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Zn++ inhibits both tumor necrosis factor-mediated DNA fragmentation and cytolysis
D Flieger1, G Riethmüller, H W Ziegler-Heitbrock
1Institute for Immunology, University of Munich, FRG.
Abstract:
The cellular events involved in direct tumor-cell destruction by tumor necrosis factor (TNF) are still incompletely understood, but a role of endonucleases has been suggested. In the present study we have analyzed in detail the effect of Zn++, an inhibitor of endonucleases, on TNF-mediated DNA-fragmentation and on cytolysis in Actinomycin-D-pre-treated WEHII 64-S cells. Beginning 2 hr after addition of TNF, a rapid degradation of cellular DNA is observed, as evidenced by release of 3H-Thymidine (TdR) label from nuclei into cytoplasm. TNF-mediated lysis of WEHII64-S cells begins at 3 hr and reaches plateau levels at 7 hr. Addition of Zn++ to TNF-treated WEHII64 cells completely abrogates DNA fragmentation at ImM. Of greater importance is the fact that Zn++ treatment also completely blocks TNF-mediated cytolysis of the target cells. Concentrations-between 0.1 and I mM ZnSO4 prevent cell death, as assessed by chromium-release and Trypan blue dye exclusion. In addition, ZnCl2, but not other divalent cations like CaCl2, MgSO4 and CuSO4 in the same concentration range, prevents cell death as well, demonstrating that the effect in fact is mediated by Zn++. Zn++ added 2 hr after TNF treatment, still effectively inhibits cell lysis, indicating that it acts at a late stage after binding of TNF to its receptor. Our data suggest that activation of endonucleases is not an accompanying effect but an essential step in TNF-mediated tumor-cell destruction.
Insights
Zinc ions (Zn++) inhibit tumor necrosis factor (TNF)-mediated cancer cell death by blocking DNA fragmentation. This suggests endonucleases are essential for TNF-induced tumor cell destruction.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The precise mechanisms of direct tumor cell destruction by tumor necrosis factor (TNF) remain unclear.
- A potential role for endonucleases in TNF-mediated cytotoxicity has been proposed.
Purpose of the Study:
- To investigate the role of endonucleases in TNF-induced tumor cell death.
- To analyze the effect of zinc ions (Zn++), an endonuclease inhibitor, on DNA fragmentation and cytolysis in WEHI 164-S cells treated with TNF.
Main Methods:
- WEHI 164-S cells were pre-treated with Actinomycin-D.
- Cells were subsequently treated with TNF and varying concentrations of Zn++.
- DNA fragmentation was assessed by 3H-Thymidine release.
- Cytolysis was measured using chromium-release and Trypan blue dye exclusion assays.
Main Results:
- TNF induced rapid DNA fragmentation and cell lysis in WEHI 164-S cells.
- Zn++ (1 mM) completely abrogated TNF-mediated DNA fragmentation.
- Zn++ also completely blocked TNF-induced cytolysis, even when added 2 hours after TNF treatment.
- Other divalent cations did not prevent cell death, confirming Zn++ specificity.
Conclusions:
- Activation of endonucleases is an essential step, not merely an accompanying effect, in TNF-mediated tumor cell destruction.
- Zn++ acts at a late stage in the TNF signaling pathway, after TNF receptor binding, to inhibit cell lysis.