I-KAPPA-B-ALPHA OVEREXPRESSION IN HUMAN BREAST-CARCINOMA MCF7 CELLS INHIBITS NUCLEAR FACTOR-KAPPA-B ACTIVATION BUT NOT TUMOR NECROSIS FACTOR-ALPHA-INDUCED APOPTOSIS

Nuclear factor-kappa B (NF-kappa B) is one of major component induced by turner necrosis factor-alpha (TNF), and its role in the signaling o f TNF-induced cell death remains controversial, In order to delineate whether the involvement of NF-kappa B activation is required for trigg ering of the apoptotic signal of TNF, we inhibited the nuclear translo cation of this transcription factor in TNF-sensitive MCF7 cells by int roducing a human MAD-3 mutant cDNA coding for a mutated I kappa B alph a that is resistant to both phosphorylation and proteolytic degradatio n and that behaves as a potent dominant negative I kappa B alpha prote in. Our results demonstrated that the mutated I kappa B alpha was stab ly expressed in the transfected MCF7 cells and blocked the TNF-induced NF-kappa B nuclear translocation. Indeed, TNF treatment of these cell s induced the proteolysis of only the endogenous I kappa B alpha but n ot the mutated I kappa B alpha. The nuclear NF-kappa B released from t he endogenous I kappa B alpha within 30 min of TNF treatment was rapid ly inhibited by the mutated I kappa B alpha. There was no significant difference either in cell viability or in the kinetics of cell death b etween control cells and the mutated I kappa B alpha transfected cells . Furthermore, electron microscopic analysis showed that the cell deat h induced by TNF in both control and mutated I kappa B alpha transfect ed cells was apoptotic. The inhibition of NF-kappa B translocation in mutated I kappa B alpha-transfected cells persisted throughout the sam e time course that apoptosis was occurring. Our data provide direct ev idence that the inhibition of NF-kappa B did not alter TNF-induced apo ptosis in MCF7 cells and support the view that TNF-mediated apoptosis is NF-kappa B independent.