Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection

Cell surface-associated anti-MUC1-derived signal peptide antibodies: implications for cancer diagnostics and therapy.

The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1's signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin

Proteasome inhibition prolongs the pSmad3 signal with a dependence on TβRI kinase activity.

<p>A, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, treated with 2ME2 or vehicle, proteasome inhibitors (MG132 and ALLN), or their combination, and stimulated with TGF-β1 for the indicated times. B, Bar graph depicts average ± SD of the proteasome inhibitor protection factor (PIP) at each time point after ligand addition. The factor is defined as PIP = 1−(pSmad3C/tSmad3/clathrin)_uninhibited/(pSmad3C/tSmad3/clathrin)_inhibited. n = 3; p<0.015, 1-tailed t-test. C, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, treated with proteasome inhibitors or vehicle and stimulated with TGF-β1. In indicated samples, SB431542 was added at 1 h after TGF-β1 stimulation.</p

2ME2 impairs the turnover and endocytosis of myc-TβRII-GFP.

<p>A, ES-2 cells, stably expressing myc-TβRII-GFP, were treated with 2ME2 or vehicle. Arrested and cycling cells were pre-treated for 20 min with cycloheximide (300 µg/ml), in combination (or not) with proteasome inhibitors (MG132 and ALLN); followed by the addition of TGF-β1 (5 ng/ml, 3 h, in the same media). Cells were subsequently lysed and immunoblotted with α-myc antibodies. Bar graph depicts average ± SEM of the 3 h/0 h ratio of the relative myc-TβRII-GFP content, n = 3; p = 0.05, 2-tailed t-test. B, ES-2 cells grown on glass coverslips were treated as in (A), cooled to 4°C, stained with α-myc/Alexa-546-GαM/DAPI and imaged by confocal microscopy. Bar graph depicts average ± SEM of the 3 h/0 h ratio of the Alexa-546 staining normalized to the DAPI staining of 18 fields of cells (10 x magnification, ∼1400 cells) for each condition; p<0.01. C, Confocal micrographs of ES-2 cells, stably expressing myc-TβRII-GFP, treated with 2ME2, hypertonic medium (0.45 M sucrose, 30 min), or vehicle and submitted to endocytosis experiment (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043459#s2" target="_blank">Materials and Methods</a>). Bar, 10 µm. Specific signals were identified through intensity-based segmentation, and co-localized signal-positive pixels were determined with Slidebook™ and employed for quantification. D, Bar graph depicts the average fold change (± SEM) in the percent of 546-α-myc that did not co-localize with 647-GαM in all experimental conditions as compared to that of cells kept at 4°C. Cycling cells show a 1.82±0.11 fold increase in internalized TβRII (n = 35; p<0.01, 1-tailed t-test); no changes in the levels of internalized TβRII were observed upon hypertonic medium treatment (0.97±0.08 fold change, n = 8; p>0.8, 2-Tailed t-test) and a slight reduction of this parameter, which failed to reach statistical significance, was induced by arrest in mitosis with 2ME2 (0.8±0.12 fold change; n = 20 cells; p>0.16, 2-tailed t-test).</p

Receptor-independent phosphorylation of Smad3 is sensitive to an increase in the volume of mitotic cells and occurs upon GFP-Smad3 over-expression.

<p>A, α-pSmad3C, α-tSmad3 and α-clathrin immunoblot of ES-2 cells treated with 2ME2 or vehicle, hypotonic medium (2 h; as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043459#s2" target="_blank">Materials and Methods</a>) or their combination. 2ME2-arrested cells treated with hypotonic medium show 0.31±0.09 fold lower levels of the (pSmad3C/tSmad3)/clathrin ratio (n = 4; p<0.05, 1-tailed t-test), and 1.94±0.23 fold increase in tSmad3/clathrin ratio (n = 4; p<0.01, 1-tailed t-test), as compared to 2ME2-arrested cells grown in isotonic medium. B, Confocal micrographs of tubulin and DNA of 2ME2-arrested ES-2 cells, treated or not with hypotonic medium. Bar, 10 µm. Numbers represent the percentage of cellular tubulin that localizes to the spindle. In arrested cells treated with hypotonic medium, this percentage was 0.44±0.09 fold lower as compared to arrested cells grown in normal medium (n = 6; p<0.03, 1-tailed t-test). C, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, transfected with GFP-Smad3 or GFP constructs and treated with SB431542 (6 h) or vehicle. D, α-pSmad3C, α-tSmad3 and α-clathrin immunoblots of ES-2 cells, transfected with GFP-Smad3 and treated with reversine (5 µM, 2 h) or vehicle. Transfected cells treated with reversine showed 0.58±0.06 fold lower levels of (pGFP-Smad3C/tGFP-Smad3)/clathrin ratio (n = 4; p<0.05, 1-tailed t-test), as compared to untreated cells.</p

