Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: Cells were cultured as described in “ ” and treated with E2 or ICI 184,780 (ICI) using EtOH as vehicle. The vehicle-only treatment served as a control. The mRNA levels of CXCL12 (A and B), CXCR4 (D and E) and CXCR7 (G and H) were quantified by real-time PCR analysis of cells treated for different periods of time with 10 −8 M E2 (A, D and G) or cells treated for 48 h with 10 −8 M E2 alone, 10 −6 M ICI alone or both E2 and ICI (B, E and H). The real-time PCR results were normalized against the internal control GAPDH and expressed as the mean CXCL12 , CXCR4 or CXCR7/GAPDH mRNA ratio ± SEM of at least three independent experiments. Protein levels of CXCL12 (C), CXCR4 (F) and CXCR7 (I) was assayed. Secreted CXCL12 protein levels after treatment with EtOH or 10 −8 M E2 for 48 h were determined by ELISA, and the values were normalized relative to the total protein concentration (C). The expression of CXCR4 and CXCR7 at the surface of MCF-7 cells was measured by flow cytometry after treatment with EtOH or 10 −8 M E2 for 48 h (F, I). Representative data from at least three experiments performed in duplicate are shown. Asterisks or different lowercase letters indicate significant differences ( p <0.05) between the control and treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: CXCL12 , CXCR4 and CXCR7 mRNA were assessed by quantitative real time PCR after 48 h treatment of ZR-75 (A) or MDA-MB-231 (B) cells to EtOH (−) or to10 −8 M E2 (+). Transcript levels were normalized against GAPDH mRNA and data were calculated as percentage of the E2 effect. Data are from triplicate samples and are representative of three separate experiments. Asterisk indicates significant differences ( p <0.05) between the control and ligand treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: The levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative real-time PCR in MCF-7 cells treated under various conditions for 48 h. Treatment with EtOH and 10 −8 M E2 served as the negative and positive controls, respectively. In each experimental assay, the cells were exposed to different concentrations of 17 α-ethynyl-estradiol (EE2) (A) and Genistein (Gen) (B). Transcript levels were normalized against GAPDH mRNA, and data were calculated as percentage of the E2 effect for each experiment. Significant differences (P<0.05) are indicated by different lowercase letters.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: (A) FAIRE assays were performed on MCF-7 cells exposed to either ETOH (−) or 10 −8 M E2 (+) for 48 h. Real-time PCR was performed to monitor enrichment of the DNA corresponding to the proximal promoters of the CXCL12 , CXCR4 and CXCR7 genes relative to input chromatin. The data are from triplicate samples and are representative of three separate experiments. Asterisks indicate significant differences ( p <0.05) between the control and treated cells. (B) The Integrated Genome Browser (Affymetrix) was used to visualize ER-binding sites in the regions surrounding the CXCL12 , CXCR4 and CXCR7 genes. Raw ChIP-chip data for ER and high confidence ER-binding sites called using the MAT algorithm are shown , . The numbered ER-binding sites correspond to bound regions in which the closest TSS is that of CXCL12 , CXCR4 or CXCR7 . Arrows indicate the orientation of the CXCL12 , CXCR4 and CXCR7 genes.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Binding Assay, ChIP-chip

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: siRNA directed against CXCL12, CXCR4 or CXCR7 was transfected into MCF-7 cells treated with EtOH (−) or 10 −8 M E2 (+). (A) After 48 h, the levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative PCR and normalized against GAPDH mRNA. The results were compared with those obtained from MCF-7 cells transfected with a nonspecific siRNA control. (B) Total protein was extracted from MCF-7 cells, and the levels of CXCL12, CXCR4 and CXCR7 were analyzed by Western blotting. (C) To determine the growth rate of the MCF-7 cells, the siRNA-transfected cells were treated with EtOH (−E2) or 10 −8 M E2 (+E2) for seven days. E2-dependent and -independent cell growth were evaluated using MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells transfected with the control siRNA and treated with EtOH (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (D) The effects of specific inhibitors for CXCL12 (Chalcon 4), CXCR4 (AMD3100) or CXCR7 (CCX771) were measured after treatment of MCF-7 cells with either EtOH (−E2) or 10 −8 M E2 (+E2) for 7 days. DMSO (vehicle) was used as the control. E2-dependent and E2-independent cell growth were then evaluated by MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells treated with the vehicle control (considered as 100%). Significant differences ( p <0.05) between treated cells in the absence of E2 are indicated by an asterisk and between treated cells in the presence of E2 by a sharp symbol.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: MCF-7 cells were transiently transfected with either a control expression vector or one containing the human CXCR7 open reading frame. (A) Total protein extracts were prepared 48 h after transfection, and a Western blot analysis was performed to confirm CXCR7 over-expression. (B) Transfected cells were cultured in the presence of EtOH (−) or 10 −8 M E2 (+) for seven days. E2-dependent and E2-independent cell growth rates were then evaluated by cell count of three independent experiments (n = 3). The results are expressed as a percentage of the relative cell number obtained from control cells treated with E2 (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (C) A proposed model for the involvement of the CXCL12 signaling axis in E2-dependent and -independent cell growth is shown. The binding of CXCL12 to CXCR4 and CXCR7 leads to the stimulation of cell growth through diverse pathways . CXCR7 can also modulate CXCL12 availability by removing the chemokine from the extracellular space (left panel). Estrogens could stimulate cell growth by favoring the activation of CXCL12 through CXCR4 and reducing the expression of CXCR7 (right panel).

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Over Expression, Cell Culture, Cell Counting, Binding Assay, Activation Assay