G1 arrest and Rb dephosphorilation is induced by ceramide 2(N-acetyl sphingosine) in cultured human keratinocytes

In the skin, the generation of intracellular ceramide may provide a link between an extracellular signal and the induction of apoptosis for the elimination of damaged cells.Previous studies have shown that cell permeant ceramides are involved in signal transduction, cell cycle regulation and apoptosis in different cell types. The aim of the present study was to investigate the intracellular apoptotic signal induced by permeant ceramides on cultured normal human keratinocytes (NHK). NHK from neonatal skin were cultured in serum-free medium with or without Ceramide 2 (CER-2, n-acelyl-sphingosine) 20 M, 40 M. Cell cycle was investigated by FACS analysis, and western blot analysis of cyclin D1 and Rb dephosphorilation. Apoptosis was studied by TUNEL staining and western blot analysis of BCL-2 protein, p53 and p21. FACS analysis demonstrated a G1 arrest 48 h after ceramide addition, accompanied by down-regulation of cyclin D1 and by Rb dephosphorilation. TUNEL staining showed the presence of CER-2-induced apoptosis after 48, 72 and 96 h of culture. Western blot analysis demonstrated that CER-2 induces a down-regulation of BCL-2 after 24 h up to 36 h, and an up-regulation of p53 after 24 h. No modifications were noted in p21 levels. These results demonstrate that in NHK CER-2 cell permeant ceramide induces cell cycle arrest in G1, which precedes apoptosis.