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Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity
P Suffys1, R Beyaert, F Van Roy
1Laboratory of Molecular Biology, State University of Ghent, Belgium.
Abstract:
We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.
Insights
Protease inhibitors block tumour necrosis factor (TNF) by affecting a late-stage protease, not TNF itself. This research clarifies the mechanism of TNF
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Tumour necrosis factor (TNF) is a cytokine with significant anti-proliferative and cytotoxic effects on various cancer cells.
- The precise molecular mechanisms underlying TNF-induced cytotoxicity and growth inhibition are not fully elucidated.
- Previous studies suggested a potential role for cellular proteases in mediating TNF's action.
Purpose of the Study:
- To investigate the role of protease activity in mediating the anti-proliferative and cytotoxic effects of TNF on mouse L929 fibrosarcoma cells.
- To identify the type of protease involved and its stage of action during TNF-induced cell death.
- To determine if TNF itself possesses proteolytic activity.
Main Methods:
- Treatment of L929 cells with TNF in the presence of various protease inhibitors, including serine-type and thiol protease inhibitors.
- Assessment of TNF-mediated cytotoxicity with and without actinomycin D or cycloheximide.
- Assays to detect proteolytic activity of TNF and its interaction with protease inhibitors.
- Time-course experiments to determine the stage of protease involvement.
Main Results:
- Several serine-type protease inhibitors, including phenylmethylsulfonyl fluoride and antipain, significantly inhibited TNF's anti-proliferative and cytotoxic effects.
- Inhibitors of thiol proteases did not affect TNF action.
- TNF itself exhibited no detectable proteolytic activity, and protease inhibitors did not interfere with TNF binding, internalization, or metabolism.
- The protective effect of protease inhibitors diminished when added later after TNF administration, indicating a late-stage protease action.
Conclusions:
- A protease activity, likely a serine protease, is involved in the late stages of TNF-induced cytolysis and growth inhibition in L929 cells.
- This protease is distinct from TNF itself and acts downstream of TNF binding and internalization.
- The findings provide crucial insights into the signaling pathway of TNF and suggest potential therapeutic targets for modulating TNF responses.