Adriana Stoica

Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland.

It has been suggested that environmental contaminants that mimic the effects of estrogen contribute to disruption of the reproductive systems of animals in the wild, and to the high incidence of hormone-related cancers and diseases in Western populations. Previous studies have shown that functionally, cadmium acts like steroidal estrogens in breast cancer cells as a result of its ability to form a high-affinity complex with the hormone binding domain of the estrogen receptor. The results of the present study show that cadmium also has potent estrogen-like activity in vivo. Exposure to cadmium increased uterine wet weight, promoted growth and development of the mammary glands and induced hormone-regulated genes in ovariectomized animals. In the uterus, the increase in wet weight was accompanied by proliferation of the endometrium and induction of progesterone receptor (PgR) and complement component C3. In the mammary gland, cadmium promoted an increase in the formation of side branches and alveolar buds and the induction of casein, whey acidic protein, PgR and C3. In utero exposure to the metal also mimicked the effects of estrogens. Female offspring experienced an earlier onset of puberty and an increase in the epithelial area and the number of terminal end buds in the mammary gland.

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

Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and heregulin-β1 (HRG-β1), can modulate the expression and activity of the estrogen receptor-α (ER-α) via the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in the ER-α-positive breast cancer cell line, MCF-7. Estradiol can also rapidly activate PI 3-K/Akt in these cells (nongenomic effect). The recent study examines whether Akt is involved in the ER-α regulation by estradiol (genomic effect). Stable transfection of parental MCF-7 cells with a dominant-negative Akt mutant, as well as the PI 3-K inhibitors wortmannin and LY 294,002, blocked the effect of estradiol on ER-α expression and activity by 70–80 and 55–63%, respectively. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of estradiol. The changes in ER-α expression and activity were abrogated in response to estradiol by an arginine to cysteine mutation in the pleckstrin homology (PH) domain of Akt (R25C), suggesting the involvement of this amino acid in the interaction between Akt and ER-α. Experiments employing selective ErbB inhibitors demonstrate that the effect of estradiol on ER-α expression and activity is mediated by ErbB2 and not by EGFR. Moreover, anchorage-dependent and -independent growth assays, cell cycle and membrane ruffling analyses showed that Akt exerts estrogen-like activity on cell growth and membrane ruffling and that a selective ErbB2 inhibitor, but not anti-ErbB2 antibodies directed to the extracellular domain, can block these effects. In the presence of constitutively active Akt, tamoxifen only partially inhibits cell growth. In contrast, in cells stably transfected with either a dominant-negative Akt or with R25C-Akt, as well as in parental cells in the presence of a selective ErbB2 inhibitor, the effect of estradiol on anchorage-dependent and -independent cell growth was inhibited by 50–75 and 100%, respectively. Dominant-negative Akt inhibited membrane ruffling by 54%; however, R25C-Akt did not have any effect, suggesting that kinase activity plays an important role in this process. Scatchard analysis demonstrated a 67% reduction in estrogen-binding capacity in cells transfected with constitutively active Akt. No change in binding affinity of estradiol to the receptor was observed upon transfection with either Akt mutant. Taken together, our results suggest that estradiol treatment results in binding to membrane ER-α and interaction with a heterodimer containing ErbB2, leading to tyrosine phosphorylation. This results in the activation of PI 3-K and Akt. Akt, in turn, may interact with nuclear ER-α, altering its expression and activity.

Effects of Arsenite on Estrogen Receptor-α Expression and Activity in MCF-7 Breast Cancer Cells1

To determine whether arsenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ERalpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 microM arsenite resulted in a 60% decrease in the amount of ERalpha and in a parallel decrease of 40% in ERalpha messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment. pS2 messenger RNA also increased 2. 1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ERalpha and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ERalpha (Ki = 5 +/- 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected with the chimeric receptors GAL-ER, which contains the hormone-binding domain of ERalpha and the DNA-binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite activates ERalpha through an interaction with the hormone-binding domain of the receptor. Transfection assays with ERalpha mutants identified C381, C447, H524, and N532 as interaction sites of arsenite with the hormone-binding domain.

