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

Abstract

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)2 D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionylleucyl-[ 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-'0 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)2 D3-induced differentiation based upon nitroblue tetrazolium reduction and Nformyl-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)2 D3: :510-1' M had no detectable effect, 10-9 M stimulated growth, and 2:10-8 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)2 D3-induced differentiation of HL-60 cells to monocytes/ macrophages is occurring via receptor-mediated events .The induced differentiation of the human promyelocytic leutamin D3 (1,25(OH]2D3), is of great current interest (1-4). kemia, HL-60, by a variety of compounds such as phorbolThe most potent inducer of differentiation in HL-60 cells is esters, DMSO, retinoic acid (RA),' and la,25-dihydroxyvivarious concentrations of KCI; NBT, nitroblue tetrazolium; PBS, 'Abbreviations used in this paper: FMLP, N-formyl-methionyl-leuphosphate-buffered saline ; PCMBS, p-chloromercuribenzene sulfonic cyl-phenylala...

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