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

Zhenzi Cai,Marie Körner,Nadine Tarantino,Salem Chouaib

doi:10.1074/jbc.272.1.96

Abstract

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

Highlights

Cytokine-dependent activation of transcription factors such as Nuclear factor-␬B (NF-␬B) is one of the mechanisms by which signals are transmitted from the extracellular surface to the nucleus to enhance the transcription of specific genes [1, 2]
In order to directly examine whether the NF-␬B activation is an essential requirement for triggering the apoptotic signal of Tumor necrosis factor-␣ (TNF), we chose an approach based on the inhibition of the translocation of this transcription factor by introducing a dominant-negative human MAD-3 gene (MAD-)3 mutant construct into the TNFsensitive MCF7 cells
The TNF-sensitive human breast carcinoma MCF7 cell line was used in this study to examine the effect of the inhibition of NF-␬B activation on its susceptibility to the cytotoxic action of TNF

Summary

Introduction

Cytokine-dependent activation of transcription factors such as NF-␬B is one of the mechanisms by which signals are transmitted from the extracellular surface to the nucleus to enhance the transcription of specific genes [1, 2]. EMSA analysis (Fig. 2B) demonstrated that the introduction of exogenous I␬B␣ mutant led to a significant suppression of TNF-induced NF-␬B activation in the four representative MAD-3-transfected clones as compared with the level of NF-␬B translocation after 90-min treatment with TNF in the control pcN-112 and pcN-183 cells.

Results

Conclusion