Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis.
Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.
Askew
D J
DJ
Departments of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, 85721 USA.
Kuscuoglu
U
U
Brunner
T
T
Green
D R
DR
Miesfeld
R L
RL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005938:Glucocorticoids; C064769:N-acetylsphingosine; D019014:fas Receptor; D003907:Dexamethasone; D020169:Caspases; D019311:Staurosporine; D013110:Sphingosine