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Biomedical subjects

E A Turley

Publications and source records attributed to E A Turley.

At least 55 records · Page 3Linked to original sources
PubMed1993

Ras-transformed cells express both CD44 and RHAMM hyaluronan receptors: only RHAMM is essential for hyaluronan-promoted locomotion.

Hyaluronan (HA) is an important regulator of cell locomotion. We show that ras-transformed cells, termed 245 cells, respond to HA with an increase in random locomotion. We show that two HA receptors, RHAMM (receptor for hyaluronan-mediated motility) and CD44, are present on these ras-transformed fibroblasts. RHAMM is expressed as a 58-kDa protein and is distributed primarily as patches over lamellae. CD44 occurs largely as an 85- to 90-kDa protein that is distributed more or less evenly over the cell surface with small amounts concentrated at the tips of lamellae. CD44 and RHAMM both bind biotinylated HA in a transblot assay, indicating that they are both potential fibroblast HA receptors. CD44 binds approximately five times more HA than RHAMM as determined by densitometric analysis of transblots, indicating that this protein is the major HA receptor on fibroblasts. We assessed the role of these receptors in mediating the stimulatory effects of HA on cell motility by using antibody neutralization. Several antibodies to CD44 were used that inhibit HA/CD44 interactions. None of these had an effect on locomotory responses to HA, indicating that CD44 is not directly involved in mediating locomotion in response to HA on ras-transformed cells. In contrast, antibodies specific to RHAMM completely inhibited locomotion, indicating that RHAMM is the primary mediator of HA-promoted locomotion of ras-transformed cells.

Animals↗
PubMed1993

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

Amino Acid Sequence↗
PubMed1993

Effects of extracellular matrix components on cell locomotion.

The extracellular matrix (ecm), which is composed of collagens, glycoproteins, and proteoglycans, has emerged as an important regulator of cell locomotion. This review describes some of the mechanisms by which the ecm may regulate locomotion, focusing primarily on cell extension and lamellae formation. Ecm-receptor interactions form an important part of cell recognition of ecm. Such interactions can result in altered cell adhesion, signal transduction, and cytoskeletal organization, all of which impact on cell locomotion. It is important to note that although the effects of single ecm components have been studied, generally, the cell is likely to perceive ecm in vivo as a macromolecular complex. It will fall to future work to define how complexes of ecm regulate cell behavior. Because of our own particular research bias, we focus on reviewing the role of fibronectin, integrins, chondroitin sulfate, hyaluronan, and hyaluronan receptors in the regulation of cell locomotion and examine their effect on adhesion, signal transduction, and cytoskeletal integrity. Cytoskeleton assembly mechanisms, particularly those that might be regulated by the ecm, are also described. These events are summarized in a working model of ecm-promoted locomotion.

Animals↗
PubMed1993

Identification of hyaluronan binding proteins using a biotinylated hyaluronan probe.

A method for detecting hyaluronan (HA)-binding proteins in transblot assays using biotinylated HA (BHA) is described. Some of the binding characteristics of a novel HA receptor termed RHAMM (Receptor for HA-Mediated Motility) are characterized using this assay. The method is also used to detect other HA-binding proteins in tissue homogenates. This method is semiquantitative, rapid, reproducible, sensitive and therefore of potential use in identifying the levels of HA-binding proteins in different cells and tissues.

Animals↗
PubMed1993

Transforming growth factor beta and fibroblast growth factor as promoters of tumor progression to malignancy.

Polypeptide growth factors are a diverse group of biological regulators. Because they are fundamentally involved in the cellular processes that are important for transformation and progression to malignancy, alterations in growth factor control and in their signal pathways are often observed in tumor cells. In this review, we consider the participation of growth factors and the mechanisms by which they effect tumor progression, using as examples members of the transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families. We explore the hypothesis that although abrogation of TGF-beta negative growth regulation is necessary for transformation, in the later stages of tumor progression, TGF-beta plays a direct role in the enhancement of invasion and metastasis as an autocrine stimulator of these processes. In addition, we present evidence that demonstrates both the potential and the importance of members of the FGF family in transformation and induction of metastasis. Several models of growth factor regulation of malignancy are presented in which we demonstrate (1) a link between TGF-beta 1 mitogenic stimulation of malignant cells and alterations in the expression of ribonucleotide reductase, a key rate-limiting step in the synthesis of DNA and in cell proliferation; (2) autocrine and/or intracrine FGF mitogenic stimulation of malignant cell proliferation and metastasis; and (3) autocrine TGF-beta regulation of malignant cell locomotion and invasion through elevated proteolytic activity and increased synthesis of hyaluronan and RHAMM, a novel hyaluronan cell surface receptor.

