Figure 2
Figure 2. XIAP inhibitors sensitize acute leukemia cells for TRAIL- or CD95-induced apoptosis. (A) Surface expression of TRAIL receptors TRAIL-R1 to -R4 and CD95 on acute leukemia cell lines was determined by fluorescence-conjugated antibodies and flow cytometry (thin line indicates cells stained with isotype control; thick line, cells stained with anti–TRAIL-R1 to -R4 or anti-CD95 antibodies). Fluorescence intensity (x-axis) is blotted against counts (y-axis). A representative experiment of 3 independent experiments is shown. (B-D) Acute leukemia cell lines were treated for 72 hours with indicated concentrations of TRAIL in the absence (□) or presence of XIAP inhibitors, control compound, or DMSO (Reh: 6 nM; Nalm-6: 60 nM; CEM: 60 nM; HL-60: 30 nM, Jurkat: 10 nM). (B,C) Apoptosis was determined by forwardside scatter analysis and flow cytometry in Reh, Nalm-6, CEM, HL-60 cells (B), and Jurkat (C) cells. (D) Apoptosis was determined in Jurkat cells by fluorescence-activated cell sorting (FACS) analysis of DNA fragmentation of propidium iodide–stained nuclei. (E) Morphologic features of apoptosis were determined by DAPI staining in Jurkat cells that were left untreated or were treated for 48 hours with 0.4 ng/mL TRAIL and 10 nM XIAP inhibitor 2, control compound, or DMSO. Arrows indicate apoptotic cells; scale bar represents 200 μm. (F) Jurkat cells transduced with XIAP or control shRNA or empty vector were analyzed for XIAP expression by Western blotting (left panel) and for apoptosis after treatment with 0.4 ng/mL TRAIL for 72 hours by forwardside scatter analysis and flow cytometry (right panel). (G) Jurkat cells transduced with empty vector or a vector containing Smac cDNA were analyzed for Smac expression by Western blotting (left panel) and for apoptosis after treatment with 0.4 ng/mL TRAIL for 72 hours by forwardside scatter analysis and flow cytometry (right panel); arrowhead indicates Flag-tagged Smac. (H) Clonogenic survival was assessed by colony assay as described in “Methods” in Jurkat cells that were left untreated or were treated with 0.4 ng/mL TRAIL in the presence or absence of 10 nM XIAP inhibitor 2, control compound, or DMSO. The percentage of colonies relative to untreated cells are shown. (I) Jurkat cells were treated for 72 hours with indicated concentrations of agonistic anti-CD95 antibodies in the absence (□) or presence of 10 nM XIAP inhibitors, control compound, or DMSO. Apoptosis was determined by forwardside scatter analysis and flow cytometry. (B-D,F-I) Mean and SD of 3 experiments each performed in triplicate (all except H) or duplicate (H) are shown. For statistical analysis, t test was performed comparing XIAP inhibitors to control compound (B-D,H-I), XIAP shRNA to control shRNA (F), or Smac to empty vector (G). #P < .05; *P < .01; **P < .001.

XIAP inhibitors sensitize acute leukemia cells for TRAIL- or CD95-induced apoptosis. (A) Surface expression of TRAIL receptors TRAIL-R1 to -R4 and CD95 on acute leukemia cell lines was determined by fluorescence-conjugated antibodies and flow cytometry (thin line indicates cells stained with isotype control; thick line, cells stained with anti–TRAIL-R1 to -R4 or anti-CD95 antibodies). Fluorescence intensity (x-axis) is blotted against counts (y-axis). A representative experiment of 3 independent experiments is shown. (B-D) Acute leukemia cell lines were treated for 72 hours with indicated concentrations of TRAIL in the absence (□) or presence of XIAP inhibitors, control compound, or DMSO (Reh: 6 nM; Nalm-6: 60 nM; CEM: 60 nM; HL-60: 30 nM, Jurkat: 10 nM). (B,C) Apoptosis was determined by forwardside scatter analysis and flow cytometry in Reh, Nalm-6, CEM, HL-60 cells (B), and Jurkat (C) cells. (D) Apoptosis was determined in Jurkat cells by fluorescence-activated cell sorting (FACS) analysis of DNA fragmentation of propidium iodide–stained nuclei. (E) Morphologic features of apoptosis were determined by DAPI staining in Jurkat cells that were left untreated or were treated for 48 hours with 0.4 ng/mL TRAIL and 10 nM XIAP inhibitor 2, control compound, or DMSO. Arrows indicate apoptotic cells; scale bar represents 200 μm. (F) Jurkat cells transduced with XIAP or control shRNA or empty vector were analyzed for XIAP expression by Western blotting (left panel) and for apoptosis after treatment with 0.4 ng/mL TRAIL for 72 hours by forwardside scatter analysis and flow cytometry (right panel). (G) Jurkat cells transduced with empty vector or a vector containing Smac cDNA were analyzed for Smac expression by Western blotting (left panel) and for apoptosis after treatment with 0.4 ng/mL TRAIL for 72 hours by forwardside scatter analysis and flow cytometry (right panel); arrowhead indicates Flag-tagged Smac. (H) Clonogenic survival was assessed by colony assay as described in “Methods” in Jurkat cells that were left untreated or were treated with 0.4 ng/mL TRAIL in the presence or absence of 10 nM XIAP inhibitor 2, control compound, or DMSO. The percentage of colonies relative to untreated cells are shown. (I) Jurkat cells were treated for 72 hours with indicated concentrations of agonistic anti-CD95 antibodies in the absence (□) or presence of 10 nM XIAP inhibitors, control compound, or DMSO. Apoptosis was determined by forwardside scatter analysis and flow cytometry. (B-D,F-I) Mean and SD of 3 experiments each performed in triplicate (all except H) or duplicate (H) are shown. For statistical analysis, t test was performed comparing XIAP inhibitors to control compound (B-D,H-I), XIAP shRNA to control shRNA (F), or Smac to empty vector (G). #P < .05; *P < .01; **P < .001.

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