ZINC INHIBITS APOPTOSIS UPSTREAM OF ICE CED-9 PROTEASES RATHER THAN AT THE LEVEL OF AN ENDONUCLEASE/

Apoptosis is commonly associated with DNA digestion, but it remains co ntroversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-depe ndent endonuclease in cell lysates, has been used frequently to sugges t this is the endonuclease involved. However, zinc has many other effe cts on cells, and here it is shown that zinc also prevents many upstre am events in apoptosis. These studies were performed in human ML-1 cel ls following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all o f these events. An upstream event in apoptosis is activation of ICE/CE D-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event eve n further upstream in apoptosis; zinc was also found to inhibit Rb dep hosphorylation. Therefore, zinc must protect cells at a very early ste p in the apoptotic pathway, and not as a direct inhibitor of an endonu clease.