Cellular Retention of TKI and Apoptosis Commitment Figure 4. Recovery kinetics of intracellular signaling pathways upon HD-TKI pulse-exposure. (A) Ba/F3-BCR-ABL cells were treated either with 25 mM imatinib or with 100 nM dasatinib for 2 hours followed by drug wash-out and consecutive replating as described in Figure 1B. Untreated cells served as positive controls for phosphorylation signals (‘‘M’’). Cells treated continuously for 2 hours or 10 hours (2 h and 10 h) served as positive controls for dephosphorylation of intracellular signalling pathways. ‘‘M’’: medium; ‘‘1x’’, ‘‘2x’’, ‘‘3x’’: Cells were treated with HD-TKI for 2 h and drug wash-out was performed 1–3x in 2 h intervals, respectively. Lysates were prepared 2 h after drug wash-out, allowing for recovery of phosphorylation signals in case of absent TKI activity. ‘‘S1’’, ‘‘S2’’, ‘‘S3’’: Lysates were prepared upon incubation of previously untreated cells for 2 h with the respective cell culture supernatants obtained during each washing procedure. (B) FACS analysis of Ba/F3-BCR-ABL cells after HD-TKI exposure followed by repetitive drug wash-out (‘‘1x’’, ‘‘2x’’, ‘‘3x’’) using phosphorylation-specific antibodies against CRKL (upper panel) and STAT5 (lower panel). Cells were treated with respective TKI for 2 h as indicated and washed as explained in Figure 1B. Two hours after each washing step cells were fixed and permeabilized. Antibody staining was performed in parallel with p-CRKL-Alexa488 and p-STAT5-Alexa647 antibody. Untreated cells served as a control for phosphorylation signals. All experiments were performed at least in triplicate and one representative experiment is shown. doi:10.1371/journal.pone.0040853.g004 ABL cells and K562 cells (a CML blast crisis cell-line) were either treated with 25 mM imatinib or 100 nM dasatinib for 2 h followed by three consecutive rounds of thorough medium exchange (each consisted of 2 washing steps) at 2 h intervals. FACS analysis for apoptotic cell death was performed at 24 h (for details see Figure 1). In line with previously published data, apoptosis measured as subG1-DNA content (Figure 2), AnnexinV-positivity and caspase3 cleavage (Figure 3), respectively was efficiently induced by HD-TKI pulse-exposure in both cell-lines (Figure 2 and Figure 3, upper panel: ‘‘1x’’) while lower TKI concentra- tions did not result in apoptotic cell death upon pulse-exposure (see Figure S2A). As a control, off-target effects were ruled out by HD-TKI pulse-exposure of parental Ba/F3 cells with either imatinib or dasatinib (see Figure S2B). Induction of apoptosis after HD-TKI pulse-exposure could also be observed in the context of other oncogenes: Ba/F3 FLT3-ITD cells and MV 4– 11 cells treated with midostaurin and Ba/F3 JAK2V617F cells treated with JAK-Inhibitor I showed similar results (data not shown). This further confirmed HD-TKI pulse-exposure induced apoptosis as being target specific and independent of cellular background. Pulse-exposure using HD-TKI was previously reported to irreversibly induce cell death. However, unexpectedly, when cells were extensively washed a second (2 h post the initial media exchange) and a third time (4 h post initial media exchange) almost all cells survived HD-TKI pulse treatment and were rescued from apoptotic cell death (Figure 2 and Figure 3, upper panel: ‘‘2x’’ & ‘‘3x’’; Figure S3A+B). Moreover, incubation of previously untreated cells with cell culture supernatants derived from the second (S2) media exchange resulted in substantial increase in the fraction of apoptotic cells (Figure 2 and Figure 3, lower panel: ‘‘S2’’; Figure S3A+B). Similar results were obtained using FLT3-ITD or JAK2V617F expressing cell-lines (Ba/F3 FLT3-ITD, MV4-11 and Ba/F3 JAK2V617F) which were treated with midostaurin or JAK-inhibitor I, respectively (data not shown). Since we employed a drug wash-out protocol that was slightly different from previously published experimental procedures, as a control, we performed a set of experiments following the drug wash-out procedure published by Shah and colleagues. [12] Employing this wash-out protocol repetitively (three times every 2 hours), we were able to confirm our findings as described above (Figure S3A+B). These results were surprising as the first extensive drug wash-out should have removed all biologically active TKI. This raised the possibility that intracellular TKI retention, leakage into the medium over time and thus prolonged presence of biologically active TKI concentrations might be responsible for induction of cell death upon HD-TKI pulse- exposure. HD-TKI Pulse-exposure Results in Prolonged Inhibition of Intracellular Signaling Nodes To test for residual TKI activity, we then investigated the kinetics of inhibition of key signaling nodes (BCR-ABL, STAT5 and CRKL) in response to HD-TKI pulse-exposure. HD-TKI exposure results in early dephosphorylation of BCR-ABL (Y412), STAT5 (Y694), and CRKL (Y207), while BCR-ABL (Y177) did not show significant dephosphorylation after 2 h TKI exposure (Figure 4A, lane 8, ‘‘2 h’’). After the first round of drug wash-out (lane 5, ‘‘1x’’), STAT5 signaling was still suppressed, while CRKL appeared to be largely rephosphorylated again. These effects were seen with both imatinib and dasatinib. Using imatinib, analysis of BCR-ABL phosphorylation (Y412) showed strong inhibition of autophosphorylation and BCR-ABL (Y412) remained dephos- phorylated after the first round of drug wash-out, while this was less evident using dasatinib (Figure 4A, ‘‘1x’’). To achieve better quantitation of signaling events upon drug wash-out, we performed multiparameter intracellular flow cytometry using phospho-specific antibodies against CRKL and STAT5. These experiments confirmed our western blot data, demonstrating strong dephosphorylation of STAT5 and only slight dephosphor- ylation of CRKL after the first drug wash-out upon HD-TKI pulse-exposure (Figure 4B, ‘‘1x’’). Recovery of STAT5-phosphorylation levels correlated well with rescue from apoptotic cell death: We observed gradual reappear- ance of STAT5-phosphorylation signals after the second round of drug wash-out (‘‘2x’’) reaching complete recovery of phosphory- lation signals in all cell-lines tested upon the third round (‘‘3x’’) of media exchange (Figure 4B). At the same time, the proportion of apoptotic cells decreased accordingly (Figure 2 and Figure 3). As an internal control, incubation of previously untreated cells with supernatants derived from the corresponding washing steps 1 (S1), 2 (S2) and 3 (S3) perfectly mirrored the results observed in washed cells: phosphorylation signals in S1 resemble those after 2 h TKI treatment, phosphorylation signals in S2 resemble those in ‘‘1x’’, and phosphorylation signals in S3 resemble those in ‘‘2x’’ (Figure 4A). These data could also be confirmed independently using K562 cells (Figure S4A). Again, observation of biological activity in supernatants 2 (S2) and 3 (S3) was surprising as the first extensive washing procedure should have removed all remaining biologically active TKI. This indicates an abundance of active TKIs in supernatants obtained after drug wash-out. Thus, in line with data presented above, these results suggest intracellular retention of TKIs which is released into the re-plating media over time. BCR-ABL Signaling Nodes Exhibit Different Kinetics upon TKI Exposure Next, we investigated biochemical dose response of phospho- tyrosine residues of BCR-ABL (Y412 and Y177), CRKL and STAT5 in Ba/F3-BCR-ABL and K562 cells upon TKI exposure PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40853