Animals↗
PubMed1992

Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-beta 1 (Ser223, 225).

An expression vector was constructed in which TGF-beta 1 was placed under the control of the metallothionein promoter. Cys223 and Cys225 in the TGF-beta 1 propeptide were converted to serines, mutations which result in dissociation of the pro-peptide and secretion of bioactive TGF-beta 1 [Brunner, A.M., Marquardt, H., Malacko, A.R., Lioubin, M.N. and Purchio, A.F. (1989) J. Biol. Chem., 264, 13660-13664]. A fibrosarcoma was transfected with this plasmid and a clone (17.18) was selected in which TGF-beta 1 mRNA was able to be induced six-fold following zinc sulphate treatment. These cells increased the secretion of bioactive TGF-beta 1 14-fold and exhibited a coincidental increase in jun-B mRNA expression, suggesting that secreted TGF-beta 1 was acting to induce this early response gene by autocrine activation. Following zinc sulphate induction, the tumor cells became progressively more motile and able to invade collagen gels. In contrast to parental tumor not bearing the TGF-beta 1 expression vector, zinc sulphate stimulation of clone 17.18 enhanced collagenase IV and procathepsin L mRNA levels and enhanced the secretion of many collagenolytic proteases into the medium. Since the action of TGF-beta generally decreases proteolysis by suppression of protease transcription, we compared the response of normal parental fibroblasts to ras-transformed fibrosarcomas and confirmed that TGF-beta could greatly enhance collagenase IV and procathepsin L mRNA levels while having little effect on non-transformed fibroblasts. These experiments indicate that induction of TGF-beta secretion can enhance motility and protease production through autocrine activation, thus increasing the invasion potential of fibrosarcomas.

Amino Acid Sequence↗
PubMed1992

Hyaluronan and cell locomotion.

Hyaluronan (HA), a glycosaminoglycan, has long been implicated in cell locomotion. We have shown that HA production regulates the locomotion of H-ras-transformed cells. This autocrine motility mechanism is mediated by a novel HA receptor termed RHAMM, an acronym for Receptor for HA Mediated Motility. HA:RHAMM interactions regulate directional locomotion of tumor cells and result in enhanced protein tyrosine phosphorylation that may be a critical messenger mechanism for initiation of locomotion.

Animals↗
PubMed1992

Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility.

A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.

3T3 Cells↗
PubMed1991

FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes.

In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1.2), suggesting a role in locomotion. On dividing multinegative thymocytes, FMC46 was found almost exclusively along the cleavage furrow, implicating it in detachment processes. We conclude that the properties of the LAM-1 molecule recognized by FMC46 are consistent with a role in detachment phases of motility and of cell interactions.

Antibodies, Monoclonal↗
PubMed1991

Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells.

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.

Carrier Proteins↗
PubMed1990

A hyaluronan-binding protein shows a partial and temporally regulated codistribution with actin on locomoting chick heart fibroblasts.

The ultrastructural distribution of a hyaluronan-binding protein (HABP) and its relationship to actin-containing microfilaments were studied with immunocytochemistry. Ultrastructural analysis localized HABP to the cell coat and demonstrated that it occurred largely in cell processes where the apical surfaces were immunopositive. The codistribution of HABP with actin-containing microfilaments in cell processes was demonstrated with double immunolabeling using monoclonal antibodies to actin and monospecific, polyclonal antibodies to HABP. Both the topological localization of HABP and its cytoskeletal coassociations were modulated by cells during different cellular phases. Thus, in cells exhibiting large lamellae and few actin fibrils, typical of rapidly locomoting cells, HABP codistributed primarily with the actin meshwork occurring in cell processes, although some codistribution between the two proteins occurred over the cell body. In cells containing prominent stress fibers and less obvious lamellae, HABP was absent in cell processes but, rather, was aligned primarily along actin fibrils occurring in the cell body. A functional association between HABP and the actin-containing cytoskeleton was suggested by the ability of cytochalasin D to coordinately alter the distribution of HABP and disrupt its coassociation with actin. As well, the addition of hyaluronan to monolayers increased the association of HABP with a Triton-insoluble cytoskeleton. The possible roles of HABP in cell motility and cytoskeletal organization are discussed.

