Involvement molecular chaperone in apoptosis signaling in human salivary gland cell line

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line(HSG)was induced by treatment of the cells with anti-Fas antibody(CH-11)，and this cell death was enhanced by pre-treatment with TNF-α。The.Enhancement of the apoptosis caused byTNF-αresulted from increased sensitivity to CH-11-mediated apoptosis due to the induction of Fas，caspase8and3proteins by TNF-αvia the activation of NFid3。Fas-mediated apoptosis was mainly inhibited by the caspase-8inhibitor。CH-11-induced apoptosis was mainly mediated by the death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8at the death-inducing signaling complex(DISC).Heat shock protein(HSP9O)is involved in the regulation of signaling cascades including apoptosis.Using a specific inhibitor for HSP90，geldanamycin(GDM)，we investigated the involvement of HSP90in CH-11-induced apoptosis。When HSG A Is were treated with GDM alone，apoptotic cell death was observed.The pretreatment with GDM prior to that with CH-11significantly increased the cell death as compared with that obtained with GDM or CH-11alone.The transfect ion of HSG cells with recombinant HSP90a significantly inhibited the CH-11and GDM-induced apoptosis.These results showed that HSP90had an anti-apoptotic activity toward HSG cells.We，next，tried to determine the target protein on the Fas signaling pathway。Immunoprecipitation with anti-human·HSP9O antibody and subsequent Western blotting analysis of these precipitates detected bands for caspase-8and FADD-like ICE inhibitory protein(FLIP)that is known to regulate Fas cascade.Therefore，caspase-8 and FLIP were shown to be target proteins of HSP90.These results suggest that HSP90inhibits apoptosis by associat ing with caspase-8 and FLIP and negatively regulating their functions.

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line (HSG) was induced by treatment of the cells with anti-Fas antibody (CH-11), and this cell death was enhanced by pre-treatment with TNF-α. The. enhancement of the apoptosis caused byTNF-α resulted from increased sensitivity to CH-11-mediated apoptosis due to the induction of Fas, caspase 8 and 3 proteins by TNF-α via the activation of NFid3. Fas-mediated apoptosis was mainly inhibited by the caspase-8 inhibitor. CH-11-induced apoptosis was mainly mediated by the death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8 at the death-inducing signaling complex (DISC).Heat shock protein (HSP9O) is involved in the regulation of signaling cascades including apoptosis. Using a specific inhibitor for HSP90, geldanamycin (GDM), we investigated the involvement of HSP90 in CH-11-induced apoptosis. When HSG A Is were treated with GDM alone, apoptotic cell death was observed. The pretreatment with GDM prior to that with CH-11 significantly increased the cell death as compared with that obtained with GDM or CH-11 alone. The transfect ion of HSG cells with recombinant HSP90a significantly inhibited the CH-11 and GDM-induced apoptosis. These results showed that HSP90 had an anti-apoptotic activity toward HSG cells. We, next, tried to determine the target protein on the Fas signaling pathway. Immunoprecipitation with anti-human・HSP9O antibody and subsequent Western blotting analysis of these precipitates detected bands for caspase-8 and FADD-like ICE inhibitory protein (FLIP) that is known to regulate Fas cascade. Therefore, caspase-8 and FLIP were shown to be target proteins of HSP90.These results suggest that HSP90 inhibits apoptosis by associat ing with caspase-8 and FLIP and negatively regulating their functions.