Effect of N-tosyl-L-phenylalanylchloromethyl ketone on tumor necrosis factor-alpha -induced NF-kappaB activation and apoptosis in U937 cell line.

To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-kappaB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-kappaB/p65 and IkappaB-alpha were observed by fluorescencemicroscopy and expression and degradation of IkappaB-alpha by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-kappaB/p65, IkappaB-alpha only localized in cytoplasm. After TNF-alpha stimulation, p65 was localized only in nuclei, and IkappaB-alpha was only localized in cytoplasm and decreased. The changes of TNF-alpha stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IkappaB-alpha protein during TNF-alpha-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-alpha induction. DNA ladder can be detected in cells treated by TNF-alpha. It is concluded that degradation of IkappaB-alpha protein and NF-kappaB/p65 translocation occur during TNF-alpha-induced apoptosis of U937 cells, suggesting the activation of NF-kappaB TPCK-sensitive protease plays an important role in the degradation of IkappaB-alpha protein induced by TNF-alpha in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-alpha.