Cycloheximide blocks TGF-beta1-induced apoptosis in murine hepatocytes.
OBJECTIVE
To study the mechanism of transforming growth factor beta1-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-beta1-induced gene expression. Thin layer chromatography was used to examine ceramide level in AML12 cells.


RESULTS
In response to TGF-beta1 treatment, AML12 cells exhibited typical chang es, which was characteristic of apoptosis, such as condensation of chromatin, disintegration of nuclei, and DNA fragmentation. TGF-beta1-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-beta1-induced PAI-1 expression. TGF-beta1 induced p53 expression but not Bax. No increase of ceramide was observed in TGF-beta1-induced apoptosis.


CONCLUSIONS
TGF-beta1-induced apoptosis requires TGF-beta1-induced gene expression.
Liao
J H
JH
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031, China.
Zhou
B H
BH
Chai
M Q
MQ
Song
J G
JG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002518:Ceramides / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D000099151:MAP Kinase Kinase Kinase 7
D002518:Ceramides; D003513:Cycloheximide; D020930:MAP Kinase Kinase Kinases; D017395:Plasminogen Activator Inhibitor 1; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D000099151:MAP Kinase Kinase Kinase 7; C494827:Bax protein, mouse; C508961:Tgfb1 protein, mouse