IκBα Overexpression in Human Breast Carcinoma MCF7 Cells Inhibits Nuclear Factor-κB Activation but Not Tumor Necrosis Factor-α-induced Apoptosis

Abstract

Nuclear factor-κB (NF-κB) is one of major component induced by tumor necrosis factor-α (TNF), and its role in the signaling of TNF-induced cell death remains controversial. In order to delineate whether the involvement of NF-κB activation is required for triggering of the apoptotic signal of TNF, we inhibited the nuclear translocation of this transcription factor in TNF-sensitive MCF7 cells by introducing a human MAD-3 mutant cDNA coding for a mutated IκBα that is resistant to both phosphorylation and proteolytic degradation and that behaves as a potent dominant negative IκBα protein. Our results demonstrated that the mutated IκBα was stably expressed in the transfected MCF7 cells and blocked the TNF-induced NF-κB nuclear translocation. Indeed, TNF treatment of these cells induced the proteolysis of only the endogenous IκBα but not the mutated IκBα. The nuclear NF-κB released from the endogenous IκBα within 30 min of TNF treatment was rapidly inhibited by the mutated IκBα. There was no significant difference either in cell viability or in the kinetics of cell death between control cells and the mutated IκBα transfected cells. Furthermore, electron microscopic analysis showed that the cell death induced by TNF in both control and mutated IκBα transfected cells was apoptotic. The inhibition of NF-κB translocation in mutated IκBα-transfected cells persisted throughout the same time course that apoptosis was occurring. Our data provide direct evidence that the inhibition of NF-κB did not alter TNF-induced apoptosis in MCF7 cells and support the view that TNF-mediated apoptosis is NF-κB independent.