Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1 alpha,25-dihydroxyvitamin D3-differentiated HL60 cells.
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 1 alpha,25-dihydroxyvitamin D3 (1 alpha,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 1 alpha,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 x 10(6) 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 1 alpha,25-(OH)2D3 for 4 days synthesized 2.17 +/- 0.07 (S.E.M.) pmol 24,25-(OH)2D3/10(6) cells per h.(ABSTRACT TRUNCATED AT 250 WORDS)
Hayes
M E
ME
Department of Medicine, University of Manchester.
Bayley
D
D
Mawer
E B
EB
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism*; D002112:Calcifediol / Q000378:metabolism*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology*; D015473:Leukemia, Promyelocytic, Acute; D013250:Steroid Hydroxylases / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D015650:24,25-Dihydroxyvitamin D 3; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D002112:Calcifediol; D007654:Ketoconazole