Biomedical subjects

G F Burns

Publications and source records attributed to G F Burns.

At least 19 recordsLinked to original sources

PubMed2005

FAT, E-cadherin, beta catenin, HER 2/neu, Ki67 immuno-expression, and histological grade in intrahepatic cholangiocarcinoma.

AIM: To identify surrogate prognostic markers in intrahepatic cholangiocarcinoma (IHCC). METHODS: Thirty one cases of IHCC were graded and immunostained for FAT, Ki67, E-cadherin, beta catenin, and HER 2/neu. RESULTS: Twenty two cases were high grade and 27 had high Ki67 counts. Strong membranous staining of HER 2/neu was found in 10 tumours and reduced membranous E-cadherin and beta catenin in 19 and 18 tumours, respectively. Nuclear localisation of beta catenin was identified in five tumours and 22 showed weak cytoplasmic staining of FAT. Strong HER 2/neu and weak FAT immuno-expression were significantly correlated with high histological grade (p=0.01) and high Ki67 index (p=0.03). Upregulation of HER 2/neu was also significantly associated with nuclear localisation of beta catenin (p=0.01). Reduced membranous beta catenin was significantly related to reduced membranous E-cadherin (p=0.03), weak staining for FAT (p=0.01), and nuclear translocation of beta catenin (p=0.04). CONCLUSIONS: Reduced immuno-expression of E-cadherin and FAT at their normal membranous location may be potential prognostic markers, and the overexpression of HER 2/neu and beta catenin nuclear translocation may have a role in cholangiocarcinogenesis.

Adult↗

PubMed2001

A CD44 survival pathway triggers chemoresistance via lyn kinase and phosphoinositide 3-kinase/Akt in colon carcinoma cells.

A major obstacle to successful treatment of colorectal cancer is chemotherapy resistance. Enhanced expression of variant CD44 isoforms has been associated with aggressive tumor behavior, prompting the question of whether signaling from this receptor might modulate drug sensitivity. Activation of variant CD44 in colon carcinoma cell lines triggered resistance to the drug 1,3-bis(2-chloroethyl)-1-nitrosurea. Resistance was induced by monoclonal antibodies directed against epitopes independent of the hyaluronate-binding region but was not triggered by identical treatment of a carcinoma line expressing the standard CD44 isoform. We observed that variant CD44 produced activation of the src-family tyrosine kinase lyn. Moreover, overexpression of dominant-active lyn recapitulated chemoresistance via a pathway shown to involve activation of phosphoinositide 3-kinase and Akt. These results establish a novel role for CD44 in determining survival of colon carcinoma cells through lyn kinase and Akt. The ability to suppress apoptosis might play a critical role in the onset and development of colorectal malignancies.

Antineoplastic Agents, Alkylating↗

PubMed2000

The integrins alpha3beta1 and alpha6beta1 physically and functionally associate with CD36 in human melanoma cells. Requirement for the extracellular domain OF CD36.

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.

Animals↗

PubMed2000

CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport.

The CD36 receptor sequence predicts two hydrophobic domains located at the N- and C-termini of the protein, but there are conflicting reports as to whether the N-terminal uncleaved leader sequence functions as a transmembrane domain. To investigate the topology of CD36, we generated a panel of mutants lacking either one or both hydrophobic regions and analyzed their folding and transport in COS-7 cells. The N- and the C-terminal hydrophobic regions were both sufficient to anchor CD36 in the membrane, and a FLAG epitope inserted at the N-terminus was located intracellularly. These results indicate that CD36 adopts a ditopic configuration. Accordingly, neither N- nor C-terminal truncation mutants were secreted. Analysis with conformation-specific monoclonal antibodies showed that the N-terminal transmembrane domain truncated molecule was slowly transported through the exocytic pathway and largely accumulated intracellularly. Thus, dual membrane insertion dictates the correct topogenesis and seems to be necessary for efficient folding and intracellular transport.

Amino Acid Sequence↗

PubMed1998

Active Lyn protein tyrosine kinase is selectively enriched within membrane microdomains of resting platelets.

