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There is a growing line of evidence that glycosylation of α and β subunits is important for the function of integrins. Integrin α3β1, from human ureter epithelium cell - line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin α3β1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.
Regulator of G-protein signalling (RGS)2 proteins critically regulate signalling cascades initiated by G-protein coupled receptors (GPCRs) by accelerating the deactivation of heterotrimeric G-proteins. Lysophosphatidic acid (LPA) is the predominant growth factor that drives the progression of ovarian cancer by activating specific GPCRs and G-proteins expressed in ovarian cancer cells. We have recently reported that RGS proteins endogenously expressed in SKOV-3 ovarian cancer cells dramatically attenuate LPA stimulated cell signalling. The goal of this study was twofold: first, to identify candidate RGS proteins expressed in SKOV-3 cells that may account for the reported negative regulation of G-protein signalling, and second, to determine if these RGS protein transcripts are differentially expressed among commonly utilized ovarian cancer cell lines and non-cancerous ovarian cell lines. Reverse transcriptase-PCR was performed to determine transcript expression of 22 major RGS subtypes in RNA isolated from SKOV-3, OVCAR-3 and Caov-3 ovarian cancer cell lines and non-cancerous immortalized ovarian surface epithelial (IOSE) cells. Fifteen RGS transcripts were detected in SKOV-3 cell lines. To compare the relative expression levels in these cell lines, quantitative real time RT-PCR was performed on select transcripts. RGS19/GAIP was expressed at similar levels in all four cell lines, while RGS2 transcript was detected at levels slightly lower in ovarian cancer cells as compared to IOSE cells. RGS4 and RGS6 transcripts were expressed at dramatically different levels in ovarian cancer cell lines as compared to IOSE cells. RGS4 transcript was detected in IOSE at levels several thousand fold higher than its expression level in ovarian cancer cells lines, while RGS6 transcript was expressed fivefold higher in SKOV-3 cells as compared to IOSE cells, and over a thousand fold higher in OVCAR-3 and Caov-3 cells as compared to IOSE cells. Functional studies of RGS 2, 6, and 19/GAIP were performed by measuring their effects on LPA stimulated production of inositol phosphates. In COS-7 cells expressing individual exogenous LPA receptors, RGS2 and RSG19/GAIP attenuated signalling initiated by LPA1, LPA2, or LPA3, while RGS6 only inhibited signalling initiated by LPA2 receptors. In SKOV-3 ovarian cancer cells, RGS2 but not RGS6 or RGS19/GAIP, inhibited LPA stimulated inositol phosphate production. In contrast, in CAOV-3 cells RGS19/GAIP strongly attenuated LPA signalling. Thus, multiple RGS proteins are expressed at significantly different levels in cells derived from cancerous and normal ovarian cells and at least two candidate RGS transcripts have been identified to account for the reported regulation of LPA signalling pathways in ovarian cancer cells.
Objectives: Angiogenesis is a process that is indispensable in cancer progression. A complex network of tumor and microenvironment stimuli regulate angiogenesis. VEGF, TGF-β1, IL-8 and TNF belong to the angiogenic factors that are key points in vessel formation. The aim of the study was to assess h-VEGF, TGF-β1, IL-8 and TNF secretion by human ovarian cell lines. Material and methods: OVA 2, OVA 4, OVA 9, OVA 11 and OVA 14 cell lines were established in our laboratory. The cells derived from primary and metastatic tumors of epithelial and non-epithelial origin. SK-OV-3, MDAH 2774, CAOV-1 and OVP-10 were the cell lines obtained from other sources. The concentration of VEGF, TGF-β1 and IL-8 was determined in culture supernatants by using the ELISA tests. Results: OVA 11 secreted all the evaluated cytokines. MDAH 2774 was the source of h-VEGF, TGF-β1, IL-8. SK-OV-3 secreted h-VEGF and IL-8. OVA 4 secreted TGF-β1 and TNF. TNF was the only studied cytokine secreted by CAOV-1, OVA 2 and OVA 9 cell lines. OVA 14 did not secret any of the cytokines. Conclusions: The investigated cell lines present heterogeneous profile of angiogenic cytokine secretion and seem to be an interesting set of models for the study of angiogenic signaling, or target therapy.
Cel: Angiogeneza jest procesem niezbędnym do progresji raka. Złożona sieć bodźców pochodzących od guza i z mikrośrodowiska reguluje angiogenezę. VEGF, TGF-β1, IL-8 i TNF należą do czynników angiogennych, które odgrywają kluczową rolę w tworzeniu naczyń. Celem pracy była ocena wydzielania h-VEGF, TGF-β1, IL-8 i TNF przez ludzkie linie raka jajnika. Materiał i metoda: Linie OVA 2, OVA 4, OVA 9, OVA 11 oraz OVA 14 zostały ustalone samodzielnie. Komórki pochodziły z pierwotnych lub przerzutowych guzów jajnika pochodzenia nabłonkowego lub nienabłonkowego. Linie SK-OV-3, MDAH 2774, CAOV-1 oraz OVP-10 pochodziły z innych źródeł. Stężenie VEGF, TGF-β1 i IL-8 określano w supernatantach hodowli komórkowych w teście ELISA. Wyniki: Linia OVA 11 wydzielała wszystkie badane cytokiny. Linia MDAH 2774 była źródłem h-VEGF, TGF-β1, IL-8. Linia SK-OV-3 wydzielała h-VEGF oraz IL-8. Linia OVA 4 wydzielała TGF-β1 i TNF. TNF był jedyną cytokiną wydzielaną przez linie CAOV-1, OVA 2 oraz OVA 9. Linia OVA 14 nie wydzielała żadnej spośród badanych cytokin. Wnioski: Badane linie komórkowe stanowią heterogenną grupę nowotworów wydzielających cytokiny o właściwościach angiogennych i wydają się interesującym panelem do badań nad procesami angiogenezy czy terapii celowanej.
Metabolomics approaches allow systematic identification and quantitation of all metabolites in biological samples analyzes. As already known metabolism is directly or indirectly related to every aspect of cell function, therefore a careful observation of every changes taking place in metabolism, for example endogenous biochemical reaction products, reflectsthe phenotype of any living cell. Monitoring the metabolite profiles using metabolomics technologies, especially nuclear magnetic resonance (NMR) spectroscopy based on cell culture, allows us to evaluate drug efficiency and outcome of experimental therapy, and most importantly, it allows us to monitor the reaction of the model cell lines to a given treatment. The continued development of metabolomic approaches, e.g. analytical technique, or chemometric software, will accelerate the widespread use of metabolomics not only in the clinical field but also in different biological fields. This work presents a use of nuclear magnetic resonance to characterize and understand the cellular metabolome in a wide range of pathophysiological and clinical contexts.
Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between β-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105 000 × g), isolated from the tumor cells was used as a source for actin quantification. The higher level of β-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of β-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. β-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-β actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) β-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and β-actin expression.
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