Results BH3 peptides induce cell death in the absence of BAX, BAK and BOK BAX or BAK are the essential regulators for permeabilization of the outer mitochondrial membrane. Previous published studies [3,18], have demonstrated that several death stimuli including anoxia, growth factor withdrawal, DNA damaging agents fail to induce cell death in the absence of BAX and BAK. These various death stimuli likely activate BH3-only proteins which bind and neutralize a fraction of pro-survival BCL-2 proteins to lower the threshold for the activation of BAX and BAK. To test negation of prosurvival BCL-2 proteins in the absence of BAX and BAK can trigger death, we treated Bax2/2/Bak2/2 MEFs with peptides spanning the BH3 domains of the BH3 only proteins. These peptides have been previously demonstrated to have high binding specificity to pro-survival proteins [19–21]. These peptides can be administered to cells at concentrations that are likely to bind to a large fraction of pro- survival BCL-2 proteins. The BH3 peptides BID, BIM and PUMA are known to bind all BCL-2 pro-survival proteins. By contrast, NOXA BH3 peptide specifically binds MCL-1 and BAD binds BCL- 2, BCL-XL, BCL-w and A1. The BH3 peptides are tagged with 8-D arginines which allows for rapid uptake by cells (Figure S1). Treatment with the BID BH3 peptide (100 mM) resulted in similar levels of cell death as measured by LDH release in wild type and Bax2/2/Bak2/2 MEFs at 24 hours (Figure 1A). As a control, cells were treated with a mutant BID BH3 peptide in which the conserved residues leucine and aspartate were mutated to alanine. The mutation of these two critical amino acids has been previously shown to disrupt binding to all pro-survival BCL-2 proteins [19,21]. As predicted, treatment with the mutant BID peptide did not induce cell death in wild type or Bax2/2/Bak2/2 MEFs. The overexpression of Bcl-XL slightly prevented BID BH3 induced cell death (Figure S2). BIM or PUMA BH3 peptides also induced cell death in wild type or Bax2/2/Bak2/2 MEFs (Figure 1B). Treatment of wild type and Bax2/2/Bak2/2 MEFs with NOXA or BAD peptides alone did not induce cell death in either cell lines, whereas the combination of both NOXA and BAD peptides induced cell death in wild type or Bax2/2/ Bak2/2 MEFs (Figure 1C). To ensure that the cell death was not due to high concentration of the peptides, we designed a mutant version of the BAD peptide, containing two mutations at the conserved residues leucine and aspartate (mutated to alanine). The mutant BAD peptide in combination with NOXA peptide did not cause cell death. To delineate the kinetics of cell death, we treated Bax2/2/Bak2/2 MEFs with BH3 peptides for 1 hour and assessed cell death by propidium iodide (PI) staining (Figure 1D). Treatment with BID and BIM BH3 peptides resulted in significant cell death in Bax2/2/Bak2/2 MEFs compared to the mutant BID BH3 peptide. In order to rule out that this finding was not restricted to embryonic fibroblasts, we tested for PI staining in an epithelial cell line derived from kidneys of BAX and BAK deficient mouse (referred to as BMK). BMK cells also displayed significant cell death after 1 hour of BID or BIM peptide treatments (Figure 1E). The BCL-2 related ovarian killer (BOK) is another pro- apoptotic protein similar to BAX and BAK proteins, in that it contains BH1, BH2 and BH3 domains [22]. In the absence of BAX and BAK we therefore evaluated whether BOK was required for BH3 peptide induced cell death. PCR and western blot analysis both revealed that BOK mRNA and protein were expressed in the Bax2/2/Bak2/2 embryonic fibroblast cells and in epithelial kidney cells (data not shown). We retrovirally infected Bax2/2/Bak2/2 MEFs with short hairpin RNA against BOK, or a control shRNA containing drosophilia HIF (DHIF) (Figure 2A). BID and BIM BH3 peptides induced death in cells containing BAX/BAK Independent Cell Death shRNA against BOK or DHIF (Figure 2B). Collectively these data demonstrate that BH3 peptides that can bind to all the pro- survival BCL-2 proteins can induce cell death independent of BAX, BAK and BOK. Caspase inhibition does not inhibit BH3 peptides induced cell death Caspases are key effectors of apoptosis. We evaluated whether the peptide induced cell death was caspase dependent. To assess whether caspase 9 was involved, we inhibited endogenous caspase- 9 of Bax2/2/Bak2/2 MEFs by the overexpressing dominant negative caspase-9 [23]. BID and BIM peptides still induced cell death in the presence of the dominant negative caspase-9 (Figure 3A). To test whether pan caspase inhibition would prevent cell death, Bax2/2/Bak2/2 MEFs were treated with the pan- caspase inhibitor quinoline-Val-Asp(ome)-CH2-O-phenoxy (Q- VD-OPh). BH3 peptides induce cell death in the presence of Q- VD-OPh (Figure 3B). These results indicate that BH3 peptides might be activating caspase independent cell death in the Bax2/2/ Bak2/2 MEFs. Caspase-independent cell death (CICD) can be prevented by the overexpression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of a pan caspase inhibitor [24]. However, BH3 peptides still induced cell death in Bax2/2/Bak2/2 MEFs overexpressing GAPDH in the presence of the broad spectrum caspase inhibitor Q-VD-OPh (Figure 3C and 3D). We also tested whether autophagy contributed to the BH3 peptide induced cell death. Autophagy induced cell death can occur in the absence of BAX and BAK [25]. To test whether Bax2/2/Bak2/2 MEFs died by activating autophagy, we utilized a well characterized inhibitor of autophagy, 3-methyladenine (3-MA). Bax2/2/Bak2/2 MEFs were pretreated with 3-MA followed by BID BH3 peptide in the presence of 3-MA. 3-MA did not protect against BID BH3 peptide cell death (Figure 3E), therefore suggesting that the BH3 peptide induced cell death in the absence of BAX and BAK was not mediated by autophagy. Collectively, these experiments demon- strate that caspases, caspase independent cell death regulated by GAPDH or autophagy are not likely contributing to the cell death. BH3 peptides do not cause the release cytochrome c in Bax2/2/Bak2/2 MEFs To evaluate whether the BID BH3 peptide induces cytochrome c release, the BID BH3 peptide was added to isolated mitochondria from wild type and Bax2/2/Bak2/2 MEFs. BID BH3 peptide induced cytochrome c release in the wild type MEFs but not in the Bax2/2/Bak2/2 MEFs. The mutant BID BH3 peptide did not cause cytochrome c release in either wild type or Bax2/2/Bak2/2 isolated mitochondria (Figure 4A). Because cytochrome c was not released from the isolated mitochondria of Bax2/2/Bak2/2 MEFs, we further evaluated whether cytochrome c was released in live cells. Wild type and Bax2/2/Bak2/2 MEFs were transfected with cytochrome c tagged with GFP and time lapse microscopy was performed. While the mutant BID peptide did not display release of cytochrome c from the mitochondria, both BID and BIM peptides induced cytochrome c release in the wild type cells as demonstrated by the diffusion pattern of the GFP throughout the cell (Figure 4B). However, Bax2/2/Bak2/2 MEFs treated with BID and BIM peptides, did not display diffused GFP within the cell, which is indicative of a lack of cytochrome c release (Figure 4C). Western blot analysis further confirmed that the mutant BID, BID or BIM BH3 peptides did not induce the release of cytochrome c in Bax2/2/Bak2/2 MEFs as detected by the retention of cytochrome c in the mitochondrial pellet fraction PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5646