Abstract
Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.
Highlights
Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet proliferative signals, which have been implicated in tumor pathogenesis
The recruitment of other death effector domain (DED)-containing proteins to the Fas-mediated death-inducing signaling complex (DISC), which has been described, modulates DISC functions. These include a family of virus-encoded proteins referred to as v-FLIP [16, 17]. v-FLIP contains two DEDs that can bind to the Fas/FADD complex to inhibit Fasmediated apoptosis by interfering with the recruitment of caspase-8 to the DISC
Three glioma cell lines (U343MG, LN-18, T98G) were selected in the study for their expression of cell surface Fas, as shown by flow cytometry, and sensitivity to agonistic Fas antibody CH-11, as determined by crystal violet assay. We stimulated these cells with 1 g/ml CH-11 for 30 min at 37 °C, and the Fas-mediated DISC was immunoprecipitated using goat anti-mouse IgM-agarose and examined by Western blotting to identify the proteins that are incorporated into the Fas-mediated DISC
Summary
Upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet proliferative signals, which have been implicated in tumor pathogenesis. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells.
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