All issues > Volume 46(7); 2003

Original Article

J Korean Pediatr Soc. 2003;46(7):702-709. Published online July 15, 2003.

Sensitization of TNFα and Agonistic FAS/CD95 Antibody-Induced Apoptosis by INFγ on Neuroblastoma Cells

Ho Il HI Bang¹, Jong Duck JD Kim¹, Du Young DY Choi¹

¹Department of Pediatrics, School of Medicine, Wonkwang University, Iksan, Korea

Correspondence Du Young DY Choi ,Email: CDY8118@wonkwang.ac.kr

Abstract

Purpose
: IFNγ sentitizes many tumor cells to TNFα and FASL-mediated apoptosis by enhancing the expression of TNF or FAS/CD95 receptor and modulating the activation of caspase and Bcl-2 family. It has been reported that IFNγ and TNFα synergistically caused differentiation and growth inhibition of neuroblastoma cells. Even though some neuroblastoma cell express FASR/FASL on the cell surface, they could not induce apoptosis by ligation of the FAS/CD95 receptor. But the treatment of IFNγ is reported to induce apoptosis in some neuroblastoma cell lines through the CD95/ CD95L autocrine circuit. In this study, we examined whether IFNγ could affect TNFα and agonistic FAS/CD95 antibody(CH-11)-induced apoptosis against neuroblastoma cell lines that had shown diverse drug sensitivity and resistance.
Methods
: CHLA-15, CHLA-90 and LA-N-2 neuroblastoma cells were cultured in IMDM and treated with recombinant IFNγ, TNFα and CH-11 antibody. Cell viability was measured by DIMSCAN with a fluorescent calcein-AM. Apoptosis was analyzed through flow cytometry using Annexin V- PE and 7-ADD staining and confirmed by pancaspase and caspase-8 blocking experiments. The expression of TNF RI and FAS/CD95 receptor was evaluated by flow cytometry using the corresponding antibody and PE-conjugated secondary antibody.
Results
: IFNγ or TNFα alone had no demonstrable cytotoxic effects, whereas both cytokines in combination induced apoptosis synergistically in CHLA-15 and CHLA-90 cells. Although there was no cytotoxicity with the ligation of CH-11 alone in CHLA-90 cells, pretreatment of IFNγ increased the sensitivity of CH-11-mediated apoptosis. The expression of TNFRI and FAS/CD95R were nonspecifically enhanced after treatment of IFNγ without relation to sensitivity to TNFα and CH-11. This finding suggest up-regulation of both receptors may contribute to sensitization of TNFα and CH-11-mediated apoptosis by IFNγ in only sensitive cell lines.
Conclusion
: IFNγ induced sensitization of TNFα and agonistic FAS/CD95 antibody-mediated apoptosis on some neuroblastoma cells through up-regulation of TNFRI and FAS/CD95 receptor.

Keywords :Neuroblastoma, IFNγ, TNFα, Agonistic FAS/CD95 antibody(CH-11)