Detachment variants of Chinese hamster cells

Barnhart, Benjamin J.; Cox, Summers H.; Kraemer, Paul M.

doi:10.1016/0014-4827(79)90360-4

Cited by 71 publications

(10 citation statements)

References 8 publications

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“…The heparin binding site for antithrombin has recently been shown to be enriched for iduronate-containing sequences (18). Our (20). Another mutant CHO cell line that is resistant to detachment from the substratum is greatly reduced in its production of hyaluronic acid, and trypsin-removed cells from this mutant cell line contained 3 times the parental level of 35S detectable as heparan sulfate (21).…”

Section: Methodsmentioning

confidence: 94%

Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

Laterra¹,

Ansbacher²,

Culp³

1980

Proc. Natl. Acad. Sci. U.S.A.

102

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Glycosaminoglycans (GAGs) and glycoprotein-derived glycopeptide from mouse BALB/c3T3 and simian virus 40-transformed 3T3 whole cells or their adhesion sites, which are left bound to the serum-coated tissue culture' substratum after detachment of cells mediated by [ethylenebis-(oxyethylenenitrilojtetraacetic sulfate) and dermatan sulfate were able to displace bound radiolabeled adhesion site GAG from the column, whereas the other GAGs had no effect. CIg has been shown to be the only adhesion-promoting activity in the serum layer of this culture system.. Because these fibroblast adhesion sites do not contain collagen, which could potentially mediate adhesion to the substratum-bound

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Section: Methodsmentioning

confidence: 94%

Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

Laterra¹,

Ansbacher²,

Culp³

1980

Proc. Natl. Acad. Sci. U.S.A.

102

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“…One subline is resistant t o detachment from the substratum and synthesizes little or n o hyaluronic acid, as well as reduced amounts of other complex carbohydrates at the cell periphery [ 6 8 , 6 9 ] . Conversely, an easily detached variant has three times more detectable label in surface hyaluronic acid than does the parent cell [69]. Furthermore, in the parent CHO cell, an anchorage-dependent Chinese hamster cell line, the synthesis and deposition of hyaluronic acid into cell surface material is stimulated by cell attachment to and growth on a solid substratum [70].…”

Section: :Tcsmmentioning

confidence: 99%

Fibronectin and proteoglycans as determinants of cell‐substratum adhesion

1979

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When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.

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“…As a surface coating, hyaluronan is primarily anti-adhesive for cells that already have it on their surface [7] and its effects on collagen gel contraction by fibroblasts in vitro are variable [8, 9]. High hyaluronan production has also been linked to detachment of cells [10, 11]. Therefore, the question of how hyaluronan controls myofibroblast adhesion, differentiation, and matrix assembly remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

et al. 2015

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The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccarides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.

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Detachment variants of Chinese hamster cells

Cited by 71 publications

References 8 publications

Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

Fibronectin and proteoglycans as determinants of cell‐substratum adhesion

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

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