1,25-Dihydroxyvitamin D₃-induced differentiation in a human promyelocytic leukemia cell line (HL-60): Receptor-mediated maturation to macrophage-like cells

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃). We investigated differentiation by monitoring 1,25(OH)₂D₃-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[³H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)₂D₃ concentrations as low as 10^-10 M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED₅₀ for 1,25(OH)₂D₃-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[³H]phenylalanine binding was 5.7 x 10^-9 M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)₂D₃: ≤10^-10 M had no detectable effect, 10^-9 M stimulated growth, and ≥10^-8 M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)₂D₃ in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)₂D₃. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)₂D₃-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)₂[³H]D₃ in culture, we found that HL-60 contains ~4,000 1,25(OH)₂D₃ receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with ~8% of that number. The concentration of 1,25(OH)₂D₃ (5 x 10^-9 M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED₅₀ for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)₂D₃-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.