Significance of apoptosis induced by tumor necrosis factor-alpha and/or interferon-gamma against human gastric cancer cell lines and the role of the p53 gene

The expression of the p53 gene in target cells plays an important role in the induction of apoptosis by tumor necrosis factor (TNF)-Alpha and interferon (IFN)-Gamma. We herein present a study suggesting the existence of a caspase-independent pathway from p53 via insulin-like growth factor binding protein 3 (IGF-BP3) which acts as an apoptosis induction mechanism. MKN-45 (wild-type p53) or MKN-28 (mutant-type p53) was cultured with TNF-Alpha or IFN-Gamma either alone or together. After 24 and 48 h, the apoptotic index (AI) was determined by Hoechst staining and then compared. To clarify whether the mechanism of apoptosis is induced by TNF-Alpha and/or IFN-Gamma, apoptosis-related genes were examined by a cDNA on microarray analysis and a Western blot analysis. (1) The AI for MKN-45 increased at 48 h in the presence of TNF-Alpha or IFN-Gamma alone. (2) In the case of combination treatment using TNF-Alpha and IFN-Gamma, the AI for MKN-45 was higher than those in the groups with a single treatment. (3) A cDNA microarray analysis showed that IGF-BP3, the TNF superfamily, and caspase 1 were all upregulated after treatment with the combination of TNF-Alpha and IFN-Gamma. (4) A Western blot analysis of MKN-45 showed a reaction with an anti-IGF-BP3 antibody. These results suggest that the induction mechanism underlying apoptosis induced by TNF-Alpha and IFN-Gamma might therefore involve the caspase-independent pathway via IGF-BP3.