Events in apoptosis - Acidification is downstream of protease activation and BCL-2 protection

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems, However, its relation to other events in the process of apoptosis is not yet characterized, In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas Ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling, Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1 beta converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation, These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.