FTI-induced inhibition of cell viability and apoptosis in CML cells is mediated by activation of caspase-3. (A) FTI-induced activation of caspase-3. Marrow cells from a patient with CML in chronic phase were cultured for 24 hours in medium alone (untreated) or treated with a representative FTI (1 μM SCH) or with SCH + caspase-3 inhibitor Z-DEVD-FMK (50 μM). Caspase-3 activity was measured by flow cytometry using the fluorogenic caspase-3–specific substrate DEVD-AMC as described in “Materials and methods.” At least 1 × 10⁴ cells were examined in each assay. (B) Caspase-3 triggering by SCH in living CML cells. Marrow cells from a representative case of CML were cultured for 24 hours in control medium (– FTI) or treated with SCH (1 μM; + FTI). Caspase-3 was measured by flow cytometry using the cell-permeable FITC-labeled peptide FAM-VAD-FMK in combination with propidium iodide to distinguish dead cells from living cells: 65% of living CML cells (lower right quadrant) are stained with FAM-VAD-FMK. (C) Caspase-3 activation in CML cells is induced early after FTI exposure. Western blot analysis of active caspase-3 in CML cells treated for 6 and 24 hours with SCH (1 μM). (D) Inhibition of caspase-3 partially reverted FTI-induced inhibition of CML cell viability, as measured by the MTT assay. Bars represent the cumulative mean ± SEM after exposure to any FTI, expressed as percentage of control: 51.7% ± 6% versus 73.2% ± 2%, in absence and presence of Z-DEVD-FMK, respectively; P = .005. (E) Z-DEVD-FMK partially blocked FTI-induced apoptosis in marrow cells. Flow cytometric detection of apoptosis from a CML patient in chronic phase who showed in vitro susceptibility to a representative FTI (SCH [1 μM]). A minimum of 6 × 10⁵ events were counted per sample. Horizontal bars indicate hypodiploid DNA peak and its percentage.