Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique

The level of estrogen receptor (ER) is a major factor regulating cell sensitivity to estrogen. We have determined the rates of ER synthesis and turnover in MCF-7 breast cancer cells by incubating cells in medium supplemented with 13C15N2H-amino acids (dense amino acids) and monitoring the shift from "old-light" (preexisting) to "new-dense" (newly synthesized) receptors by velocity sedimentation on 0.4-M KCl, 5-20% sucrose gradients prepared in buffered deuterium oxide. Cytosol or unoccupied nuclear ER prepared from control cells grown in the presence of 12C14N1H-amino acids (normal amino acids) and labeled in vitro with [125I]iodoestradiol sediments as a single, normal density peak. When cells were incubated in medium supplemented with dense amino acids for various periods of time, the [125I]iodoestradiol-labeled receptor showed a progressive shift with time to a denser species; by 3 h, a more rapidly sedimenting shoulder was observed on the normal density peak; by 6 h, 40% of the receptor sedimented at the normal density rate, with 60% at a more rapid rate; by 8 h, the predominant peak (70%) was at the more dense position, and at 15 h, all of the sites sedimented as the denser form. Thus, the magnitude of the receptor peak of normal density as a function of time in dense medium indicates that the receptor in control cells has a half-life of 4.0-4.5 h. To determine whether estradiol or the antiestrogens nafoxidine (U11,100A; (1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy] ethyl)pyrrolidine hydrochloride) or CI628 affected the turnover rate of the receptor, measurements were performed on cells grown in the continuous presence of estradiol or antiestrogen (steady state conditions). Exposure of cells to 10 nM estradiol was found to increase the turnover rate of the nuclear receptor, while exposure to 200 nM nafoxidine or CI628 (alpha-(p-[2-(1-pyrrolidino)ethoxy]phenyl)4-methoxy-alpha'- nitrostillbene) did not substantially alter the turnover rate of the nuclear receptor. The half-lives of receptor were found to be 4.02 +/- 0.23 and 4.47 +/- 0.26 h for control unoccupied cytosol and nuclear receptors, 3.00 +/- 0.38 h for estradiol-occupied nuclear receptors, 4.90 +/- 0.66 h for CI628-occupied nuclear receptors, and 3.43 +/- 0.37 h for nafoxidine-occupied nuclear receptors. These results indicate that ER turns over rapidly, with a half-life of 3-5 h, in the presence or absence of estradiol or antiestrogen, and that receptor synthesis is also rapid, with the rate of appearance of newly synthesized receptor being 0.3-0.5 pmol/mg DNA . h. These rates provide the cell with the capacity for dynamic and rapid regulation of its ER levels.