IGF-1 treatment does not protect MCF-7 cells from 4OH-tamoxifen-induced cell death.

<p>(A) MCF-7 cells were co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence. 48 h after transfection, cells were pre-incubated for 6 h in the absence or presence of PUGNAc+GlcN. Cells were incubated for 10 min with coelenterazine and then stimulated with 100 nM IGF-1. Light acquisition at 480 nm and 530 nm started immediately after IGF-1 addition. A typical experiment is shown. (B) PUGNAc+GlcNAc and IGF-1-induced BRET (BRET above basal at the plateau). Results are the means ± S.E.M. of at least 5 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey’s post-test. *, P< 0.05; **, P< 0.01; NS, not significant. (C) MCF-7 cells were cultured in presence of 1% FBS during 24 h in the absence or presence PUGNAc (100 µM), Glucosamine (GlcN, 5 mM), 4-OH-tamoxifen (10 µM) and IGF-1 (100 nM). Cell population growth in each well was evaluated using an Uptiblue-based-assay as the ratio of final fluorescence over the initial one (broken line) in the same well. Each determination corresponds to measurements performed in triplicate wells. Results are the mean±SEM of 5 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey’s post-test. *, P< 0.05; **, P< 0.01; NS, not significant.</p

Inhibition of ERα expression level by O-GlcNAcylation-inducing treatments.

<p>Cells were cultured for 24 h in the absence or presence of PUGNAc+GlcN. (A) RNA were extracted and the expression of ERα mRNA was evaluated by RT-qPCR. (B) Cells were lysed and the expression of ERα protein was analysed by western-blot. GAPDH expression level was used as loading control. (C) ERα/GAPDH signals quantified by densitometric analysis of the autoradiograms of western-blots from 6 independent experiments. Statistical analysis was performed using unpaired t test. *, P< 0.05; ***, P< 0.001.</p

Inhibition of OGT expression sensitizes MCF-7 cells to tamoxifen-induced apoptosis.

<p>MCF-7 cells were transfected with control (siNEG) or anti-OGT siRNA (siOGT, sequence: TGGCATCGACCTCAAAGCA). (A) OGT protein expression and global O-GlcNAc levels were analysed 48h later by western-blot. (B) 48h after transfection with siRNA, cells were cultured for 24 hours in absence or presence of 4-OH-tamoxifen (10 µM) and then analysed by FACS after Annexin V-FITC/Propiduim Iodide staining. Statistical analysis was performed using ANOVA followed by Tukey’s post-test *, P< 0.05. Results correspond to the mean±SEM of 5 independent experiments.</p

O-GlcNAc-inducing treatments stimulate the PI-3 kinase/Akt pathway in MCF-7 cells.

<p>(A) Principle of the BRET assay to measure PIP₃ production in living cells. Activation of PI-3 kinase induces the phosphorylation of phosphatidyl-inositol 2 phosphate (PIP₂) into phosphatidyl-inositol 3 phosphate (PIP₃) and subsequent recruitment of Akt to the plasma membrane through its pleckstrin homology (PH) domain. To monitor the production of PIP₃ induced by receptor activation, cells are co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence. (B) 48 h after transfection, cells were pre-incubated for 6 h in presence of PUGNAc, GlcN or both. Cells were incubated for 10 min with coelenterazine, then light acquisition at 480 nm and 530 nm started. A typical experiment is shown. (C) PUGNAc and GlcNAc-induced BRET (BRET above basal at the plateau). Results are the means ± S.E.M. of 4 to 8 independent experiments. Statistical analysis was performed using ANOVA followed by Dunnet’s post-test. **, P< 0.01 when compared to untreated control. (D) Effect of O-GlcNAcylation-inducing treatment on Akt phosphorylation. MCF-7 cells were incubated in the absence or presence of PUGNAc+GlcN for 6h or 24 h and lysed. The phosphorylation of Akt was evaluated by western-blot using anti-phospho-S473-Akt antibody.</p

The hexosamine biosynthetic pathway controls O-GlcNAc-modification of proteins.

<p>Cytosolic and nuclear O-GlcNAc glycosylation constitutes a dynamic process that regulates the activity, the localisation or the stability of the modified proteins. O-GlcNAc glycosylation of proteins on serine and threonine residues depends on the flux of glucose through the hexosamine biosynthetic pathway (HBP). A fraction (2 to 5%) of the glucose entering the cell is directed into this pathway for the biosynthesis of UDP-GlcNAc. OGT uses UDP-GlcNAc as a substrate to add GlcNAc on serine or threonine residues, and its activity is tightly dependent on the concentration of UDP-GlcNAc in the cell. These modifications can be reversed by O-GlcNAcase (OGA), which removes the O-GlcNAc moiety from O-GlcNAc-modified proteins. Experimentally, the level of O-GlcNAc in proteins can be increased by incubating cells with glucosamine (which bypasses allosteric inhibition of the rate limiting enzyme GFAT (Glutamine Fructose 6-P amidotransferase)), or with PUGNAc (O-[2-acetamido-2-deoxy-D-glucopyranosylidene] amino-N-phenylcarbamate), which inhibits deglycosylation by OGA.</p

Protection of MCF-7 cells from 4-OH-tamoxifen-induced cell death is not abrogated by inhibition of the PI-3 kinase/Akt pathway.

