cells to apoptosis. This effect was evident upon pulse treatment for only 20 min –4 h.[12–14] It was proposed that depth, rather than duration of kinase inhibition, is the critical determinant for TKI efficacy. [12,13] However, the molecular mechanism for apoptosis induction after HD-TKI pulse-exposure has remained elusive. In our present work, we demonstrate that dramatic intracellular drug retention mediates apoptotic cell death upon HD-TKI pulse- exposure. In line with this, over-expression of ABC transporters prevented cell death upon HD-TKI pulse-exposure. These findings will be useful to rethink our current framework of pharmacokinetic requirements of TKIs for CML and other diseases. In addition, these studies refine the molecular concept of TKI-induced apoptosis. Materials and Methods Ethics Statement Patient blood samples were drawn after written informed consent was obtained. Experimentation on human material was performed according to the premises of the Helsinki declaration and was approved by the local ethics committee (University Medical Center, Otto-von-Guericke University Magdeburg, Germany). Cell-lines and Patient Samples Hematopoietic Ba/F3 cells (DSMZ, Braunschweig, Germany) either parental or stably expressing p210-BCR-ABL (Ba/F3-BCR- Cellular Retention of TKI and Apoptosis Commitment ABL) [15] were used. K562 cells were obtained from DSMZ (Braunschweig, Germany). K562-Dox cells (referred herein as K562-ABCG1 cells) were kindly provided by J. Melo (Department of Haematology, Centre for Cancer Biology, University of Adelaide, Australia). [16] K562-ABCG2 cells were kindly provid- ed by Sheng Zhou (Division of Experimental Hematology, Department of Hematology, St. Jude Childrens Research Hospi- tal, Memphis, USA). [17] Cells were cultured in RPMI1640 containing 4 mM L-glutamine and 10% FCS at 37uC in humidified air containing 5% CO2. Media for parental Ba/F3 cells was supplemented with 10% WEHI-conditioned media. Primary CML samples (MNC) as well as CD34+ cells from normal controls were isolated and stored in liquid nitrogen. Upon thawing, cells were cultured in RPMI1640 supplemented with 20% FCS, 50 U/ml penicillin, and 50 mg/ml streptomycin. Reagents Cells were treated either with dasatinib (Selleck Chemicals LLC, Houston, TX, USA) or imatinib (kindly provided by Novartis, Basel, Switzerland). A 10 mM stock solution was prepared in DMSO and stored at 220uC. Stocks were further diluted in cell culture medium. The final DMSO concentration was #0.35%, depending on the inhibitor concentration used. DMSO exposed cells were used as controls in all experiments. For some experiments 10 mM of the ABCB1 inhibitor PSC833 (Tocris, Missouri, USA) were used. Figure 1. Experimental set-up for analysis of induction of apoptosis upon HD-TKI exposure. (A) Cells were seeded at a density of 56104 cells/ml in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS. Cells were treated for 2 h with TKI as indicated. Then, cells were washed twice with 2 ml PBS at room temperature and replated in fresh cell culture media (2 ml final volume). Cells exposed to 0.35% DMSO served as controls. 24 h after start of TKI exposure percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining. (B) To analyze for residual TKI activity upon HD-TKI pulse exposure, a second and third drug wash-out procedure (each consisting of 262 ml PBS washing) was performed: Cells were treated with TKI for 2 h. Cells initially pulse-exposed to HD-TKI were washed twice with 2 ml PBS at room temperature and replated in 2 ml fresh media (density: 56104 cells/ml) as described in (a) (‘‘1x’’). To test for residual TKI-activity, the cell culture supernatant was transferred to previously untreated cells (‘‘S1’’), which were subsequently incubated for 24 h. Two hours after replating, a second drug wash-out was performed (262 ml PBS). Cells were again replated in 2 ml fresh media (‘‘2x’’). Again supernatants were transferred to previously untreated cells (‘‘S2’’). This procedure was repeated for a third time (‘‘3x’’, ‘‘S3’’). doi:10.1371/journal.pone.0040853.g001 PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40853