Actin Cytoskeleton↗
PubMed1989

Hyaluronic acid stimulates protein kinase activity in intact cells and in an isolated protein complex.

The addition of hyaluronate to intact chick embryonic heart fibroblasts enriched with a hyaluronate-binding protein (HABP) stimulated phosphorylation of tyrosine and serine/threonine residues in cellular proteins. A protein complex containing a hyaluronate-binding protein (cell-HABP) was isolated from the cultured heart fibroblasts. The isolated complex (Mr approximately 1 x 10(6] contained phosphoproteins that exhibited protein kinase activity specifically stimulated by hyaluronate. Both tyrosine and serine residues in the protein complex were phosphorylated in response to this glycosaminoglycan. The hyaluronate-stimulated protein kinase activity was tightly associated with cell-HABP in vitro; enzyme activity co-immunoprecipitated with cell-HABP using a monospecific anti-HABP antibody and co-eluted with cell-HABP when chromatographed on a column of Sephacryl S-1000 in 2.0 M guanidine hydrochloride. The uniqueness of the cell-HABP-associated protein kinase activity was suggested by both its specific response to hyaluronate, relative to related glycosaminoglycans such as heparin and chondroitin sulfate or to growth factors such as epidermal growth factor or insulin, and its antigenic distinction from other protein kinases such as growth factor receptors. These results point to a new mechanism by which glycosaminoglycans, such as hyaluronate, may modify cell behavior.

Animals↗
PubMed1989

The role of a cell-associated hyaluronan-binding protein in fibroblast behaviour.

In a model culture system hyaluronan has been shown to promote cell ruffling and random cell locomotion. At the same time it promotes both protein tyrosine phosphorylation and phospholipid breakdown. These results indicate that this glycosaminoglycan, in addition to its previously described role as a regulator of adhesion and cytoskeletal organization, may also influence cell behaviour via second messenger formation. A cell-associated hyaluronan-binding protein (cell-HABP) has been isolated from locomoting fibroblasts that may represent one binding site that transduces these effects of hyaluronan. This protein is concentrated in the lamellae and ruffles of migrating fibroblasts but is lost from the cell surface as cell locomotion slows. It is a large (molecular mass estimated at 1-2 x 10(6) daltons) complex of proteins that includes a hyaluronan-binding site and a protein kinase. The protein kinase is responsive to hyaluronan and is related, antigenically, to the pp60src protein kinase. The function of cell-HABP has not been characterized but its location, regulated distribution and enzyme characteristics suggest that it may be involved in hyaluronan-regulated cell locomotion.

Animals↗
PubMed1989

A model system that demonstrates interactions among extracellular matrix macromolecules.

Binding interactions among fibronectin (FN), glycosaminoglycan chains (GAG) and hyaluronate binding proteins (HABP) have been examined using a bead aggregation assay. 3H-FN binds to Dowex beads coated with chondroitin sulfate GAG chains (CS) but not to beads coated with hyaluronic acid (HA). Assays of bead agglutination indicate that FN has multiple binding sites for CS. Binding of 3H-FN to HA-beads is slightly promoted by exogenous divalent cations but this interaction does not aggregate Dowex-HA. The interaction between FN and CS reduces the previously reported ability of Dowex-CS to associate with HA-beads (Turley, E.A. and Roth, S. 1980, Nature Vol. 283, 268-271). HABP bind to HA and to a lesser extent CS. These proteins both stabilize the interaction between HA- and CS-beads in a manner reminiscent of the stabilization of HA-proteoglycan interactions by link protein and permit aggregation of Dowex-CS-FN with Dowex-HA. This simple bead aggregation assay allows rapid characterization of properties that may help to clarify how macromolecular constituents spontaneously assemble into an interstitial matrix.

Agglutination Tests↗
PubMed1987

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells.

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The effects of epidermal growth factor on neural crest cells in tissue culture.

Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the 3H-labeled proteoglycan. Furthermore, EGF stimulates [3H]thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.

Animals↗
PubMed1985

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices.

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