Comparative proteome analysis of human epithelial ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer.</p> <p>Results</p> <p>The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior.</p> <p>Conclusion</p> <p>The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.</p

The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism

Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo. Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosageand RNA-dependent manner. In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro. Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632. Furthermore, overexpression of RBP29 suppresses the temperaturesensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles. These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA

One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells

Homozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma

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Western blot validation of selected proteins identified by iTRAQ and/or 2DE analysis

<p><b>Copyright information:</b></p><p>Taken from "Comparative proteome analysis of human epithelial ovarian cancer"</p><p>http://www.proteomesci.com/content/5/1/16</p><p>Proteome Science 2007;5():16-16.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2072939.</p><p></p> Equal amounts, 25 μg, of protein extracts from TOV-112D and TOV-81D cell lines were loaded onto a 12 % SDS-PAGE and processed for Western blotting with the indicated antibodies. Protein expression differences were quantified using a Chemilmager 4000 imaging system and AlphaEase software 3.3 (Alpha Innotech Corporation). The data are expressed as relative integrated density value (IDV). Each point represents the mean +/- SE from three independent experiments. Data were analysed by Student's unpaired test. Representative blots for each analysis are depicted

Zoomed sections from TOV-112D and TOV-81D gels demonstrating differential expression of proteins listed in Table 2

<p><b>Copyright information:</b></p><p>Taken from "Comparative proteome analysis of human epithelial ovarian cancer"</p><p>http://www.proteomesci.com/content/5/1/16</p><p>Proteome Science 2007;5():16-16.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2072939.</p><p></p> (A) Selected TOV-112D spots with undetectable TOV-81D matching spots. (B) Selected TOV-81D spots with undetectable TOV-112D matching spots. (C) Selected spots differentially expressed with a minimum deregulation ratio of 2.0