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ABSTRACT

The constitutive expression of 25-hydroxyvitamin D₃-24-hydroxylase (25-(OH)D₃-24-hydroxylase) activity has been studied in an adherent variant (Ad-HL60) of the human promyelomonocytic leukaemia cell line HL60. The Ad-HL60 cells have a more differentiated phenotype than the non-adherent cells from which they were derived, and synthesized 1.88±0.07 (±s.e.m.) pmol 24,25-(OH)₂D₃/h per 10⁶ cells following culture in RPMI-1640 medium containing <0.02 nm 1α,25-(OH)₂D₃. They also synthesized 1.66±0.05 pmol 24,25-(OH)₂D₃/h per 10⁶ cells following culture in 1α,25-(OH)₂D₃-free medium supplemented with 1 g bovine serum albumin/l instead of 10% serum. In contrast, non-adherent HL60 cells required exposure to 10–100 nm 1α,25-(OH)₂D₃ to induce equivalent 24,25-(OH)₂D₃ synthesis. The 25-(OH)D₃-24-hydroxylase expressed by Ad-HL60 cells had an apparent Michaelis constant of 1 μm and maximal rate of 20 pmol/h per 10⁶ cells with substrate concentrations from 0.012 to 1.2 μm/incubation (5–500ng/ml). Furthermore, 24,25-(OH)₂D₃ synthesis was inhibited in a dose-dependent manner by ketoconazole (0.01–10 μm), suggesting that the enzyme is cytochrome P-450 dependent.

Ad-HL60 cells expressed approximately 3500 specific receptors for 1α,25-(OH)₂D₃/cell with a dissociation constant of 40 pm. Following exposure to 0.1–100 nm 1α,25-(OH)₂D₃, Ad-HL60 cell proliferation was significantly inhibited compared with controls grown in medium containing <0.02 nm 1α,25-(OH)₂D₃ for 96h. Expression of 25-(OH)D₃-24-hydroxylase was also inhibited in a dose- and time-dependent manner; however, expression of non-specific esterase was not induced. Both of these findings are contrary to those previously demonstrated for non-adherent HL60 cells, whereas the dose-dependent inhibition of cell proliferation by 1α,25-(OH)₂D₃ occurs in both adherent and non-adherent phenotypes. These observations on Ad-HL60 cells represent the first description of a cell type in which 1α,25-(OH)₂D₃ appears to inhibit 25-(OH)D₃-24-hydroxylase activity. The Ad-HL60 cells also constitutively metabolized 1α,25-(OH)₂D₃ in a manner consistent with formation of 1α,24,25-(OH)₃D₃ without previous exposure to 1α,25-(OH)₂D₃. In contrast, many other cell types, including non-adherent HL60 cells, require exposure to 1α,25-(OH)₂D₃ to induce metabolism of 1α,25-(OH)₂D₃ to 1α,24,25-(OH)₃D₃, a reaction that represents the initial step for catabolism of 1α,25-(OH)₂D₃ to calcitroic acid.

ABSTRACT

Regulation of the metabolism of [³H]25-hydroxyvitamin D₃ ([³H]25-(OH)D₃) in vitro to material with the characteristics of [³H]24,25-dihydroxyvitamin D₃ ([³H]24,25-(OH)₂D₃) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)₂D₃ was induced in a dose-dependent manner in cells pretreated with 0·1–100 nm 1α,25-dihydroxyvitamin D₃ (1α,25-(OH)₂D₃) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [³H]24,25-(OH)₂D₃ from [³H]25-(OH)D₃ and the expression of NSE activity both responded to changes in concentration of 1α,25-(OH)₂D₃ in the culture medium in a parallel manner. Synthesis of [³H]24,25-(OH)₂D₃ was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12×10⁶ cells/incubation. The optimum substrate concentration for its synthesis was 125 nm, giving an apparent Michaelis constant of 360 nm. The identity of the [³H]24,25-(OH)₂D₃ synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)₂D₃ on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nm 1α,25-(OH)₂D₃ for 4 days synthesized 2·17±0·07 (s.e.m.) pmol 24,25-(OH)₂D₃/10⁶ cells per h. This synthesis was inhibited in a dose-dependent manner over a concentration range of 0·01–1 μm by the drug ketoconazole, an antimycotic imidazole which is a known inhibitor of certain cytochrome P-450 enzyme systems, suggesting that the HL60 25-(OH)D₃-24-hydroxylase is also a P-450-dependent enzyme system.