Analysis of Cytotoxic Mechanism of Tumor Necrosis Factor

In view of further elucidating the cytotoxic mechanism of the tumor necrosis factor (TNF), the relationship between the number of TNF receptors and susceptibility to its cytotoxicity on TNF-sensitive tumor cells and on certain normal cells was firstly studied by a specific binding assay. A fairly good correlation was shown between the receptor number and the sensitivity of the tumor cells. However, for normal cells, despite the existence of TNF receptors, no cytotoxic effect was observed. Furthermore, certain normal diploid cells underwent a proliferation as a result of TNF stimulation. The internalization process and intracellular distribution of 125I labeled TNF in L-M cell (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) and HEL cell (human embryonic lung cells, non-sensitive to TNF cytotoxicity), were elucidated by pulse labeling and Percoll density gradient centrifugation. In both L-M and HEL cells, receptor-bound 125I-TNF was rapidly internalized and delivered to lysosomes within 15-30 min., followed by a degradation and release into the culture medium. The impairment of the cytotoxic process by cytoskeletal disrupting or lysosomotropic agents also indicated that internalization of TNF into cells, followed by the fusion of pinosomes with lysosomes, is essential in the cytolytic mechanism of TNF. Effect of TNF treatment on the RNA and protein synthesis of target cells was then studied using 3H-UDR and 35S-methionine incorporation. RNA synthesis and protein synthesis were markedly enhanced by TNF treatment in L-M cells, but not in HEL cells. By treating the L-M cells with actinomycin D or cycloheximide which inhibit RNA or protein synthesis, their susceptibility to TNF was rather increased. And even in TNF non-sensitive HEL cells, the cytotoxic activity of TNF was newly evoked by using actinomycin D or cycloheximide. These results suggest that TNF, bound on the surface receptors of tumor cells, is rapidly internalized into the cells and delivered to lysosomes, and that the lysosomal enzyme seemed to play an essential role in the cytolytic reaction since lysosomotropic agents impair the cytotoxic activity of TNF. Normal cells, on the other hand, always are equipped with a defence system which protects them from cytotoxic attack of TNF. Although tumor cells are also provided with this defence system, produced by the stimulation of TNF, they eventually undergo cytolysis by the attack of TNF due to its relative insufficiency