Alternative splicing further increases the number of the Bcl-2 family members. Thus, the bcl-x gene is expressed as a long antiapoptotic form (Bcl-xL) and a short proapoptotic form (Bcl-xS) [14]. We have recently described Bcl-xAK (atypical killer), a new proapoptotic splice product which encloses BH2, BH4 and TM. It completely lacks the BH3 domain, which has been regarded so far as indispensable for the proapoptotic function [15]. For unraveling the mechanism of Bcl-xAK-mediated apoptosis and exploring its possible therapeutic potential, we constructed an adenoviral vector, which mediates its efficient and conditional expression. We show that Bcl-xAK clearly activated the mitochon- drial pathway, and its activity was critically controlled by both pro- and anti-apoptotic Bcl-2 proteins, despite the lack of BH3. Thus, a new model is suggested, in which Bcl-xAK acts as an atypical killer to trigger Bax/Bak-dependent apoptosis. Materials and Methods Cell culture and cell lines Three representative human melanoma cell lines, SK-Mel-13 [16], Mel-2a and A-375 [17] were investigated. For analyzing the function of Bax and Bak, the prostate carcinoma cell line DU145 (DSMZ, Braunschweig, Germany) and the colon carcinoma cell line HCT116 (ATCC, Maryland, MD, USA) were used. Parental DU145 cells are deficient for Bax and reveal only moderate expression of Bak. The cells had been reconstituted by EGFP-tagged Bax or Bak, resulting in DU145-EGFP-Bax and DU145-EGFP-Bak, as described previously [18]. HCT116 parental cells express both Bax and Bak. Isogenic sublines with either Bax knockout or Bak knockdown as well as Bax2/Bak2 double knockdown cells had been kindly provided by B. Vogelstein (John Hopkins Cancer Center, Baltimore) [18]. Subclones of A- 375 melanoma cells resulted from stable tansfection of a pIRES- Bcl-2 plasmid (A375-Bcl-2) or the pIRES empty plasmid (A375- Mock), as previously described [13]. The pIRES plasmid originated from Clontech (Palo Alto, California, USA). Cell lines were cultured at 37uC, 5% CO2 in DMEM (Gibco, Karlsruhe, Germany) supplemented with 10% FCS and antibiotics (Biochrom, Berlin, Germany). For caspase inhibition, cells were preincubated for 1 h with 10 mM of the pancaspase inhibitior zVAD-fmk (R&D Systems, Wiesbaden, Germany), which binds the active sites of caspase-like proteases. Construction of Bcl-xAK adenovirus Bcl-xAK full-length cDNA [15] was subcloned into the Ad5 adenoviral vector pAd5-tTA, according to a strategy described previously [19]. In brief, the cDNA was inserted into the TRE- containing pHVAd2 shuttle vector. The resulting TRE-Bcl-xAK expression cassette was then inserted into pAd5-tTA by homol- ogous recombination, thereby replacing the E1 region and creating pAdV-AK DNA (Fig. 1A). This was transfected into HEK293 cells, and adenoviral plaques corresponding to AdV-AK were propagated. Expression of Bcl-xAK after AdV-AK transduc- tion was suppressed by addition of 1 mg/ml doxycycline to the culture medium (OFF condition), whereas omitting doxycycline resulted in promoter induction (ON condition). An adenoviral vector for expression of myc-tagged Bik/Nbk (Ad5-myc-Nkb- tTA = AdV-Nbk), used here as control, had been described previously [19]. A luciferase-encoding adenovirus (Ad5-CMV- Luc) served as mock control for adenovirus transduction and was applied at the same MOI [20]. Regulation of Bcl-2 Proteins Independent of BH3 Apoptosis, cytotoxicity, cell proliferation and viability For quantification of apoptosis, cell cycle analyses were carried out, and apoptotic cells corresponded to cell populations with hypodiploid nuclei [21]. Therefore, cells were seeded in 24-well plates (50,000 cells per well). After incubation, cells were harvested by trypsinisation, washed with ice-cold phosphate-buffered saline (PBS) and incubated for 1 h with the staining buffer, containing 0.1% sodium citrate, 0.1% triton X-100 and propidiumiodide (PI; 40 mg/ml; Sigma-Aldrich, Taufkirchen, Germany). The DNA content of nuclei was determined by using flow cytometry (FACSCalibur and CellQuest software; Becton Dickinson, Heidel- berg, Germany). As a second assay for quantification of apoptosis, a cell death detection ELISA (Roche Diagnostics, Mannheim, Germany) was applied, which detects mono and oligonucleosomes formed in apoptotic cells. Cytotoxicity was determined in parallel by a cytotoxicity detection assay (Roche Diagnostics), which measures LDH activity in culture fluids. As positive controls for induced cytotoxicity, cells were completely lysed by triton X-100 or were treated with doxorubicin (500 nM, 72 h). Protocols for apoptosis ELISA and LDH release were according to the manufacturer with minor modifications [22]. Cell proliferation (as a product of cell number and mitochon- drial activity) was quantified according to the cleavage of the water-soluble tetrazolium salt WST by mitochondrial dehydroge- nases in viable cells (WST-1 assay, Roche Diagnostics). Cells were seeded in a density of 10,000 per 100 ml in 96-well plates, and treatments started after 24 h. At the time of analysis, WST-1 reagent was added and absorbance (450 nm) was determined in an ELISA reader. Data were reported in percent of non-treated controls. Cell viability at the single cell level was monitored by the life-cell labeling dye calcein-AM. Briefly, 105 cells were incubated with calcein (4 mM; eBioscience, Frankfurt, Germany) in serum- free growth medium (60 min, 37uC). After PBS washing, cell viability was determined by flow cytometry, comparing calcein- stained (viable) and unstained (dead) cells. For identification of chromatin condensation and nuclear fragmentation in course of apoptosis, cells were harvested by trypsinisation, centrifuged on cytospins and fixed for 30 min in 4% formaldehyde. Cytospins were stained with bisbenzimide (Hoechst-33258; Sigma, Taufkirchen, Germany; 1 mg/ml, 30 min) and examined by fluorescence microscopy. Apoptotic cells were identified by fragmented nuclei or by bright blue-stained nuclei with condensed chromatin. For quantitative evaluation, fields with 100–200 cells were assessed in triplicates. Cell transfection Melanoma cells were seeded in six-well plates with 26105 cells/ well. For transient transfection, cells at a confluence of 50% were washed with serum-free Opti-MEM medium (Life Technologies, Carlsbad, CA, USA), followed by incubation at 37uC in Opti- MEM for 4 h with plasmid DNA (2.5 or 5 mg/ml) and 0.1% DMRIE-C (Life Technologies). Detailed protocols for transient cell transfection had been described previously [22]. Plasmid constructs of pcDNA3 (Invitrogen, Eugene, OR, USA) were used for transient transfection to express full length Bcl-xL and Bcl-xAK. Mitochondrial membrane potential and ROS For determination of the mitochondrial membrane potential (Dym), the fluorescent dye JC-1 (5,59,6,69-tetrachloro-1,19,3,39- tetraethyl-benzimidazolyl carbocyanine iodide) or the dye TMRM+ (Tetramethyl rhodamine methyl ester perchlorate) were used (both from Sigma-Aldrich). Cells were harvested by trypsinisation and stained for 15 min at 37uC with JC-1 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34549