Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes

Background Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by three different major cellular responses: downregulation of cell proliferation, induction of cell differentiation and apoptosis. In the skin, the generation of intracellular ceramide may also provide a link between an extracellular signal and the induction of the apoptosis programme for the elimination of damaged cells. Objectives We investigated the effect of ceramides capable of entering cells on cultured keratinocytes. Methods Human keratinocytes from neonatal skin were cultured in serum-free medium with or without increasing concentrations of ceramide 2 (CER-2; N-acetyl sphingosine) (5, 10, 20 and 40 mu mol L-1). Proliferative effects were studied either by cell counts or by H-3-thymidine incorporation and flow cytometric analysis. Apoptosis was studied by TUNEL staining and Western blot analysis of Bcl-2 protein. Results Cell counts and DNA synthesis were reduced in a dose-dependent manner following CER-2 treatment. TUNEL staining showed CER-2-induced apoptosis at 48, 72 and 96 h. Western blot analysis showed that CER-2 induces downregulation of Bcl-2, at 24-96 h. Conclusions These results demonstrate that CER-2 inhibits cell proliferation and induces apoptosis, possibly via a Bcl-2-dependent mechanism.