A missense mutation was detected at codon 96, GCT (Alanine) to GTT (Valine), of the caspase 8 gene in one of the NB cell lines lacking caspase 8 expression.
Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme.
Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.
The combined data suggest that FOXO3 is activated by 5-azadC treatment and triggers expression of caspase-8 in caspase-8-negative neuroblastoma, which may have important implication for metastasis, therapy, and death resistance of this childhood malignancy.
Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma.
Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines.
These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation.
Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells.
Exogenous interferon-γ upregulated expression of caspase-8 in 3 of 4 neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against 2 of the 3 lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-interferon-γ neutralizing antibody.
A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.
These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.
As frequent methylation of the CASP8 gene has also been reported in neuroblastoma, we investigated whether RASSF1A and CASP8 methylation were independent or related events.
However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria.
We propose the simultaneous analysis of hypermethylation of APAF1, TMS1 and CASP8 apoptotic genes on primary NB tumour as a good prognostic factor of disease progression.
Subsequently others have also shown that caspase-8 is silenced by methylation in neuroblastoma and peripheral neural ectodermal tumors, and that the caspase-9 regulator Apaf-1 is silenced by methylation in melanoma cell lines and patient samples.
Re-expression of caspase-8 in neuroblastoma cells lacking endogenous caspase-8 expression was found to promote cell adhesion to extracellular matrix and to activate adhesion-dependent signaling pathways, such as the Erk kinase cascade.
Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity.
Contrarily, the presence of the -652 6N Del or the CASP8 302His variant was reported to be an unfavorable prognostic factor in colorectal cancer or neuroblastoma.
We established N-myc-downregulated IMR-32 cells using shRNA lentiviral particles targeting N-myc and examined the effect the N-myc inhibition on TRAIL susceptibility in human neuroblastoma IMR-32 cells expressing caspase-8.
The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression.