Biomedical subjects

B Boilly

Publications and source records attributed to B Boilly.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regulation of cell proliferation and urokinase plasminogen activation of human breast epithelial cells by carrageenans.

Carrageenans, a family of polysulphated carbohydrates, are able to inhibit the binding to cells of growth factors such as transforming growth factor 1 (TGF 1), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor (PDGF) and so modulate cell invasion and proliferation. We studied the effects of carrageenans on the proliferation and on the uPA/PAI-1 system in breast epithelial cells. Carrageenans were able to inhibit the proliferation of both normal breast epithelial cells (NBEC) and breast epithelial cancer cell lines (MDA-MB-231 and MCF-7) but could only inhibit the uPA activity in the MDA-MB-231 cells. Moreover, carrageenans inhibited FGF-2 binding in all three cell types, suggesting that they regulate cell proliferation and uPA/PAI-1 system through two distinct mechanisms. These molecules could be considered as potentially useful anti-cancer agents.

Anticoagulants↗

PubMed1997

Production of sulfated proteoglycans by human breast cancer cell lines: binding to fibroblast growth factor-2.

The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [35S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (Kav 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (Kav 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind 125I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties.

Breast Neoplasms↗

PubMed1997

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level.

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.

Breast↗

PubMed1997

Nerve-blastema interactions induce fibroblast growth factor-1 release during limb regeneration in Pleurodeles waltl.

Previous studies have shown that both fibroblast growth factor (FGF)-1 and nerves play an important function during limb regeneration, but no correlation between these two regeneration factors has yet been demonstrated. In the present study we first establish that exogenous FGF-2, a member of the FGF family that binds to the same high-affinity receptors as FGF-1, is able to stimulate both [3H]-thymidine incorporation and the mitotic index in the mesenchyme and the epidermal cells of denervated blastemas. We then use cocultures of spinal cord and blastema on heparin-coated dishes, an in vitro system mimicking the in vivo interactions during limb regeneration, to show that interactions between nerve fibers from the spinal cord and the blastema enhance the release of bioactive FGF-1. Release of this growth factor seemed to correlate with nerve fiber regeneration, as it decreased in the presence of the dipeptide Leu-Ala, known to inhibit neurite outgrowth, while the inverse dipeptide Ala-Leu was inactive. Therefore, these results support our hypothesis that the interaction between nervous tissue and blastema is permissive for the release of FGF-1, which in turn stimulates blastema cell proliferation.

Animals↗

PubMed1997

Activation of protein kinase C during newt limb regeneration: effect of denervation.

Blastema cell proliferation during newt limb regeneration is a nerve-dependent process. The present study was undertaken to determine whether or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles walt. Analysis included evaluation of PKC activity and its subcellular localization at various stages of regeneration, both in vivo and in vitro. The data reveal an increase in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correlates with blastema cell proliferation rate. Denervation significantly reduces blastema cell proliferation and also causes a reduction in membrane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic reduction in activity 96 h after denervation. In contrast, PKC activity in the epidermal cap did not change. Cultured whole blastemas likewise express a decrease in particulate PKC activity and therefore mimic denervated blastemas in this parameter. Co-culture of blastemas with spinal ganglia partially reduces the decline in PKC activity, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimulating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regeneration.

Animals↗

PubMed1997

[Proteoglycans and breast cancer].

Proteoglycans (PG) are complex sulphated macromolecules composed of linear polysaccharide chains of glycosaminoglycans (GAG) covalently attached to a core protein. These GAG chains contain sulphate groups at various positions, giving them a high density of negative charges, and allowing them to interact with extracellular matrix molecules, including various growth factors. In the developing mammary gland, sulphated proteoglycans participate in morphogenesis and interact with extracellular matrix components in order to constitute a functional matrix. In breast pathogenesis, qualitative or quantitative changes in PG may have important consequences on cell proliferation and/or differentiation. Thus, several studies showed large variations in the nature and distribution of PG/GAG in breast cancer. Accumulation of chondroitin sulfate proteoglycans was described in the stromal compartment of mammary biopsy sections, and content in heparan sulfate proteoglycans, which were more specifically distributed in the epithelial compartment, increased with the level of malignancy and invasiveness of breast cancer tissues. Furthermore, heparan sulfate proteoglycans seem to be involved in control of the growth-promoting activity of numerous growth factors such as fibroblast growth factors also named Heparin-Binding Growth Factors (HBGF). The implication of PG in growth factor activity suggest that PG may have prognostic value in breast cancer. In future, structural studies into the specific HS-sequences involvement in growth factors binding could allow the development of new antiproliferative strategies.

Breast Neoplasms↗

PubMed1996

Heparan sulfate proteoglycans play a dual role in regulating fibroblast growth factor-2 mitogenic activity in human breast cancer cells.

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of 125I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10-30 mM; higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.

