ALTERATION OF THE SPHINGOMYELIN CERAMIDE PATHWAY IS ASSOCIATED WITH RESISTANCE OF HUMAN BREAST-CARCINOMA MCF7 CELLS TO TUMOR NECROSIS FACTOR-ALPHA-MEDIATED CYTOTOXICITY/

The interference of tumor necrosis factor-alpha (TNF) signaling proces ses with the acquisition of tumor resistance to TNF was investigated u sing the TNF-sensitive human breast carcinoma MCF7 cell line and its e stablished TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expr ession and in partial restoration of TNF signaling, including nuclear factor-kappa B (NF-kappa B) activation, However, the transfected cells remained resistant to TNF-induced apoptosis, Northern blot analysis r evealed a comparable induction of manganous superoxide dis mutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cel ls, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomita nt sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it f ailed to trigger these events in TNF-resistant p55-transfected cells, In addition, the basal SM content was significantly higher in sensitiv e MCF7 as compared to the resistant counterparts, Furthermore, the TNF -resistant cells tested could be induced to undergo cell death after e xposure to exogenous SMase or cell-permeable C-6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-t ransfected resistant cells, suggesting that an alteration of phospholi pase A(2) activation may be associated with MCF7 cell resistance to TN F. Our findings strongly suggest a role of ceramide in the mechanism o f cell resistance to TNF-mediated cell death and may be relevant in el ucidating the biochemical nature of intracellular messengers leading t o such resistance.