CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma.
OBJECTIVE
Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status.


METHODS
CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays.


RESULTS
CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP(L)) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP(L) down-modulation at the DISC level. Down-regulation of c-FLIP(L) with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP(L) in apoptosis resistance. Removal of c-FLIP(L) block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP(L) involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP(L) expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP(L) and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status.


CONCLUSIONS
The inhibitory protein c-FLIP(L) is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.
Unit of Molecular Therapies, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy. delia.mezzanzanica@istitutotumori.mi.it
Delia
D
Mezzanzanica
Emanuela
E
Balladore
Fabio
F
Turatti
Elena
E
Luison
Paola
P
Alberti
Marina
M
Bagnoli
Mariangela
M
Figini
Alessandra
A
Mazzoni
Francesco
F
Raspagliesi
Maria
M
Oggionni
Silvana
S
Pilotti
Silvana
S
Canevari
D017209:Apoptosis; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D015536:Down-Regulation; D004351:Drug Resistance; D004789:Enzyme Activation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000378:metabolism*; D019014:fas Receptor / Q000096:biosynthesis*
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D016376:Oligonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D045304:Cytochromes c; D003513:Cycloheximide; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases