E₂-ERα signaling pathway interferes with CIITA pIV activity in MC2.

<p>VC5 and MC2 cells were cultured in E₂-depleted media followed by transfection with CIITA pIV luciferase constructs. On the following day, cells were treated with vehicle (ethanol), E₂ (10⁻⁹ M) and/or ICI (10⁻⁶ M), and stimulated or not with IFN-γ (100 U/ml) for 12 hours. Data are expressed as fold induction over the PGL2 Basic empty plasmid after controlling for transfection efficiency using cells dual transfected with GFP (Green Florescent Protein). The effect of ERα on the transcription activation of CIITA PIV was determined from relative luciferase activities in transfected MC2. Error bars represent the mean ± SEM of three independent experiments (**p<0.01).</p

Coordinate downregulation of IFN-γ inducible HLA-II expression by E₂ is reversed by ICI-mediated degradation of ERα in MC2 cells.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) followed by stimulation with IFN-γ (100 U/ml) for 96 hours. HLA-II expression was analyzed by surface flow cytometry using (A) anti-DR, (L243), and intracellular flow cytometry using (B) anti-DM (Map.DM1) and (C) anti-Ii (LN2). Bar graphs represent the MFI ± SEM of three independent experiments. (*p<0.05, **p<0.01). (D) Western blot analysis was performed on whole cell extracts using for HLA-DRα (TAL 1B5), HLA-DM (TAL18.1) and Ii (LN2); GAPDH (Ab8245) is the protein loading control. Bar graphs show the ratio of band intensities, normalized to GAPDH band intensities and represent the mean ± SEM ratio of three independent experiments: (E) HLA-DRα/GAPDH (F) HLA-DM/GAPDH, and (G) Ii/GAPDH (* p<0.05, ** p<0.01).</p

E₂-ERα signaling down regulates CIITA protein and mRNA expression in ER⁺ BCCL.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) and stimulated or not with IFN-γ (100 U/ml) for 24 and 4 hours, for CIITA protein and mRNA expression, respectively. (A) Western blot analysis was performed on cytoplasmic and nuclear extracts for CIITA (antiserum #21) and ERα (HC-20). (B) Cytoplasmic CIITA and nuclear CIITA were normalized to GAPDH and P84 respectively; bar graphs represent the mean ± SEM ratio of three independent experiments (**p<0.01). (C) CIITA mRNA was relatively quantified by real time PCR using Taqman gene expression assay. GAPDH was used as an endogenous control and the data were expressed relative to a control B cell line (RAJI). Bar graphs represent the mean ± SEM of three replicate assays (**p<0.01).</p

E₂ differentially modulates inducible HLA-DR expression in ERα⁺ and ERα⁻ breast cancer cell lines.

<p>MDA-MB-231, SK-BR-3, MCF-7, BT-474, and T47D were cultured in E₂-depleted media, treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A & E) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B & F) Bar graphs represent the MFI (mean florescence intensity) ± SEM for HLA-DR expression of three independent experiments. (C & G) Western blot analysis was performed on cytoplasmic and nuclear extracts for ERα expression (HC-20) and on cytoplasmic extracts for HLA-DRα (TAL 1B5). Protein loading controls included α-tubulin (B-7) and P84 (5E10) for cytoplasmic and nuclear proteins, respectively. (D & H) Bar graphs show the ratio of band intensity for HLA-DRα, normalized to the α-tubulin band intensity and represent the mean ± SEM of three independent experiments (*p<0.05, **p<0.01, ***p<0.001).</p

Mutation of putative ERE sites in CIITA pIV does not enhance CIITA pIV activation in MC2.

<p>(A) CIITA pIV nucleotide sequence from −346 to +50 with the GAS and IRF1 binding sites (shaded hexagon) and the predicted ERE (clear rectangles) were identified using online transcription factor prediction software, (<a href="http://tfbind.hgc.jp/" target="_blank">http://tfbind.hgc.jp/</a>, <a href="http://alggen.lsi.upc.es/" target="_blank">http://alggen.lsi.upc.es/</a> and <a href="http://www.cbrc.jp/index.eng.html" target="_blank">http://www.cbrc.jp/index.eng.html</a>). Site directed mutagenesis was used to perform deletion of the predicted ERE. (B) VC5 and MC2 were transfected with CIITA pIV constructs, then treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated with IFN-γ (100 U/ml) for 12 hours, followed by determination of luciferase activity. Bar graphs represent the mean ± SEM of three independent experiments (**p<0.01, ***p<0.001).</p

E₂ differentially down regulates IFN-γ signaling and IFN-γ induced proteins in endogenous ER⁺ breast cancer cell lines.

<p>(A) MCF-7, (B) BT-474, (C) T47D, (G) MDA-MD-231, and (H) SK-BR-3 were cultured in E₂-depleted media, transfected with 8 X GAS binding sequence construct, then treated with vehicle (ethanol), E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 6 hours. Firefly luciferase activities in samples were normalized to Renilla luciferase activities in the same samples and expressed as fold induction over the un-stimulated mock. (D) MCF-7, (E) BT-474, (F) T47D, (I) MDA-MB-231 and (J) SK-BR-3 were cultured in E₂-depleted media, treated with vehicle (ethanol), or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. Western blot analysis of cytoplasmic extracts was performed for expression of IFN-γ inducible proteins: STAT1 (06-501), IRF1 (BD-20), IRF9 (C-20), GILT (T-18). Each figure represents one of three independent experiments.</p

IFN-γ inducible HLA-DR is down regulated in the ERα⁺ transfected breast cancer cell line, MC2.

<p>MDA-MB-231 clone 10A (MDA-231 c10A), VC5 (MDA-231 c10A, transfected with the empty plasmid vector) and MC2 (MDA-231 c10A, transfected with wild type <i>ESR1</i>) were cultured in E₂-depleted medium and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B) Bar graphs represent the MFI ± SEM for HLA-DR expression of three independent experiments (***p<0.001). (C) Western blot analysis was performed on whole cell lysates for HLA-DRα (TAL 1B5) and ERα (HC-20).</p

Silencing ERα with ESR1 siRNA reversed the inhibitory effect of E₂ on CIITA expression.

<p>(A) ERα was silenced (<i>ESR1</i> siRNA) or not (scrambled siRNA) in MC2; VC5 served as an ERα negative cell control. Cells were treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 24 hours. Nuclear lysates were prepared and probed for CIITA (anti-serum #21), ERα (HC-20), and p84. Each figure represents one of three individual experiments. (B) <i>ESR1</i> siRNA and scrambled siRNA transfected MC2 cells were treated with either vehicle (ethanol) or E₂ (10⁻⁹ M) followed by stimulation with or without IFN-γ (100 U/ml) for 4 hours and CIITA mRNA was relatively quantified by real time PCR using Taqman gene expression assay. GAPDH was used as an endogenous control and the data were expressed relative to a control B cell line (RAJI). Bar graphs represent the mean ± SEM of three replicate assays (*** p<0.001).</p