Regulation of Bcl-2 Proteins Independent of BH3 Figure 2. Activation of caspases and mitochondria. (A) Processing of caspase-3, -8 and -9 is shown in Mel-2a cells at 24 h and at 48 h after transduction with AdV-AK (MOI = 50). Expression of Bcl-xAK was switched on in the absence of doxycycline (ON) or shut off with doxycycline (OFF). Equal protein loading (20 mg/lane) was confirmed by GAPDH. The whole experiment was performed twice. (B) Inhibition of apoptosis by preincubation with the pancaspase inhibitor zVAD-fmk (1 h, 10 mM) is shown. SK-Mel-13 cells had been transduced with AdV-AK (MOI = 100, 48 h). Means and SDs of triplicate values of a representative experiment (one of two) are shown. (C) Decrease of the mitochondrial membrane potential (Dym) is shown for Mel-2a cells at 48 h after transduction of AdV-AK, as determined by flow cytometry after JC-1 or TMRM+ staining. Cultures with doxycycline (OFF, grey) are compared to cultures grown in the absence of doxycycline (ON, open graphs). The experiment was performed three times, resulting in highly comparable results. (D) ROS levels were determined in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK under ON and OFF conditions (flow cytometry after H2DCFDA staining). Below, parallel cultures were pre-treated for 1 h with 200 mM NAC before transduction. (E) Relative DNA-fragmentation rates (apoptosis) at 48 h with or without NAC were determined in parallel. Non-transduced cells (2/ +NAC) are shown as additional controls (open bars). Values had been normalized with regard to non-treated controls, set to 1. Means and SDs of triplicate values of a representative experiment are shown (two independent experiments). (F) Expression levels of Bcl-2 proteins, of p53 and Survivin were determined by Western blot analysis in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK (ON and OFF conditions). Equal protein loading (20 mg/lane) was confirmed by GAPDH. doi:10.1371/journal.pone.0034549.g002 Bcl-xAK full length cDNA under control of a Tet-OFF promoter inserted into the adenoviral E1 region. The tetracycline/ doxycycline repressible transactivator tTA was located in the adenoviral E3 region (Fig. 1A). The construct mediated high expression of Bcl-xAK in melanoma cell lines as shown for SK- Mel-13, A-375 and Mel-2a, when doxycycline was omitted (ON condition), whereas addition of doxycycline almost completely abolished Bcl-xAK expression (OFF condition, Fig. 1B). Significant induction of apoptosis, as determined by counting hypodiploide sub-G1 cells, was seen in melanoma cell lines after transduction and promoter activation, whereas doxycyline strongly diminished apoptosis (Fig. 1D, examples shown in 1C left panel). Kinetic analyses revealed a delayed induction of apoptosis in the three cell lines, which increased to 12%–23% at 48 h and to 17%– 37% at 72 h after transduction (Fig. 1D). In contrast, other proapoptotic Bcl-2 proteins induced apoptosis already at 24 h, as shown here for the BH3-only protein Bik/Nbk subcloned in the same adenoviral background (Fig. 1G). The delay in apoptosis induction by Bcl-xAK occurred despite its adenovirus-mediated high expression already at 6 h after transduction (Fig. 1H). Comparable results concerning increased DNA fragmentation were obtained by an apoptosis ELISA (data not shown). In parallel with DNA fragmentation, clearly visible effects indicating apoptosis were evident, as reduced cell numbers, rounded and detached cells (Fig. 1C, middle panel). Chromatin condensation and nuclear fragmentation, typical hallmarks in apoptosis, were seen after bisbenzimide staining (Fig. 1C, right panel). At 48 h after transduction of Bcl-xAK, the cell numbers with atypical nuclei increased from 4% (Off) to 33% (On). LDH release monitoring loss of plasma membrane integrity was determined to exclude early necrotic cell death. Indeed, LDH release was not significant at 48 h, when apoptosis was already induced, and it was less affected at 72 h, as compared to cytotoxicity controls (Fig. 1E). As determined by WST-1 assay, PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34549