Glycosaminoglycans in the substrate adhesion sites of normal and virus-transformed murine cells

Rollins, Barrett J.; Culp, Lloyd A.

doi:10.1021/bi00568a022

Cited by 154 publications

(100 citation statements)

References 45 publications

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“…Despite this striking parallel, it is known that in CHO cells only a fraction of cellular GAGs are present on the surface and thus are directly relevant for attachment (33). The precise Effect of incubation with/~-o-xyloside on the species of GAG synthesized.…”

Section: Discussionmentioning

confidence: 99%

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

Preston¹,

Regula²,

Sager³

et al. 1985

The Journal of Cell Biology

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[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two-to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate.Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.In their pioneering work on glycosaminoglycan (GAG) ~ synthesis by cultured cells, Kraemer and Tobey (14) showed that a significant fraction of trypsin-releasable (i.e., surface) GAGs was shed during mitosis. Although the Chinese hamster ovary (CHO) cells analyzed were grown in suspension, viewed from the perspective of recent studies of cell adhesion (e.g., reference 32), it seemed likely that the shedding of GAGs was related to the familiar ease with which mitotic cells are detached from monolayers. We hypothesized that any effect of shedding would be reinforced by a coordinate reduction in GAG synthesis that would delay replacement of surface GAGs, and that a rapid resumption of GAG synthesis in early G i would be required for cell reattachment.A parallel motivation for our studies arose from the accumulating evidence that membrane vesicle-mediated transport is depressed during mitosis. This is shown clearly in work from our laboratory demonstrating depression of endocytosis ( 1, 2) and transferrin recycling (35) and from that of Warren and colleagues showing a decrease in surface transferrin receptors (41), and in depression of histamine secretion (9). These studies focus primarily on fusion and budding events at the plasma membrane. In addition, the insertion of the G t Abbreviations used in this paper. CHO, Chinese hamster ovary; GAG, glycosaminoglycan.protein of vesicular stomatitis virus is also depressed during mitosis (42), which suggests a defect in its processing through the Golgi apparatus. Since carbohydrate chains of GAGs are largely assembled within the Golgi apparatus by multiple enzymes (31), a general defect in processing should affect GAG synthesis. Moreover, the apparent localization of sulfation to the trans-Gol~ apparatus (7, 44) would serve to focus on later steps of the processing sequence.We describe here the rates of labeled sulfate incorporation into CHO cells during mitosis, in their subsequent movement into G1, and in late G2. We followed incorporat...

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Section: Discussionmentioning

confidence: 99%

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

Preston¹,

Regula²,

Sager³

et al. 1985

The Journal of Cell Biology

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“…Mouse BALB/c 3T3 cells (clone A31) and 3T3 cells transformed by simian virus 40 (SV40) (clone SVT2) were grown Mycoplasma free as described (11). The preparation of protein-free polysaccharides from cell or SAM fractions has also been described (12). Briefly, cells were grown in medium containing D-[6-3H]glucosamine (29.0 Ci/mmol; Amersham; 1 Ci = 3.7 X 10"' becquerels) at 5.0 ,uCi/ml during 72 hr of exponential growth and detached from the substratum with 0.5 mM EGTA in phosphate-buffered saline (12).…”

Section: Methodsmentioning

confidence: 99%

“…The preparation of protein-free polysaccharides from cell or SAM fractions has also been described (12). Briefly, cells were grown in medium containing D-[6-3H]glucosamine (29.0 Ci/mmol; Amersham; 1 Ci = 3.7 X 10"' becquerels) at 5.0 ,uCi/ml during 72 hr of exponential growth and detached from the substratum with 0.5 mM EGTA in phosphate-buffered saline (12). After the substratum had been rinsed with phosphate-buffered saline twice and distilled water once, SAM was quantitatively solubilized with 0.2% sodium dodecyl sulfate.…”

Section: Methodsmentioning

confidence: 99%

“…The final polysaccharide pellets were dissolved in 0.05 M Tris-HCl (pH 7.4), 1 mM MgCl2, and 1 mM CaC12 (TMC buffer). Biochemical identification of glucosamine-radiolabeled polysaccharides utilized chondroitinase ABC or AC (Miles) digestion, followed by gel filtration or paper chromatography (12). The N-sulfated sequences of heparan sulfate were determined by their sensitivity to nitrous acid degradation (13).…”

Section: Methodsmentioning

confidence: 99%

“…SAM is greatly enriched in fibronectin-and GAG-containing proteoglycans (9,11,12).. Heparan sulfate proteoglycans are enriched in newly formed adhesion sites, whereas hyaluronate, dermatan, and the chondroitin sulfates (the latter two as proteoglycans) become associated with these sites as cells spread and initiate movement. This and other evidence suggest that the two specific functions of adhesion site stabilization and destabilization are mediated by specific subclasses of GAG-containing macromolecules (1,8).…”

mentioning

confidence: 99%

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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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Reattachment Rate of Human Retinal Pigment Epithelium to Layers of Human Bruch's Membrane

Priore

Tezel²

1998

Arch Ophthalmol

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To determine the reattachment rate of human retinal pigment epithelium (RPE) to different layers of human Bruch's membrane (BM). Methods: Explants of BM were prepared from 10 human cadaver eyes by removing native RPE. The RPE basal lamina, inner collagenous layer, elastin layer, and outer collagenous layer were exposed by sequentially removing each apical layer by mechanical or enzymatic means. First-passage human RPE was plated onto the surface and the RPE reattachment rate to each layer of BM was determined. Results: Retinal pigment epithelial cell reattachment was highest to the inner aspects of BM and decreased as deeper layers of BM were exposed (ie, reattachment rate to basal lamina was higher than to the inner collagenous layer, which was higher than to the elastin layer, which was higher than to the outer collagenous layer). The reattachment rate to the inner collagenous layer, elastin layer, and outer collagenous layer harvested from elderly donors (age Ͼ60 years) was less than the reattachment rate to corresponding layers harvested from younger (age Ͻ50 years) donors. Conclusions: Retinal pigment epithelial cell reattachment depends on the anatomical layer of BM present in the host tissue. Age-related changes in BM may interfere with RPE reattachment. Our observations may have implications for understanding the pathogenesis of agerelated macular degeneration and its potential treatment with RPE transplantation techniques.

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Glycosaminoglycans in the substrate adhesion sites of normal and virus-transformed murine cells

Cited by 154 publications

References 45 publications

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

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

Reattachment Rate of Human Retinal Pigment Epithelium to Layers of Human Bruch's Membrane

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