Biomedical subjects

G F Burns

Publications and source records attributed to G F Burns.

At least 37 records · Page 2Linked to original sources

PubMed1995

A fibroblast elongation factor purified from colon carcinoma cells shares sequence identity with TIMP-1.

We have previously reported that human colon cancer cells secrete a factor(s) which induces elongation of colon fibroblasts in vitro. Isolation of this factor led to the identification of a 55kD protein with fibroblast stretching activity. Two internal amino acid sequences identified in this protein (YEI; GFQALGDAADI) share complete homology with tissue inhibitor of metalloproteinase (TIMP-1).

Amino Acid Sequence↗

PubMed1995

Macrophage colony-stimulating factor induces thrombospondin 1 production by cultured human macrophages.

The role of colony-stimulating factors (CSFs) in regulating the synthesis of thrombospondin 1 (TSP1) by cultured human macrophages is investigated. Macrophage (M)-CSF is shown rapidly and transiently to induce two predominant species of TSP1 mRNA. One of these species was 3.2 kb in size and appeared to be specific to M-CSF-stimulated macrophages. Adherent M-CSF-treated macrophages are also shown to express abundant surface cell-associated TSP rapidly when examined by indirect immunofluorescence staining. Granulocyte-macrophage (GM)-CSF induced TSP1 mRNA at a later time point, and this was attributable to the effects of endogenous M-CSF induced by the GM-CSF; the GM-CSF-treated cells did not display surface-associated TSP after 3 hr of treatment. Analysis of the TSP1 protein synthesised by the M-CSF-treated macrophages revealed the expected trimeric form of the molecule. In addition, an unidentified 95-kDa protein was found to be covalently associated with immunoreactive TSP1, and this appeared to be specific to the macrophages as it was not found in TSP1 precipitated from other cell types. It is suggested that the induction of TSP1 by M-CSF may play an important role in the major physiological functions of macrophages.

Cells, Cultured↗

PubMed1995

Involvement of integrins in cell survival.

Apoptosis is a regulated process of cell death by which cells actively participate in their own destruction. In multicellular organisms, the balance between cell proliferation and apoptosis provides homeostatic control, and a regulatory failure of either event can contribute to oncogenesis. The extracellular matrix (ECM) is known to play a regulatory role in cellular growth and differentiation, but only more recently has it been recognized as a regulator of apoptosis. In these processes the major transmitters of ECM-derived signals to the cell are members of the integrin family, although the mechanical process of cell spreading also plays a role. Both in vivo and in vitro the loss of adhesion to specific components of the ECM can lead to cell death, and such apoptosis can be induced experimentally by blocking integrin binding. Heterotypic and homotypic cell-cell adhesion can also protect from adhesion-dependent apoptosis and there is evidence to suggest that this too in integrin mediated. In addition, some integrin mediated signaling appears to promote apoptosis. The downstream mechanisms of integrin signaling causing cell death have not been greatly explored, but there is evidence from two different systems that the induction of ICE transcription and nuclear translocation of p53 are candidate processes. Alterations in integrin expression or signaling therefore are likely to contribute to tumor development by enabling escape from apoptosis. Also, the recognition of the importance of cell-cell adhesion in tumor cell survival offers the potential of developing improved drug regimes for the treatment of malignancy.

Amino Acid Sequence↗

PubMed1995

Effect of TGF-beta on differentiated organoids of the colon carcinoma cell line LIM 1863.

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.

Carcinoma↗

PubMed1995

Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment.

Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication.

Antibodies, Monoclonal↗

PubMed1994

Phorbol ester-induced transcription of an immediate-early response gene by human T cells is inhibited by co-treatment with calcium ionophore.

