Teitz et al. reply—Our previous manuscript1 agrees with Banelli et al. on essentially all major points. In particular, we agree that there is minimal or no expression of caspase-8 in a substantial portion (25–35%) of NB cell lines and patient samples1. Furthermore, as we also described, Banelli et al. and others have observed a strong correlation between methylation of the CASP8 5′ UTR and CASP8 expression in more than 30 NB cell lines and 150 NB patient samples2,3,11,12,13,14. Thus, methylation of this region could potentially be used as a diagnostic tool in conjunction with protein analysis, given that a few examples of discordant protein and RNA expression have been described (by Banelli et al. above and others14).
We also observed higher levels of CASP8 methylation in MYCN-amplified and over-expressing cell lines and patient samples1,3. In particular, we demonstrated partial methylation in stage 1, 2 and 3 NBs, whereas complete methylation occurred almost exclusively in stage 4 NBs, especially those with amplified MYCN (67%)1. We do not yet know the biological significance of this observation.
We recognize that the region of CASP8 we analyzed was not a classical CpG island and that it was not the promoter, as others have identified CASP8 exons 5′ upstream to this sequence14,16. Thus, we described this sequence as a CpG-rich region and 5′-flanking sequence. Nevertheless, the correlation between the loss of expression and methylation of this region is notable and suggests a role for these sequences in CASP8 silencing. Whereas the ability of 4HPR and IFN-γ to trans-activate CASP8 in vivo is certainly of interest16, until more is known about the regulation of caspase-8 expression it is not necessarily inconsistent with the possibility that gene silencing involves a mechanism associated with this methylated region.
In contrast to Banelli et al., we interpret the studies cited in their letter2,3 as consistent with our results. Moreover, there may be a mechanism for the functional inactivation of caspase-8 in NBs expressing the protein, emphasizing the potential importance of caspase-8 in NB tumorigenesis2.
Notably, caspase-8 expression and methylation of the CASP8 5′ UTR region have also been tightly linked to other neural crest tumors, including rhabdomyosarcoma, medulloblastoma, retinoblastoma and neuroendocrine lung carcinomas14. Silencing of CASP8 in a variety of neural crest tumors warrants further study of the associated mechanisms.
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Teitz, T., Lahti, J. & Kidd, V. Reply to “Expression and methylation of CASP8 in neuroblastoma: Identification of a promoter region”. Nat Med 8, 1335 (2002). https://doi.org/10.1038/nm1202-1335
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DOI: https://doi.org/10.1038/nm1202-1335
