In vitro and in vivo effects of NPI-0052. (A) NPI-0052 treatment causes DNA fragmentation in Jurkat, K562, and ML-1 cells. Cells were treated with increasing doses (0–1 μM) of NPI-0052 for 24 hours and stained with PI. DNA fragmentation was assessed by flow cytometry using the FL-3 channel of a Becton Dickinson FACS Calibur. Inset: Jurkat cells were treated with 0, 10, 50, or 200 nM NPI-0052 for 6 hours, and early apoptotic cells were detected by monitoring FITC-conjugated Annexin V binding to the cell surface (x-axis) and PI staining (y-axis) by flow cytometry. Shown are representative dot plots of 3 separate experiments. Percentages indicate cells that were Annexin V positive and PI negative. (B) NPI-0052 inhibits the chymotrypsin-like activity at the same doses it induces DNA fragmentation. Cells treated for DNA fragmentation with NPI-0052 in Figure 2A also were harvested after only 1 hour of exposure and assessed for chymotrypsin-like activity as described in Figure 1 in triplicate. P < .05 for 1 nM dose and P < .001 for 10 nM dose, compared with control. (C) NPI-0052 triggers DNA fragmentation in a time-dependent manner. Jurkat cells were exposed to 200 nM or 1 μM NPI-0052 for indicated times. DNA fragmentation was determined by flow cytometric analysis of PI staining (P < .05 for 24 hours compared with 0 hours). (D) Total white blood cell counts in NPI-0052–treated mice. CB.17/SCID mice (n = 6/group) were inoculated with 2 × 10⁷  ML-1 cells by tail-vein injection. Mice were administered 0.15 mg/kg NPI-0052 or vehicle alone, IP twice weekly, starting on day 2. Blood samples were collected by tail-vein bleeds at days 17, 21, 23, 32, and 35 and analyzed for complete cell blood counts. Shown are white blood cell counts in 10³ /μL over time and linear regression analysis of control (solid line) versus treatment (dotted line). Each data point represents data obtained from one mouse at the indicated time.