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Abstract
The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.
Highlights
Effects of TPA on Estradiol Binding to the Estrogen Receptor—We and others have demonstrated that treatment of MCF-7 cells with TPA results in a decrease in the concentration of estrogen receptor (ER) protein and ER mRNA [14, 15]
To determine whether the decrease and subsequent recovery of ER protein corresponds to a similar change in estradiolbinding sites, the concentration of ER protein and binding capacity were measured simultaneously (Fig. 1B)
The concentration of ER protein returned to control values, the number of estrogen-binding sites remained at approximately 20% of control values
Summary
Several ER variants, including point mutations and alternately spliced forms, have been described in tumors and in breast cancer cell lines [6, 7] Some of these variants lack the ability to bind estradiol, whereas other variants are active in the absence of ligand. We and others have shown that treatment of the breast carcinoma cell line MCF-7 with TPA, an activator of protein kinase C, results in a decrease in the level of ER [14, 15]. This effect is mediated by a post-transcriptional destabilization of the ER mRNA [14]. The inhibitory effect of TPA on ER activity is due to the induction/activation of a factor that blocks the binding of estradiol to the ER
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