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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.

ABSTRACT

The receptor for the active metabolite of vitamin D, 1,25(OH)₂D₃, known as the vitamin D receptor (VDR), belongs to the steroid hormone nuclear receptor superfamily. We have developed novel methods for detection of VDR mRNA and protein within a human promyelomonocytic cell line, HL-60.

Using the newly developed technique of in situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use of a novel immunogold cytochemical technique has allowed clear and sensitive detection of VDR protein expression within the HL-60 cells.

Further development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver.

These techniques will be useful to localise specifically the VDR within cell types that contain low levels of mRNA and protein, and will permit further investigation of the role played by 1,25(OH)₂D₃ in cellular regulatory mechanisms.