American Society of Hematology

Figure 2.

$Figure 2. L744832 rapidly and highly synergistically potentiates UCN-01-mediated lethality in human leukemia cells, accompanied by mitochondrial dysfunction, caspase activation, and PARP cleavage. (A) U937 and Jurkat cells were exposed to 10 μM L744832 (L) with or without UCN-01 (UCN or U, U937, 100 nM; Jurkat, 150 nM). At the indicated intervals, cells were harvested and the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright-Giemsa-stained cytospin preparations. (B) U937 and Jurkat cells were incubated for 18 hours with the indicated concentration of UCN-01 in either the presence or absence of 10 μM L744832, after which the percentage of apoptotic cells was determined as described in panel A. (C) U937 and Jurkat cells were exposed to the indicated concentration of L744832 in either the presence or absence of UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which the percentage of apoptotic cells was determined as described in panel A. (D) U937 (upper panel) and Jurkat (lower panel) cells were exposed to a range of L744832 (5-25 μM) and UCN-01 (50-250 nM) concentrations alone and in combination at a fixed ratio (eg, U937, 100:1; Jurkat, 66.7:1) for 18 hours. At the end of this period, the percentage of cells exhibiting annexin V positivity was determined for each condition. Fractional effect values were determined by comparing results with those of untreated controls and median dose effect analysis employed to characterize the nature of the interaction between L744832 and UCN-01. Combination index (CI) values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (E) U937 and Jurkat cells were treated with 10 μM L744832 in either the presence or absence of UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which cells were lysed and subjected to Western blot to assess activation of caspase-associated cascades using the indicated primary antibodies.Alternatively, cytosolic fractions (S-100) were prepared, and expression of cytochrome c, Smac/DIABLO, and AIF was monitored by Western blot. (F) U937 cells were incubated with 10 μM L744832 (L) + 100 nM UCN-01 (U) for 8 hours in either the presence or absence of 20 μM BOC-D-fmk, after which the percentage of apoptotic cells was determined by annexin V-FITC/flow cytometry (upper panel). **Significantly lower than values for cells exposed to L744832 + UCN-01 in the absence of BOC-D-fmk (P < .01). Alternatively, S-100 fractions were prepared and subjected to Western blot analysis to monitor cytosolic expression of cytochrome c, Smac/DIABLO, and AIF (lower panel). For panels A-C and F (upper panel), results represent the means ± SD for 3 separate experiments performed in triplicate. For panels E and F (lower panels), each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. The results of a representative experiment are shown; an additional study yielded equivalent results. CF indicates cleavage fragment.$

L744832 rapidly and highly synergistically potentiates UCN-01-mediated lethality in human leukemia cells, accompanied by mitochondrial dysfunction, caspase activation, and PARP cleavage. (A) U937 and Jurkat cells were exposed to 10 μM L744832 (L) with or without UCN-01 (UCN or U, U937, 100 nM; Jurkat, 150 nM). At the indicated intervals, cells were harvested and the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright-Giemsa-stained cytospin preparations. (B) U937 and Jurkat cells were incubated for 18 hours with the indicated concentration of UCN-01 in either the presence or absence of 10 μM L744832, after which the percentage of apoptotic cells was determined as described in panel A. (C) U937 and Jurkat cells were exposed to the indicated concentration of L744832 in either the presence or absence of UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which the percentage of apoptotic cells was determined as described in panel A. (D) U937 (upper panel) and Jurkat (lower panel) cells were exposed to a range of L744832 (5-25 μM) and UCN-01 (50-250 nM) concentrations alone and in combination at a fixed ratio (eg, U937, 100:1; Jurkat, 66.7:1) for 18 hours. At the end of this period, the percentage of cells exhibiting annexin V positivity was determined for each condition. Fractional effect values were determined by comparing results with those of untreated controls and median dose effect analysis employed to characterize the nature of the interaction between L744832 and UCN-01. Combination index (CI) values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (E) U937 and Jurkat cells were treated with 10 μM L744832 in either the presence or absence of UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which cells were lysed and subjected to Western blot to assess activation of caspase-associated cascades using the indicated primary antibodies.Alternatively, cytosolic fractions (S-100) were prepared, and expression of cytochrome c, Smac/DIABLO, and AIF was monitored by Western blot. (F) U937 cells were incubated with 10 μM L744832 (L) + 100 nM UCN-01 (U) for 8 hours in either the presence or absence of 20 μM BOC-D-fmk, after which the percentage of apoptotic cells was determined by annexin V-FITC/flow cytometry (upper panel). ^**Significantly lower than values for cells exposed to L744832 + UCN-01 in the absence of BOC-D-fmk (P < .01). Alternatively, S-100 fractions were prepared and subjected to Western blot analysis to monitor cytosolic expression of cytochrome c, Smac/DIABLO, and AIF (lower panel). For panels A-C and F (upper panel), results represent the means ± SD for 3 separate experiments performed in triplicate. For panels E and F (lower panels), each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. The results of a representative experiment are shown; an additional study yielded equivalent results. CF indicates cleavage fragment.

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