Figure 5.

The free radical scavenger, NAC, protects from NPI-0052–induced apoptosis. (A) NAC attenuates NPI-0052 activation of caspase-3. Jurkat cells pretreated with 24 mM NAC for 15 minutes prior to exposure of 200 nM NPI-0052 for 6 hours. Caspase-3 activity was assessed by measuring the cleavage of DEVD-amc. A significant reduction (P > .001) in caspase-3 activity was observed in the presence of NAC. Inset: Pretreatment of Jurkat cells with 24 mM NAC for 15 minutes prior to 100 nM NPI-0052 exposure reduces superoxide levels as measured by HEt staining and flow cytometric analysis on the FL3 channel. (B) NAC confers protection from NPI-0052–induced apoptosis. DNA fragmentation was assessed by PI staining of Jurkat cells treated with 200 nM NPI-0052 alone or in combination with 24 mM NAC (P > .001) for 24 hours. (C) NPI-0052 induces oxidant-dependent apoptosis in a Ph + ALL patient sample. Lymphocytes from patient material were separated by centrifugation on a double-density Histopaque gradient. Cells were incubated in the presence/absence of 24 mM NAC and 500 nM, 1 μM or 750 nM NPI-0052. After 24 hours, apoptosis was assessed by PI staining and subsequent flow cytometric analysis. (D) Effects of combination treatment with NPI-0052 and NAC on the chymotrypsin-like activity. Chymotrypsin-like activity was determined in Jurkat cells exposed to 1 μM NPI-0052 and/or 24 mM NAC after 1 hour. (E) NAC does not protect from NPI-0052 activation of caspase-8. Cell lysates from Jurkats treated with 24 mM NAC, 200 nM NPI-0052, and 200 ng/mL CH-11 (positive control) for 6 hours were subjected to immunoblotting for caspase-8 and actin. (F) Neither a pan-caspase nor caspase-8 inhibitor protects from NPI-0052–induced ROS generation. HEt staining was performed in Jurkat cells treated with 200 nM NPI-0052 and/or 50 μM zVAD-fmk or 50 μM IETD-fmk for 14 hours to detect intracellular O² levels.