Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker

<p>Abstract</p> <p>Background</p> <p>Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.</p> <p>Results</p> <p>In the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.</p> <p>Conclusion</p> <p>Serum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.</p

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

<p>Abstract</p> <p>Background</p> <p>TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.</p> <p>Results</p> <p>Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).</p> <p>Conclusion</p> <p>Interestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.</p

Cholesterol uptake and efflux are impaired in human trophoblast cells from pregnancies with maternal supraphysiological hypercholesterolemia.

Maternal physiological (MPH) or supraphysiological hypercholesterolaemia (MSPH) occurs during pregnancy. Cholesterol trafficking from maternal to foetal circulation requires the uptake of maternal LDL and HDL by syncytiotrophoblast and cholesterol efflux from this multinucleated tissue to ApoA-I and HDL. We aimed to determine the effects of MSPH on placental cholesterol trafficking. Placental tissue and primary human trophoblast (PHT) were isolated from pregnant women with total cholesterol <280 md/dL (MPH, n = 27) or ≥280 md/dL (MSPH, n = 28). The lipid profile in umbilical cord blood from MPH and MSPH neonates was similar. The abundance of LDL receptor (LDLR) and HDL receptor (SR-BI) was comparable between MSPH and MPH placentas. However, LDLR was localized mainly in the syncytiotrophoblast surface and was associated with reduced placental levels of its ligand ApoB. In PHT from MSPH, the uptake of LDL and HDL was lower compared to MPH, without changes in LDLR and reduced levels of SR-BI. Regarding cholesterol efflux, in MSPH placentas, the abundance of cholesterol transporter ABCA1 was increased, while ABCG1 and SR-BI were reduced. In PHT from MSPH, the cholesterol efflux to ApoA-I was increased and to HDL was reduced, along with reduced levels of ABCG1, compared to MPH. Inhibition of SR-BI did not change cholesterol efflux in PHT. The TC content in PHT was comparable in MPH and MSPH cells. However, free cholesterol was increased in MSPH cells. We conclude that MSPH alters the trafficking and content of cholesterol in placental trophoblasts, which could be associated with changes in the placenta-mediated maternal-to-foetal cholesterol trafficking

L'autre transparence : Oeuvres holographiques, 6 artistes du Québec : Boissonnet, Cantin, Cossette, Dyens, Evergon, Mathieu

For the presentation of holograms by six Québec artists, Boissonnet and Fischer give evidence of the evolution of holography as an artistic form. Brief artists' statements. Biographical notes

Transforming growth factor (TGF)-βinduces an epithelial-to-mesenchymal transition (EMT) in the BRI-JM01 cell line

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Immunofluorescence microscopy demonstrating the altered localization of E-cadherin, N-cadherin and β-catenin, the rearrangement of F-actin and vimentin filaments, and the disappearance of ZO-1 that accompany the TGF-β1 induced EMT (see text for details). Flow cytometry of nonfixed live cells monitoring the presence of E-cadherin at the cell surface of BRI-JM01 cells that were exposed to TGF-βfor up to 120 hours. Nonspecific isogenic IgG (HA) and cells grown in the absence of TGF-β(CTL) were used as a negative and positive control, respectively. Western blot analysis determining the total cellular content of E-cadherin, N-cadherin and β-catenin in BRI-JM01 cells cultured for 24, 72 and 120 hours in the absence (CTL) or presence of TGF-β(see text for details). BRI-JM01 cells were treated with TGF-βfor 48 hours (BRI-JM01T) and passaged several times in the absence of this growth factor. When seeded in Matrigel these 'TGF-βprimed' BRI-JM01 cells formed larger three-dimensional structures than did the nontreated cells

Evaluation of the activated Neu and transforming growth factor-β type II receptor antisense RNA (TβRII-AS) gene expression in mouse mammary tumor virus (MMTV) transgenic mouse mammary tumors and the derived cell lines

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Activated Neu and TβRII-AS transgene expression was determined by RT-PCR in four independent MMTV/activated Neu + TβRII-AS mammary tumors (left panel), in one MMTV/activated Neu mammary tumor (right panel), and the BRI-JM01, BRI-JM04 and BRI-JM05 cell lines. First strand reactions were carried out in the presence or absence of reverse transcriptase (± RT). Genomic DNA obtained from the BRI-JM01, BRI-JM04, and BRI-JM05 cell lines was evaluated by PCR for the presence of the TβRII-AS (middle) and activated Neu (right) transgenes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as positive control (parts a and c, left panels)

Growth response of the three cell lines to recombinant human epidermal growth factor (hEGF), heregulin (HRG)-β, and transforming growth factor (TGF)-β

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> [H]thymidine incorporation measured in BRI-JM01, BRI-JM04, and BRI-JM05 cell lines after 24 hours of exposure to serial dilutions of hEGF, HRG-β, or TGF-β. Experiments were conducted in quadruplicate and repeated three times with similar results (for details see text). Western blot analysis of whole cell lysates (20 μg total protein/lane) evaluating ErbB-1, ErbB-2, ErbB-3, and ErbB-4 expression. Additional ErbB-2/Neu immunoprecipitations showed this receptor to be highly phosphorylated in the BRI-JM04 and BRI-JM05 cells. Affinity cross-link labeling of the TGF-β type I, II, III receptors expressed on the BRI-JM01, BRI-JM04 and BRI-JM05 cells using [I]TGF-βin the absence (-) or presence (+) of 100× excess of unlabeled TGF-β. Enzymatic deglycosylation using both heparinitase I/II/III and chondroitinase ABC collapses the diffusely migrating TGF-β type III receptor band to a core protein (approximately 115 kDa, indicated as TβRIII**) labeled with either [I]TGF-βor [I]TGF-β. TGF-β responsive Mv1Lu cells served as a positive control in the TGF-β growth assay and the cell surface cross-link affinity labeling studies. RT-PCR analysis of BRI-JM01 mRNA using two different primer sets (set 1, set 2) confirmed the presence of TGF-β type III receptor transcripts in the BRI-JM01 cells. TGF-β type III receptor-expressing BRI-JM05 cells (part e) were used as a positive control for TGF-β type III receptor expression, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Experiments were repeated twice with similar results