15 research outputs found

    Molecular Mechanisms of Selective Estrogen Receptor Modulator Activity in Human Breast Cancer Cells: Identification of Novel Nuclear Cofactors of Antiestrogenā€“ERĪ± Complexes by Interaction Proteomics

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    Estrogen receptor alpha (ERĪ±) is a ligand-activated transcription factor that controls key cellular pathways <i>via</i> proteinā€“protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERĪ± ligands are classified as agonists (17Ī²-estradiol/E<sub>2</sub>), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERĪ± conformations, characterized by specific surface docking sites for functional proteinā€“protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERĪ± interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E<sub>2</sub>)- vs antagonist (Tam, Ral or ICI)-bound ERĪ± interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogenā€“ERĪ± complexes in human BC cell nuclei. In particular, the E<sub>2</sub>-dependent nuclear ERĪ± interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds

    Molecular Mechanisms of Selective Estrogen Receptor Modulator Activity in Human Breast Cancer Cells: Identification of Novel Nuclear Cofactors of Antiestrogenā€“ERĪ± Complexes by Interaction Proteomics

    No full text
    Estrogen receptor alpha (ERĪ±) is a ligand-activated transcription factor that controls key cellular pathways <i>via</i> proteinā€“protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERĪ± ligands are classified as agonists (17Ī²-estradiol/E<sub>2</sub>), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERĪ± conformations, characterized by specific surface docking sites for functional proteinā€“protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERĪ± interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E<sub>2</sub>)- vs antagonist (Tam, Ral or ICI)-bound ERĪ± interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogenā€“ERĪ± complexes in human BC cell nuclei. In particular, the E<sub>2</sub>-dependent nuclear ERĪ± interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds

    Analysis of FRTL-5 cell response to UV-C irradiation following long-term BPA treatment.

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    <p>(<b>A</b>) Proliferation rate analysis of FRTL-5 cells treated for 28 days with 10<sup>āˆ’9</sup> M BPA and then subjected to UV-C irradiation. Cells were counted every 24 hrs until 144 hrs post-irradiation. Population doubling was calculated as described in Materials and Methods. Data are reported as mean Ā± standard deviation of three independent experiments. (<b>B</b>) Quantification of DNA damage by comet assay. Data are reported as mean Ā± standard deviation of the tail intensity of around 100 cells analyzed for each point. (<b>C</b>) qRT-PCR analysis of the pattern of <i>p21</i> transcript levels following UV-C irradiation. Data are reported as the ratio between <i>p21</i> transcript levels in irradiated and control cells. The mean Ā± standard deviation of three independent experiments is reported. *<i>p</i>-value <0.05; **<i>p</i>-value <0.01. (<b>D</b>) Quantification of apoptotic cells by TUNEL staining. Data are reported as percentage of TUNEL positive cells per total cell number identified by DAPI staining. The results are expressed as mean Ā± standard deviation of several fields analyzed in three independent experiments.</p

    Summary of BPA mechanisms of toxicity.

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    <p>(<b>A</b>) Cells exposure to high-dose of BPA can strongly deregulates the expression of a single (a) or few genes in the same pathway (b), impairing a cellular function. (<b>B</b>) Low-dose BPA exposure can induce a slight deregulation of many genes in the same pathway without compromising a specific phenotype. If a second injury is applied, the damage is highlighted by phenotypic changes potentially representing a hazard for health.</p

    Transcript inhibition in long-term BPA-exposed FRTL-5 cells.

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    <p>(<b>A</b>) Proliferation rate analysis of FRTL-5 cells exposed for 28 days to 10<sup>āˆ’9</sup> M BPA. Cells were counted every 7 days and the population doubling calculated as described in Material and Methods. Data are reported as mean Ā± standard deviation of three independent experiments. (<b>B</b>) qRT-PCR analysis of <i>Atf4</i>, <i>Ddit3</i>, <i>Tp53</i> and (<b>C</b>) of <i>Cops4</i>, <i>Cops5</i>, <i>Cops6</i>, <i>Cops8</i> and <i>Ddb1</i> in FRTL-5 cells treated for 28 days. Data are reported as the ratio between mRNA levels in 10<sup>āˆ’9</sup> M BPA-treated and control samples. The mean Ā± standard deviation of three independent experiments is reported. *<i>p</i>-value <0.05; **<i>p</i>-value <0.01.</p

    Time-dependent transcriptome perturba4tion induced by low-dose BPA in FRTL-5 cells.

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    <p>Volcano plots of microarray data after 1-day (<b>A</b>), 3-day (<b>B</b>) and 7-day (<b>C</b>) treatment with 10<sup>āˆ’9</sup> M BPA compared to untreated cells. The <i>y</i>-axis value is the negative logarithm (base 10) of the corrected <i>p</i>-value. A green horizontal line on the plot represents the significant threshold for <i>p</i>-value. The <i>x</i>-axis is shown as the logarithm (base 2) of the FC in expression level between treated and control cells. The vertical green lines on the plot represent the thresholds for FC. Red dots are up-regulated probes; green dots are down-regulated probes. The number of down/up-regulated genes for each Volcano plot is reported in the underlying table. (<b>D</b>) Venn Diagram showing the gene set overlap between 3- and 7-day treatments.</p

    Time-dependent inhibition of genes in FRTL-5 cells treated with BPA verified by qRT-PCR.

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    <p>qRT-PCR analysis of some microarray down-regulated genes in FRTL-5 cells after 1-, 3-, and 7-day 10<sup>āˆ’9</sup> M BPA treatment. In (<b>A</b>) and (<b>B</b>), genes enriched in the ā€œDNA replication, recombination, and repair, developmental disorder, hereditary disorderā€ network, predicted in the 7-day gene set, are reported. In (<b>C</b>) and (<b>D</b>), transcripts leading to the prediction of p53 inhibition are shown. Data are reported as the ratio between mRNA content in BPA-treated and control samples. The mean Ā± standard deviation of three independent experiments is reported. *<i>p-value</i> <0.05; **<i>p-value</i> <0.01.</p

    Tp53 and TF regulation following BPA treatment in FRTL5 cells.

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    <p>(<b>A</b>) qRT-PCR analysis of Tp53 transcript in FRTL-5 cells after 1-, 3-, and 7-day treatment with 10<sup>āˆ’9</sup> M BPA. Data are reported as the ratio between transcript levels in BPA-treated and control samples. The mean Ā± standard deviation of three independent experiments is reported. *<i>p-value</i> <0.05; **<i>p-value</i> <0.01. (<b>B</b>) Western blot analysis of Tp53 nuclear protein levels in FRTL-5 cells after 1-, 3-, and 7-day treatment with 10<sup>āˆ’9</sup> M BPA. (<b>C</b>) Schematic representation of <i>Tp53</i> promoter (-300/+130bp), depicting binding sites for TF predicted modulated by IPA. (<b>D</b>) Western blot analysis of p65 and c-Myc nuclear protein levels in FRTL-5 cells after 1-, 3-, and 7-day treatment with 10<sup>āˆ’9</sup> M BPA. Topoisomerase I was used as loading control. Data are representative of three independent experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151618#pone.0151618.s004" target="_blank">S4 Fig</a>). (<b>E</b>) Western blot analysis of p-Akt (Ser 473) and Akt in FRTL-5 cells after 7-day treatment with 10<sup>āˆ’9</sup> M BPA. Ī²-actin was used as loading control. Data are representative of three independent experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151618#pone.0151618.s004" target="_blank">S4 Fig</a>).</p
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