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PubMed1995

CCAAT/enhancer-binding protein-alpha-dependent transactivation of CYP2C12 in rat hepatocytes.

Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP alpha protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP alpha expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP alpha response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP alpha. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP alpha and C/EBP beta in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP alpha. We conclude that C/EBP alpha is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP alpha, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP alpha or via enhanced DNA-binding activity of this transcription factor.

Animals↗
PubMed1991

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter.

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.

Animals↗
PubMed1997

Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung.

During mouse lung morphogenesis, the distal mesenchyme regulates the growth and branching of adjacent endoderm. We report here that fibroblast growth factor 10 (Fgf10) is expressed dynamically in the mesenchyme adjacent to the distal buds from the earliest stages of lung development. The temporal and spatial pattern of gene expression suggests that Fgf10 plays a role in directional outgrowth and possibly induction of epithelial buds, and that positive and negative regulators of Fgf10 are produced by the endoderm. In transgenic lungs overexpressing Shh in the endoderm, Fgf10 transcription is reduced, suggesting that high levels of SHH downregulate Fgf10. Addition of FGF10 to embryonic day 11.5 lung tissue (endoderm plus mesenchyme) in Matrigel or collagen gel culture elicits a cyst-like expansion of the endoderm after 24 hours. In Matrigel, but not collagen, this is followed by extensive budding after 48-60 hours. This response involves an increase in the rate of endodermal cell proliferation. The activity of FGF1, FGF7 and FGF10 was also tested directly on isolated endoderm in Matrigel culture. Under these conditions, FGF1 elicits immediate endodermal budding, while FGF7 and FGF10 initially induce expansion of the endoderm. However, within 24 hours, samples treated with FGF10 give rise to multiple buds, while FGF7-treated endoderm never progresses to bud formation, at all concentrations of factor tested. Although exogenous FGF1, FGF7 and FGF10 have overlapping activities in vitro, their in vivo expression patterns are quite distinct in relation to early branching events. We conclude that, during early lung development, localized sources of FGF10 in the mesoderm regulate endoderm proliferation and bud outgrowth.

Animals↗
PubMed2003

PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression.

It was previously reported that a midregion domain of parathyroid hormone-related protein (PTHrP), that is, [67-86]-amide, is able to restrain growth and promote matrigel penetration by the 8701-BC cell line, derived from a biopsy fragment of a primary ductal infiltrating carcinoma of the human breast, and that cell invasion in vitro is drastically impaired by inactivation of urokinase-plasminogen activator (uPa). In this study we started a more detailed investigation of the possible effects on gene expression arising from the interaction between PTHrP [67-86]-amide and 8701-BC breast cancer cells by a combination of conventional-, differential display-and semi-quantitative multiplex-polymerase chain reaction (PCR) assays. We present here the first evidence that the upregulation of some stress-related genes, most noticeably heat shock factor binding protein-1 (hsbp1) and heat shock protein 90 (hsp-90), is involved in the acquisition of an in vitro more invasive phenotype by cells treated with midregion PTHrP. This is conceivably accomplished by sequestering and inactivating heat shock factor-1 (hsf1) which is able to recognize Ets transcription-factor-binding sites present in some gene promoters, such as those of uPa and matrix metalloprotease-1 (MMP-1). In fact, our data show that incubation of PTHrP [67-86]-amide-treated cells with either antisense hsbp1-oligonucleotide or geldanamycin, an hsp90-inactivating antibiotic, results in downregulation of uPa and upregulation of MMP-1, and in a prominent inhibition of cell invasion in matrigel-containing Transwell chambers. Alternatively, incubation of untreated 8701-BC cells with quercetin, a flavonoid known to decrease the amount of free hsf1, is found to induce upregulation of uPa and downregulation of MMP-1, and an increase of matrigel invasion by cells, thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression and on cell invasive behaviour. These studies confirm a previous postulate that over-secretion of uPa, rather than of other extracellular proteases, is a primary condition for the increase of invasive activity triggered by PTHrP [67-86]-amide in vitro, and support a role for midregion forms of PTHrP in potentially affecting pathological mammary growth and differentiation. They also identify two new key protagonists in the complex scenario of breast tumor cell invasiveness in vitro, that is, hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for anti-breast cancer treatments.

Breast Neoplasms↗
PubMed1994

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion.

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

Anti-Bacterial Agents↗
PubMed1995

Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro.

Galectin-3 is a member of a closely related family of beta-galactoside-binding soluble proteins found in many vertebrate epithelial and myeloid cell types. The developmentally regulated presence of galectin-3 in tissues, for example kidney, and an affinity for many cell-surface and matrix glycoproteins indicate its importance in extracellular biological processes. Since a polarised expression and secretion of galectin-3 was observed in monolayer-cultured MDCK cells, an understanding of the secretion and distribution of this lectin in a three-dimensional in vitro model would help to uncover its role(s) in the interplay between cell-surface adhesion molecules and extracellular matrix components occurring during cell aggregation and polarisation in tissue formation. In this study, the cellular distribution and secretion of galectin-3 were examined in MDCK cells cultured within a gel matrix. MDCK cells were cultured within type I collagen or Matrigel to obtain multicellular cysts, and tubule formation was induced in collagen gels with hepatocyte growth factor. Immunofluorescent staining of these structures using antibodies against galectin-3 and other cell-surface domain markers was carried out either in situ or on cryosections and was visualised by confocal and conventional epifluorescence microscopy. Our results show that MDCK cells suspended in hydrated collagen gels or Matrigel exhibit differential and polarised galectin-3 expression on the baso-lateral surface domains of cells lining the cysts. The lectin is colocalised with laminin on the basal surface. In tubule-forming cysts, galectin-3 is excluded from the initial spikes and the progressing tips of the tubules although its basolateral expression on the cyst body remains. Galectin-3 added exogenously to cultures, as well as antibodies against laminin subunits and integrin beta 1 subunit, exerted an inhibitory effect on cyst enlargement of MDCK cells in 3-D Matrigel while galectin-3-specific antibodies could promote this process. The results suggest that galectin-3 exerts its effect on MDCK cells in a three-dimensional environment through modulation of both cell-cell and cell-substratum adhesions, and the interplay between these adhesions is important in the growth of multicellular aggregates and extensions occurring during normal kidney tubulogenesis.

Animals↗
PubMed1996

Role of laminin-1 and TGF-beta 3 in acinar differentiation of a human submandibular gland cell line (HSG).

Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P < or = 0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-beta 3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary gland-specific protein expression in vitro.

Apoptosis↗
PubMed1997

The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis.

In vivo, normal mammary epithelial cells utilize hemidesmosome attachment devices to adhere to stroma. However, analyses of a potential role for hemidesmosomes and their components in mammary epithelial tissue morphogenesis have never been attempted. MCF-10A cells are a spontaneously immortalized line derived from mammary epithelium and possess a number of characteristics of normal mammary epithelial cells including expression of hemidesmosomal associated proteins such as the two bullous pemphigoid antigens, alpha 6 beta 4 integrin and its ligand laminin-5. More importantly, MCF-10A cells readily assemble mature hemidesmosomes when plated onto uncoated substrates. When maintained on matrigel, like their normal breast epithelial cell counterparts, MCF-10A cells undergo a branching morphogenesis and assemble hemidesmosomes at sites of cell-matrigel interaction. Function blocking antibodies specific for human laminin-5 and the alpha subunits of its two known receptors (alpha 3 beta 1 and alpha 6 beta 4 integrin) not only inhibit hemidesmosome assembly by MCF-10A cells but also impede branching morphogenesis induced by matrigel. Our results imply that the hemidesmosome, in particular those subunits comprising its laminin-5/integrin 'backbone', play an important role in morphogenetic events. We discuss these results in light of recent evidence that hemidesmosomes are sites involved in signal transduction.

Antibodies↗
PubMed2000

Reconstituted basement membrane induces glandular-like morphogenesis but no difference in ACTH synthesis of anterior pituitary cells.

Basement membrane, a thin extracellular matrix, promotes tissue integrity and differentiated phenotype. This study was performed in order to investigate the effect of basement membrane components on adrenocorticotropin (ACTH) synthesis and to observe its relationship with cell morphology. Rat anterior pituitary cells were cultured on plastic culture plate coated with either Matrigel or poly-D-lysine. Phase-contrast microscopy and scanning electron microscopy showed that cells cultured on Matrigel appeared as a three-dimensional glandular-like cell aggregate, while those cultured on plastic showed a flat, confluent monolayer. Reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis revealed that ACTH synthesis in the Matrigel culture was not significantly different from that in the plastic culture. Our results suggest that the relationship between the morphological changes caused by cell-substrate interaction and pituitary hormone synthesis does not exist in all pituitary cell types and that other factors associated with cell-substrate interaction influence the hormone synthesis of some pituitary cells.

Adrenocorticotropic Hormone↗
PubMed1997

The effects of basic fibroblast growth factor, transforming growth factor-alpha, and transforming growth factor-beta on mesenchymal cells in the rabbit phallus: an in-vitro study.

Effects of bFGF, TGFalpha and TGFbeta on cultured mesenchymal cells of rabbit phalli were investigated. A remarkable cell proliferation reaching confluency was only obtained from the media containing bFGF by the first week of culture. By subsequent 4 weeks culture using such confluent cells in the media containing bFGF, TGFalpha or TGFbeta, respectively, cells cultured in the media containing bFGF showed an active transformation to endothelial precursor cells determined by positive immunoreactions for factor VIII-related antigen at 2 weeks. By electron microscopy, such vasoformative cells appeared in contact with each other, occasionally forming a solid cell cord. The use of Matrigel's basement membrane matrix (Matrigel) in each culture bed promoted the capillary ingrowth, implying that certain extracellular matrix (ECM) components included in Matrigel may accelerate the formation of neocapillaries. Enhancement in the production of ECM components and transformation to smooth muscle cells determined by positive immunoreactions for actin were also seen in cells cultured in the media containing TGFalpha and TGFbeta, respectively. The present results indicate that the above growth factors play crucial roles in the differentiation of the rabbit cavernous body.

Animals↗
PubMed2006

Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair: conversion of cell columns into fibrovascular bundles.

The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently become a topic of intense scrutiny. Here, we characterized the behavior of MC/Mph in cellular infiltrates, with emphasis on their spatial organization and localization in newly formed microvessels. To this end, we studied MC/Mph migration and assembly in basic fibroblast growth factor-supplemented Matrigel plugs placed in transgenic Tie2-beta-galactosidase mice for up to 4 weeks. In these plugs, along with Nile Red-positive adipocytes, we found MC/Mph distributed in cell cords, also containing various mature and progenitor tissue cells; and functional Tie2-positive or -negative microvessels embedded in bundles of fibrillar collagen surrounded by F4/80-positive MC/Mph. At earlier stages of infiltration, we found tubular destruction of the matrix (tunnels) and MC/Mph-lined capillary-like structures occasionally containing erythrocytes, indicating their propensity for endothelial trans-differentiation. We also analyzed in vitro the MCP-1-induced chemotactic migration of fluorescently labeled peritoneal MC/Mph incorporated in Matrigel-containing fluorescent protease substrates. Many of these MC/Mph produced MMP-12- and TIMP-1-dependent tunnels coupled with acquisition of a lumen. In conclusion, long-term implantation of Matrigel plugs qualifies as a novel experimental model of tissue regeneration, in which neovascularization intimately couples with fibrosis and organogenesis and in which cells of MC/Mph phenotype play a key structural role.

Animals↗
PubMed2006

Involvement of endothelial CD44 during in vivo angiogenesis.

CD44, a cell-surface receptor for hyaluronan, has been implicated in endothelial cell functions, but its role in the formation of blood vessels in vivo has not been established. In CD44-null mice, vascularization of Matrigel implants and tumor and wound angiogenesis were inhibited. Leukocyte accumulation during tumor growth and wound healing in wild-type and CD44-null mice were comparable, and reconstitution of CD44-null mice with wild-type bone marrow did not restore the wild-type phenotype, suggesting that impairments in angiogenesis in CD44-deficient mice are due to the loss of endothelial CD44. Although the cell proliferation, survival, and wound-induced migration of CD44-null endothelial cells were intact, these cells were impaired in their in vitro ability to form tubes. Nascent vessels in Matrigel implants from CD44-null mice demonstrated irregular luminal surfaces characterized by retracted cells and thinned endothelia. Further, an anti-CD44 antibody that disrupted in vitro tube formation induced hemorrhage around Matrigel implants, suggesting that antagonism of endothelial CD44 undermined the integrity of the endothelium of nascent vessels. These data establish a role for CD44 during in vivo angiogenesis and suggest that CD44 may contribute to the organization and/or stability of developing endothelial tubular networks.

Animals↗
PubMed2000

Morphology of luminal and glandular epithelial cells from pig endometrium grown on plastic or extracellular matrices.

Luminal (LE) and glandular epithelial (GE) cells from d-13 pregnant pigs were cultured on plastic, matrix secreted from endometrial stroma, and EHS matrix (Matrigel) in culture medium (RPMI-1640) supplemented with 20 and 10% fetal bovine serum, respectively. After culture for 7 and 14 d, GE and LE cells were prepared for transmission and GE cells for scanning electron microscopy. The two types of endometrial epithelial cells displayed different morphological characteristics when grown on different culture substrates. On plastic, the GE and LE cells formed flattened monolayers. However, stroma-secreted matrix directed the polarization of endometrial epithelia. The GE and LE cells reacted differently to thick Matrigel coatings; LE cells formed a colony after 7 d of culture and then proliferated further to form a colony with a cavity, but GE cells organized to form a colony with a shallow depression in the center at 7 d and developed duct-like structures after 14 d in vitro. Luminal epithelial cells grown on either diluted or thin-coated Matrigel and grown on stroma-secreted matrix formed a monolayer but no three-dimensional structures.

Animals↗
PubMed1991

Extracellular matrix mediated growth and differentiation in human pigment epithelial cell line 0041.

Efforts to grow differentiated pigment epithelial cells have led to a characterization of the growth kinetics of spontaneously established, continuously growing, human retinal pigment epithelial (PE) cell line 0041 on several biomatrices. These substrates were prepared from (a) placental and amniotic membrane, (b) commercially available basement membrane matrix (Matrigel), (c) dishes coated with extracellular matrix secreted by endothelial cells (ECM), (d) dishes coated with collagen IV and/or laminin, (e) dishes coated with collagen I and/or fibronectin. Our findings suggest that tissue culture plastic and dishes coated with collagen IV alone promote higher cell densities, while highest plating efficiency (24 hrs) was seen on tissue culture plastic and Matrigel. The highest degree of differentiation (epithelioid appearance, apical villi and junctional complexes) was seen in cells plated on dishes coated with collagen IV and extracellular matrix secreted by endothelial cells. Cells were epithelioid and polarized on those two substrates; they expressed fine finger-shaped villi and the highest degree of cell contact (in the form of junctions). Cells grown on Matrigel looked like fibroblasts and became deeply pigmented; however, the nature of the pigment remains to be determined. Collagen IV and ECM coated dishes, therefore, are most suitable for cultures of human PE cell line 0041 because they provide higher cell densities while retaining the differentiated state. This is the first report where an established pigmented epithelial cell line has been induced to become differentiated by use of extracellular matrices and extracellular matrix components.

Adolescent↗
PubMed2001

The role of vascular endothelial growth factor in the tissue specific in vivo growth of prostate cancer cells.

Despite the fact that cancer cells can be found in many vascular beds, continued growth of the metastatic tumor focus exhibits a significant degree of 'organ tropism', with only certain organs exhibiting the ravages of metastatic disease. Since a limiting factor to the growth of metastases beyond 2 mm in diameter, may be a lack of angiogenesis, we sought to determine whether tumor overexpression of vascular endothelial growth factor (VEGF), a potent angiogenic factor related to prostate cancer metastasis, is causally related to organ specific tumor growth in a prostate cancer xenograft model. LnCaP-C4-2 is a subline of the human prostate cancer cell line LnCaP which unlike its parent, has a predilection for growth in bone, a common site for human prostate cancer metastasis. LnCaP-C4-2, is tumorigenic when injected intrafemorally in mice but requires co-injection of stromal components (Matrigel) to be tumorigenic in the subcutaneous site. Because of this site-specific tumorigenicity profile and relatively low VEGF mRNA and protein expression, this line was transfected with a full length cDNA encoding the 165 isoform of VEGF. Cells either overexpressing or not expressing the transfected gene were selected for study in vivo and in vitro. Overexpression of VEGF did not seem to affect in vitro cell growth. Such overexpression did affect tumorigenicity and in vivo tumor growth rates when cells were inoculated in the subcutaneus site. Interestingly, the dependency of subcutaneous tumorigenicity on Matrigel co-inoculation was still observed in cells overexpressing VEGF. In contrast to the impact that VEGF overexpression has on subcutaneous tumorigenicity, no such effect was observed when cells were inoculated in orthotopic/prostate (primary) or intrafemoral (metastatic) sites. In view of the importance of tumor-stromal interactions in growth of xenografts, we sought to determine if the host strain is important to the observed tumorigenicity effects of VEGF overexpression. No differences in subcutaneous tumorigenicity as a function of either Matrigel use or VEGF expression levels were observed when SCID/bg and RAG/pfp mouse strains were compared. In conclusion, our data indicate that the biological impact of prostate tumor VEGF overexpression is organ/site specific, leading to the speculation that it may play a part in the observed organ tropism of metastatic spread. In addition, these results highlight the importance of the tumor microenvironment in determining the biological impact of transfected and overexpressed genes in the study of tumor biology.

