Cycloheximide blocks TGF-β1-induced apoptosis in murine hepatocytes

AIM: To study the mechanism of transforming growth factor beta (1)-induced apoptosis in cultured hepatocytes. METHODS: DNA fragmentation and fluorescent microscopy were used to characterize cell apoptosis. Crystal violet staining was used to assess cell viability. Immunoblotting was used to detect Tak1, p53, and Bax. Dual luciferase assay was used to determine TGF-beta (1)-induced gene expression. Thin layer chromatography was used to examine ceramide level in AML12 cells. RESULTS: In response to TGF-beta (1) treatment, AML12 cells exhibited typical changes, which was characteristic of apoptosis, such as condensation of chromatin, disintegration of nuclei, and DNA fragmentation. TGF-beta (1)-induced apoptosis in AML12 cells was completely blocked in the presence of cycloheximide. The inhibitory effect of cycloheximide was accompanied with down-regulation of Tak1 expression and TGF-beta (1)-induced PAI-1 expression. TGF-beta (1) induced p53 expression but not Bax. No increase of ceramide was obeserved in TGF-beta (1)-induced apoptosis. CONCLUSION: TGF-beta (1)-induced apoptosis requires TGF-beta (1)-induced gene expression.