Regulation of Bcl-2 Proteins Independent of BH3 Figure 4. Bax and Bak activation after Bcl-xAK overexpression. (A) For investigation of Bax and Bak clustering, DU145 cells stably transfected for expression of EGFP-Bax or EGFP-Bak were transduced with AdV-AK and cultured for 48 h under OFF or ON conditions. Doxorubicin-treated cells (2 mM, 24 h) were used as positive controls. Examples of fluorescence microscope images taken at 48 h after transduction and promoter induction are shown. (B) A quantitative evaluation of Bax and Bak clustering was performed (means and SDs of triplicate values of a representative experiment). A second experiment revealed comparable results. (C) Bax and Bak activation upon Bcl-xAK expression was determined in Mel-2a at 48 after AdV-AK transduction (50 MOI), by flow cytometry after staining with conformation-specific antibodies against Bax and Bak N termini (Bax/Bak NT). The bars indicate the populations counted as positive for activated Bax and Bak, respectively. (D) A quantification of triplicate values (one experiment of two independent) is shown. Transduction with AdV-Nbk (50 MOI) is shown for comparison. doi:10.1371/journal.pone.0034549.g004 In a complementary approach, a DU145 prostate carcinoma cell model was applied. Parental cells are deficient for Bax and reveal only moderate activity of Bak. They had been reconstituted for either Bax or Bak expression by using EGFP-tagged copies (Fig. 3C). Parental DU145 cells were clearly non-responsive to AdV-AK, possibly indicating an endogeneous non-functional Bak. However, the reconstitution of either Bax or Bak strongly enhanced Bcl-xAK-mediated apoptosis, resulting in each case in more than 50% apoptotic cells. This again showed that Bcl-xAK can induce apoptosis via both Bax and Bak (Fig. 3D). Formation of Bax/Bak clusters has been reported as related to proapoptotic function [23]. For monitoring this step, DU145 cells were used that had been stably transfected with EGFP-Bax and EGFP-Bak, respectively. In agreement with the function of both Bax and Bak, Bcl-xAK expression resulted in visible clustering of both EGFP-Bax and EGFP-Bak at 48 h after transduction. Clustering induced by Bcl-xAK was comparable to the effects of doxorubicin (2 mM, 24 h), used as positive control (Fig. 4A). Evaluations revealed Bax/Bak clusters in 20%–30% of cells, similar to apoptosis inductions at these conditions (Fig. 4B). In course of Bax/Bak activation, conformational changes may lead to exposure of their N- termini. Flow cytometry with N-terminus-specific antibodies (Bax- NT, Bak-NT) showed activation of Bax and Bak in 30% of Mel-2a cells in response to Bcl-xAK expression (Fig. 4C, 4D). Abrogation of Bcl-xAK-mediated apoptosis by antiapoptotic Bcl-2 proteins To address the role of antiapoptotic Bcl-2 proteins, A-375 melanoma cells stably transfected for Bcl-2 overexpression (A375- Bcl-2) were applied. These cells were completely protected against the proapoptotic effects of Bcl-xAK, whereas mock-transfected cells (A375-Mock) revealed about 30% apoptotic cells at 48 h of transduction with AdV-AK (Fig. 5A). A similar result was obtained after Bcl-xL overexpression. Transient transfection of a Bcl-xAK expression plasmid significantly enhanced apoptosis in SK-Mel-13 melanoma cells at 48 h, whereas the co-transfection of a Bcl-xL expression plasmid almost completely prevented Bcl-xAK-induced apoptosis (Fig. 5B). Thus, either one or these antiapoptotic proteins was sufficient to block Bcl-xAK-mediated apoptosis. Loss of Dym was also seen in A375-Mock, which was completely prevented by Bcl-2 overexpression in A375-Bcl-2 (Fig. 5C). Mitochondrial translocation of Bcl-xAK is not prevented by Bcl-2 Hallmarks in mitochondrial apoptosis pathways are transloca- tion of Bax and release of mitochondrial factors. Significant cytochrome c release was seen in Mel-2a and in A375-Mock at 48 h after AdV-AK transduction (Fig. 6A). Also higher levels of PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34549