Role of insulin-like growth factor-I in regulating estrogen receptor-α gene expression

The role of insulin‐like growth factor‐I (IGF‐I) in regulating estrogen receptor‐α (ER‐α) gene expression and activity was investigated in the human breast cancer cell line MCF‐7. Treatment of cells with 40 ng/ml IGF‐I resulted in a 60% decrease in ER‐α protein concentration by 3 h, and the amount of ER‐α remained suppressed for 24 h. A multiple‐dose ligand‐binding assay demonstrated that the decrease in ER‐α protein corresponded to a similar decrease of 50% in estradiol‐binding sites with no effect on the binding affinity of ER‐α. The dissociation constant of the estradiol‐ER‐α complex in the absence of IGF‐I (Kd = 3 × 10−10 ± 0.5 × 10−10 M) was similar to the dissociation constant in the presence of IGF‐I (Kd = 6 × 10−10 ± 0.3 × 10−10 M). The decrease in ER‐α protein concentration was paralleled by an 80% decrease in the steady‐state amount of ER‐α mRNA by 3 h. The IGF‐I induced decrease in ER‐α mRNA was due to the inhibition of ER‐α gene transcription. When an 128‐base pair ER‐α‐promoter‐CAT construct was transfected into MCF‐7 cells, treatment with IGF‐I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER‐α, treatment with IGF‐I induced two endogenous estrogen‐regulated genes, progesterone receptor and pS2, by 4‐ and twofold, respectively. The pure antiestrogen ICI‐164,384 blocked this induction, suggesting that ER‐α mediates the effects of IGF‐I. Transient co‐transfections of wild‐type ER‐α and an estrogen response element‐CAT reporter into COS‐1 cells demonstrated that IGF‐I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3‐kinase inhibitors blocked the IGF‐I effects on ER‐α expression and activity, suggesting that these kinases may be involved in the cross‐talk between the IGF‐I and ER‐α pathways. J. Cell. Biochem. 76:605–614, 2000.

Regulation of estrogen receptor‐α gene expression by 1,25‐dihydroxyvitamin D in MCF‐7 cells

This report describes an investigation of the role of 1,25‐dihydroxyvitamin D (VD3) in the regulation of estrogen receptor‐α (ER) in the ER‐positive breast cancer cell line, MCF‐7. Treatment of cells with 10 nM VD3 resulted in a 50% decline in the concentration of ER protein at 24 h. Scatchard analysis showed a corresponding decrease in the number of estradiol binding sites and no alteration in the binding affinity of estradiol for the ER (Kd = 0.08 nM in VD3‐treated cells compared with Kd = 0.07 nM in control cells). Vitamin D treatment also caused a 50% decrease in the steady state amount of ER mRNA, which was maximal by 18 h. In vitro transcription run‐on experiments demonstrated a decrease of approximately 60% in transcription of the estrogen receptor gene. Transient transfections using an ER promoter‐CAT construct also demonstrated a 40% decrease in CAT activity after VD3 treatment. Sequence analysis identified a potential vitamin D response element (nVDRE) within the ER promoter. When this element was mutated, the ability of VD3 to block transcription from the ER promoter was lost. When the nVDRE was placed upstream of a heterologous promoter, nVDRE‐SV40‐CAT, treatment with VD3 resulted in a 50% decrease in CAT activity. Interestingly, co‐transfection of either the ER promoter‐CAT or the nVDRE‐SV40‐CAT construct and a vitamin D receptor expression vector into COS‐1 or CV‐1 cells showed an approximately 4‐fold increase in CAT activity after VD3 treatment. Taken together these data suggest that VD3 inhibition of ER gene transcription is mediated through a nVDRE in the ER promoter. Inhibition appears to be cell specific. J. Cell. Biochem. 75:640–651, 1999.

