Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex

The Bcl-2 family member Bcl-x(L) has often been correlated with apoptosis resistance, We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing sig naling complex (DISC) and by increased expression of Bcl-x(L) (Peter, M. E., Kischkel, F. C., Scheuerpflug, C. G., Medema, J. P., Debatin, K.-M., and Krammer, P. H. (1997) Eur. J. Immunol. 27, 1207-1212), This raises the possibility that Bcl-x(L) directly prevents caspase-8 activation by the DISC, To test this hypothesis a cell line in which CD95 signaling was inhibited by overexpression of Bcl-x(L) was used, In these MCF7-Fas-bcl-x(L) cells Bcl-x(L) had no effect on the recruitment of caspase-8 to the DISC, It did not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10, In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, was inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells, To test whether Bcl-x(L) would inhibit active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed, Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-x(L) antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-x(L) was found, Moreover, overexpression of Bcl-x(L) did not inhibit the activity of the caspase-8 active subunits p18/p10, Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-x(L), Bcl-x(L) acts independently and downstream of caspase-8.