Circulating platelets are primed to respond very rapidly to thrombogenic stimuli, but most platelets complete their lifespan without ever becoming activated. Platelet activation is accompanied by waves of sequential tyrosine phosphorylation thought to involve members of the Src family of protein tyrosine kinases (PTKs). We show here that resting platelets contain highly active pp53/56(Lyn) PTK within membrane microdomains (rafts) isolated biochemically with or without the use of detergent. This fraction is also greatly enriched in the transmembrane glycoprotein CD36, known to associate with Lyn PTK, but in transfection studies we could find no evidence to suggest that CD36 affects the distribution or function of Lyn. Upon platelet activation Lyn activity remains constant or diminishes and pp60(c-src) PTK within this fraction becomes highly activated, indicating the dynamic nature of the membrane microdomains. It is suggested that the function of active Lyn PTK in the resting platelet is to allow prolonged survival of this anucleate cell.

Animals↗

PubMed1998

Antibody binding to individual short consensus repeats of decay-accelerating factor enhances enterovirus cell attachment and infectivity.

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.

Animals↗

PubMed1998

Engagement of variant CD44 confers resistance to anti-integrin antibody-mediated apoptosis in a colon carcinoma cell line.

The LIM 1863 colon carcinoma cell line grows as structured organoids around a central lumen, and we have previously demonstrated that the three-dimensional arrangement protects the individual cells from apoptosis induced by an anti-alpha v integrin antibody, 23C6 (Bates et al., 1994). Here we show that the intercellular forces which drive spheroid formation can be overcome by exposure of the cells to a collagen substrate, or more specifically through ligation of the CD44 receptor by a monoclonal antibody. Binding to immobilized anti-CD44 antibody induced a monolayer morphology which is accompanied by fibronectin production and secretion, and expression of the integrin alpha v beta 6. Significantly, the cells of the monolayer acquired resistance to 23C6 antibody-mediated apoptosis over time and this property was sustained even after removal from the monolayer. We provide data to show that this resistance is not dependent on monolayer morphology, constant engagement of the CD44 receptor, loss of the 23C6 antigen, or elevation of Bcl-2 or Bcl-XL protein. The CD44 expressed by LIM 1863 is shown to be the metastatic variant of the molecule therefore these results provide a possible explanation for the selective advantages conferred by expression of this variant for metastasizing colon cancer cells. Overall, the findings of this study support a model for the development of malignancy through the production of specific survival and growth signals as a direct consequence of a signaling event induced by stimulation of an epithelial variant of CD44.

Animals↗

PubMed1997

CD36 forms covalently associated dimers and multimers in platelets and transfected COS-7 cells.

CD36 is a transmembrane glycoprotein expressed on the surface of a number of cell types. The analysis of CD36 from platelets using immunoblotting, gel filtration, and native PAGE indicated the presence of high molecular complexes exceeding the Mr of monomeric CD36. Experiments using transfected COS-7 cells revealed these complexes were homodimers and -multimers of CD36. The multimers could be dissociated by treatment with a reducing agent, indicating they were formed by intermolecular cysteine-bridging. Mutagenesis of the cDNA for CD36 implicated the cysteines in the extracellular domain of the molecule. The potential physiological roles of CD36 multimerisation are discussed.

Animals↗

PubMed1997

TLiSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of the immunoglobulin superfamily exhibiting distinctive regulation of expression.

T lineage-specific activation antigen 1 (TLiSA1) antigen was initially described as a T lineage-specific activation antigen involved in the differentiation of human cytotoxic T cells. Subsequently, the antigen was identified on platelets and was shown to be involved in platelet activation, hence it was renamed platelet and T cell antigen 1 (PTA1), although identity between the two antigens was not established. In the present study we have cloned the cDNA encoding TLiSA1 from Jurkat cells and show it to be a novel member of the immunoglobulin superfamily with the unusual structure of two V domains only. Identity between TLiSA1 and platelet PTA1 is established by immunological criteria, by internal peptide sequences obtained from the purified platelet glycoprotein and by sequencing the platelet transcript after reverse transcriptase-polymerase chain reaction. In Jurkat cells, TLiSA1/PTA1 mRNA and surface protein expression is greatly stimulated by treatment of the cells with phorbol ester, but the T cell proliferative signal of phorbol ester and ionophore combined greatly reduces or abrogates this response, and this suppressive effect of the ionophore is not reversed by incorporating FK506 to inhibit calcineurin. Together with the known signaling role of PTA1, these data substantiate the notion that this molecule is implicated in T cell differentiation, perhaps by engagement of an adhesive ligand.

Amino Acid Sequence↗

PubMed1997

Stimulation of platelet activation and aggregation by a carboxyl-terminal peptide from thrombospondin binding to the integrin-associated protein receptor.

Thrombospondin, a major secretory product of the alpha-granules of activated platelets, is a large trimeric glycoprotein that plays an important role in platelet aggregation. On resting platelets, thrombospondin binds to a single receptor in a cation-independent manner, but upon platelet activation it binds at least two further, distinct receptors that are both dependent upon divalent cations. Each of these receptors on the platelet surface binds to different regions of the thrombospondin molecule, and such binding may be responsible for the multifunctional role of thrombospondin in aggregation. We show here that a peptide from the carboxyl terminus of thrombospondin, RFYVVMWK, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 microM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the RFYVVMWK-containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.

Adenosine Diphosphate↗

PubMed1997

Formation of spheroid structures in a human colon carcinoma cell line involves a complex series of intercellular rearrangements.

The structural remodelling of tissues that occurs in vivo during animal morphogenesis can often prove difficult to study. Here we investigate the organizational processes of the LIM 1863 colon carcinoma cell line as it transforms from a single-cell stage into multicellular spherical structures called 'organoids'. The organoids can be dissociated into a viable single-cell suspension when cultured in calcium-depleted medium, and then induced to reform the organoid structure by the readdition of calcium. Previous studies have shown that initial cell attachment under these conditions is characterized by a novel mechanism of cell engulfment termed 'clutching'. This investigation reveals the subsequent appearance of junctional complexes between groups of 'clutched' cells prior to lumen formation, and the ultimate 'declutching' of entrapped cells as a means of cell rearrangement. Intact actin filaments but not microtubules were required for the initial clutching events, while inhibition of microtubule polymerization resulted in aberrant apical protein polarization, but did not affect the development of a luminal space within the spheroids. Single cells exhibited pools of intracellular microvilli contained in vacuolar apical compartments, which were resistant to the effects of cytoskeleton-disrupting drugs. However, these structures did not seem to be responsible for the swift development of the luminal surface observed in these cells. Two other cell lines, MDCK and DU 4475, were found to exhibit similar clutching conformations when induced to form three-dimensional structures, suggesting that this may be a widespread mechanism of cell rearrangement that reflects the process of organ morphogenesis in vivo.

Carcinoma↗

PubMed1997

Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21.

Competitive viral binding assays have revealed previously that coxsackievirus A21 (CAV21) and human rhinovirus 14 (HRV14) share a common cell surface receptor. More recently, intercellular adhesion molecule-1 (ICAM-1) has been identified as the cellular receptor for HRV-14. Also, anti-ICAM-1 monoclonal antibodies (MAbs) blocked infection by HRV14, CAV13, CAV18, and CAV21, suggesting that these viruses share this receptor; however, this has never been established by more direct methods. In this study we show conclusively that CAV21 binds to ICAM-1 and that MAbs directed against the N-terminal domain of the molecule inhibit this attachment. Furthermore, we show that the specific interaction between ICAM-1 and 160S CAV21 virions induces formation of 135S A particles. Finally, we show transfection of normally nonsusceptible mouse L cells with human ICAM-1 cDNA renders them susceptible to infection by CAV21.

Animals↗

PubMed1997

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.

Animals↗

PubMed1996

Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36.

Here we describe the isolation and characterization of a Triton X-100-insoluble fraction isolated from lysates of platelets by flotation in sucrose gradients. Transmission electron microscopy of the insoluble material revealed a heterogeneous population of vesicles ranging in size from 20 to 1000 nm, and Western blot analyses of platelet lysates for the caveolae structural coat protein, caveolin/VIP21, were negative. Biochemical characterization of the Triton X-100-insoluble fraction showed it to be cholesterol-rich, greatly and specifically enriched in the plasma membrane glycoprotein CD36, and also to contain Src and the Src-related kinase, Lyn. CD36 within this fraction is shown to be palmitoylated, but the fraction itself is not generally enriched in palmitoylated platelet proteins. These results suggest that this fraction represents caveolin-negative, CD36-rich microdomains in the resting platelet membrane. CD36 can form associations with certain Src-related kinases and can signal to activate platelets. These results suggest the possibility that such microdomains are implicated in platelet activation.

