Regulation of Bcl-2 Proteins Independent of BH3 Figure 3. Bcl-xAK-mediated apoptosis depends on Bax or Bak. (A, C) Expression of Bax and Bak is shown by Western blot analysis in subclones of HCT116 and DU145, respectively. Equal loading was confirmed by incubation with b-actin. Two independent series of protein extracts revealed largely comparable expression. (B) HCT116 parental cells (Bax+/Bak+) as well as subclones (Bax2/Bak+), (Bax+/Bak2) and (Bax2/Bak2) were transduced with AdV-AK (MOI = 50) and cultured under OFF or ON conditions. Relative DNA fragmentation values (apoptosis ELISA) were normalized according to the values of parental cells under OFF conditions (set to 1). (D) DU145 parental cells (Bax2/EGFP-Bak2) as well as subclones (Bax2/EGFP-Bak+) and (EGFP-Bax+/EGFP-Bak2) were transduced with AdV-AK (MOI = 50, 100) and cultured under OFF or ON conditions. The percentages of apoptotic cells (sub-G1 populations) are shown, as determined by flow cytometry at 48 h after transduction. (B, D) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Statistical significance as determined by Student’s t-test is indicated by asterisks (*, p,0.05; **, p,0.005), when comparing parental cells and subclones under ON conditions. doi:10.1371/journal.pone.0034549.g003 cell proliferation of Mel-2a cells was strongly decreased, reaching a loss of 60% at 72 h (Fig. 1F). Also cell viability, determined by calcein staining, was decreased (38% in Mel-2a at 72 h), as compared to 6% under Off conditions (Fig. 2I, J). Thus, Bcl-xAK triggered delayed but efficient induction of apoptosis in melanoma cells. Activation of caspases and mitochondria through adenovirus-encoded Bcl-xAK Targeting of the caspase cascade was investigated in Mel-2a cells by Western blot analyses for the initiator caspases 8 and 9 as well as for the main effector caspase 3. Under conditions of high adenovirus-mediated expression of Bcl-xAK and strong apoptosis induction, also significant processing of these caspases was evident at 48 h of transduction (Fig. 2A). Underlining the role of caspases, Bcl-xAK-induced apoptosis was almost completely blocked by the pancaspase inhibitor zVAD-fmk (10 mM; Fig. 2B). The effects on mitochondrial proapoptotic pathways were monitored by two distinct mitochondrial membrane potential (Dym)-dependent dyes. Both JC-1 and TMRM+ revealed the same result, namely decrease of Dym upon Bcl-xAK expression. Interestingly, loss of Dym appeared already at 24 h after AdV- AK transduction, thus proving this as an early step in Bcl-xAK signal transduction, before apoptosis became evident (Fig. 2C). Reactive oxidative species (ROS) are regarded as an additional step in apoptosis regulation. Increased ROS levels were deter- mined by flow cytometry after H2DCFDA staining and found in Mel-2a cells at 48 h but not at 24 h after transduction, thus characterizing this step likely as a consequence of apoptosis (Fig. 2D). Thus, increased ROS may further enhance the apoptotic effect, which was proven by pretreatment for 1 h with the antioxidant N-acetyl cysteine (NAC). Neutralization of ROS by NAC (Fig. 3D) resulted in a two-fold decrease of Bcl-xAK- induced apoptosis (Fig. 2E). Despite the clear involvement of the mitochondrial pathway, levels of other Bcl-2 proteins remained rather stable after transduction with AdV-AK, as shown by Western blot analysis at 24 h and at 48 h for Bcl-2, Mcl-1, Bax, Puma and Noxa. Similarly, there were no significant changes of the levels of p53 or Survivin (Fig. 2F). Dependency on Bax and Bak To address the relation of Bcl-xAK-induced cell death to Bax and Bak, we used a HCT116-derived colon carcinoma cell model. This consisted of parental Bax+/Bak+ cells and sublines with either Bax knockout or Bak knockdown as well as Bax2/Bak2 double knockdown cells (Fig. 3A). AdV-AK (50 MOI, 48 h) revealed strong apoptosis induction in parental cells, whereas both Bax and Bak single knockdown significantly diminished apoptosis, indicat- ing that both proteins may be engaged by Bcl-xAK. In accordance, Bcl-xAK-induced apoptosis was completely abrogated in the double knockdown cells (Fig. 3B). PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34549