Human T cells require two discrete signals to initiate their proliferation. In Jurkat T cells the first signal can be provided by the phorbol ester TPA and the second by the calcium ionophore A23187. We have isolated a cDNA from Jurkat T cells representing mRNA induced by TPA but inhibited by simultaneous treatment of the cells with antibody, lectin, or A23187. Sequencing revealed identity of the Jurkat clone to a cDNA, termed ETR101, recently isolated from HL60 promyelocytic leukaemia cells and shown to be an immediate early gene expressed upon TPA stimulation of these cells [Shimizu et al.: J Biol Chem 266:12157, 1991]. The gene is also induced very rapidly upon TPA treatment of Jurkat cells and is superinduced by co-treatment with cycloheximide. The predicted amino acid sequence encoded by ETR101 has weak homology to JunB and JunD, therefore it is of some interest that these three genes share the chromosomal localization, 19p13.2. The divergent effects of TPA treatment upon cell proliferation and differentiation in different circumstances allow some speculation about a possible role for the ETR101 gene product upon cellular differentiation.

Base Sequence↗

PubMed1994

Apoptosis induced by inhibition of intercellular contact.

The LIM 1863 colon carcinoma cell line grows as structural organoids of goblet and columnar cells around a central lumen and provides a model for the development of stem cells in the normal colon. The organoid structure can be disrupted by removal of calcium from the medium, resulting in a suspension of single cells. Upon readdition of calcium, the cells reform the organoid structure over a period of 24 h, and ultrastructural examination of the reforming cells reveals that this involves a complex process that we have termed clutching. To determine the adhesion molecules involved in organoid formation we attempted to block this process by single cell suspensions of LIM 1863 reseeded in the presence of monoclonal antibodies. An anti-integrin antibody directed against a conformational epitope on the alpha v subunit totally inhibited organoid reformation. As a consequence of this inhibition of cell contact the colon carcinoma cells rapidly underwent apoptosis. Investigations of the apoptotic pathway involved suggested an induction mechanism since the onset of apoptosis in the contact-inhibited cells showed specific increased synthesis of 68- and 72-kD proteins. In addition, immunoblotting of cytosolic and nuclear extracts of the cells revealed the rapid translocation of the tumor suppressor gene product, p53 to the cell nucleus upon induction of apoptosis. These results suggest that cell-cell adhesion may be a vital regulator of colon development overcome in tumor cells by loss of adhesion molecules or of functional p53 protein.

Apoptosis↗

PubMed1993

Scanning tunnelling microscopy of collagen I reveals filament bundles to be arranged in a left-handed helix.

Collagen I is the most abundant protein found in the body. Its quaternary structure has been extensively characterised and consists of filaments arranged into bundles. In this study we used scanning tunnelling microscopy to image the collagen I filaments and fibrils directly. The images suggested that filaments are arranged into a left-handed helical structure with a periodicity of about 10nm. Such a structure potentially adds increased tensile strength to the collagen fibrils.

Animals↗

PubMed1993

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

Antibodies, Monoclonal↗

PubMed1993

A re-examination of the molecular basis of cell movement.

A model for cell movement is presented. It is suggested that cells do not migrate on collagen using their VLA (very late antigen) integrins that bind this extracellular matrix protein. Rather, the cells utilize alpha v integrins to bind endogenously produced fibronectin, which binds to the underlying collagen. It is envisaged that cells proceed by a process of engagement and disengagement of alpha v integrins to the extracellular matrix, somewhat analogous to the motion of a monkey climbing a tree. Secretion of isoforms of the adhesion modulator, thrombospondin, regulates disengagement of the integrin from its ligand in migrating cells. The integrin disengagement signal is mediated by thrombospondin cross-linking the alpha v integrin to an integrin accessory molecule and thus activating protein kinases. The cross-linked receptor complex undergoes recycling back along actin stress fibres, guided by the integrin beta-subunit. After endocytosis and protein sorting the alpha v integrin is transported back to the leading edge off migrating cells in vesicles guided by the tubulin-binding capabilities of an integrin accessory molecule. Direct attachment to collagen required for processes, such as matrix contraction, is mediated by VLA integrins which displace alpha v integrins from points of attachment during integrin recycling, possibly through an alpha v beta 1 intermediary receptor.

Animals↗

PubMed1992

Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36).

