Two discrete types of tumor necrosis factor-resistant cells derived from the same cell line

From the murine fibrosarcoma cell line L929s, which is sensitive to tumor necrosis factor (TNF)-mediated cell lysis, two discrete types of TNF-resistant variants were derived by TNF selection. Cells of the first type (named L929r1) were not sensitized to TNF cytotoxicity by cotreatment with either inhibitors of protein or RNA synthesis, or gamma-interferon, despite the presence of a functional gamma-interferon response. L929r1 constitutively produced TNF in the supernatant and expressed membrane-bound TNF, which was not bound to the TNF receptor. In fact, TNF receptors could not be demonstrated on L929r1 cells, not even after low pH treatment and/or incubation with antiserum to TNF. L929r1 exhibited a stable TNF-resistant phenotype in the absence of further TNF selection. No evidence could be obtained that TNF acted as an autocrine growth factor for these cells. L929r2, the second type of TNF-resistant L929 cells, became sensitive to TNF lysis in the presence of RNA or protein synthesis inhibitors, or in the presence of gamma-interferon. TNF induced the secretion of interleukin 6 in these cells, additionally showing that functional TNF signaling in these cells indeed takes place, but does not lead to cell lysis under normal conditions. L929r2 did not produce TNF, also not upon stimulation with exogenous TNF. The number and binding affinity of TNF receptors were not consistently different between L929s and L929r2 cells. In the absence of further TNF selection, L929r2 gradually reverted to TNF sensitivity. This sensitivity was not reversible to TNF resistance by the gene-regulatory agents 5-azacytidine or sodium butyrate. Treatment with these agents also did not affect the TNF sensitivity of L929s cells nor the TNF resistance of L929r1 and L929r2 cells. In summary, our results suggest the existence among cells of the same cell line of discrete mechanisms for acquisition of resistance to TNF-mediated cell lysis.