Fas-mediated induction of hepatocyte apoptosis in a neuroblastoma and hepatocyte coculture model

Background We have previously demonstrated an increase in hepatocyte apopto sis when they are cocultured with neuroblastoma cells. Death receptors in t he tumor necrosis factor (TNF) family such as TNFR1 and Fas have been ident ified as regulators of apoptosis and may be responsible for the altered reg ulation of apoptosis seen in our coculture model. To evaluate the effects o f released factors and remove the potential alterations induced by direct c ontact, a noncontact coculture system was used to study the interaction bet ween hepatocytes and neuroblastoma cells. Methods. Human Chang hepatocytes (HC) were plated onto Falcon cell culture inserts with 0.45-mu m pores in the permeable membrane. Human neuroblastoma cells (NB-IMR-32) were seeded into wells of the Falcon companion plate. Af ter 24 h, inserts containing NC were placed into wells containing NE cells and incubated for 4 days. This provided a coculture environment without act ual cellular contact. Immunohistochemical staining for TNF alpha, Fas, and Fas Ligand (Fas-L) was performed. Apoptosis was detected via the TUNEL meth od; Images were analyzed with ImagePro-Plus. Statistical analyses were done with significance determined at P < 0.05. Results. Chang hepatocytes demonstrated a significant increase in the level s of TNF, Fas, and Fas-L when cocultured with neuroblastoma cells (P < 0.00 5). In addition, the cocultured hepatocytes had a 20-fold increase in the a poptotic rate (P < 0.001). Neuroblastoma cells had no demonstrable level of Fas or TNF when grown alone and in cocultures. Neuroblastoma cells that we re grown alone had an elevated level of Fas-L, but this level diminished by 44% when cocultured with hepatocytes (P < 0.001). Conclusion. An upregulated TNF/Fas receptor-ligand system may be responsibl e for increased apoptosis in hepatocytes when cocultured with neuroblastoma . This upregulation may be due to release of neuroblastoma-derived Fas liga nd into the media. Tumors may alter the regulation of apoptosis in surround ing tissues via the death receptors. (C) 1999 Academic Press.