Fetal embryonic fibroblasts attach and spread on thrombospondin (TSP). Adhesion is tight and focal adhesion plaques and "spots" are formed. We have investigated the receptors responsible for this adhesion. Unstimulated cells express the vitronectin receptor on their surface and this beta 3 integrin molecule contributes to adhesion. Another putative receptor for TSP, termed glycoprotein (GP) 88, which exists as a cytoplasmic pool in unstimulated cells becomes surface expressed when these cells are plated on TSP and localizes to areas of cell adhesion. Western blot analysis of cell lysate confirms GP88 as a TSP binding protein. Studies with fucoidan indicate that the heparan sulfate proteoglycan, known to function as a receptor for TSP, appears to contribute substantially to the TSP attachment of these cells and may be the receptor most important in the initial phases of TSP interaction.

Antibodies, Monoclonal↗

PubMed1991

Individual embryonic fibroblasts express multiple beta chains in association with the alpha v integrin subunit. Loss of beta 3 expression with cell confluence.

The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity.

Base Sequence↗

PubMed1991

GMP-140 binding to neutrophils is inhibited by sulfated glycans.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.

Antibodies, Monoclonal↗

PubMed1991

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.

Amino Acid Sequence↗

PubMed1991

A monoclonal antibody to a 67 kD cell membrane glycoprotein directly induces persistent platelet aggregation independently of granule secretion.

The transition from reversible to persistent platelet aggregation has been difficult to study because of interference both from preceding primary aggregation and from the events associated with granule secretion during secondary aggregation. As a result it remains unclear whether the persistence of aggregation involves some secretion-independent specific platelet surface reactions. Here we show that a monoclonal antibody (MAb), LeoAl, against a newly described 67 kD platelet membrane glycoprotein induced active platelet aggregation consisting of two distinct phases. The first secretion-independent phase was in several respects (extracellular protein, divalent cation, and pH dependence) different from primary aggregation, but closely resembled the transition from primary to secondary aggregation observed at certain concentrations of physiological agonists. The second, faster phase was indistinguishable from secretion-dependent aggregation to various stimulants. It was shown that p67, GPIIb-IIIa and FC gamma RII are all involved in the observed aggregation, probably through their close topographical association. It is suggested that LeoAl-induced aggregation can be used as a model to study the receptors, ligands and metabolic pathways specifically involved in the transition from reversible to persistent platelet aggregation.

Antibodies, Monoclonal↗

PubMed1990

Identification of a novel integrin beta subunit expressed on cultured monocytes (macrophages). Evidence that one alpha subunit can associate with multiple beta subunits.

The vitronectin receptor (VnR) is one member of a subset of cell adhesion receptors within the integrin supergene family which shares the beta 3 subunit (IIIa). We show here that the VnR is absent from the surface of monocytes freshly isolated from blood but is expressed on these cells after a period of in vitro culture. Such cultured monocytes (macrophages) from a patient with type I Glanzmann's thrombasthenia, however, failed to express the VnR. Instead, immunoprecipitation with a monoclonal antibody directed to the VnR alpha chain (alpha v) revealed a novel integrin comprising alpha v associated noncovalently with a 100-kDa beta subunit (beta 3b), immunologically unrelated to the VnR beta subunit (beta 3a). This same novel integrin complex was also identified on 10-day-old macrophages from healthy donors, but on these cells, the beta 3b subunit was co-expressed with the classical VnR complex of alpha v beta 3a. The novel beta 3b subunit was not identified by monoclonal or polyclonal antibodies to IIIa (beta 3a) nor by a monoclonal antibody to the classical VnR complex. The beta 3b subunit could be distinguished from beta 3a by its relatively greater migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction, by its distinct isoelectric point upon two-dimensional gel electrophoresis, and by one-dimensional peptide mapping. Neither platelets nor B lymphoblasts from this patient with Glanzmann's thrombasthenia expressed any VnR on their surface, whereas control cells from a normal donor expressed the classical VnR but not the beta 3b subunit. The two beta chains, and hence also the combined receptor complexes, appeared to be differentially regulated. These findings provide the first example of an integrin alpha chain complexed with more than a single beta chain in the same cell. Furthermore, the differential regulation of expression of the different beta subunits that associate with the VnR alpha chain on cultured monocytes suggests a role for the novel receptor complex during monocyte/macrophage differentiation.