Breast Neoplasms↗

PubMed1996

Ductal cyst formation in collagen-embedded adult human islet preparations. A means to the reproduction of nesidioblastosis in vitro.

Neogenesis of endocrine islets from ductal epithelium termed nesidioblastosis has been described in vivo after various experimental conditions (90% pancreatectomy or pancreas wrapping in the rodent) and in clinical pathologies. In the adult regenerating pancreas, a proliferation and organization of ductal epithelium into tubular structures precedes its differentiation into endocrine cells. Reproduction of nesidioblastosis in vitro may provide a novel approach to human islet propagation in vitro. With this aim, adult human islet preparations were cultured in diverse three-dimensional (3D) gels in the presence of serum. After 3-5 days in rat tail collagen gels, proliferating (bromodeoxyuridine-positive) cystic structures appeared associated with islets and as isolated spheres. Percentage labeling indexes of the cysts were 4.1, 18.7, 15.4, and 13.3% after 3, 5, 7, and 10 days of culture, respectively. Immunohistochemistry confirmed the ductal (carbohydrate antigen 19-9) and epithelial (keratin-1) nature of the cysts. No cysts were formed in agarose gels or Vitrogen 100, whereas the cyst number was increased by the quantity of serum (20% > 10%) and gels rich in extracellular matrix components and growth factors (Matrigel). The latter lead to tubular networks. Single endocrine islet cells were observed in the ductal cysts after 7 (2.8%) to 10 (5.6%) days in rat tail collagen. Our observations paralleled the changes characteristic of the regenerating pancreas in vivo. 3D culture may permit the identification of matrix and media constituents promoting the neogenesis of islets and may be the means to increase the mass of endocrine tissue obtained from adult cadaveric pancreases for transplantation.

Adult↗

PubMed1996

[Involvement of sulfated proteoglycans in the control of proliferation of MCF-7 breast cancer cells].

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.

Breast Neoplasms↗

PubMed1996

Nerve growth factor induced neurite outgrowth from amphibian neuroepithelial precursor cells is prevented by dipeptides inhibiting ubiquitin-mediated proteolysis.

The effect of dipeptides known to inhibit the ubiquitin-mediated proteolysis has been examined on growth factor induced neurite outgrowth from amphibian neuroepithelial precursor cells in primary culture. Nerve growth factor (NGF) stimulated neuritogenesis from these cells but fibroblast growth factor 2 (FGF-2) only increased the number of melanophores. The neurite outgrowth induced by NGF was inhibited by the dipeptides blocking the ubiquitin mediated proteolysis (Leu-Ala and Leu-Gly) whereas the inactive control dipeptides (Ala-Leu and Ala-His) had no effect. This suggests that ubiquitin-mediated proteolysis involving the ubiquitin ligase E3 is necessary for growth factor induced neuronal differentiation during the development of the central nervous system.

Animals↗

PubMed1996

Experimental evidence for FGF-1 control of blastema cell proliferation during limb regeneration of the amphibian Pleurodeles waltl.

During regeneration, blastema cell proliferation depends on several different factors which are, as yet, not fully understood. Previous studies showing the presence of FGF-1 and FGF receptors in the limb blastema make FGF-1 a potentially important molecule for limb regeneration but they do not demonstrate that this factor is active during the process. In the present study, we have first of all confirmed the presence of FGF-1 in limb blastemas of the amphibian Pleurodeles waltl using immunochemistry. Second, we provide evidence in vivo that FGF-1 controls blastema cell proliferation by using different reagents which interfere with FGF activity. Sulfated polysaccharides which bind FGFs, such as heparin, iota-carrageenan and pentosan polysulfate, are able to decrease both 3H-thymidine incorporation and the mitotic index in regeneration blastemas. In addition, suramin which inhibits the binding of growth factors to their receptors, induces the same effect. The presence of receptors in blastema cells is also demonstrated by using the FGF-saporin complex which is known to bind to FGF receptors and to kill cells bearing these receptors. This complex decreases the mitotic index in mesenchyme, while saporin alone did not influence cell proliferation. Finally, results obtained using a neutralizing monoclonal antibody against FGF-1 which was able to specifically reduce blastema cell proliferation, suggests that FGF-1 plays an important function in limb regeneration.

Animals↗

PubMed1996

[Two-dimensional electrophoresis of proteins from breast cancer cells MCF-7. Changes in synthesis induced by FGF-2].

Two-dimensional electrophoresis analysis of proteins from breast cancer cells MCF-7. Modifications of synthesis induced by FGF-2. Using high resolution two-dimensional electrophoresis, we have separated more than 1,000 proteins from the breast cancer cell line MCF-7. Computer assisted analysis of gels allowed us to classify these proteins in function of their isoelectric point, molecular weight and relative quantity. This data-base will now be used as a powerful tool to identified proteins which synthesis is regulated in various experimental or pathological situations. Thus we studied modifications induced by FGF-2. This growth factor induces the synthesis of four polypeptides which are not detected in cells not stimulated by this factor. In addition, intensity of nine other polypeptides was found increased in presence of FGF-2.

