Activation of the human estrogen receptor by the antiestrogens ICI 182,780 and tamoxifen in yeast genetic systems: implications for their mechanism of action

Abstract

The antiestrogens tamoxifen and ICI 182,780 have been portrayed as competitive antagonists of the estrogen binding site of the alpha-form of the human estrogen receptor (ER). However, in functional studies, neither compound has consistently been able to block estradiol-induced transcription. In this report, three yeast genetic systems were used to investigate the effects of tamoxifen and ICI 182,780 on ER dimerization, transcriptional activation, and the interaction of the receptor with a coactivator, RIP140. Tamoxifen and ICI 182,780 were able to induce ER dimerization and ER-dependent transcription, albeit at up to 15,000-fold higher concentrations than that of estradiol. In the presence of RIP140, the transcription response maximum was increased up to 30-fold for estradiol and both antiestrogens. Whole yeast cell [(3)H]estradiol binding studies demonstrated that tamoxifen could displace the estradiol from the ER, whereas ICI 182,780 treatment resulted in a 4-fold increase in [(3)H]estradiol binding to the receptor. No antagonism of estradiol was observed with tamoxifen or ICI 182,780 in any of the yeast models employed. We have concluded that the antiestrogen activity of compounds like tamoxifen and ICI 182,780 is not caused by their ability to competitively antagonize estradiol binding to the hormone binding site, but possibly by their ability to induce ER-dependent transcription, which in mammalian systems would result in receptor down-regulation. Compounds such as tamoxifen act through the hormone binding site, whereas ICI 182,780 may cause receptor activation through an allosteric binding site.

Figure 1

Dose response for E2, tamoxifen, and ICI 182,780. (A) PCY2 yeast. (B) RS188N yeast. The figure is representative of three to four independent experiments. The points shown are means ± SEM. The curves areas follows: E2 (●), ICI 182,780 (▴), and tamoxifen (■). Point C is the response in the absence of added E2.

Figure 2

Effect of antiestrogens on E2 dose response. (A) RS188N yeast. The curves are as follows: E2 (●); + ICI 182,780: 0.3 μM (♦), 1.0 μM (▾), 3.0 μM (▴), and 10 μM (■). (B) RS188N/RIP140 yeast. The curves are as follows: E2 (●); + 10.0 μM ICI 182,780 (▴) or + 10.0 μM tamoxifen (■). Point C is the response in the absence of added E2. The values shown are means ± SEM from a single experiment.

Figure 3

Analysis of [³H]estradiol binding in the PCY2 yeast. (A) Saturation binding. (B) Scatchard analysis. The K_d value was 3.00 ± 0.24 nM and B_max was 9.39 ± 3.5 fmol per million cells. Both figures are representative of three separate experiments with each concentration of [³H]estradiol run in quadruplicate.

Figure 4

Whole cell competition binding studies for [³H]estradiol in the PCY2 yeast. (A) E2 and tamoxifen. The curves are as follows: E2 (●); tamoxifen (■). The K_i values are 3.47 ± 0.12 nM for E2 and 2.61 ± 0.19 μM for tamoxifen. (B) ICI 182,780. The ED₅₀ for increasing [³H]estradiol binding was 52.3 ± 12.4 μM. The curves are representative of three to four independent experiments, with compound concentrations run in quadruplicate. The points shown are means ± SEM.