In vivo activity of CD437 in the SCID/NB4 model of acute promyelocytic leukemia. (A) SCID mice (6 animals/experimental group) were inoculated intraperitoneally with 2 × 10⁶ NB4 cells. Two days later, treatment was started and continued for 3 weeks with 1 daily intraperitoneal injection of vehicle or CD437, as indicated. The number in parentheses represents the median survival time for each experimental group. Combined results from 2 separate experiments are presented. In the first experiment (solid line for the vehicle), animals were treated with 5 and 15 mg/kg CD437 or vehicle alone. In the second experiment (dashed line for the vehicle), animals were injected with 30 mg/kg CD437 or vehicle alone. Increases in survival time for the retinoid-treated groups were analyzed by the log-rank test: CD437 5 mg/kg (P < .05); CD437 15 mg/kg and CD437 30 mg/kg (P < .01). (B-D) SCID mice (4 animals/experimental group) were inoculated intraperitoneally with 2 × 10⁶ NB4 cells. Twenty days later, animals were treated with a single intraperitoneal injection of vehicle (control) or CD437 (30 mg/kg). Sixteen hours after treatment, the ascitic fluid was withdrawn and the leukemic cells were subjected to various types of analysis. The results, summarized by the bar graph data, are the mean ± SD of 4 separate animals. In B the total number of cells was counted after staining with erythrosin to determine the level of viability (control, 98% ± 1%; CD437, 80% ± 2%; mean ± SD of 4 animals). In C the percentage of apoptotic cells showing signs of nuclear fragmentation, after staining with DAPI (left panel) or showing cytoplasmic membrane phosphatidylserine externalization (annexin-V positivity and PI negativity; right panel), was evaluated. For annexin-V, the cytofluorometric analysis of 1 representative vehicle-treated and 1 representative CD437-treated animal, from which the data summarized in graphic form were derived, is also shown. The lower right quadrant contains apoptotic cells (annexin-V⁺/PI⁻); the lower left quadrant contains viable cells (annexin-V⁻/PI⁻; control, 92% ± 1%; CD437, 68% ± 6%; mean ± SD of 4 animals). In this analysis, the upper right quadrant contains necrotic cells (annexin-V⁺/PI⁺; control, 1% ± 1%; CD437, 4 ± 2%; mean ± SD of 4 animals). In D the percentage of nonproliferating viable cells was evaluated by biparametric flow cytometry after staining with an anti-PCNA antibody and counterstaining with PI. The cytofluorometric analysis of 1 representative vehicle-treated and 1 representative CD437-treated animal, from which the data summarized in graphic form were derived, is also shown. The left quadrant contains apoptotic cells and cell debris and was not considered in the analysis. The 2 right quadrants contain viable PCNA⁺ or PCNA⁻ cells in the G1, S, or G2/M phase of the cycle as indicated by the content of DNA determined after PI staining. The percentage of viable PCNA⁻cells is 3% ± 1% for control animals and 45% ± 4% for CD437-treated animals (mean ± SD; n = 4).