Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells.

Abstract

Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 1251 and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 1251-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 x 10-10 M and 3.2 x 10-10 M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 370C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.Tumor necrosis factor (TNF) was originally described as a protein found in the serum of animals sensitized with Bacillus Calmette-Guerin or Corynebacterium parvum and challenged with bacterial endotoxin (1). TNF is functionally defined as a protein producing hemorrhagic necrosis of some tumors in experimental animals and cytolytic or cytostatic effects in tumor cells in culture. Since the major cellular source of TNF is the macrophage (1-6), TNF is thought to be a mediator of the cytotoxic activity of macrophages against tumor cells (7). Recently, human TNF was purified to homogeneity (8) and cDNA for human TNF was cloned, sequenced, and expressed in Escherichia coli (9-11). As a result of these advances, highly purified recombinant TNF has become available for experimental'studies.One of the most intriguing properties of TNF is the apparent selectivity of its cytotoxic and cytostatic activities. TNF is active against many types of tumor cells, whereas untransformed cells generally remain unaffected (1, 11). The mechanism of this selectivity as well as the mechanisms by which TNF exerts its cytolytic or cytostatic activity are unknown. The action of polypeptide hormones, growth factors, and cytokines is generally initiated by their binding to specific cellular receptors (12). This binding is usually followed by internalization of the receptor-ligand complex, through receptor-mediated endocytosis, and the eventual degradation of the ligand by lysosomal hydrolases (13).In this study we examined the presence of high-affinity receptors on two cell lines-one highly sensitive (L929) and one completely resistant (FS-4)

Alteration of the Sphingomyelin/Ceramide Pathway Is Associated with Resistance of Human Breast Carcinoma MCF7 Cells to Tumor Necrosis Factor-α-mediated Cytotoxicity

“…Measurement of Internalization and Degradation of Cell-bound TNF-TNF ligand internalization and degradation were estimated, as described previously by Tsujimoto et al (30). Cells (1 ϫ 10 6 ) were exposed to 125 I-TNF (Amersham) at 4°C for 2.5 h and then washed with ice-cold medium and shifted to 37°C by adding prewarmed medium and further incubated at 37°C.…”

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function

“…Therefore, understanding of the molecular and biochemical mechanisms of tumor cell resistance to the cytocidal action of TNF may ultimately provide new approaches to enhance the therapeutic e cacy of TNF against human malignancies. Although expression of the TNF-receptor alone is necessary for triggering the biological response, it is not su cient to induce the cytolytic process Tsujimoto et al, 1985). The cytotoxic e ect of TNF towards tumor cells can be a ected by both intrinsic and acquired cell resistance.…”

Heparan Sulfate Plays a Central Role in a Dynamic in Vitro Model of Protein-losing Enteropathy

“…Basis of HS Loss, IFN␥, and TNF␣ Synergy-Because IFN␥ alone had no effect on protein leakage but enhanced TNF␣-induced protein leakage, we asked whether this synergism is caused by an IFN␥-mediated up-regulation of the TNFR1 receptor (24). To address this question, we To actually determine the combined effects of these three factors, IFN␥ (10 ng/ml) and TNF␣ (2 ng/ml) were added to the cells 24 and 12 h before measuring albumin flux, respectively.…”