Loss of Mcl-1 Protein and Inhibition of Electron Transport Chain Together Induce Anoxic Cell Death

Abstract

How cells die in the absence of oxygen (anoxia) is not understood. Here we report that cells deficient in Bax and Bak or caspase-9 do not undergo anoxia-induced cell death. However, the caspase-9 null cells do not survive reoxygenation due to the generation of mitochondrial reactive oxygen species. The individual loss of Bim, Bid, Puma, Noxa, Bad, caspase-2, or hypoxia-inducible factor 1β, which are potential upstream regulators of Bax or Bak, did not prevent anoxia-induced cell death. Anoxia triggered the loss of the Mcl-1 protein upstream of Bax/Bak activation. Cells containing a mitochondrial DNA cytochrome b 4-base-pair deletion ([rho⁻] cells) and cells depleted of their entire mitochondrial DNA ([rho⁰] cells) are oxidative phosphorylation incompetent and displayed loss of the Mcl-1 protein under anoxia. [rho⁰] cells, in contrast to [rho⁻] cells, did not die under anoxia. However, [rho⁰] cells did undergo cell death in the presence of the Bad BH3 peptide, an inhibitor of Bcl-X_L/Bcl-2 proteins. These results indicate that [rho⁰] cells survive under anoxia despite the loss of Mcl-1 protein due to residual prosurvival activity of the Bcl-X_L/Bcl-2 proteins. Collectively, these results demonstrate that anoxia-induced cell death requires the loss of Mcl-1 protein and inhibition of the electron transport chain to negate Bcl-X_L/Bcl-2 proteins.

SUPPLEMENTAL MATERIAL

This work was supported in part by National Institutes of Health grants (GM60472-07 and P01HL071643-03004) and an American Heart Association grant (0350054N) to N.S.C. J.K.B. and E.H.S. are supported by National Institutes of Health predoctoral training grants and grants CA009560 and HL076139, respectively. A.S. is supported by NHMRC (Canberra), NIH, the Leukemia and Lymphoma Society of America, and JDRF/NHMRC.

We thank I. de Coo (University Hospital Rotterdam) for the original cytochrome b mutant fibroblasts. We thank Aly Karsan for the LNCX containing the human Mcl-1 cDNA. We thank Michael Murphy for providing MitoQ.