H90 induces granulocytic differentiation of HL60 and NB4 cells.
(A,C) Increased expression of lineage differentiation antigens. The expression of CD11b, CD14, and CD15 was measured as described in “Materials and methods” and in the legend to Figure 1A. Time and dose-dependency curves are shown. ▪: control; ▴: + H90; ▵: + RA. (B,D) May-Grünwald Giemsa–stained cytosmears of HL60 cells (B) and NB4 cells (D) treated with H90, 10−7 M/L RA, or J173 (controls). Both H90-treated HL60 and NB4 cells showed a segmented nucleus, condensed chromatin, rare nucleoli, and low nucleus-cytoplasmic ratio, all typical for differentiated granulocytic cells (metamyelocytes and band cells) and also observed in RA-treated cells. Magnification: × 630. (E) Degradation of PML-RARα in H90-treated NB4 cells; 24, 48, and 72 hours after addition of H90 (20 μg/mL), PML-RARα and RARα were revealed by successive incubation, first with a 1:2000 dilution of an anti-RAR polyclonal antibody and second with peroxidase-labeled goat antirabbit antibody for detection with ECL chemoluminescence system. The PML-RARα band (approximately 110 kd), present in control NB4 cells, was greatly decreased following addition of H90 (20 μg/mL). This decrease was time-dependent. A similar decrease was observed in RA-treated NB4 cells. The wild-type RARα band (50 kd) was also decreased.
