|Budget Amount *help
¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Objective To understand the mechanism of apoptosis in neuroblastomas (NBs) in vivo, we investigated expression profiles of Fas, Fas ligand (FasL) and caspase-3 in primary NB tissues.
Methods Immunoperoxidase staining for Fas and FasL/caspase-3 was performed in paraffin sections of 32 and 46 tumor specimens, respectively, using tyramide signal amplification. Tissue lysates of ten NBs were analyzed in immunoblotting for Fas, FasL and caspase-3 expression and in the substrate hydrolytic assay using Ac-Asp-Glu-Val-Asp (DEVD) -MCA for caspase-3-like activity. In the tumors showing caspase-3-like activity, inhibition assay using Ac-DEVD-H peptide was further carried out.
Results Eighty-eight percent of NBs exhibited no or only faint Fas immunostaining, although-70% of them did moderate to strong immunoreactivities for FasL and caspase-3. In the few NBs showing moderate Fas immunoreactivity, the protein expressing cells did not exist nearby apoptotic foci. Consistent with the immunostaining results, immunoblot analyzes showed no Fas expression in all tested tumors, whereas they expressed FasL and caspase-3 at various degrees. Four of ten NBs showed significant caspase-3-like activities that were markedly suppressed by the caspase-3 specific inhibitor Ac-DEVD-H (-90% inhibition).
Conclusion NBs hardly express Fas, but considerably caspase-3, which can lead to DNA fragmentation, in, at least in part, activated form, suggesting that non-Fas-mediated pathway (s), such as that involving p53 and/or Bcl-2 family proteins, may regulate apoptosis in these tumors.