Cellular Retention of TKI and Apoptosis Commitment Figure 3. AnnexinV- and Cleaved Caspase3-staining after high-dose TKI pulse-exposure. To confirm results obtained with propidium iodide staining, AnnexinV-PE staining (A) and Cleaved Caspase3-Alexa488 staining (B) of Ba/F3-BCR-ABL cells was performed at 24 hours after HD-TKI pulse-exposure using either imatinib or dasatinib as indicated followed by repetitive wash-out as described in Figure 2. Experiments were performed in triplicate and one representative experiment is shown. doi:10.1371/journal.pone.0040853.g003 caspase3(asp715)-Alexa488 (Cell Signaling Technology, Danvers, MA, USA). Flow cytometry analysis was done using FACSDiva-Software on a FACS Canto II cytometer (Becton Dickinson, Heidelberg, Germany). Final data analysis was performed using FlowJo Software (Tree Star, Ashland, Oregon, USA). Radioactive Imatinib Uptake and Release Assay For uptake of 14C-labeled imatinib (Novartis, Basel, Switzer- land) 56104 cells/ml were incubated in the presence of 25 mM 14C-labeled imatinib for 2 h at 37uC. Cellular and aqueous phases were subsequently separated by centrifugation. The supernatant was prepared for analysis. The cell pellet was washed twice with 1 ml PBS/56104 cells as described above and samples were either subjected to replating in TKI-free cell culture media or prepared for analysis. Prior to measurement of intracellular imatinib concentration 56104 cells were washed twice with 1 ml PBS and subsequently resuspended into 1 ml of media and then mixed with 3 ml scintillation liquid (luma safe plus, Perkin Elmer, Rodgau, Germany). Specific activity of 14C-labeled imatinib was measured using a Packard TriCarb 1600TR liquid scintillation analyzer (Perkin Elmer, Rodgau, Germany). Concentrations of imatinib in cells and supernatants were calculated using a standard curve. HPLC Measurements of TKIs An Extrelut H NT 3 tube (Merck KGaA, Darmstadt, Germany) was used for the imatinib and midostaurin extraction from medium. 3 ml of a sample were added to the extraction tube and after 10 minutes the extraction tube was washed with 15 ml of diethyl-ether/ethyl-acetate (50:50, v/v) and then with 15 ml of chloroform/isopropanol/ammonia (84:15:1, v/v/v). The solvents were collected in a glass reaction tube and evaporated until dry. The residue was redissolved in 200 ml of the mobile phase. For the dasatinib extraction from medium, the same method was used with the exception that a mixture of 2990 ml of a sample and 10 ml of 1 mg/l D-5 Fentanyl (internal standard) was added to the extraction tube. Imatinib quantification in cell culture supernatants was performed using an Agilent HPLC 1200 Series system with a diode array detector operated at 265 nm. For midostaurin the DAD was operated at 286 nm and the additional detection with a FLD was accomplished with excita- tion/emission wavelengths set at 286/386 nm. Data acquisition and integration was performed by ChemStation for LC 3D Systems. A Lichrospher 100-5 RP8, 25064 mm column (Ma- cherey-Nagel GmbH & Co. KG, Du¨ ren, Germany) maintained at 3061uC for imatinib and at 4061uC for midostaurin was used for the separation. 50 ml of a prepared sample were injected into the HPLC and eluted in a mobile phase of 0,05 M H3PO4/KH2PO4 - CH3CN (7:3, v/v) for imatinib or of CH3OH-H2O-NH3 (89,7:10:0,3, v/v/v) for midostaurin at a flow rate of 1 ml/min. For dasatinib quantification an Agilent HPLC 1100 Series with a PE Sciex API 2000 LC/MS/MS System was used. The detector was operated in the MRM scan type with the 488.1/401.0 precursor/product ion for dasatinib and 342.4/188.2 for D-5 Fentanyl. Data acquisition and integration was performed by the Analyst 1.5 software. A Zorbax SB-C18, 4.6650 mm, 5 micron column (Agilent Technologies Deutschland GmbH, Bo¨blingen, Germany) maintained at 4561uC was used for the separation. In total, 20 ml of a prepared sample were injected into the HPLC and eluted in a mobile phase consisting of A: CH3OH and B: 50 ml 0,1 M CH3COONH4+50 ml CH3CN +950 ml H2O +1 ml CH3COOH with a A:B ratio of 7:3, v/v and a flow rate of 0,3 ml/min. Determination of Cell Size Ba/F3-BCR-ABL cells were identified and photographed under a bright field fluorescence microscope (Axio Observer A1, Zeiss, Jena, Germany) and cell diameter was measured using Meta- MorphH image analysis software. The mean diameter of 100 cells was used to calculate cellular volume, assuming that Ba/F3-BCR- ABL cells are spheric. Intracellular imatinib concentration was calculated using the following formula: CTKI supernatant~CTKI intracellular|Vcells|1=Vsupernatant Intracellular TKI-concentration of a single cells can be calculated as follows: CTKI intracellular~CTKI supernatant|Vsuperntant|1=Vcells Statistical Analyses Data are presented as means + SEM if not otherwise stated. Results HD-TKI Pulse-exposure does not Alter Kinetics of Apoptosis In previous studies, the underlying molecular mechanism for cell death upon high-dose TKI (HD-TKI) pulse-exposure remained elusive.[12–14] We speculated whether HD-TKI pulse-exposure may alter kinetics of cell death which would result in earlier onset of irreversible commitment to apoptosis. Thus, we treated BCR-ABL (p 210) expressing Ba/F3 cells (Ba/F3 BCR- ABL) with high-dose and conventional dose of either imatinib or dasatinib. Protein lysates were obtained at 0 h, 2 h, 4 h, 6, 8 h, 10 h, 12 h, and 24 h of treatment and monitored for caspase3 cleavage by immunoblotting. A rather uniform onset of caspase3 cleavage was observed after 6 to 8 h of TKI treatment, independent of TKI concentration or inhibitor used (see Figure S1). Thus, apparently, onset and kinetics of cell death program upon HD-TKI pulse-exposure was not altered. Therefore, it appeared to be unlikely that an alternative apoptotic pathway was activated by HD-TKI pulse-exposure. Repetitive Drug Wash-out Prevents Apoptotic Cell Death Upon High-dose TKI Pulse-exposure To clarify, whether the wash-out regimen used affected drug efficacy, we set up experiments with serial drug wash-out procedures to observe the effects on cell death: Ba/F3 BCR- PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40853