Estrogen Response Element Binding Research Articles

High passage T47D human breast cancer cells: altered endocrine and 2,3,7,8-tetrachlorodibenzo- p-dioxin responsiveness

Low passage (L) T47D cells cultured for up to 12 months in media containing fetal bovine serum, fetal bovine serum plus 1 nM 17β-estradiol and estrogen-deficient media gave high passage cells denoted H, H(E +) and H(E −) cells, respectively, which exhibited differential responsiveness to 17β-estradiol, various growth factors, tamoxifen, 2,3,7,8-tetrachlorodibenzo- p-dioxin, and their combinations. Moreover, the altered mitogen/antimitogen responsiveness was paralleled by changes in hormone receptor levels, cellular architecture and ploidy. Estrogen receptor binding levels in the H, L and H(E +) cells varied from 36 to 155 fmol/mg protein; in contrast, the estrogen receptor binding in H(E −) cells exhibited a time-dependent increase from 81 to 1229 fmol/mg after culturing in estrogen-deficient media for approximately 12 months. Gel mobility shift assays of the nuclear estrogen receptor extracts from high and low passage cells with 32P-labeled estrogen responsive element showed that levels of the estrogen receptor-estrogen responsive element retarded band were lower in all the high passage cells compared to the low passage cells. These studies further illustrate the genetic instability of the T47D human breast cancer cell line and the resulting changes in mitogen and antimitogen responsiveness. In addition, the high passage H(E −) cells which express high estrogen receptor but low estrogen responsive element binding represent a unique model system for investigating the cellular and molecular biology of breast cancer cells which appear to be estrogen receptor-positive but are insensitive to antiestrogens.

Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA

The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E 2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5′-GGTCAgagTGACC-3′) but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E 2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E 2-ER binding to AT-rich EREs, not cooperative. The affinity of E 2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E 2-ER binding to closely spaced EREs is more stable ( K d =0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing ( K d =2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E 2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E 2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E 2-ER binding and may be critical for cooperative binding to stereoaligned EREs.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Inhibition of Estrogen-Responsive Gene Activation by the Retinoid X Receptor β: Evidence for Multiple Inhibitory Pathways

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Human estrogen receptor forms multiple protein-DNA complexes.

A baculovirus expression system was used to overproduce the human estrogen receptor in insect cells. The estrogen receptor made in this system is full-length, binds estrogen specifically, and is recognized by a monoclonal antibody to the human estrogen receptor. The recombinant estrogen receptor binds the estrogen response element (ERE) in both the absence and presence of estrogen if the binding is carried out in the absence of Mg2+. In the presence of Mg2+, the estrogen receptor binds the ERE in a hormone-dependent fashion. This effect is more pronounced at higher temperatures. Tamoxifen, a nonsteroidal anti-estrogen, is able to stimulate ERE binding to the same extent and under the same conditions as estradiol. Estradiol stimulates formation of an estrogen receptor-ERE complex with an increased mobility in native gels as compared with the complex formed without hormone or with tamoxifen. These results demonstrate that specific DNA binding of the estrogen receptor is not absolutely dependent on the presence of hormone and that estradiol but not tamoxifen is able to induce a change in the estrogen receptor. This differential effect of estradiol and tamoxifen may be important in understanding the role of the receptor to activate target genes differentially.