Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing.
OBJECTIVE
Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L are members of the TNFalpha family that can trigger apoptosis in susceptible cells via respective death receptors (DRs). FasL cross-links its receptor Fas, resulting in recruitment and proteolytic activation of caspase-8, which initiates the downstream apoptotic cascade. TRAIL signals through its receptors DR4 and DR5, which can activate caspase-8 as well. This study was undertaken to investigate the functional status of the FasL and TRAIL apoptotic pathways in retinoblastoma (Rb) cells.


METHODS
The human Rb cell lines Y79 and WERI-Rb1 were evaluated for their response to the Fas cross-linking antibody CH11 and recombinant TRAIL, as well as for cell surface presence and mutational status of Fas, DR4, and DR5 by flow cytometry and genomic DNA sequencing, respectively. The expression of caspase-8 and its inhibitor FLIP, as well as their recruitment to the DR signaling complex were studied by immunoblot analysis.


RESULTS
Rb cells express Fas, DR4, and DR5 on their surfaces, yet were resistant to DR-mediated apoptosis. This was not due to DR mutations or secretion of the soluble decoy Fas, antiapoptotic NF-kappaB activity, or FLIP overexpression, but to the absence of caspase-8 expression. The demethylating agent 5-aza-2'-deoxycytidine restored caspase-8 expression and sensitivity to DR-mediated apoptosis.


CONCLUSIONS
Rb cells are resistant to DR-mediated apoptosis because of a deficiency in caspase-8 expression secondary to epigenetic gene silencing by overmethylation. The data help delineate the apoptotic pathways in Rb cells and suggest that the combination of demethylating agents with DR-activating modalities, such as TRAIL receptor monoclonal antibodies, may benefit patients with retinoblastoma.
Poulaki
Vassiliki
V
Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA. poulakiv@hotmail.com
Mitsiades
Constantine S
CS
McMullan
Ciaran
C
Fanourakis
Galinos
G
Negri
Joseph
J
Goudopoulou
Athina
A
Halikias
Ioannis X
IX
Voutsinas
Gerassimos
G
Tseleni-Balafouta
Sophia
S
Miller
Joan W
JW
Mitsiades
Nicholas
N
D017209:Apoptosis; D001374:Azacitidine / Q000494:pharmacology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics*; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D019572:Retinal Neoplasms / Q000201:enzymology / Q000473:pathology*; D012175:Retinoblastoma / Q000201:enzymology / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; D019014:fas Receptor; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D001374:Azacitidine