Fig. 2

ALDH^Bright cells are resistant to BTZ but sensitive to DSF/Cu²⁺, and BTZ-resistance confers increased resistance to Ara-C in CMY cells. CMY cells were treated with 5 nM BTZ and processed for ALDH activity using ALDELUOR^TM assay and flow cytometry. Dot plot shows the percentage of ALDH-positive cells (FITC) on the x-axis, and the sideward scatter (SSC-A) on the y-axis. The gated cell populations were created using the ALDH inhibitor DEAB provided with the kit. One percent of the ALDH^Bright cell population was resistant to BTZ (a). Because these cells may play a role in BTZ resistance, this subpopulation was flow-sorted and subsequently subjected to drug dose response assays of either DSF/Cu²⁺ or a re-exposure to BTZ. The BTZ-resistant ALDH^bright subpopulation of CMY cells was still resistant to BTZ (IC₅₀ 7.7 nM) compared to untreated, unsorted CMY cells (IC₅₀ 2 nM), but were sensitive to DSF/Cu²⁺ (IC₅₀ 56 nM vs 52 nM for untreated, unsorted CMY cells) (b). BTZ-resistant variants from CMY and CMK cell lines (CMY-BR and CMK-BR) were generated by exposure to stepwise increasing concentrations of BTZ up to 200 nM for CMY, and 100 nM for CMK. These cells were treated with different doses of Ara-C, VP-16 and daunorubicin. Dose response curves were plotted in comparison to the CMY and CMK parent cell lines. Approximately 25% of the CMY-BR cells remained viable after treatment with high Ara-C doses for 72 h, but the parental cells completely died at the same doses. The CMK cell line and its BTZ-resistant variant (CMK-BR) were both equally sensitive to Ara-C, VP-16 and daunorubicin (c)