Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: Comparison of immunocytochemical data with biochemical measurements

Abstract

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF‐7 cells under estrogenic and anti‐estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER‐EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E2 and DES) and pure anti‐estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4‐OH‐TAM and RU 39,119). E2‐induced ER down‐regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down‐regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre‐labeling of the cells with [3H]TAZ indicated that all investigated ligands eliminate pre‐existing ER through binding to newly synthetized receptors, since [3H]TAZ co‐valently associates with ER; E2 and RU 58,668 were more effective than 4‐OH‐TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre‐exposed to [3H]TAZ. Nuclear location of [3H]TAZ‐ER complexes may explain this discrepancy, since pure AE‐ER complexes were reported to be incapable of nuclear translocation. Int. J. Cancer 78:760–765, 1998. © 1998 Wiley‐Liss, Inc.

Estrogen Receptor Alpha: Impact of Ligands on Intracellular Shuttling and Turnover Rate in Breast Cancer Cells

“…cycloheximide, puromycin) do not affect the turnover rate of unliganded ER [188] and also fail to modify receptor down regulation induced by pure antiestrogens [186,188,192]. On the other hand, these inhibitors totally abrogate estradiol-induced ER down regulation [188].…”

“…Estrogens and pure antiestrogens provoke in less than 6 hours a drastic ER depletion, which can be demonstrated by both immunocytochemistry and Western blotting analysis of cell extracts [192]. Proteasomal degradation is not the sole mechanism leading to ER down regulation.…”

Stimulatory effect of genistein and apigenin on the growth of breast cancer cells correlates with their ability to activate ER alpha

“…After 3 days of culture, genistein and apigenin were added to the flasks and the culture pursued for 6 h. Separated cell extracts (nuclear and cytosol extracts) were prepared as previously described [28,29], and ERa contents of these fractions were measured by the Abbott enzyme immunoassay (ER-EIA) according to manufacturer's recommendations. Data were expressed per mg of protein measured by the Coomassie protein reagent assay (Pierce; Rockford, IL, USA).…”

Pharmacological Profile of 6,12-Dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one, a Novel Human Estrogen Receptor Agonist.

“…37) Down-regulation of ERs in the whole-cell assay, i.e., ligand induced degradation leading to its progressive elimination, [38][39][40][41] The assessment of luciferase induction in MVLN cells confirms this view of an intracellular association of 1 with ER. We found 53% enhancement for compound 1 at 10 Ϫ6 M (control, 100%; E2, 178% at 10 Ϫ10 M; RU 58668, 34% at 10 Ϫ7 M), suggesting an estrogenic effect (Fig.…”