Colchicine induces membrane-associated activation of matrix metalloproteinase-2 in osteosarcoma cells in an S100A4-independent manner

Loennechen, Thrina; Mathisen, Berit; Hansen, Janne; Lindstad, Rune I.; El-Gewely, Sara Ann; Andersen, Kristin; Mælandsmo, Gunhild Mari; Winberg, Jan

doi:10.1016/j.bcp.2003.08.014

Cited by 22 publications

(41 citation statements)

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“…In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35 S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.…”

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confidence: 77%

“…Secretion of these endogenous proteases was studied in the transfected cells, in both the absence and presence of the phorbol ester phorbol 12-myristate 13-acetate (PMA), previously reported to enhance the secretion of MMP-9 in MDCK cells [14]. Results presented show that the secretion levels of proteases correlate with the levels of serglycin mRNA, HisTrap isolated 35 S-labelled serglycin and serglycin core protein detected by western blotting. The MDCK system with transfected serglycin is potentially a useful model to study PGs in relation to secretion of enzymes important in physiological and pathological conditions.…”

mentioning

confidence: 77%

“…It has previously been shown that the major fraction of 35 S-labelled macromolecules in MDCK cells are of PG nature [16]. The level of 35 S-labelled macromolecules therefore indicates the level of PG synthesis. As can be seen in Fig.…”

Section: S-labelled Macromoleculesmentioning

confidence: 93%

“…To investigate the extent to which expression of serglycin influenced total PG biosynthesis, cells were labelled with 35 S sulfate for 24 h. The labelled macromolecules were separated from unincorporated 35 S-labelled sulfate by Sephadex G-50 Fine gel chromatography. It has previously been shown that the major fraction of 35 S-labelled macromolecules in MDCK cells are of PG nature [16]. The level of 35 S-labelled macromolecules therefore indicates the level of PG synthesis.…”

Section: S-labelled Macromoleculesmentioning

confidence: 99%

“…One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35 S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting.…”

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Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells

et al. 2006

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Studies on proteoglycans (PGs) have, to a large extent, focused on molecules located in the extracellular matrix and on cell surfaces [1][2][3], and their roles in, for example, the regulation of cell adhesion, cell migration, proliferation and wound healing. However, PGs located in different intracellular locations are receiving increasing attention [4,5]. In particular, PGs in storage and secretory granules in cells of the haematopoietic lineage have been the subject of several recent studies, as, for instance, in the mast cell, where heparin PG is stored in secretory granules together with histamine and proteases. Generation of mice with a deleted version of the gene for the heparinsynthesizing enzyme N-deacetylase ⁄ N-sulfotransferase-2 (NDST-2) resulted in the appearance of mast cells with large changes in secretory granule morphology and in greatly reduced levels of the proteases normally confined to these granules [6,7]. Heparin PG in mast cells, accordingly, seems to be of fundamental importance for the generation of storage granules. Recently, serglycin knockout mice were generated [8]. They developed normally and were fertile, but their mast cells were affected in a manner similar to that of the NDST-2 knockout mice. Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35 S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.

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confidence: 77%

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confidence: 77%

Section: S-labelled Macromoleculesmentioning

confidence: 93%

Section: S-labelled Macromoleculesmentioning

confidence: 99%

mentioning

confidence: 99%

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Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells

et al. 2006

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Metastasis‐associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells

Lai

Morris

Pang

et al. 2007

Molecular Carcinogenesis

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The development of metastatic cancer is associated with overexpression or downregulation of specific genes and cell regulatory pathways. Some of these genes and pathways may be involved in invasion and dissemination of tumor cells, while others may promote seeding, survival or growth of cells at specific distant sites. In this investigation, gene expression profiles of nonmetastasizing tumors generated by injecting mouse pheochromocytoma cells (MPCs) subcutaneously were compared to those of liver tumors generated by injecting the cells intravenously. Both were compared to the cultured parental cell line. Tumors in the liver have a route of spread, anatomical distribution, and growth environment similar to naturally metastasizing pheochromocytomas, while intravenous injection of cells bypasses the initial steps of metastasis occurring spontaneously from a primary tumor. Eight genes were upregulated in liver tumors, 15 in subcutaneous tumors and seven in both compared to the cultured cells. Using quantitative real-time PCR, expression of five genes (Metap2, Reck, S100a4, Timp2, and Timp3) was verified as significantly lower in liver tumors than in subcutaneous tumors. Downregulation of these genes has been previously been associated with malignancy of pheochromocytomas. These findings indicate that different microenvironments can differentially affect the expression of metastasis-related genes in pheochromocytomas, and that overexpression or underexpression of these genes need not be present when the tumor cells are initially disseminated. The hepatic localization of tumors formed by intravenously injected MPC cells and the tumors' gene expression profile resembling that of naturally occurring pheochromocytoma metastases support the use of this model to study pheochromocytoma metastasis.

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Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

Hadler‐Olsen

Winberg

2019

The Extracellular Matrix

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To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localise them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrade a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin degrading proteases in tissue extracts. This method uses fluorescence labelled gelatin and therefore, we also present an easy, fast and cheap method for labelling gelatin with 2-Methoxy-2,4-Diphenyl-3(2H)-Furanone (MDPF).

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Colchicine induces membrane-associated activation of matrix metalloproteinase-2 in osteosarcoma cells in an S100A4-independent manner

Cited by 22 publications

References 50 publications

Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells

Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells

Metastasis‐associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells

Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

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