Cellular Retention of TKI and Apoptosis Commitment immediately after 2 h incubation with 25 mM 14C-labeled imatinib represents the amount of imatinib present in the incubation medium (‘‘EOE’’). Intervals 0, 15, 30, and 120 min represent the time elapsed after initial drug wash-out and replating in fresh cell culture media. doi:10.1371/journal.pone.0040853.g006 (Figure 5 and Figure S4B). Using Western blotting and intracellular phospho-flow analysis, we observed a rapid down- regulation of STAT5-phosphorylation after 20 min exposure to low TKI concentrations (0.25 mM imatinib or 1 nM dasatinib) with complete inhibition of STAT5-phosphorylation occurring at higher concentrations (Figure 5). At the same time, CRKL- phosphorylation remained almost unchanged following exposure to low imatinib (0.25 mM) or dasatinib (1 nM) concentrations (Figure 5). A slight decrease in CRKL-phosphorylation was observed upon exposure to 2.5 mM imatinib or 10 nM dasatinib. Complete dephosphorylation of CRKL was achieved upon exposure to very high concentrations of imatinib (25 mM) or dasatinib (100 nM) only (Figure 5). When investigating later time-points (2 h and 12 h), we did not observe obvious changes in CRKL-phosphorylation levels as compared to the 0.3 h time-point (Figure 5). Looking at BCR-ABL-phosphorylation itself, we confirmed our previous observation that BCR-ABL (Y412) behaves similarly to STAT5, while BCR-ABL (Y177) exhibits only minor phosphorylation changes at least at the 2 h time-point examined by Western blot in this study (Figure 5). In summary, our data indicate that suppression of BCR-ABL kinase activity, as measured by dephosphorylation of BCR-ABL (Y412) and STAT5 (Y694), does not necessarily result in immediate suppression of either CRKL (Y207)- or BCR-ABL (Y177)-phosphorylation. Thus, in our hands, the phosphorylation status of CRKL and BCR-ABL (Y177) did not correlate well with the presence or absence of biologically active TKI concentrations. Taken together, these results suggest that dephosphorylation of CRKL and BCR- ABL (Y177) are inadequate surrogates for inhibition of BCR-ABL kinase activity in the setting of experimental drug wash-out. TKIs Show Intracellular Accumulation and are Released in a Time-dependent Fashion upon HD-TKI Pulse-exposure The findings described above suggest that the effects observed upon HD-TKI pulse-exposure are attributable to intracellular TKI retention resulting in prolonged inhibition of intracellular signaling pathways. To confirm whether redistribution of TKIs from pulse-treated cells into the re-plating medium occurs, TKI concentrations (imatinib and dasatinib) were analyzed intracellu- larly and in cellular supernatants. As an initial approach, we treated K562 cells with 25 mM 14C- labeled imatinib. Measurement of the cellular phase immediately after our standard drug wash-out protocol (Figure 6A, ‘‘EOE’’) revealed a dramatic cellular drug uptake: given a mean cellular volume of 2.487610212 L/cell (see Table S1B), the intracellular imatinib concentration (based on ‘‘EOE’’, Figure 6A) was calculated to be about 4.2 mM. We then re-plated the cells in cell culture medium and subsequently measured intracellular and extracellular imatinib concentrations simultaneously at predefined time-points (0, 15, 30, and 120 min). This resulted in almost complete removal of imatinib in the extracellular compartment (time-point 0 min, Figure 6B), while the intracellular drug concentration remained relatively high (time-point 0 min, Figure 6A). Importantly, we then observed a time-dependent increase of extracellular imatinib concentration reaching a mean final concentration of 0.5360.18 mM at 120 min after drug wash- out (Figure 6B). This was paralleled by a time-dependent decrease in cellular imatinib concentration (Figure 6A). Thus, these findings indeed demonstrated significant TKI release from cells into the medium. To control for technical quality of drug wash-out, we performed up to 4 serial washing steps and measured imatinib concentration in cell culture supernatants immediately after resuspension of cells: We found that increasing washing steps beyond 2 did not result in further relevant decrease of imatinib concentration (see Figure S5). Imatinib remained detectable even after as much as 4 washing steps suggesting continuous drug release from intracellular reservoirs. Next, we set up an HPLC-based method for measurement of extracellular and intracellular TKIs to be able to measure different TKIs without being dependent on the availability of isotope labeled TKIs. First, we measured imatinib in cell culture supernatants and cellular lysates to test whether it is technically feasible to confirm the results obtained with radioactively labeled imatinib. We found close correlation between HPLC-data and those obtained using 14C-labeled imatinib (Figure 7A, panel I and Table S1A). To test whether this mechanism is also operative in an independent BCR-ABL driven cell model and in primary human cells, we then investigated imatinib release upon HD-TKI pulse-exposure using different cell populations: Ba/F3-BCR-ABL cells as well as mononuclear cells from untreated CML patients and normal CD34+ hematopoietic progenitor cells from healthy donors exhibited the same kinetics as previously demonstrated for K562 cells (Figure 7A, panels II–IV). Next, we evaluated a method for extracellular and intracellular detection of dasatinib. Similarly to imatinib, dasatinib exhibited massive intracellular accumulation as well as slow time-dependent drug release into cell culture media following drug wash-out. In Ba/F3-BCR-ABL cells, dasatinib reached a final mean concentration of 1.22 nM in the extracellular media 2 h after drug wash-out (Figure 7B, panel I). This is well above the reported in vitro IC50 level of 0.5–1 nM. [20,21] A similar result with even higher dasatinib levels was obtained in normal CD34+ cells from healthy donors (Figure 7B, panel II). Together, these findings provide experimental evidence that biologically active TKI is retained intracellularly upon HD-TKI pulse-exposure in BCR-ABL positive cell-lines and in primary human cells. Overexpression of ABCB1 Prevents Apoptosis upon Imatinib Pulse-exposure ABC-family transporters have been reported to be involved in active TKI efflux and TKI resistance.[22–24] For imatinib, it has been shown that ABCB1 and ABCG2 play a major role in drug efflux-mediated drug resistance.[24–28] If intracellular TKI accumulation and retention indeed mediates apoptosis upon HD-TKI pulse-exposure, we hypothesized that ABC-transporters may prevent cells from apoptosis upon HD-TKI pulse-exposure. Indeed, overexpression of either ABCB1 or ABCG2 in K562 cells resulted in prevention of apoptotic cell death upon HD-imatinib pulse-exposure (Figure 8). Addition of the ABCB1-specific inhibitor PSC833 restored susceptibility to HD-imatinib pulse- exposure (Figure 8 A), while treatment of K562-ABCB1 cells with PSC833 alone did not result in any significant induction of apoptosis (see Figure S6). Discussion Induction of apoptosis upon HD-TKI pulse-exposure has been demonstrated by several groups.[12–14] Based upon these findings, a concept of irreversible commitment to apoptosis upon PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40853