Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes

Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y
Source: Nat Cell Biol
Publication Date: (2004)
Issue: 6(12): 1221-1228
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic fibroblast, mouse (MEF) immort
Species: mouse
Tissue Origin: embryo
Nucleofector® I/II/2b
Primary and SV40 T antigen-transformed wild-type and Bax/Bak–/– mouse embryonic fibroblasts (MEFs), as well as APG5+/+ and APG5−/− MEFs were transfected and/or cotransfected with plasmids expressing human Bax, human Bak, human Bcl-2, human Bcl-xL, or GFP–LC3. The cells were also transfected with siRNAs to mouse Bcl-x, mouse Bcl-2, mouse Bax, mouse Beclin 1, or mouse APG5. In the case of transfection with complementary DNA and siRNA, cDNAs were transfected, and after 24 h siRNAs were introduced.
Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.