restores ABT-737 sensitivity in Fbw7-deficient T-Cell Acute Lymphoblastic Leukemia cells, indicating that elevated Mcl-1 expression is important for Fbw7-deficient cells to evade apoptosis [17]. We have previously shown that by treating melanoma cells with 9.2.27PE, the Mcl-1 protein level is rapidly decreased due to its short half-life [11]. We therefore hypothesized that by combining 9.2.27PE and ABT-737, synergistic cell cytotoxicity could be achieved in melanoma cells with high apoptotic resistance. We have assessed the effect of combining the 9.2.27PE immunotoxin with the BH-3 mimetic ABT-737 in a panel of melanoma cells. The results suggest that the combination treatment causes strong synergistic cytotoxic effects. Importantly, the combination of the two drugs caused a cytostatic effect on the growth of human melanoma xenograft in mice. Materials and Methods Immunotoxins and reagents The immunotoxins 9.2.27PE and 425.3PE, the mAb 9.2.27 and the toxin Pseudomonas Exotoxin A (PE) have previously been described [11]. The agents were diluted in PBS 0.1% human serum albumin (Octapharma, Stockholm, Sweden). Control cells were given 0.1% human serum albumin. ABT-737 and its enantiomer A-793844 (kindly provided by Abbott Laboratories, Abbott Park, IL), were dissolved in DMSO (Sigma-Aldrich, St. Louise, MO), and stored at 220uC until used. For the in vivo studies, ABT-737 was dissolved as previously described [18]. The pan-caspase inhibitor Z-VAD-FMK, the cathepsin B/L inhibitor Z-FA-FMK and the caspase-3 inhibitor Z-DEVD-FMK were from Calbiochem (La Jolla, CA). Cycloheximide (CHX) and Staurosporine (STS) were from Sigma-Aldrich, and Tunicamycin was from Sigma Chemical (Castle Hill, Australia). Control cells were given dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Antibodies The following antibodies were used; anti-a-tubulin (Calbio- chem, La Jolla, CA), anti-GAPDH (Applied Biosystems, Mulgrave, Australia), anti-PARP (Calbiochem and BD Bioscience, San Jose, CA), anti-caspase-3 (R&D Systems, Minneapolis, MN), anti-BAX, anti-peIF2a, anti-eIF2a (Cell Signaling Technology, La Jolla, CA), anti-Mcl-1, anti-GRP78/BiP and anti-CHOP/GADD 153 (Santa Cruz Biotechnology, Santa Cruz, CA). Cell culture The FEMX, Melmet-1, Melmet-5 and Melmet-44, previously described [19,20], were kept in RPMI-1640 medium supplemented with 8% heat inactivated fetal calf serum, Hepes and Glutamax (Gibco, Paisley, UK) at 37uC. The MM200 and MelRM (kindly provided by P. Hersey, Calvary Mater Newcastle Hospital, Australia, [21,22]), were kept in DMEM (Sigma-Aldrich, Castle Hill, Australia) supplemented with 5% fetal calf serum (Sigma- Aldrich), supplemented with 2 mg/ml Sodium Bicarbonate (Chem- Supply, Thermo Scientific, Scoresby, Australia), 20 mg/ml Genta- micin (Pfizer Australia, West Ryde, Australia) at 37uC (100% humidity, 5% CO2, 95% air). For all in vitro experiments, cells were seeded one day prior to start of the experiments, and the cells were in growth phase and never below 60% confluent at start of treatment. The cells were treated with 100 ng/ml 9.2.27PE or 10 mM ABT- 737, unless otherwise indicated. All cell lines were routinely tested and found to be free from contamination with Mycoplasma species. Transduction with short hairpin RNA Mcl-1 were silenced in MelRM cells (MelRMshMcl-1) by transduction using short hairpin RNA (clone ID NM_021960.3- 664s1c1, Sigma-Aldrich) according to manufacturer’s instructions. 9.2.27PE and ABT-737 in Melanoma Control cells (MelRMshCtr) were generated using non-target control sequence (Product no. SH002, Sigma-Aldrich). The expression level of Mcl-1 was determined by Western Blot. Cell viability assay Cell viability of the FEMX, Melmet-1, Melmet-5, Melmet-44, MelRM and MM200 after treatment for 24 or 48 h with 9.2.27PE (1–1000 ng/ml), ABT-737 (0.1–20 mM) or a combination of 9.2.27PE and ABT-737 was measured using CellTiter 96HAQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI) as described previously [11] or the VisionBlueTM Quick Cell Viability Assay (BioVision, Mountain View, CA). We rule out that there was a difference between the two cell viability assays, as the cell viability results of FEMX cells treated with 9.2.27PE and ABT-737 were similar using the two different agents. For this cell line, data from both the methods/agents were used to calculate the cell viability results. Cell viability of 9.2.27PE6ABT-737 treated MelRM, MelRMshCtr and MelRMshMcl-1 (12 and 24 h) was determined in a separate experiment. To determine whether caspases and cathepsins were involved in the decreased cell viability observed, FEMX cells were pre-treated for 45–60 min with Z-VAD-FMK (50 mM)6Z-FA-FMK (50 mM) prior to treatment with 9.2.27PE6ABT-737. Assays were performed in triplicate and repeated at least three times. Furthermore, FEMX and MelRM cells were treated with A- 793844, the enantiomer of ABT-737, with concentrations up to 10 mM for 24 and 48 h (48 h: FEMX cells only). Assays were performed in triplicate and repeated three times. Caspase activity assay The activity of caspase-3/7 in FEMX and Melmet-5 cells were measured using the Caspase-Glo 3/7 (Promega, Madison, WI) as described previously [20]. The cells were pre-treated for 45 min with Z-DEVD-FMK (50 mM) prior to treatment with 9.2.27PE6ABT-737, STS (1 mM) or CHX (10 mg/ml). The activity was measured after 24 h according to the manufacturer’s instructions. Assays were performed in triplicate and repeated three times. Mitochondrial Membrane Potential The mitochondrial membrane potential (Dym) in 9.2.27PE6ABT-737 treated FEMX and Melmet-5 cells using the MitoProbeTM JC-1 Assay Kit (Molecular Probes, Eugene, OR) was performed as described previously [11]. In addition FEMX, Melmet-5 and MelRM were treated with ABT-737 for 24, 32 and 48 h. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at a final concentration of 50 mM was used as depolarization control. The experiments were repeated three times. MelRM, MelRMshCtr and MelRMshMcl-1 cells were also treated with 9.2.27PE+ABT- 737 for 12 h. This experiment was repeated 3 times. Western Blot Analysis FEMX, Melmet-5 and MelRM cells were treated as indicated in the figures and subjected to Western Blot analysis as previously described [11]. Equal amounts of protein (20 mg) were separated by NuPAGE Bis-Tris (Invitrogen, Carlsbad, CA) or SDS-PAGE gels, transferred by electrophoresis to Immobilon membrane (Millipore, Bedford, MA), and probed as previously described [11]. When necessary, the membranes were stripped using RestoreTM Western Blot Stripping Buffer (Thermo Scientific Pierce Protein Research Products, Rockford, IL) and re-probed with the desired primary antibody. PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24012