Animals↗
PubMed1999

B1 integrin activation inhibits in vitro tube formation: effects on cell migration, vacuole coalescence and lumen formation.

Human endothelial cells (EC), when plated onto gels of extracellular matrix proteins such as Matrigel or collagen form capillary tubes in a process thought to mimic angiogenesis. We have shown previously that the extent of tube formation and the phenotype of the lumen are regulated by integrins (Gamble et al 1993) and lumen formation occurs through a process of vacuolization, coalescence and ultimate directional fusion of these vacuoles with the plasma membrane (Meyer 1997 et al). We now show here that activation of beta1 integrins on endothelial cells inhibits tube formation. On collagen gels, endothelial cells treated with 31 activating antibody 8A2 failed to migrate into the gel and tube formation was inhibited. Although several integrins mediate EC attachment to collagen alpha2beta1 is the chief determinant of EC behaviour since a blocking antibody to (alpha2beta1 reversed the effect of 8A2. On Matrigel tube formation was also inhibited by 8A2 treatment although cell alignment and sprout formation was still evident. Electron microscopy revealed the organisation of normal numbers of cells into solid sprouts and the formation of small intracellular vacuoles suggesting that initial stages of tube formation including cell migration were unaffected. However, beta1 integrin activation inhibited the coalescence of these small vacuoles into larger vacuoles, the recruitment of more cells into the sprout and the subsequent formation of mature lumen. The inhibition of capillary tube formation by beta1 activation was time dependent and long lasting. The critical time for activation of the beta1 integrin was the initial 1-2h after plating in order to inhibit tube formation although once activated, the beta1 mediated inhibition on Matrigel was still evident 4 days later. Our results suggest that beta1 integrins are critical in capillary tube formation in at least two phases. beta1 integrins are essential for migration of EC through collagen gels. Independently, beta1 integrins, although not involved in initial vacuole formation, are involved in the process of vacuole coalescence and subsequent lumen formation since beta1 integrin activation inhibits these processes.

Antibodies, Monoclonal↗
PubMed1997

Role of integrins and evidence for two distinct mechanisms mediating human colorectal carcinoma cell interaction with peritoneal mesothelial cells and extracellular matrix.

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Co115 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-beta 1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell-mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.

Cell Adhesion↗
PubMed1994

Basement membrane invasion of glioma cells mediated by integrin receptors.

Basement membrane invasion precedes meningeal dissemination and systemic metastasis of glioma cells. In order to investigate the invasive ability of glioblastomas and the functional role of extracellular matrix receptors, the authors performed in vitro invasion assays where the number of cells was determined from freshly resected tumors (primary cultures and fifth passages) and from cell lines (U-138 MG, U-373 MG, and GaMg) that had migrated through a filter coated with a reconstituted basement membrane (Matrigel). The involvement of integrin adhesion molecules was examined by preincubation of glioma cells with blocking antibodies to specific integrin chains. Cells from all of the glioblastomas had migrated through the Matrigel after 4 to 24 hours; the number of invasive cells was highest in the cell lines. Invasion of U-138 MG cells was reduced with antibodies to alpha 7, alpha v, beta 1, and beta 3 integrin chains and markedly increased by anti-alpha 5, while invasion of U-373 MG cells was reduced by antibodies to alpha 3, alpha v, beta 1, and beta 3 and increased by anti-alpha 6. It is concluded that: 1) glioma cells are able to penetrate Matrigel, indicating that the basement membrane is not a resistant barrier for infiltrating cells; and 2) basement membrane invasion is mediated by integrins in a complex manner. Some integrins promote while others inhibit basement membrane invasion. Furthermore, the integrins involved may differ between various glioma cells.

Antibodies↗