Abstract

Tumor Necrosis Factor-α (TNF) is a cytokine with a broad range of biological activities. This study has focused on one of these activities; TNF's selective in vitro cytotoxicity for a subset of mouse and human tumors. For this purpose, we established subclones of the TNF-sensitive mouse sarcoma SA-1 and derived TNF-resistant counterparts to these cells by repeated exposure to TNF. This cell panel was used to identify and evaluate gene products that could be associated with the TNF sensitive (S) or resistant (R) phenotype. Comparison of SA-1 S vs. R cells in pairs, using two dimensional gel electrophoresis, revealed no consistent difference in proteins expressed by them, but TNF treatment resulted in the induction of significantly more proteins in S cells as compared to R cells. Using differential display analysis, we identified a gene product, designated 7.1, whose expression correlated with TNF resistance. A functional analysis of this gene by transfection experiments, however, did not demonstrate a primary role for 7.1 in TNF sensitivity/resistance. The possible involvement of a range of other gene products in the S/R phenotype was analyzed, including TNF receptors, TNF or IL-6 induction by TNF, MnSOD, EGF, SMase, PKC and Ca ⁺⁺. In contrast to other model systems, however, we were unable to find consistent correlative changes in these parameters in our S/R cell panel. We propose that the relative decrease in TNF responsiveness that correlates with TNF resistance indicates a receptor proximal site for the regulation of TNF signal transduction in our cell panel and that the effector mechanism utilized for TNF cytotoxicity is likely to have remained intact. We further suggest that our inability to correlate the involvement of previously published cytokines and second messengers in TNF mediated SA-1 cytotoxicity is indicative of the heterogeneity of intracellular signals utilized by different cell types in response to TNF.