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Signalling pathway of tumor necrosis factor in normal and tumor cells
N Watanabe1, H Neda, Y Ohtusuka
1Department of Internal Medicine, Sapporo Medical College, Japan.
Abstract:
Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r = 0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound 125I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15-30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF. As observed by [3H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.
Insights
Tumor necrosis factor (TNF) cytotoxicity in tumor cells correlates with receptor number. TNF internalization and lysosomal delivery are essential for its cell-killing effects, but it stimulates RNA synthesis in sensitive cells.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Tumor necrosis factor (TNF) is a cytokine with known cytotoxic effects.
- Understanding the precise mechanism of TNF-induced cytotoxicity is crucial for therapeutic applications.
- The role of TNF receptors and cellular uptake in TNF activity requires further elucidation.
Purpose of the Study:
- To investigate the cytotoxic mechanism of tumor necrosis factor (TNF).
- To determine the relationship between TNF receptor number and cellular sensitivity.
- To explore the process of TNF internalization and its impact on cellular functions.
Main Methods:
- Specific binding assays to quantify TNF receptors on tumor and normal cells.
- Pulse-labeling and Percoll density gradient centrifugation to track TNF internalization.
- Assessment of RNA synthesis via [3H]uridine incorporation.
- Use of cytoskeletal disrupting and lysosomotropic agents to inhibit TNF effects.
Main Results:
- A strong correlation (r=0.855) was found between TNF receptor number and sensitivity in tumor cells.
- Normal cells showed no cytotoxicity from TNF, despite receptor presence, and some exhibited proliferation.
- TNF was rapidly internalized and degraded in lysosomes in sensitive cells, a process inhibited by specific agents.
- TNF stimulated RNA synthesis in sensitive tumor cells (L-M) but not in resistant cells (L-R) or normal cells (HEL).
Conclusions:
- TNF-induced cytotoxicity is closely linked to its receptor number on tumor cells.
- Internalization of TNF and its subsequent lysosomal degradation are critical for its cytotoxic mechanism.
- TNF exhibits differential effects on RNA synthesis, stimulating it in sensitive tumor cells while having no effect on resistant or normal cells.