Date Published: November 2, 2016
Publisher: Public Library of Science
Author(s): Katharine J. Goodall, Megan L. Finch-Edmondson, Joanne van Vuuren, George C. Yeoh, Ian E. Gentle, James E. Vince, Paul G. Ekert, David L. Vaux, Bernard A. Callus, Dhyan Chandra.
Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.
The role of Bcl-2 as an inhibitor of cell death was first established in FDC-P1 cells, an IL-3 dependent mouse myeloid cell line . These cells undergo apoptosis when growth factor is removed, but when growth factor was removed from cells over-expressing Bcl-2, they arrested, but did not die.
Initially, a dose-response experiment was performed to determine the concentration of cycloheximide (CHX) that caused FDM cells to die. CHX induced a dose-dependent decrease in viability of wild-type (WT) FDM cells for concentrations above 1 μg/ml with greater than 90% of cells killed at 20 μg/ml by 24 h (Fig 1A). CHX induced cell death by this time point was dependent on the expression of Bax or Bak because bax/bak deficient FDM cells derived from bax/bak double knockout (DKO) mice were profoundly resistant to CHX treatment (Fig 1A). This resistance was confirmed by treating bax/bak DKO cells for 96 hours with CHX, at which the majority of the cells were still viable, in contrast to the rapid cell death of the WT cells (Fig 1B) From these experiments a concentration of 20 μg/ml CHX was chosen and was used in all subsequent experiments.
It has generally been assumed that initiation of Bax/Bak dependent apoptosis requires the production or activation of one of more of the BH3-only members of the Bcl-2 family . The activation of BH3-only proteins has been shown to occur via transcriptional or post-translational mechanisms in response to a number of external and intracellular signals. For example, DNA damage can promote stabilization of p53, which directly transactivates the puma locus [15–17], Bid can be activated by proteolytic cleavage , and Bim can be regulated by phosphorylation [19–22]. Once active BH3-only proteins are produced, they are believed to trigger Bax and Bak in either of two ways. The “direct activators” such as Bim, Puma and Bid can directly bind to Bax and/or Bak and cause them to form multimers in the mitochondrial outer membrane that allow exit of proteins including cytochrome c. Alternatively, the “indirect activators” such as Bad, Hrk, Bmf, and Bik bind to the anti-apoptotic Bcl-2 family members, and thereby prevent them from binding to and inhibiting Bax and Bak, and perhaps also by displacing Bim, Puma or Bid from the anti-apoptotic Bcl-2 family members so they are free to activate Bax or Bak. By either of these scenarios it is widely assumed that the BH3-only proteins perform an obligatory role in Bax/Bak activation [8, 10]. Consistent with this, mice deficient in Bim, Bid and Puma phenotypically resemble bax/bak DKO mice, suggesting they are essential for Bax/Bak activation .