Differential Regulation of Smad3 and of the Type II Transforming Growth Factor-β Receptor in Mitosis: Implications for Signaling

<div><p>The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.</p> </div

ES-2 ovarian cancer cells have a mesenchymal phenotype and respond to TGF-β1.

<p>A, Panels depict confocal micrographs of different ovarian cancer cell lines (as specified) immuno-stained against e-cadherin, MUC1 and vimentin; or labeled for their actin content using rhodamine-conjugated phalloidin. Note the difference in the pattern of expression of phenotypic markers amongst epithelial-like cells (Ovcar3 and Caov3) and mesenchymal-like cells (ES-2 and HEY). Bar, 10 µm. B, ES-2 and Caov3 cells were stimulated with TGF-β1 (5 ng/ml, here and throughout the manuscript) or vehicle for the indicated times and immunoblotted with α-pSmad3C, α-tSmad3 and α-tubulin (employed here and throughout the manuscript as a protein loading control) antibodies. C, ES-2 and Skov3 cells were stimulated with TGF-β1 or vehicle and immunoblotted with α-pSmad3C, α-tSmad2/3 and α-clathrin (employed here and throughout the manuscript as a protein loading control) antibodies. D, qRT-PCR of Smad7, PAI-1, SnoN and Fibronectin. Bar graph depicts the average ± SEM fold change in normalized transcript content in TGF-β1-treated cultures (2 h) as compared to untreated. Smad7∶ 8.4±2.82, n = 8; p<0.01; PAI1∶ 1.82±0.27, n = 6; p<0.06; SnoN: 4.54±0.77, n = 8; p<0.01; fibronectin: 1.7±0.28, n = 9; p<0.03; all 1-tailed t-tests. E, Growth curve of ES-2 cells treated with TGF-β1 or vehicle. Cells were grown in medium supplemented with 1% or 10% FCS, with or without TGF-β1. Graph depicts the average ± SEM optical density at each time point of a typical experiment (O.D.₅₉₅, n = 6).</p

Localization of Smad3, Smurf2, Ski and Mps1 to the mitotic spindle and co-immunoprecipitation of phosphorylated Smad3, Ski and Smurf2 in 2ME2-arrested cells.

<p>A–D, Panels depict a single confocal mid-plane of imaged cells. Bar, 10 µm in upper panel and 1 µm in inset images. The Pearson’s Correlation Coefficient (PCC) of images acquired with 473 nm and 561 nm illumination was calculated with the JaCop plugin of the ImageJ™ software (presented as PCC ± SEM). A, Confocal micrographs of tSmad3, α-tubulin and DNA, of unperturbed-mitotic or 2ME2-arrested ES-2 cells. B, Confocal micrographs of tSmad3, Mps1 and DNA of unperturbed-mitotic or 2ME2-arrested ES-2 cells. C, Confocal micrographs of Smurf2, α-tubulin and DNA of unperturbed-mitotic or 2ME2-arrested ES-2 cells. D, Confocal micrographs of tSmad3, Ski and DNA of unperturbed-mitotic or 2ME2-arrested ES-2 cells. E, Immunoprecipitation with α-pSmad3C or sepharose beads alone (control) of lysates of cells treated with TGF-β1 (1 h) or 2ME2 (16 h). Left panel: Immunoprecipitates were separated by SDS-PAGE and immunoblotted with α-Ski, α-Smurf2 and α-tSmad2/3 antibodies. In 2ME2-arrested cells, Ski and Smurf2 co-immunoprecipitated with pSmad3C at 7.7±1.7 fold and 2±0.25 fold (respectively) higher levels than in TGF-β- stimulated cells, (n = 3; p<0.02, 1-tailed t-test). Right panel: 5% of the lysates, prior to immunoprecipitation, were immunoblotted with α-pSmad3C, α-tSmad3, α-Ski and α-Smurf2 antibodies.</p