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 examin ed the effect of BCL-2 overexpression on acidification mediated by cyc loheximide treatment or Fas Ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and ap optosis detected by annexin V labeling, Acidification and phosphatidyl serine externalization were found to occur concurrently. We also exami ned the requirement for protease activation for cytoplasmic acidificat ion to occur and found that inhibition of interleukin-1 beta convertin g enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoro methylketone, 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 activat ion is also upstream of acidification.