Purification and characterization of biologically active scatter factor from <i>ras</i>-transformed NIH 3T3 conditioned medium

Coffer, Arnold I.; Fellows, Jane; Young, San-Lin; Pappin, Darryl; Rahman, Dinah

doi:10.1042/bj2780035

Cited by 11 publications

(2 citation statements)

References 25 publications

(34 reference statements)

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“…As mutations at the Nf1 locus in Schwann cells result in increased activation of Ras, it is of some interest that constitutive activation of Ras can facilitate tumor angiogenesis. Activated Ras upregulates expression of angiogenic factors, including HGF, bFGF, and vascular endothelial growth factor (VEGF), in fibroblasts (11,20,23,51). In a tumor cell line, VEGF secretion is suppressed by disruption of mutant Ras activity by FPTI (53).…”

Section: Discussionmentioning

confidence: 99%

Nf1-Deficient Mouse Schwann Cells Are Angiogenic and Invasive and Can Be Induced To Hyperproliferate: Reversion of Some Phenotypes by an Inhibitor of Farnesyl Protein Transferase

Kim

Ling

Ratner

1997

Molecular and Cellular Biology

135

120

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We have developed a potential model of Schwann cell tumor formation in neurofibromatosis type 1 (NF1). We show that mouse Schwann cells heterozygous or null at Nf1 display angiogenic and invasive properties, mimicking the behavior of Schwann cells from human neurofibromas. Mutations at Nf1 are insufficient to promote Schwann cell hyperplasia. Here we show that Schwann cell hyperplasia can be induced by protein kinase A activation in mutant cells. Removal of serum from the culture medium also stimulates hyperplasia, but only in some mutant cells. After serum removal, clones of hyperproliferating Schwann cells lose contact with axons in vitro, develop growth factor-independent proliferation, and exhibit decreased expression of the cell differentiation marker P0 protein; hyperproliferating cells develop after a 1-week lag in Schwann cells heterozygous at Nf1. The experiments suggest that events subsequent to Nf1 mutations are required for development of Schwann cell hyperplasia. Finally, an anti-Ras farnesyl protein transferase inhibitor greatly diminished both clone formation and hyperproliferation of null mutant cells, but not invasion; farnesyl transferase inhibitors could be useful in treating benign manifestations of NF1.Neurofibromatosis type 1 (NF1) is one of the most common inherited human autosomal dominant diseases, with a worldwide incidence of 1 in 3,500 individuals (56). Benign manifestations frequently associated with NF1 include pigmented lesions of the skin (café au lait spots), hamartomas of the iris (Lisch nodules), learning disabilities, formation of optic pathway gliomas, and neurofibromas (22). Neurofibromas are one of the major defining features of NF1. Cutaneous neurofibromas (benign peripheral nerve sheath tumors) are associated with small nerve branches, plexiform neurofibromas develop along major peripheral nerves, and malignant peripheral nerve sheath tumors (MPNST) develop in about 4% of NF1 patients (56). Genetic studies show that malignant tumors in NF1, including MPNSTs, contain cells with mutations in both the constitutionally inactivated and previously normal somatic allele, consistent with NF1 acting as a tumor suppressor (19,36,64). The mechanism of neurofibroma formation is less clear. While recent studies demonstrated loss of heterozygosity (LOH) at the NF1 locus in some neurofibromas (12, 60), LOH could be associated with either of the major cell types in the benign tumors, fibroblasts or Schwann cells.Schwann cells may be the primary pathogenic cells in neurofibromas. The majority (40 to 85%) of cells in neurofibromas are Schwann cells (18,49), and in contrast to Schwann cells in normal nerve, neurofibroma Schwann cells are found without association with axons (28, 50). Furthermore, in vitro, Schwann cells, but not fibroblasts, from neurofibromas show angiogenic and invasive properties (63). Because the genetic status of neurofibroma Schwann cells is unknown, it remains unclear if a single NF1 mutation is sufficient for manifestation of some neurofibroma Schwann cell phenotype...

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Section: Discussionmentioning

confidence: 99%

Nf1-Deficient Mouse Schwann Cells Are Angiogenic and Invasive and Can Be Induced To Hyperproliferate: Reversion of Some Phenotypes by an Inhibitor of Farnesyl Protein Transferase

Kim

Ling

Ratner

1997

Molecular and Cellular Biology

135

120

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“…Murine HGF was purified to homogeneity from medium conditioned by ms transformed NIH-3T3 cells by a two-step procedure described in details elsewhere (Coffer et al, 1991). The factor was precipitated by ammonium sulphate at 60% saturation, dissolved in 50 mM MES, 0.1 M NaCI, pH 6, loaded onto a Mono S column (Pharmacia Fine Chemicals) and eluted by a linear gradient of NaCI (0.1-1.0 M).…”

Section: Purification Of Human and Murine Hgfmentioning

confidence: 99%

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Bussolino¹,

Renzo²,

Ziche³

et al. 1992

The Journal of Cell Biology

Self Cite

1,242

714

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Abstract. Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190 MEt) made of a 50 kD (o0 subunit disulfide linked to a 145-kD (~) subunit. HGF binding to endothelial ceils identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190 MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

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Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

et al. 1992

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The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single‐chain biologically inactive precursor (pro‐HGF/SF), mostly found in a matrix‐associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum‐dependent proteolytic cleavage. In vitro, pro‐HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type‐1 and protease nexin‐1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro‐HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.

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Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium

Cited by 11 publications

References 25 publications

Nf1-Deficient Mouse Schwann Cells Are Angiogenic and Invasive and Can Be Induced To Hyperproliferate: Reversion of Some Phenotypes by an Inhibitor of Farnesyl Protein Transferase

Nf1-Deficient Mouse Schwann Cells Are Angiogenic and Invasive and Can Be Induced To Hyperproliferate: Reversion of Some Phenotypes by an Inhibitor of Farnesyl Protein Transferase

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

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