INDUCTION OF UNRESPONSIVENESS TO TUMOR-NECROSIS-FACTOR (TNF) AFTER AUTOCRINE TNF EXPRESSION REQUIRES TNF MEMBRANE RETENTION

Tumor necrosis factor (TNF) has a specific gene-inducing activity on m any cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated th at introduction of an exogenous TNF gene in the TNF-sensitive cell lin e L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF, This resistance required biologically activ e TNF and was correlated with complete down-modulation of the TNF rece ptors on the cell surface. We have now characterized this process in m ore detail. The role of expression of the membrane-bound TNF preform a nd its subsequent proteolytic processing in the induction of TNF unres ponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TN F, but not in induction of TNF resistance. On the other hand, expressi on of non-secretable, membrane-bound TNF generated complete TNF unresp onsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was rep laced by the membrane anchor of trimeric chicken hepatic lectin. Expre ssion of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsivene ss only involves its function as a membrane anchor, which permits olig omerization of the TNF molecule into a biologically active homotrimer.