Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor beta in fetal rat hepatocytes in primary culture.
We have previously reported that transforming growth factor-beta (TGF-beta) induces apoptosis in fetal hepatocytes in primary culture. This effect was found to be associated with an increase in intracellular reactive oxygen species (ROS) and a lowering of total cellular reduced glutathione (GSH). In this study, we investigated whether protein synthesis plays a role in these TGF-beta-induced effects. When fetal hepatocytes were incubated in the presence of cycloheximide, a specific protein synthesis inhibitor, TGF-beta-induced apoptosis was completely blocked. The overall intracellular oxidized state of the cells, when measured using either 2',7'-dichlorofluorescein diacetate (DCFH) by laser-scanning confocal microscopy, or both DCFH and hydroethidine (DHE) by flow cytometric analysis, was increased transiently after the addition of TGF-beta. This increase was abolished by incubation of the cells in the presence of cycloheximide. Furthermore, the decrease in the total cellular GSH content induced by TGF-beta in these cells was not observed when cycloheximide was present. Cycloheximide effect was not associated with an enhancement of cysteine and restoration of cellular glutathione level, because inhibition of GSH synthesis with buthionine sulfoximine (BSO) did not prevent the cycloheximide protective effect. Experiments performed to check whether Fas (APO-1) ligand might be the protein that needs to be synthesized have indicated that this possibility can be excluded. All these findings suggest that apoptosis elicited by TGF-beta in fetal hepatocytes requires the synthesis of an unknown protein before ROS production, glutathione loss, and oxidative stress.
Sánchez
A
A
Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica (C.S.I.C./U.C.M.), Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Alvarez
A M
AM
Benito
M
M
Fabregat
I
I
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology*; D053222:Fas Ligand Protein; D005333:Fetus; D005978:Glutathione / Q000378:metabolism*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D011500:Protein Synthesis Inhibitors; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; D003513:Cycloheximide; D005978:Glutathione