Figure 3
Figure 3. Overexpression of Bcl-2 suppresses LAQ824- and LBH589-mediated apoptosis and therapeutic efficacy. (A) Eμ-myc, Eμ-myc/bcl-2, and Eμ-myc/bcl-xL lymphoma cells were incubated for 24 hours with the indicated concentrations of LAQ824 or LBH589. Cell viability was determined by staining with PI and FACS analysis. (B) Whole cell lysates were prepared from Eμ-myc/bcl-2 and Eμ-myc/bcl-xL lymphoma cells that had been treated for 2 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM). Western blot analysis was performed with antibodies specific for acetylated histone H3. Equivalent protein loading was confirmed by probing for β-actin. (C) Eμ-myc/bcl-2 and Eμ-myc/bcl-xL lymphoma cells were treated with vehicle, LAQ824 (25 nM), or LBH589 (4 nM) for 24, 48, or 72 hours. The extent of apoptosis was measured by flow cytometric analysis of Δψm and exposure of phosphatidylserine on the cell surface (annexin V staining). Each dose or time point represents the mean value of 3 individual experiments ± SE. (D) Eμ-myc/bcl-2 and Eμ-myc/bcl-XL lymphoma cells were treated for 24 to 72 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM), and cell-cycle analysis was performed by FACS after staining fixed/permeabilized cells with PI. The percentages of cells residing in the G0/G1, S, and G2/M phases of the cell cycle and those cells with less than 2n DNA content (sub-G1) are indicated. Cells treated with vehicle are represented by the gray histograms in the middle and bottom panels. (E) C57BL/6 mice bearing palpable Eμ-myc/bcl-2 lymphomas were injected intravenously with LAQ824 (75 mg/kg) or LBH589 (80 mg/kg), and lymph nodes were harvested at the indicated times. Histologic sections were assessed by hematoxylin and eosin staining (top panel), immunohistochemistry using antibodies to acetyl histone H3 (middle panel), and TUNEL staining (bottom panel). Each time point is of an individual mouse. (F) C57BL/6 mice bearing an Eμ-myc/bcl-2 lymphoma were treated with vehicle (200 μL 10% lactic acid/D5W; n = 10), LAQ824 (75 mg/kg; n = 10), or LBH589 (80 mg/kg; n = 10). Kaplan-Meier survival curves of mice treated with vehicle (black line), LAQ824 (blue line), or LBH589 (red line) are shown. (G) WBC counts of mice bearing an Eμ-myc/bcl-2 lymphoma (n = 3) were taken 3 days after treatment with vehicle, LAQ824, or LBH589l. Each point represents the mean value of 3 individual mice ± SE (*P = .1; #P = .3).

Overexpression of Bcl-2 suppresses LAQ824- and LBH589-mediated apoptosis and therapeutic efficacy. (A) Eμ-myc, Eμ-myc/bcl-2, and Eμ-myc/bcl-xL lymphoma cells were incubated for 24 hours with the indicated concentrations of LAQ824 or LBH589. Cell viability was determined by staining with PI and FACS analysis. (B) Whole cell lysates were prepared from Eμ-myc/bcl-2 and Eμ-myc/bcl-xL lymphoma cells that had been treated for 2 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM). Western blot analysis was performed with antibodies specific for acetylated histone H3. Equivalent protein loading was confirmed by probing for β-actin. (C) Eμ-myc/bcl-2 and Eμ-myc/bcl-xL lymphoma cells were treated with vehicle, LAQ824 (25 nM), or LBH589 (4 nM) for 24, 48, or 72 hours. The extent of apoptosis was measured by flow cytometric analysis of Δψm and exposure of phosphatidylserine on the cell surface (annexin V staining). Each dose or time point represents the mean value of 3 individual experiments ± SE. (D) Eμ-myc/bcl-2 and Eμ-myc/bcl-XL lymphoma cells were treated for 24 to 72 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM), and cell-cycle analysis was performed by FACS after staining fixed/permeabilized cells with PI. The percentages of cells residing in the G0/G1, S, and G2/M phases of the cell cycle and those cells with less than 2n DNA content (sub-G1) are indicated. Cells treated with vehicle are represented by the gray histograms in the middle and bottom panels. (E) C57BL/6 mice bearing palpable Eμ-myc/bcl-2 lymphomas were injected intravenously with LAQ824 (75 mg/kg) or LBH589 (80 mg/kg), and lymph nodes were harvested at the indicated times. Histologic sections were assessed by hematoxylin and eosin staining (top panel), immunohistochemistry using antibodies to acetyl histone H3 (middle panel), and TUNEL staining (bottom panel). Each time point is of an individual mouse. (F) C57BL/6 mice bearing an Eμ-myc/bcl-2 lymphoma were treated with vehicle (200 μL 10% lactic acid/D5W; n = 10), LAQ824 (75 mg/kg; n = 10), or LBH589 (80 mg/kg; n = 10). Kaplan-Meier survival curves of mice treated with vehicle (black line), LAQ824 (blue line), or LBH589 (red line) are shown. (G) WBC counts of mice bearing an Eμ-myc/bcl-2 lymphoma (n = 3) were taken 3 days after treatment with vehicle, LAQ824, or LBH589l. Each point represents the mean value of 3 individual mice ± SE (*P = .1; #P = .3).

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