Whole-cell uptake and nuclear localization of 1,25-dihydroxycholecalciferol by breast cancer cells (T47 D) in culture

Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] 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)₂D₃ 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)₂D₃ by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH)₂D₃ concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5nm-1,25-(OH)₂D₃ 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)₂D₃ by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca²⁺ transport and bone resorption in vivo. Order of potency was 1,25-(OH)₂D₃>(24R)-1,24,25-trihydroxycholecalciferol »25-hydroxycholecalciferol>(25R)-24,25-dihydroxycholecalciferol »(25R)-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH)₂D₃-depleted state, 80% of the 1,25(OH)₂D₃ 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 [³H]1,25-(OH)₂D₃, 59% of the specific 1,25-(OH)₂D₃ 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)₂D₃ 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)₂D₃ action.