Transmembrane TNF-α (tmTNF-α) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-α (tmTNF-α) at high level was resistant to soluble TNF-α (sTNF-α)-induced cytotoxicity, accompanied by constitutive NF-κB activation. In contrast, MCF-7 cells expressing tmTNF-α at very low level were sensitive to sTNF-α-induced cell death and had no detectable NF-κB activation. Consistently, siRNA-mediated tmTNF-α knockdown blocked NF-κB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-α, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-α containing LS constitutively activated NF-κB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-α lacking the cytoplasmic segment of LS neither activated NF-κB nor affected the sensitivity. However, NF-κB inhibitor PDTC suppressed NF-κB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-κB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)2-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-κB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-κB and resistance to sTNF-α-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-α-induced resistance in the breast cancer cells. © 2008 Springer Science+Business Media, LLC.