Regulation of the Estrogen Receptor in MCF-7 Cells by Estradiol

Abstract

The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.

The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

“…The 293T/17 cells with stable integration of the SV40 large T antigen were supplied by Dr. Debu Chakravarti (Northwestern University, Chicago, IL) and were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 100 mg/mL streptomycin. All cells were incubated in a humidified 5% CO 2 / 95% air atmosphere at 37 C. Estrogen receptor levels in MCF7 cell lines were previously reported by Saceda et al 43 …”

Effect of estradiol on estrogen receptor-α gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway

“…At 80% confluence, the medium was replaced with phenol-red-free IMEM containing 5% charcoaltreated calf serum (CCS) (Berthois et al, 1986). The calf serum was pretreated with sulfatase and dextran-coated charcoal to remove endogenous steroids (Saceda et al, 1988). After 2 days, the medium was changed into serum-free, phenol-red-free IMEM supplemented with fibronectin, glutamine, HEPES, trace elements, and transferrin, after which 10 À9 m estradiol or 100 ng/ml epidermal growth factor (EGF) was added.…”

“…The probe for the ER, pOR-300, was constructed by subcloning a 300-bp restriction fragment of pOR3 into the pGem4 polylinker regions using the restriction enzymes PstI and Eco RI (Saceda et al, 1988). The clone 36B4 was constructed by subcloning a 220-bp fragment of 36B4 into the PstI restriction site of the pGem polylinker (Saceda et al, 1988).…”

“…The clone 36B4 was constructed by subcloning a 220-bp fragment of 36B4 into the PstI restriction site of the pGem polylinker (Saceda et al, 1988). In addition, the clones for pS2 (Garcia-Morales et al, 1994), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (McLeskey et al, 1993), and PR (Stoica et al, 2000c) were used as previously described.…”

“…The concentration of receptor protein was determined using an enzyme immunoassay kit from Abbott Laboratories (North Chicago, IL, USA) (Saceda et al, 1988). To obtain total receptor protein, the cells were homogenized by sonication in a high salt buffer (10 mm Tris, 1.5 mm EDTA, 5 mm Na 2 MoO 4 , 0.4 m KCl, and 1 mm monothioglycerol with 2 mm leupeptin).…”