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Anatoliy Samoylenko,
Bozhena Vynnytska-Myronovska,
Nadiya Byts,
Nina Kozlova,
Olga Basaraba,
Ganna Pasichnyk,
Kseniya Palyvoda,
Yaroslav Bobak,
Maryna Barska,
Oksana Mayevska,
Yuriy Rzhepetsky,
Halyna Shuvayeva, Valeriy Lyzogubov,
Vasyl Usenko,
Volodymyr Savran,
Nataliya Volodko,
Vladimir Buchman,
Thomas Kietzmann,
Lyudmyla Drobot
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ABSTRACT: The adaptor protein regulator for ubiquitous kinase/c-Cbl-interacting protein of 85kDa (Ruk/CIN85) was found to modulate HER1/EGFR signaling and processes like cell adhesion and apoptosis. Although these features imply a role in carcinogenesis, it is so far unknown how and by which molecular mechanisms Ruk/CIN85 could affect a certain tumor phenotype. By analyzing samples from breast cancer patients, we found high levels of Ruk(l)/CIN85 especially in lymph node metastases from patients with invasive breast adenocarcinomas, suggesting that Ruk(l)/CIN85 contributes to malignancy. Expression of Ruk(l)/CIN85 in weakly invasive breast adenocarcinoma cells deficient of Ruk(l)/CIN85 indeed converted them into more malignant cells. In particular, Ruk(l)/CIN85 reduced the growth rate, decreased cell adhesion, enhanced anchorage-independent growth, increased motility in both transwell migration and wound healing assays as well as affected the response to epidermal growth factor. Thereby, Ruk(l)/CIN85 led to a more rapid and prolonged epidermal growth factor-dependent activation of Src, Akt and ERK1/2 and treatment with the Src inhibitor PP2 and the PI3K inhibitor LY294002 abolished the Ruk(l)/CIN85-dependent changes in cell motility. Together, this study indicates that high levels of Ruk(l)/CIN85 contribute to the conversion of breast adenocarcinoma cells into a more malignant phenotype via modulation of the Src/Akt pathway.
Carcinogenesis 07/2012; 33(10):1976-84. · 5.70 Impact Factor
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ABSTRACT: Protein kinase 2 (CK2) is a ubiquitous and constitutively active serine/threonine protein kinase with various cell functions. It typically forms tetrameric complexes consisting of two catalytic (alpha and/or (alpha') and two regulatory (beta) subunits. The aim of this study was to produce monoclonal antibodies (MAbs) against the CK2beta subunit and to characterize their suitability for Western blotting, immunoprecipitation, and immunohistochemical applications. Bacterially expressed CK2beta-6His-GST recombinant protein has been used as an antigen. Balb/c mice were immunized and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells using PEG 2000. The fused cells were then selected in the HAT-RPMI medium. Anti- CK2beta high-titer antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in HT-RPMI medium supplemented with 20% fetal bovine serum (FBS). A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2beta subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. The results obtained clearly indicate that several clones produce antibodies that recognize specifically recombinant and endogenous CK2beta subunit in Western blotting and immunoprecipitation, and are suitable for immunohistochemical analysis. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.
Hybridoma (2005) 09/2005; 24(4):206-10. · 0.42 Impact Factor
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Natalya Markeeva,
Igor Lisovskiy, Valeriy Lyzogubov,
Vasilij Usenko,
Maria Soldatkina,
Serhey Merentsev,
Serhey Zaitsev,
Yuriy Kondratskii,
Anatoliy Tofan,
Serhey Osinskiy,
Petro Pogrebnoy
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ABSTRACT: The present work was AIMED on the study of patterns of human beta-defensin-2 (hBD-2) expression in human gastric tumors.
17 samples of surgically resected human gastric adenocarcinoma paired with respective normal tissue controls have been analyzed for the expression of hBD-2 by semi-quantitative RT-PCR and by immunohistochemical analysis using anti-hBD-2 monoclonal antibodies (mAbs) generated against recombinant hBD-2 by standard hybridoma technology.
The hyperexpression of hBD-2 on mRNA and protein levels was registered in 6 from 17 gastric tumor samples compared to respective controls; however, hBD-2 expression patterns seems not be related to the differentiation grade and stage of tumors and the presence of anti-Helicobacter pylori-antibodies in the blood serum of the patients.
In human gastric adenocarcinomas expression of hBDs possesses heterogeneic character; the research on larger cohort of the patients with gastric cancer should be further performed.
Experimental oncology 07/2005; 27(2):130-5.
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ABSTRACT: To assess the correlation between the expression profiles of ribosomal protein S6 kinase (S6K1/2), Ki-67 nuclear antigen (Ki-67) and proliferating cell nuclear antigen (PCNA) in human breast adenocarcinomas.
The expression pattern of S6K1/2, Ki-67 and PCNA has been investigated by immunohistochemical analysis of formalin fixed paraffin embedded sections of 40 human breast adenocarcinomas. Analysis was performed using specific monoclonal and polyclonal antibodies directed against S6K2, Ki-67 and PCNA followed by semi-quantitative measurements.
Positive correlation between nuclear staining for S6K2 and PCNA (p < or = 0.01) or Ki-67 (p < or = 0.01) in breast cancer cells has been detected. No significant correlation in nuclear staining was observed between S6K1 and PCNA or Ki-67. In addition, we have detected an increase in S6K2 expression in the nuclei of cancer cells localized predominantly in peripheral areas of the tumor contacting with normal tissue.
The accumulation of S6K2, but not S6K1, in the nuclei of cancer cells and the correlation with the expression of PCNA and Ki-67 suggest the involvement of S6K2 in the regulation of malignant growth.
Experimental oncology 06/2005; 27(2):141-4.
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Alexander Zhyvoloup,
Ivan Nemazanyy,
Ganna Panasyuk,
Taras Valovka,
Tim Fenton,
Heike Rebholz,
Mong-Lien Wang,
Richard Foxon, Valeriy Lyzogubov,
Vasylij Usenko,
Ramziya Kyyamova,
Olena Gorbenko,
Genadiy Matsuka,
Valeriy Filonenko,
Ivan T Gout
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ABSTRACT: CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.
Journal of Biological Chemistry 01/2004; 278(50):50316-21. · 4.77 Impact Factor
