24 research outputs found

    Immunohistochemical staining of Src expression and phosphorylation in primary breast tumors and Kaplan-Meier survival estimates relative to the expression of Src at the plasma membrane.

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    <p>Representative pictures of weak (A) and strong (B) immunohistochemical staining of total Src and of weak (C) and strong (D) immunohistochemical staining of phosphorylated Src (Tyr<sup>416</sup>) expressed in primary breast tumors. Kaplan-Meier survival plots demonstrating percentage disease-free (E) and overall (F) survival in patients with tumors with or without Src expressed at the plasma membrane.</p

    Effect of dasatinib, tamoxifen and fulvestrant on growth of parental and resistant T47D cells.

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    <p>(A, B) Parental and resistant cell lines were grown for 5 days with vehicle (0.1% DMSO), 1 μM dasatinib (Das), 100 nM fulvestrant (Fulv) or 1 μM tamoxifen (Tam) alone or in the indicated combinations. (C) Tamoxifen resistant cell lines were treated for five days with 100 nM fulvestrant and dasatinib at the indicated concentrations. Cell number was measured by a crystal violet colorimetric assay and expressed as percentage of untreated control. A representative experiment out of three with mean ± SD is shown. * P<0.05 for treated vs untreated cells; # P<0.05 for cells treated with dasatinib vs cells treated with a combination of dasatinib and tamoxifen or fulvestrant.</p

    Effect of dasatinib on expression of Src and downstream signaling in parental and resistant cells and identification of Src dimers.

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    <p>(A) Western blot showing expression of total and phosphorylated Src (Tyr<sup>416</sup>) in lysates from parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182<sup>R</sup>-1 and 182<sup>R</sup>-2) resistant cell lines grown under their standard conditions. β-actin was used as loading control. (B) Western blot showing expression of total and phosphorylated Akt (Ser<sup>473</sup>), Erk (Thr<sup>202</sup>/Tyr<sup>204</sup>) and Src (Tyr<sup>416</sup>) in lysates from parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182<sup>R</sup>-1 and 182<sup>R</sup>-2) resistant cell lines grown with or without dasatinib (1 μM) for 1 hour. Hsp70 was used as loading control. (C) Src dimers in parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182R-1 and 182R-2) resistant cell lines grown under their standard conditions. Src protein was immunoprecipitated from 1 mg total protein lysates. Western analysis for HER receptors, Src and ER was performed on both total protein lysates and immunoprecipitated samples. A representative experiment out of two is shown. β-actin was used as loading control.</p

    Identification of dasatinib as a kinase driving growth of tamoxifen and fulvestrant resistant breast cancer cell lines.

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    <p>(A, C) Parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182<sup>R</sup>-1 and 182<sup>R</sup>-2) resistant cell lines were treated for 5 days with a kinase inhibitor library comprising 195 different kinase inhibitors (1 μM). Cell number was assessed by a CellTiter-Glo Luminescent Cell Viability Assay. Volcano plots are displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells. The boxes indicate kinases with more than two-fold preferential growth inhibition of the resistant cell lines compared to their parental T47D cells and P<0.05. (B, D) Mean cell numbers of parental and resistant cells treated with 1 μM dasatinib shown as percentage of untreated control. The results are from the kinase inhibitor screens. (E, F) Parental and resistant cell lines were treated for 5 days with the indicated concentrations of dasatinib. Cell number was measured by a colorimetric assay and expressed as percentage of untreated control (designated C). A representative experiment with mean ± SD is shown. * P<0.05 for dasatinib treated samples vs control.</p

    Effect of dasatinib, tamoxifen and fulvestrant alone or in the indicated combinations on long-term propagation of parental and tamoxifen resistant cell lines.

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    <p>T47D/S2 (A, B), TR-1 (C, D) and TR-2 (E, F) cells were seeded with 4x10<sup>5</sup> cells in T25-flasks and treated for one week with 1 μM dasatinib (Das), 1 μM tamoxifen (Tam) or 100 nM fulvestrant (Fulv) alone or in combination as indicated in the figure. Control cells were propagated in medium containing DMSO and/or ethanol (vehicle) corresponding to the same amount as treated cultures. Growth rate was determined as the ratio between cell number after one week treatment and number of seeded cells. After cell number determination, 4x10<sup>5</sup> cells were reseeded in T25-flasks and allowed to grow for additional two weeks with weekly split and determination of cell number. In cases with fewer than 4x10<sup>5</sup> cells, the total number of cells were added and used for the calculations. w/o; without.</p

    Effect of dasatinib on the morphology of parental and resistant cells.

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    <p>Representative pictures of parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-1) and fulvestrant (182<sup>R</sup>-1 and 182<sup>R</sup>-2) resistant cell lines treated for five days with dasatinib (1 μM) or DMSO (control).</p

    Validation of the basal <i>BRCA1</i> signature and lumB <i>BRCA2</i> signature in independent datasets.

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    <p>A) The basal <i>BRCA1</i> signature was validated using basal-like tumor samples obtained from the NKI dataset and Jönsson dataset, respectively. The panels show the <i>BRCA1</i> probability estimates of basal-like <i>BRCA1</i> samples (red) and basal-like sporadic samples (grey). B) The lumB <i>BRCA2</i> signature was validated using lumB tumor samples obtained from the Jönsson dataset. The panel shows the <i>BRCA2</i> probability estimates of lumB <i>BRCA2</i> samples (blue) and lumB sporadic samples (grey). Probability estimates were obtained by leave-one-out cross-validation. Dashed lines indicate the <i>BRCA1/2</i> probability cutoff. Samples with probabilities ≥0.5 are classified as <i>BRCA1/2</i>, while samples with probabilities <0.5 are classified as sporadic tumors. Samples have been “jittered” in the vertical direction to spread them out for better visualization.</p

    Validation of gene signatures in independent datasets.

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    <p>The basal BRCA1 signature and lumB BRCA2 signature was validated in two public available datasets. Classification performances were assessed by leave-one-out cross-validation. TP, true positive; TN, true negative.</p>a<p>Mean balanced accuracy.</p>b<p>Fisher's exact test.</p
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