Studies on an adherent variant of the human promyelomonocytic HL60 leukaemia cell line that constitutively expresses 25-hydroxyvitamin D3-24-hydroxylase activity which is inhibited by 1α,25-dihydroxyvitamin D3

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 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 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.