Publications

Abstract

The early biochemical events following exposure of human target cells to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been difficult to study due to the lack of suitable in vitro systems. We have studied the T47 D human breast cancer cell line, which possesses specific high affinity receptors for 1,25-(OH)2D3 and responds to the hormone with changes in cellular replication, as a functional model of 1,25-(OH)2D3-responsive human cells. Specific uptake and binding of [3H]1,25-(OH)2D3 by T47 D cells follows a time course of relatively slow rise and decline. The time of peak binding is delayed relative to that of other steroid hormones in the same system. We have studied the kinetics of 1,25-(OH)2D3 specific uptake and binding in intact cells and Scatchard analysis in broken cell preparations of three T47 D sublines: the wild-type and the 10 and M1 sublines. Binding parameters varied among the three sublines. Thus, peak binding capacity was highest in the wild-type cell and lowest in the M1 subline; ranging from 10-65 fmol/10(6) cells in intact cell studies and from 23-148 fmol/mg protein (approximately 2.8-12.7 fmol/10(6) cells) in broken cell preparations. Receptor affinity in the three sublines ranged from 1.1-2.8 x 10(-11) M. The half-life of occupied receptor was estimated in intact cells of all three sublines by incubation in the presence of the protein synthesis inhibitor puromycin. Similar analyses were made of apparent half-life of unoccupied receptor in broken cell preparations of the wild-type and T47-10 cells. The half-life of occupied 1,25-(OH)2D3 receptor was estimated to be 2.8 +/- 0.4 h in intact wild-type T47 D cells compared with 3.1 +/- 0.7 and 1.4 +/- 0.2 hours in intact T47 D-10 and T47 D-M1 cells, respectively. The half-life of unoccupied 1,25-(OH)2D3 receptor was estimated in broken cell preparations to be 4.1 +/- 0.7 h in wild-type T47 D and 4.2 +/- 0.6 h in the T47 D-10 cells. Receptor half-lives in the wild-type and T47 D-10 cells were not different in either intact or broken cell preparations; however, receptor half-life was 40% longer (P less than 0.05) in broken cell preparations (4.2 +/- 0.4 h) than in the intact cells (3.0 +/- 0.4 h) of the two cell sublines.(ABSTRACT TRUNCATED AT 400 WORDS)