CR4 inhibits Ph⁺ ALL cell growth but not normal bone marrow differentiation. (A) Structure of (E, E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrite—CR4. (B) Ph⁺ ALL cells (Z119) were cultured in colony-forming assays in the presence of increasing concentrations of CR4, as indicated. ALL colony numbers were counted after 9 days. The solvent for CR4 (DMSO/Iscove) alone did not affect cell growth (not shown). Similar results were obtained with the Ph⁺ cell lines Z181 and Z33. (C) Single-cell suspensions of normal human bone marrow were prepared by Percoll gradient centrifugation. BM cells were plated in CFU-GEMM semisolid media cultures in the presence of CR4 as indicated. Colony type and numbers were assessed after 14 days of culture at 37°C, 5% CO₂. BM cells differentiated into normal erythroid (BFU-E; □) and granulocyte/monocyte/macrophage (CFU-C; ▦) colonies. Mixed colony numbers (erythroid/granulocyte/monocyte/macrophage) were also normal (not shown). (D) CD34⁺ hematopoietic stem cells were isolated from bone marrow with anti-CD34 magnetic beads (Miltenyi Biotec) and cultured as for complete bone marrow (shown in panel C). □ indicates BFU-E colonies; and ▦, CFU-C colonies. In each case the average of 2 independent experiments is shown with standard errors.