Effects of fibronectin-related peptides on cell spreading

Silnutzer, Janet; Barnes, David W.

doi:10.1007/bf02620918

Cited by 25 publications

(18 citation statements)

References 25 publications

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“…Peptides containing the sequence Arg-Gly-Asp-Ser (RGDS), originally identified in the cell-binding domain of fibronectin, have been shown to be competitive, reversible inhibitors of cellular adhesion to extracellular matrix components (6)(7)(8). A relatively strict requirement for the particular sequence ofamino acids in these peptides has been established as a result of extensive analysis in vitro (6,7,9,10).…”

Section: Introductionmentioning

confidence: 99%

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

Humphries¹,

Yamada²,

Olden³

1988

J. Clin. Invest.

155

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The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide GlyArg-Gly-Asp-Ser (GRGDS) derived from the central cellbinding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F1O cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F1O cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F1O cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F1O cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, deriva-

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Section: Introductionmentioning

confidence: 99%

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

Humphries¹,

Yamada²,

Olden³

1988

J. Clin. Invest.

155

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“…Cell types tested were: human hepatoma (HepG2), mammary carcinoma (MCF71, human mammary epithelium (HBLlOO), cervical carcinoma (HeLa), bladder carcinoma (T.241, fibrosarcoma (HT1080), epidermoid carcinoma (A431), choriocarcinoma (BeWo), rhabdomyosarcoma (RD), three strains of diploid human embryonic fibroblasts M38, MRC5 and IMRgO), two cultures of cells from human amniotic fluid, two lines of monkey kidney origin (BSC and CVl), and primary cultures of human embryonic kidney, human mammary carcinoma, adult human dermal fibroblasts, basal cell carcinoma and normal human epidermal cells. Many of these cell types are reported to synthesize fibronectin or laminin (Hynes and Yamada, 1982;Grotendorst et al, 1982;Yamada, 1983), and we have previously reported a number of these lines to exhibit a spreading response to SF and fibronectin (Barnes and Sato, 1979;198Ob;Barnes, 1982;Silnutzer and Barnes, 1985).…”

Section: Sf Synthesis By Hepg2 Cells In Culturementioning

confidence: 91%

“…4,5, Pate1 and Lodish, 19841, suggesting that the mechanism by which serum promotes HepG2 cell spreading in culture may be solely through the action of SF. Interestingly, although fibronectin does not promote HepG2 cell spreading, peptides derived from the proposed cell-binding site of fibronectin are capable of inhibiting SF-promoted spreading of HepG2 cells as well as other cell types (Silnutzer and Barnes, 1985).…”

Section: Sf Synthesis By Hepg2 Cells In Culturementioning

confidence: 99%

Human spreading factor: Synthesis and response by HepG2 hepatoma cells in culture

Barnes

Reing

1985

Journal Cellular Physiology

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Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.

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“…Synthetic peptides related to regions of attachment factors thought to interact with cells or fragments derived proteolytically from these factors are capable of inhibiting cell spreading on substratum-bound spreading factors if presented to cells in sufficient concentration in soluble form (16,18,20). Assays of this type are carried out by including the soluble peptides in the cell suspension that is plated on the precoated dish (step 4 of procedure A or step 3 of procedure C).…”

Section: IVmentioning

confidence: 99%

Assay of cell attachment and spreading factors

Barnes

1986

Journal of Tissue Culture Methods

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SUMMARY: Methods are presented for the quantitative assay of proteins influencing cell attachment and spreading. These include assays in which cell-substratum interactions are quantitated directly and a serum-free assay in which cell growth is dependent on attachment under substratum-limited conditions. Procedures are also presented for the identification of active sites of substratum molecules through the use of antibodies and synthetic peptides that are inhibitory for cell attachment.

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Effects of fibronectin-related peptides on cell spreading

Cited by 25 publications

References 25 publications

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

Human spreading factor: Synthesis and response by HepG2 hepatoma cells in culture

Assay of cell attachment and spreading factors

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