Bcl-2 antibodies induce hemoglobin release by red blood cells loaded with in vitro translated Bcl-2 and its cleaved fragment

Apoptosis induced by proteasome inhibitor in human THP-1 leukemia cells is associated with the cleavage of Bcl-2 into a shortened fragment, Bcl-2/Delt a 34. Both Bcl-2 and its cleaved fragment were located exclusively on the m itochondria of THP-1 cells. No translocation of Bcl-2 or Bcl-2/Delta 34 to the cytosolic fraction was detected during apoptosis. Treatment of isolated mitochondria with recombinant caspase-3 induced the same cleavage of Bcl-2 in vitro and triggered the release of cytochrome c from the mitochondria. The ability of Bcl-2/Delta 34 in regulating the opening of membrane "pores" was investigated using a sheep red blood cell (RBC) model with in vitro tr anslated Bcl-2/Delta 34 and Bcl-2 proteins. Bcl-2 and Bcl-2/Delta 34 genera ted in vitro were relocated rapidly to sheep RBC but caused no hemoglobin r elease in either case. Addition of anti-Bcl-2 antibodies directly to the RB C that had been loaded with either Bcl-2 or Bcl-2/Delta 34 resulted in a ra pid release of hemoglobin from the blood cells. Treatment of the sheep RBC with anti-Bcl-2 or anti-sheep RBC antibodies alone did not trigger hemoglob in release from the RBC. Based on these findings, we proposed that, upon "e nforced aggregation," both Bcl-2 and Bcl-2/Delta 34 can form "pores" in mem branes, which may contribute to the release of cytochrome c in apoptosis. ( C) 2000 Academic Press.