Bcl-2 Antibodies Induce Hemoglobin Release by Red Blood Cells Loaded with in Vitro Translated Bcl-2 and Its Cleaved Fragment1

Abstract

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/Δ34. Both Bcl-2 and its cleaved fragment were located exclusively on the mitochondria of THP-1 cells. No translocation of Bcl-2 or Bcl-2/Δ34 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/Δ34 in regulating the opening of membrane “pores” was investigated using a sheep red blood cell (RBC) model with in vitro translated Bcl-2/Δ34 and Bcl-2 proteins. Bcl-2 and Bcl-2/Δ34 generated in vitro were relocated rapidly to sheep RBC but caused no hemoglobin release in either case. Addition of anti-Bcl-2 antibodies directly to the RBC that had been loaded with either Bcl-2 or Bcl-2/Δ34 resulted in a rapid 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 hemoglobin release from the RBC. Based on these findings, we proposed that, upon “enforced aggregation,” both Bcl-2 and Bcl-2/Δ34 can form “pores” in membranes, which may contribute to the release of cytochrome c in apoptosis.