Interferon-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis by up-regulating Fas receptors and caspase-8.
BACKGROUND
Osteosarcoma is the third most frequent neoplasm in adolescents. Although chemotherapy, frequently used in pre- and post-operative settings, has resulted in significant improvement in disease-free survival, some patients show little sensitivity to chemotherapy and alternative therapeutic strategies are needed. Because the Fas ligand/Fas receptor (CD95, APO-1) apoptosis pathway is a potential therapeutic target in osteosarcomas, we examined the effect of IFN-gamma on Fas-induced apoptosis in four osteosarcoma cell lines.


RESULTS
As measured by flow cytometry, all cell lines expressed cell surface IFN-gamma receptors, and when cultured for 2 days in the presence of IFN-gamma, all cell lines exhibited a significant increase in expression of Fas receptors. By flow cytometric detection of intracellular fragmented DNA as a marker of apoptosis, all cell lines cultured with either IFN-gamma or anti-Fas antibody (clone CH-11) alone showed only moderate apoptosis, whereas significantly high levels of apoptosis occurred in cells cultured with both IFN-gamma and CH-11. Western blotting analysis also revealed that IFN-gamma caused up-regulation of caspase-8 in all cell lines, but no change in Fas-associated death domain protein (FADD/MORT1) or caspase-3. Both caspase-8 and caspase-3 were activated when apoptosis was induced with both IFN-gamma and CH-11. Addition to cultures of z-IETD-fmk, an inhibitor of caspase-8, significantly blocked this apoptosis.


CONCLUSIONS
IFN-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis through up-regulation of Fas receptor and caspase-8. Combined immunotherapy with IFN-gamma and either anti-Fas monoclonal antibody or cytotoxic T cells that bear Fas ligand might be a useful adjunctive therapy for patients with osteosarcoma.
Inaba
Hiroto
H
Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan, USA. hiroto.inaba@stjude.org
Glibetic
Maria
M
Buck
Steven
S
Ravindranath
Yaddanapudi
Y
Kaplan
Joseph
J
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000032:analysis / Q000235:genetics*; D045744:Cell Line, Tumor; D004357:Drug Synergism; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D012516:Osteosarcoma / Q000473:pathology*; D015854:Up-Regulation / Q000187:drug effects; D019014:fas Receptor / Q000235:genetics* / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D019014:fas Receptor; D007371:Interferon-gamma; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases