Figure 7
Figure 7. Expression of mutant NPM1 enhances sensitivity of primary AML cells to NSC348884 and/or ATRA. (A) OCI-AML3 vector and FLT3-ITD overexpressing cells were treated with the indicated concentrations of NSC348884 for 24 hours. After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent the SEM. Inset shows the expression of FLT3-ITD and NPM1 levels in OCI-AML3 vector and FLT3-ITD transfected cells. The expression levels of β-actin in the lysates served as the loading control. (B) OCI-AML3 vector and FLT3-ITD overexpressing cells were treated with the indicated concentrations of NSC348884 and/or ATRA for 48 hours. After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments. Bars represent the SEM. * indicates values significantly greater (P < .05) in the combination compared with treatment with either agent alone. † indicates values significantly less (P < .01) in OCI-AML3 cells with FLT3-ITD overexpression. (C,D) Primary patient-derived AML cells expressing wild-type (n = 4) or Mt NPM1 (n = 3), Mt-NPM1/FLT3-ITD (n = 2), and normal CD34+ progenitor cells (n = 3) were treated with the indicated concentrations of NSC348884 for 48 hours (C), or treated with the indicated concentrations of NSC348884 and/or ATRA for 48 hours (D). After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent the SEM. In panel C, * indicates values significantly greater (P < .01) in the primary AML cells with Mt NPM1 compared with primary AML cells with WT NPM1 and primary CD34+ progenitor cells. † indicates values significantly less (P < .01) in primary AML cells with Mt NPM1/FLT3-ITD compared with AML cells with Mt NPM1 alone. In panel D, * indicates % values significantly greater (P < .05) in the combination treatments compared with treatment with either agent alone. † indicates values significantly less (P < .01) in Mt NPM1/FLT3-ITD AML cells compared with AML cells with Mt NPM1 alone in the combination treatments.

Expression of mutant NPM1 enhances sensitivity of primary AML cells to NSC348884 and/or ATRA. (A) OCI-AML3 vector and FLT3-ITD overexpressing cells were treated with the indicated concentrations of NSC348884 for 24 hours. After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent the SEM. Inset shows the expression of FLT3-ITD and NPM1 levels in OCI-AML3 vector and FLT3-ITD transfected cells. The expression levels of β-actin in the lysates served as the loading control. (B) OCI-AML3 vector and FLT3-ITD overexpressing cells were treated with the indicated concentrations of NSC348884 and/or ATRA for 48 hours. After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments. Bars represent the SEM. * indicates values significantly greater (P < .05) in the combination compared with treatment with either agent alone. † indicates values significantly less (P < .01) in OCI-AML3 cells with FLT3-ITD overexpression. (C,D) Primary patient-derived AML cells expressing wild-type (n = 4) or Mt NPM1 (n = 3), Mt-NPM1/FLT3-ITD (n = 2), and normal CD34+ progenitor cells (n = 3) were treated with the indicated concentrations of NSC348884 for 48 hours (C), or treated with the indicated concentrations of NSC348884 and/or ATRA for 48 hours (D). After treatment, cells were stained with annexin V and propidium iodide and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent the SEM. In panel C, * indicates values significantly greater (P < .01) in the primary AML cells with Mt NPM1 compared with primary AML cells with WT NPM1 and primary CD34+ progenitor cells. † indicates values significantly less (P < .01) in primary AML cells with Mt NPM1/FLT3-ITD compared with AML cells with Mt NPM1 alone. In panel D, * indicates % values significantly greater (P < .05) in the combination treatments compared with treatment with either agent alone. † indicates values significantly less (P < .01) in Mt NPM1/FLT3-ITD AML cells compared with AML cells with Mt NPM1 alone in the combination treatments.

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