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The lack of the N-alpha-glucosaminidase (Naglu) is responsible for the incidence of a rare disease - mucopolysaccharidosis, type IIIB (MPS IIIB). To date, studies have been conducted based on cells derived from patients suffering from MPS or using in vivo MPS mouse models. These limitations have allowed for defining our research goal - to create and characterize the first in vitro murine cellular MPS IIIB model. In the current work we present a new, stable cell line with confirmed accumulation of glycosaminoglycans. The line stability was achieved by immortalization using a lentivirus carrying the T-antigens of SV40. The Naglu −/− cells were confirmed to produce no Naglu enzyme. To confirm the proper functioning of the in vitro MPS IIIB model, we determined the activity and expression of cystathionine γ-lyase, rhodanese and 3-mercaptopyruvate sulfurtransferase, as well as the level of low molecular-weight thiols (reduced and oxidized glutathione, cysteine and cystine). The results were referred to our earlier findings originating from the studies on the tissues of the Naglu −/− mice that were used to create the lines. The results obtained in the Naglu −/− cells were in accordance with the results found in the mouse model of MPS IIIB. It suggests that the presented murine Naglu −/− cell lines might be a convenient in vitro model of MPS IIIB. • The Naglu −/− cell lines may be an in vitro model of mucopolysaccharidosis type IIIB. • The cysteine level is elevated in the Naglu −/− cells. • The Naglu −/− cells have a weak antioxidant potential. • endo- and exogenous heparan sulfate demonstrate different biological activity. [ABSTRACT FROM AUTHOR]

The interaction between oppositely charged membranes and polycations causes cell aggregation, loss of membrane fluidity, and membrane degeneration and may cause an increase of its permeability. Unfortunately, the interaction is the reason why the use of polycations in medicine is severely limited. Therefore, in this paper, we share our observations related to the action of 40-kDa dextran modified using glycidyltrimethylammonium chloride, resulting in increased fibroblast cell proliferation. Using viability and proliferation tests [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, crystal violet, 3H-thymidine incorporation], we have observed that cationic dextran derivatives exert a positive impact on nonepithelial cell proliferation in vitro. This phenomenon has been noted for human and mouse fibroblasts and several other nonepithelial cell lines. However, the effect seems to be most pronounced for fibroblast cell lines. The presented studies allow to examine the impact of the polymer structure and the methods of its cationic modification on this newly observed phenomenon. The observation is unique because positively charged macromolecules usually exhibit high toxicity in all cell types in vitro. [ABSTRACT FROM AUTHOR]

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala76:Val77 within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity. [Copyright &y& Elsevier]

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10ng/ml) and IL-6 (10ng/ml). After 24h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera. [Copyright &y& Elsevier]