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  • Title: Whole-cell uptake and nuclear localization of 1,25-dihydroxycholecalciferol by breast cancer cells (T47 D) in culture.
    Author: Sher E, Eisman JA, Moseley JM, Martin TJ.
    Journal: Biochem J; 1981 Nov 15; 200(2):315-20. PubMed ID: 6896147.
    Abstract:
    Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH)(2)D(3)] have been described recently in broken-cell preparations of several cultured human breast cancer cell lines including the T47 D line. It was necessary to determine whether intact breast cancer cells in culture would bind 1,25-(OH)(2)D(3) specifically and whether the next step in the proposed scheme of action, i.e. nuclear translocation, occurred. The following results were obtained. (1) Specific uptake of 1,25-(OH)(2)D(3) by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH)(2)D(3) concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5nm-1,25-(OH)(2)D(3) in the medium, at least 4h are required to reach maximum compared with less than 1h for oestradiol binding. (3) Estimation of binding characteristics by Scatchard analysis indicates a single class of binding sites with K(d) of 68pm and 11800 binding sites/cell, which are similar results to those obtained with broken-cell preparations. (4) Inclusion of various vitamin D metabolites in the incubation medium decreased specific binding of 1,25-(OH)(2)D(3) by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca(2+) transport and bone resorption in vivo. Order of potency was 1,25-(OH)(2)D(3)>(24R)-1,24,25-trihydroxycholecalciferol >>25-hydroxycholecalciferol>(25R)-24,25-dihydroxycholecalciferol >>(25R)-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH)(2)D(3)-depleted state, 80% of the 1,25(OH)(2)D(3) receptor is found in the cytosol fraction of the cells even when the subcellular fractionation is performed under low-salt conditions. By contrast after incubation with [(3)H]1,25-(OH)(2)D(3), 59% of the specific 1,25-(OH)(2)D(3) binding is found in the partially purified nuclei fraction. These data indicate that nuclear translocation of the receptor-hormone complex takes place in the intact T47 D cell. The results also support the hypothesis that the 1,25-(OH)(2)D(3) receptor is functional in this cultured breast cancer cell line, which may provide a useful model for further study of the early biochemical events in 1,25-(OH)(2)D(3) action.
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