Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis.
OBJECTIVE
Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype.


METHODS
Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage.


RESULTS
Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector).


CONCLUSIONS
Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction.
Lamboley
Christelle
C
Inserm U. 327, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, BP416, 75870 Cedex 18, Paris, France.
Bringuier
Annie-France
AF
Camus
Emmanuel
E
Lardeux
Bernard
B
Groyer
André
A
Feldmann
Gérard
G
D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics; D053938:DNA Fragmentation / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms; D010641:Phenotype; D017354:Point Mutation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000502:physiology; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism*
D002843:Chromatin; D029968:Escherichia coli Proteins; D011506:Proteins; D019014:fas Receptor; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003066:Coenzyme A Ligases; C085934:acyl-coenzyme A synthetase, E coli