Breast Neoplasms↗

PubMed1996

Expression of c-ets-1 and uPA genes is associated with mammary epithelial cell tubulogenesis or neoplastic scattering.

Although the inductive interactions which trigger epithelial morphogenesis have been extensively described, little is known about the transcription factors involved in these processes. During mammary gland morphogenesis, we report the expression of the transcription factor c-ets-1 and one of its target genes uPA in mesenchymal cells during early stages of epithelial invasion, and later in epithelial cells themselves. In vitro studies show that both c-ets-1 and uPA mRNAs can be induced in cultured normal mammary epithelial cells in response to medium conditioned by MRC-5 fibroblasts. In contrast, invasive tumorigenic cell lines from the mammary epithelium express constitutively c-ets-1 and uPA while non-invasive tumorigenic cells do not. In three dimensional co-cultures in collagen gels, a preferential expression of these genes is detected in epithelial cells migrating through the gel either at the tips of normal ducts or in cancerous cells which are scattering. These genes are also expressed in the neighboring fibroblasts. In MRC-5 fibroblasts, conditioned media from tumorigenic epithelial cells induce more efficiently c-ets-1 and uPA mRNA accumulation than do conditioned medium from normal cells. These results suggest that epithelial-mesenchymal interactions trigger c-ets-1 and uPA expression in both compartments during mammary gland morphogenesis. The expression of the genes correlates with invasiveness of epithelial cells irrespective of their being normal or cancerous.

Animals↗

PubMed1995

Structure of four acidic oligosaccharides from the jelly coat surrounding the eggs of Xenopus laevis.

Novel acidic oligosaccharides were released by reductive beta-elimination from the jelly coat eggs of the Anuran Xenopus laevis. According to the structural analysis of these oligosaccharide-alditols, the following structures are proposed: [sequence: see text] where Kdn, 3-deoxy-D-glycero-D-galactononulosonic acid. These results confirm the species specificity of the glycanic structures present in the secretion of amphibian oviducts, and may form the basis of a specific egg-sperm recognition process.

Animals↗

PubMed1995

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

Cell Separation↗

PubMed1995

Primary structure of 12 neutral oligosaccharide-alditols released from the jelly coats of the anuran Xenopus laevis by reductive beta-elimination.

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Xenopus laevis were analysed by 1H-NMR spectroscopy. Among the 12 neutral oligosaccharide-alditols which have been characterized, three of them possess the following unusual structures: [sequence: see text] As previously observed for six other amphibian species, the carbohydrate chains of the jelly coat of Xenopus eggs display a high species specificity which could support a biological role during the fertilization processes.

Animals↗

PubMed1995

Differential responsiveness of human breast cancer cells to basic fibroblast growth factor: a cell kinetics study.

The effects of basic fibroblast growth factor (bFGF) on breast cancer cells are still contradictory and not fully understood. We have studied the effect of bFGF on the cell cycle kinetics of two breast cancer cell lines (MCF-7 and MDA-MB-231) and an immortalized cell line (HBL-100). The methodology included use of microscopic image analysis with cell numeration, Feulgen staining, Proliferating Cell Nuclear Antigen/Ki-67 immunodetection and bromodeoxyuridine incorporation. We show that bFGF is mitogenic for MCF-7 cells via a mechanism of recruitment of G0 phase cells to reenter into the cell cycle and by decreasing the G1 phase length. No effect of bFGF on cell cycle parameters has been found with either highly metastatic MDA-MB-231 cells or immortalized HBL-100 cells. These results reveal differences in bFGF responsiveness of breast epithelial cells.

Breast Neoplasms↗

PubMed1992

Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor.

We have investigated the ability of glycosaminoglycans from embryonic chick brain (15 days old) to interact with basic fibroblast growth factor (bFGF). 35SO4 metabolically labeled glycosaminoglycans were purified and separated on DEAE-cellulose chromatography. Material which eluted between 0.20 and 0.35 M NaCl displaced the binding of [125I]bFGF to brain membrane. This activity was dose-dependent and on the basis to its heparinase sensitivity and chondroitinase insensitivity, has been attributed to heparan sulfate. CL-6B-Sepharose chromatography of this material revealed two glycosaminoglycans of molecular masses of about 15,000 and 65,000. Incubation with [125I]bFGF followed or not by heparinase and chondroitinase treatment of electrotransfert from SDS-PAGE revealed that both of these forms correspond to heparan sulfate chains and bind bFGF. In vitro, embryonic brain-derived heparan sulfate inhibited both bFGF induced [3H]thymidine incorporation in CCL39 cells and neurite outgrowth in PC12 cells. These results suggest that heparan sulfate play an important function in the control of the biological activity of bFGF during brain development.

Animals↗