Adult↗

PubMed1990

Blood leukocytes bind platelet glycoprotein (IIb-IIIa)' but do not express the vitronectin receptor.

Within the integrin family of Arg-Gly-Asp(RGD)-binding adhesion receptors, the subfamily defined by the beta chain known as beta-3 or glycoprotein (GP)IIIa is known to contain two individual receptors. These are the GPIIb-IIIa complex of platelets, where the alpha chain of the heterodimer is GPIIb, and the vitronectin receptor (VnR) containing the alpha V subunit. The presence of either GPIIb-IIIa and/or the VnR on blood leukocytes has been controversial. We have investigated this problem by performing immunoprecipitation and immunoblotting studies with rabbit and monoclonal antibodies (mAb) to each of the subunits of GPIIb-IIIa and the VnR. On cultured cells of different origin, it was established that almost all expressed the VnR but none had GPIIb-IIIa, and the only polypeptide associated with beta 3 was alpha V. Platelets expressed predominantly GPIIb-IIIa, and weakly, the VnR. Monocytes and neutrophils freshly isolated from blood did not express the VnR but bore on their surface a modified form of GPIIb-IIIa. This molecule appeared identical to GPIIb-IIIa but an epitope on GPIIb was masked on the intact cell and was only revealed after immunoblotting. We have termed this modified form of GPIIb-IIIa, GP(IIb-IIIa)'. With differentiation in culture, monocytes rapidly lost surface GP(IIb-IIIa)' and concurrently began to express the VnR. Evidence is presented that GP(IIb-IIIa)' is derived from particles released by activated platelets and is bound firmly to the leukocyte membrane. Its primary function does not seem to be to mediate attachment to matrix proteins; thus, although U937 cells bearing platelet-derived GP(IIb-IIIa)' bound fibrinogen in an RGD-dependent manner, isolated blood monocytes did not. It is suggested that this transfer of membrane proteins from platelets to monocytes and neutrophils may regulate the expression of the leukocyte VnR and also serve as a means of facilitating leukocyte procoagulant activity.

Amino Acid Sequence↗

PubMed1989

Characterization of a novel membrane glycoprotein involved in platelet activation.

When platelets bind certain specific ligands they are induced to secrete the contents of their cytoplasmic granules and to aggregate. Studies of the molecular events accompanying this vital physiological response have led to a greater understanding of cell activation in general since the pathways involved are common to a number of cell types. By contrast most of the information about the cell surface molecules that initiate signal transduction has emerged from work on T lymphocyte activation, a process essential to the initiation of the immune response. We have described an activation antigen on T lymphocytes that is involved in the differentiation of these cells. In the present report it is demonstrated that the antigen is expressed on the platelet membrane with about 1,200 copies/platelet. A monoclonal antibody detecting this antigen stimulates platelet secretion and aggregation with a half-maximal response at approximately 10(-8) M. Characterization of the antigen, termed PTA1, reveals a glycoprotein of Mr 67,000 showing extensive N-linked carbohydrate, much of which appears to be heavily sialated. The amino-terminal sequence of PTA1, EEVLWHTSVPFAEXMSLEXVYPSM, indicates that the protein has not previously been characterized. Preliminary investigation of the mechanism by which PTA1 mediates platelet activation suggests involvement of protein kinase C and the 47-kDa protein of platelets is rapidly phosphorylated upon antibody-mediated activation. During this process PTA1 is also phosphorylated, as it is following platelet activation by the other agonists, collagen, thrombin, and 12-O-tetradecanoylphorbol 13-acetate. These results provide the first example of a cell surface glycoprotein that is directly involved in both platelet and T lymphocyte activation.

Amino Acid Sequence↗