- Paper Report
- Open Access
BNIP3: a new cell death pathway regulating necrosis?
- Corinne Guingamp1
© Current Science Ltd 2000
- Published: 12 September 2000
Several apoptotic pathways involve mitochondria. Caspase activation, a major event in the induction of cell death, is regulated by mitochondria. Apoptosis inducing factor (AIF) is a mitochondria associated protein involved in DNA fragmentation when translocated to the nucleus. Permeability transition (PT) pore opening occurs in both apoptosis and necrosis. Necrosis is distinguished by an early loss of plasma membrane integrity, which is preserved during apoptosis. To identify which pathway resulting in cell death is activated by the mitochondria associated protein BNIP3.
In contrast to most of the cells transduced with tBID, cytochrome c release prior to apoptosis was not observed in BNIP3 transfected cells. In addition, BNIP3-induced cell death was unaffected by caspase inhibitors, whereas they inhibited apoptosis of tBID or caspase 9/Apaf 1 transfectants. BNIP3 overexpression in mutant cells which were not expressing Apaf 1, caspase 9 or caspase 3 reversed their resistance to cell apoptosis. No AIF translocation to the nucleus was observed. However, DNA fragmentation was noted. In contrast to cells transfected with tBID, BAX, or caspase 9/Apaf 1, plasma membrane permeability increased very early post-transfection, without increase in annexin V. Many rounded mitochondria with destroyed internal cisternae, extensive cytoplasmic vacuoles, and autophagosomes were observed. BNIP3 was almost as efficient as tBID at suppressing ??m, ROS generation, and inducing cell death. All these effects were reversed by treatment of BNIP3 transfectants with cyclosporin A, bongkrekic acid, or when Bcl-2 and Bcl-XL were overexpressed.
Overexpression of BNIP3, tBID, or capase 9 plus Apaf 1 after transfer of the respective genes into different cell lines resulted in cell death. Cytochrome c release was evaluated by immunofluorescence and western blot. Contribution of Apaf-1, caspase 9, or caspase 3 in BNIP3-induced cell death was studied in cells deficient in these genes. The caspase inhibitors Ac-zVAD-fmk and AC-zDEVD-fmk were used to determine the role of caspases in BNIP3-induced cell death. Immunofluorescence and immunoblotting were used to check AIF translocation to the nucleus. DNA fragmentation was shown using TUNEL and agarose gel electrophoresis. Plasma membrane integrity and annexin V binding were also studied. Evaluation of PT pore opening, loss of membrane potential of mitochondria (??m) and radical oxygen species (ROS) production were used to measure the protective effect of cyclosporin A, bongkrekic acid, overexpression of Ncl-2 and Bcl-XL.