Effects of Uremic Serum on 1, 25-Dihydroxyvitamin D₃-Induced Differentiation of a Human Promyelocytic Leukemia Cell Line, HL-60

Abstract

The mechanism by which resistance to 1, 25 dihydroxyvitamin D₃ (1, 25-(OH)₂D₃) occurs in patients with chronic renal failure was studied. This agent induces differentiation and 1, 25-(OH)₂D₃-24-hydroxylase activity in the mitochondria of the human promyelocytic leukemia cell line, HL-60, via a steroid-hormone receptor mechanism. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% normal or uremic serum. Treatment of these cells with 10^-8M 1, 25-(OH)₂D₃ for 5 days in a medium containing 10% uremic serum from 4 patients with chronic renal failure resulted in a maturation of the cells amounting to 30.3 ± 18.7% (mean ± SD) and 32.5 ± 11.2%, as obtained by NBT reduction assay and NSE assay, respectively. These values were significantly lower than those obtained with 10% serum from 3 normal controls (66.6 ± 12.8%, 58.3 ± 10.9%, p<0.02). The treatment of HL-60 cells with 1, 25-(OH)₂D₃ in a mixture of 5% normal plus 5% uremic serum caused cell differentiation to an extent similar to that in 10% uremic serum, which suggests the presence of a substance(s) having 1, 25-(OH)₂D₃-inhibitory activity in the uremic serum. Exposure of HL-60 cells to uremic serum significantly impaired their responsiveness to 1, 25-(OH)₂D₃ as assessed by the induction of the cell's ability to hydroxylate the C-24 position of 1, 25-(OH)₂ [³H] D₃. The mechanism by which uremic serum confers an impaired cellular response to 1, 25-(OH)₂D₃ seemed to be due, in part, to a decrease in 1, 25-(OH)₂D₃ receptor levels. A significant positive correlation was observed between intracellular cAMP levels and 1, 25-(OH)₂D₃-induced HL-60 cell maturation. In summary, the mechanism by which uremic serum confers 1, 25-(OH)₂D₃ resistance upon HL-60 cells seemed to be due to the presence of 1, 25-(OH)₂D₃-inhibitory activity in uremic serum, which may modulate cellular responsiveness to 1, 25-(OH)₂D₃ by such mechanisms as reducing 1, 25-(OH)₂D₃ receptor levels in the cells, in part through alteration in cAMP metabolism.