Effects of selenite on estrogen receptor-? expression and activity in MCF-7 breast cancer cells

To determine whether selenite has estrogen‐like activities, the effects of this compound on estrogen receptor‐α (ER‐α) and other estrogen‐regulated genes were measured in the human breast cancer cell line MCF‐7. Treatment of cells with 1 uM of sodium selenite resulted in a 40% decrease in the amount of estrogen receptor‐α and in a parallel decrease of 40% in ER‐α mRNA. Progesterone receptor concentration increased 2.6‐fold and pS2 mRNA increased 2.4‐fold after selenite treatment. The induction of progesterone receptor and pS2 was blocked by the anti‐estrogen ICI‐182,780. In transient co‐transfection experiments of Wild‐type ER‐α and an estrogen response element‐reporter construct, selenite stimulated CAT activity. In binding assays, selenite blocked the binding of estradiol to ER‐α (Ki = 23 ± 17 nM, n = 3) suggesting that this compound interacts with the hormone binding domain of the receptor. To determine whether interaction of selenite with the hormone binding domain results in receptor activation, COS‐1 cells were transiently co‐transfected with the chimeric receptors GAL‐ER, which contains the hormone binding domain of ER‐α and the DNA binding domain of the transcription factor GAL4, and a GAL4‐responsive CAT reporter gene. Treatment of cells with estradiol or selenite resulted in a three‐ to five‐fold increase in CAT activity. The effects of selenite on the chimeric receptor were blocked by the antiestrogen, suggesting that selenite activates ER‐α through an interaction with the hormone binding domain of the receptor. Transfection assays with ER‐α mutants identified C381, C447, H524, and N532 as interaction sites of selenite with the hormone binding domain. J. Cell. Biochem. 79:282–292, 2000.

The Role of Transforming Growth Factor-β in the Regulation of Estrogen Receptor Expression in the MCF-7 Breast Cancer Cell Line1

The role of transforming growth factor-β1 (TGFβ1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pm TGFβ1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nm in TGFβ1-treated cells compared to 0.155 nm in control cells. Treatment with TGFβ1 did not influence the half-life of the ER. In TGFβ1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFβ1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (∼75%) at 6 h. By 24...

Heregulin-β1 regulates the estrogen receptor-α gene expression and activity via the ErbB2/PI 3-K/akt pathway

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-α (ER-α) pathways. Treatment of MCF-7 cells with 10−9 M heregulin-β1 (HRG-β1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-β1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-β1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-α into these cells restored Akt phosphorylation by HRG-β1, suggesting the requirement of ER-α. HRG-β1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-β1 on ER-α expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-β1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-β1 on ER-α expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-β1 on ER-α regulation. Taken together, our data suggest that HRG-β1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-α, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-α phosphorylation, thereby altering its expression and transcriptional activity.

Activation of Estrogen Receptor-α by the Anion Nitrite

In this study, the ability of nitrite and nitrate to mimic the effects of estradiol on growth and gene expression was measured in the human breast cancer cell line MCF-7. Similar to estradiol, treatment of MCF-7 cells with either 1 mumol/L nitrite or 1 mumol/L nitrate resulted in approximately 4-fold increase in cell growth and 2.3-fold to 3-fold increase in progesterone receptor (PgR), pS2, and cathepsin D mRNAs that were blocked by the antiestrogen ICI 182,780. The anions also recruited estrogen receptor-alpha (ERalpha) to the pS2 promoter and activated exogenously expressed ERalpha when tested in transient cotransfection assays. To determine whether nitrite or nitrate was the active anion, diphenyleneiodonium was used to inhibit oxidation/reduction reactions in the cell. The ability of diphenyleneiodonium to block the effects of nitrate, but not nitrite, on the induction of PgR mRNA and the activation of exogenously expressed ERalpha suggests that nitrite is the active anion. Concentrations of nitrite, as low as 100 nmol/L, induced a significant increase in PgR mRNA, suggesting that physiologically and environmentally relevant doses of the anion activate ERalpha. Nitrite activated the chimeric receptor Gal-ER containing the DNA-binding domain of GAL-4 and the ligand-binding domain of ERalpha and blocked the binding of estradiol to the receptor, suggesting that the anion activates ERalpha through the ligand-binding domain. Mutational analysis identified the amino acids Cys381, His516, Lys520, Lys529, Asn532, and His547 as important for nitrite activation of the receptor.