REGULATION OF THE FAS RECEPTOR AND ITS LIGAND IN PEDIATRIC TUMORS

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Fas is a cell surface receptor that induces apoptosis upon activation by its own ligand or a specific monoclonal antibody. We have studied Fas mRNA levels in neuroblastoma (NB) and peripheral primitive neuroectodermal tumor (PNET) cell lines by RT-PCR and found consistently high levels in PNET and low levels in NB. Furthermore, the anti-Fas antibody induced apoptosis in PNET but not in NB cell lines. However, previous studies have shown that a variety of cytotoxic and differentiating agents lead to apoptosis in NB cell lines. We therefore hypothesized that the apoptotic machinery in NB is intact and that the failure of NB cell lines to undergo anti-Fas induced apoptosis may be due to the low levels of the Fas receptor which lead to deficient interaction with the anti-Fas antibody. Previous studies have shown that interferon-gamma increases Fas expression in peripheral blood mononuclear cells and augments the cytolytic activity of the anti-Fas antibody in colon and breast carcinoma cell lines. We will therefore attempt to upregulate the Fas receptor with interferon-gamma and investigate the response of NB cells to the anti-Fas antibody. It is known that binding of the Fas receptor to its ligand results in clustering of intracytoplasmic proteins, such as the FADD protein, which bind to the cytosolic portion of Fas and transduce the signal to downstream molecules. We will investigate the localization of the FADD and the Fas proteins in control untreated NB cells, as well as in cells pre-treated with interferon-gamma and subsequently challenged with the apoptosis inducing anti-Fas antibody by double immunofluorescence labelling. Caping of the FADD protein and co-localization with the Fas protein is expected in cells challenged with the anti-Fas antibody in which sufficient Fas protein of normal sequence is available for optimal binding. In vivo, apoptosis is mediated by the Fas ligand, which is normally expressed in T lymphocytes, macrophages and a few other cells. Upregulation of the Fas ligand in a cell type could theoretically lead to apoptosis of this cell type with a positive feedback loop. We will investigate possible induction of the Fas ligand and its receptor in NB cells as the result of treatment with cytotoxic agents, such as cis-platin and adriamycin. We will also investigate the possible participation of the Fas receptor-ligand pathway in the cytotoxic drug-induced apoptosis using an anti-Fas blocking antibody.