<p>MCF-7 cells were cultured in presence of 1% FBS during 24 hours in the absence or presence PUGNAc (100 μM), Glucosamine (GlcN, 5 mM), 4-OH-tamoxifen (10 μM) and either DMSO (vehicle) or the PI-3 kinase inhibitor LY294002 (LY) at a concentration of 10 or 20 µM. The growth of the cell population in each well was evaluated using Uptiblue-based-assay as the ratio of final fluorescence over the initial one (broken line) in the same well. Each determination corresponds to measurements performed in triplicate wells. Results are the mean±SEM of 3 independent experiments. Statistical analysis was performed using ANOVA followed by Dunnet’s post-test. *, P< 0.05 when compared to corresponding untreated control; NS, not significant.</p

PUGNAc+GlcNAc treatment inhibited the effect of 4-OH-tamoxifen on <i>p21</i> and <i>Egr1</i> gene expression.

<p>Cells were cultured for 24h in absence or presence of 4-OH tamoxifen or in presence of 4-OH-Tamoxifen and PUGNAc+GlcN. (A) RNA were extracted and the level of p21 and Egr1 mRNA was evaluated by RT-qPCR and normalised using Cyclophilin A expression levels. Results are the mean ±SEM of 3 independent experiments. Statistical analysis was performed by ANOVA followed by Dunnet’s post-test. *, P< 0.05; **, P< 0.01 when compared to untreated control.</p

O-GlcNAcylation-inducing treatments protect from tamoxifen-induced cell death.

<p>MCF-7 cells were cultured in presence of 1% FBS during 24 h (A) or 48 h (B), in the absence or presence PUGNAc (100 µM), Glucosamine (GlcN, 5 mM) and 4-OH-tamoxifen (10 µM). The cell population growth in each well was evaluated using an Uptiblue-based-assay as the ratio of final fluorescence over the initial one (broken line) in the same well. Each determination corresponds to measurements performed in triplicate wells. Results are the mean±SEM of at least 7 independent experiments. Statistical analysis was performed using ANOVA followed by Dunnet’s post-test. *, P< 0.05; **, P< 0.01 when compared to untreated control; NS, not significant. (C) MCF-7 cells were cultured in presence of 1% FBS during 24 h, in the absence or presence of PUGNAc+GlcN and 4-OH-tamoxifen (10 µM) and then analysed by FACS after Annexin V-FITC/Propidium Iodide staining. Statistical analysis was performed using ANOVA followed by Tukey’s post-test *, P< 0.05; **, P< 0.01. Results correspond to the mean±SEM of at least 4 independent experiments.</p

Time-course of inhibition of ERα expression by O-GlcNAcylation-inducing treatments.

<p>Cells were cultured in absence or presence of PUGNAc+GlcN for 6, 12, 24 and 36 h. (A) RNA was extracted and the expression of ERα mRNA was evaluated by RT-qPCR. Statistical analysis was performed using unpaired <i>t</i> test. *, P< 0.05; **, P< 0.01; ***, P< 0.001. Results correspond to the mean±SEM of at least 4 independent experiments. (B) Cells were lysed at the indicated times and the expression of ERα protein was analysed by western-blot. GAPDH expression level was used as loading control. The effect of PUGNAc+GlcN treatment on O-GlcNAcylation of proteins was controlled using anti-O-GlcNAc antibody. Results are representative of at least 3 independent experiments.</p

Effect of ERα gene inactivation on <i>in vitro</i> testicular response to BPA.

<p>Testes from homozygous 12.5 dpc (ERα−/−), heterozygous (ERα+/−) and wild-type (ERα+/+) ERα-deficient fetuses were cultured on floating filters for 48 h. One testis from each animal was cultured in control medium and the other one in medium containing 10⁻⁵ M BPA. Values are means ± SEM of testosterone secreted in the medium during the 2 days of culture by BPA-treated testes referred to testosterone secreted by the respective contralateral control testes; n = 6 (ERα+/+), 13 (ERα+/−) and 5 (ERα−/−), * p<0.05 in the statistical comparison between BPA-treated and control testes using the Wilcoxon’s parametric paired t test.</p