Fas-mediated induction of hepatocyte apoptosis in a neuroblastoma and hepatocyte coculture model.
BACKGROUND
We have previously demonstrated an increase in hepatocyte apoptosis when they are cocultured with neuroblastoma cells. Death receptors in the tumor necrosis factor (TNF) family such as TNFR1 and Fas have been identified as regulators of apoptosis and may be responsible for the altered regulation of apoptosis seen in our coculture model. To evaluate the effects of released factors and remove the potential alterations induced by direct contact, a noncontact coculture system was used to study the interaction between hepatocytes and neuroblastoma cells.


METHODS
Human Chang hepatocytes (HC) were plated onto Falcon cell culture inserts with 0.45-micrometer pores in the permeable membrane. Human neuroblastoma cells (NB-IMR-32) were seeded into wells of the Falcon companion plate. After 24 h, inserts containing HC were placed into wells containing NB cells and incubated for 4 days. This provided a coculture environment without actual cellular contact. Immunohistochemical staining for TNFalpha, Fas, and Fas ligand (Fas-L) was performed. Apoptosis was detected via the TUNEL method. 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 levels of TNF, Fas, and Fas-L when cocultured with neuroblastoma cells (P < 0.005). In addition, the cocultured hepatocytes had a 20-fold increase in the apoptotic rate (P < 0.001). Neuroblastoma cells had no demonstrable level of Fas or TNF when grown alone and in cocultures. Neuroblastoma cells that were grown alone had an elevated level of Fas-L, but this level diminished by 44% when cocultured with hepatocytes (P < 0.001).


CONCLUSIONS
An upregulated TNF/Fas receptor-ligand system may be responsible for increased apoptosis in hepatocytes when cocultured with neuroblastoma. This upregulation may be due to release of neuroblastoma-derived Fas ligand into the media. Tumors may alter the regulation of apoptosis in surrounding tissues via the death receptors.
Chen
M K
MK
Division of Pediatric Surgery, University of Medicine and Dentistry of New Jersey, Camden, New Jersey, 08103, USA.
Strande
L F
LF
Beierle
E A
EA
Kain
M S
MS
Geldziler
B D
BD
Doolin
E J
EJ
D017209:Apoptosis / Q000502:physiology*; D018920:Coculture Techniques; D053222:Fas Ligand Protein; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism / Q000502:physiology*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor