Inhibition of NF-kappaB by mutant IkappaBalpha enhances TNF-alpha-induced apoptosis in HL-60 cells by controlling bcl-xL expression.

BACKGROUND

The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism.

METHODS

The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM).

RESULTS

Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner.

CONCLUSIONS

NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.