND RESULTS: 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). CONCLUSIONS: Ceramide abrogates endothelial cell proliferation independently of apoptosis or necrosis at low concentrations (