Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.
Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.
Fulda
S
S
University Children's Hospital, Prittwitzstr. 43, D-89075 Ulm, Germany.
Küfer
M U
MU
Meyer
E
E
van Valen
F
F
Dockhorn-Dworniczak
B
B
Debatin
K M
KM
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000494:pharmacology; D000077209:Decitabine; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D004790:Enzyme Induction / Q000187:drug effects; D018014:Gene Transfer Techniques; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008745:Methylation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000188:drug therapy / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D000077209:Decitabine; D015254:DNA Modification Methylases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D001374:Azacitidine