Zn++ inhibits both tumor necrosis factor‐mediated DNA fragmentation and cytolysis

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 WEH1164‐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 WEH1164‐S cells begins at 3 hr and reaches plateau levels at 7 hr. Addition of Zn⁺⁺ to TNF‐treated WEH1164 cells completely abrogates DNA fragmentation at 1mM. 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 ZnSO₄prevent cell death, as assessed by chromium‐release and Trypan blue dye exclusion. In addition, ZnCI₂, but not other divalent cations like CaCI₂, 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.