Blood Platelets↗

PubMed1996

Factor H co-purifies with thrombospondin isolated from platelet secretate.

Thrombospondin is a trimeric glycoprotein that has several known functions, including roles in platelet aggregation, phagocytosis and an inhibitor of angiogenesis. Typically the molecule is isolated from platelet secretate by heparin affinity followed by sizing chromatography. In this study, purity is analysed by 7.5% SDS-PAGE under reducing conditions when thrombospondin monomers run as a band at around 180 kDa. Under nonreducing conditions of 7.5% SDS-PAGE, thrombospondin does not penetrate beyond the stacking gel; however, under these conditions a major contaminating band can be seen which, upon reduction, merges into the thrombospondin band. Further purification of this contaminating protein was achieved by DEAE chromatography and it was identified as Factor H by peptide sequencing and immunoblotting. Factor H function was demonstrated by the ability of the protein to function as a cofactor in the Factor-I-mediated cleavage of C3b. Since Factor H has several known functions, such contamination could confound functional studies of thrombospondin thus purified and a pre-elution step of the heparin affinity column is recommended.

Amino Acid Sequence↗

PubMed1996

CD36 is spatially associated with glycoprotein IIb-IIIa (alpha IIb beta 3) on the surface of resting platelets.

Platelet activation and aggregation induced by agonists such as thrombin are accompanied by the phosphorylation of several proteins on tyrosine. Such tyrosine phosphorylation is dependent upon activation and ligand engagement of the major integrin receptor on the surface of platelets, glycoprotein (GP) IIb-IIIa (alpha IIb beta 3), but how this is accomplished is not known. The only platelet membrane GP known to associated with non receptor tyrosine kinases is CD36 (GPIV) which forms associations with pp60Fyn, pp62Yes, and pp54/58Lyn, and antibodies directed against CD36 activate platelets in a process dependent upon GPIIb-IIIa. These and other data suggest a physical association between the two membrane GPs, IIb-IIIa and CD36. By the use of immunoprecipitation of lysates of platelets that have been surface labeled and chemically crosslinked we show here that CD36 and GPIIb-IIIa are spatially associated on the surface of resting platelets.

Antibodies, Monoclonal↗

PubMed1996

A common epitope on platelet integrin alpha IIb beta 3 (glycoprotein IIbIIIa; CD41b/CD61) and alpha M beta 2 (Mac-1; CDIIb/CD18) detected by a monoclonal antibody.

Evidence is presented that the mAb 25E11, directed against the platelet integrin alpha IIb beta 3 (glycoprotein IIbIIIa;CD41b/CD61) also binds the distinct myeloid cell integrin alpha M beta 2 (Mac-1;CDIIb/CD18). The Ab is shown to identify only the alpha IIb beta 3 integrin complex and not the individual subunits in crossed Ab immunoelectrophoresis of platelet lysate. From cultured human macrophages, sequential immunoprecipitation of labeled glycoproteins indicated that 25E11 also bound the Mac-1 (CD11b/CD18) complex. This was confirmed using COS-7 and WOP cells doubly transfected with alpha M (CD11b) and beta 2 (CD18) or with alpha L (CD11a) and beta 2 when it was found that the Ab bound only the alpha M beta 2 transfectants. Studies with these cells and the RC2A myeloid cell line stimulated with tetradecanoyl phorbol acetate or FMLP indicated that the 25E11 epitope on Mac-1 did not depend on cell activation for its expression. The rationale for this cross-reactivity is not known, but since the 25E11 Ab inhibits the function of both platelets and myeloid cells, it is likely that this shared epitope is important to integrin function. Given the expression of this epitope on IIbIIIa and Mac-1, the dominant integrins of platelets and granulocyte/macrophage cells, but not on other integrins, a role of this epitope in the early events of inflammation is suggested.

Antibodies, Monoclonal↗

PubMed1995

Capture by chemical crosslinkers provides evidence that integrin alpha IIb beta 3 forms a complex with protein tyrosine kinases in intact platelets.

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.

Blood Platelets↗