Resistance to Fas-induced apoptosis in hepatocytes: role of GSH depletion by cell isolation and culture.
The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal.
Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôpital Saint-Luc, Montréal, H2X 1P1, Canada H3C 3P8.
Lina
L
Musallam
Chantal
C
Ethier
Pierre Selim
PS
Haddad
Francine
F
Denizeau
Marc
M
Bilodeau
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D004338:Drug Combinations; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D008099:Liver / Q000378:metabolism; D008297:Male; D008715:Methionine / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D051020:bcl-X Protein; D019014:fas Receptor / Q000502:physiology*
D051023:BH3 Interacting Domain Death Agonist Protein; C494812:Bcl2l1 protein, mouse; C494815:Bid protein, mouse; D002352:Carrier Proteins; D003470:Culture Media; D004338:Drug Combinations; D004792:Enzyme Precursors; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051020:bcl-X Protein; D019014:fas Receptor; D008715:Methionine; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D005978:Glutathione; D003545:Cysteine