Reduced tumorigenicity of rat glioma cells in the brain when mediated by hygromycin phosphotransferase.

OBJECT: A variant of C6 glioma cells, C6R-G/H cells express hygromycin phosphotransferase (HPT) and appear to have reduced tumorigenicity in the embryonic brain. The goal of this study was to investigate their reduced capacity to generate tumors in the adult rat brain. METHODS: Cell lines were implanted into rat brains and tumorigenesis was evaluated. After 3 weeks, all rats with C6 cells showed signs of neurological disease, whereas rats with C6R-G/H cells did not and were either killed then or allowed to survive until later. Histological studies were performed to analyze tumor size, malignancy, angiogenesis, and cell proliferation. Cells isolated from rat brain tumors were analyzed for mutation to HPT by testing their sensitivity to hygromycin. CONCLUSIONS: The results indicate that HPT suppresses tumor formation. Three weeks after implantation, only 44% of animals implanted with C6R-G/H cells developed tumors, whereas all animals that received C6 glioma cells developed high-grade gliomas. The C6R-G/H cells filled a 20-fold smaller maximal cross-sectional area than the C6 cells, and exhibited less malignant characteristics, including reduced angiogenesis, mitosis, and cell proliferation. Similar results were obtained in the brain of nude rats, indicating that the immune system did not play a significant role in suppressing tumor growth. The combination of green fluorescent protein (GFP) and HPT was more effective in suppressing tumorigenesis than either plasmid by itself, indicating that the GFP may protect against inactivation of the HPT. Interestingly. hygromycin resistance was lost in tumor cells that were recovered from a group of animals in which C6R-G/H cells formed tumors, confirming the correlation of HPT with reduced tumorigenicity.

Radial glial cell line C6-R integrates preferentially in adult white matter and facilitates migration of coimplanted neurons in vivo.

C6-R is a cell line derived from C6 glioma cells that exhibits key properties of radial glia including the ability to support neuronal migration in culture. To explore its potential use in promoting neuronal migration in vivo, we analyzed the behavior of C6-R cells in the intact and injured adult rat CNS. At 6-11 days postimplantation at the splenium of the corpus callosum, green fluorescent protein-labeled C6-R cells were observed primarily in either the corpus callosum or the hippocampus in the brain, and in the spinal cord they migrated more extensively in the white matter than in the grey matter. To determine whether C6-R cells retain their ability to promote neuronal migration in vivo, they were coinjected with labeled neurons into adult brain. When rat embryonic neurons were coimplanted with C6-R cells, the neurons and C6-R cells comigrated through a much larger volume than neurons alone or neurons coimplanted with fibroblasts. In brains preinjured with ibotenic acid, C6-R cells as well as coimplanted neurons distributed widely within the lesion site and migrated into adjacent brain tissue, while transplants with neurons alone were restricted primarily to the lesion site. The results suggest that radial glial cell lines can serve as a scaffold for neuronal migration that may facilitate development of experimental models for neural transplantation and regeneration.

Molecular events implicated in brain tumor angiogenesis and invasion.

We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis.

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng-CAM.

Mammalian L1 and avian Ng-CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng-CAM. Our results indicate that Ig domains 1-4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng-CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1-4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1-6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo.

Thalidomide and a thalidomide analogue inhibit endothelial cell proliferation in vitro.

Angiogenesis is a crucial process in inflammatory reactions as well as in tumor implantation and growth. Tumors with high rates of invasion and recurrence such as gliomas, are specially dependent on neovascularization. This suggests that inhibition of angiogenesis might reduce the growth of these tumors. Thalidomide has been previously shown to inhibit angiogenesis induced by basic fibroblast growth factor in vivo, using the rabbit corneal micropocket assay. Therefore, the effect of thalidomide and a thalidomide analogue (cc-1069) on the proliferation in vitro of endothelial and glioma cells was tested. We observed a decrease in endothelial cell proliferation in cultures treated with thalidomide or the thalidomide analogue cc-1069. The analogue inhibited endothelial cell proliferation more efficiently than thalidomide. The inhibition occurred in association with a marked decrease in the activity of the nuclear factor SP1 and a moderate inhibition of NF-kappaB activation in nuclear extracts of endothelial cells. The drugs did not impair cell viability. There was no effect of thalidomide or the thalidomide analogue on the proliferation of the glioma cell line (U251) in vitro.

In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis.

Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for alpha-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.

Generation of a radial-like glial cell line.

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase beta, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (approximately 60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (approximately 30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration.

Tenascin-C expression in the cyst wall and fluid of human brain tumors correlates with angiogenesis.

OBJECTIVE: Tenascin-C (TN) is an extracellular matrix glycoprotein with a characteristic six-armed structure. The aim of this study was to determine whether the concentration of TN in the cyst fluid of brain tumors can be used as a marker for angiogenesis and glioma grade. METHODS: We investigated the expression of TN in the cyst wall and cyst fluid of human brain tumors by immunohistochemistry, immunoprecipitation, and immunoblotting. The tumors included 12 astrocytomas (5 glioblastoma multiforme tumors, 1 anaplastic astrocytoma, 1 low-grade astrocytoma, 4 juvenile pilocytic astrocytomas, and 1 mixed glioma), 2 dysembryoplastic neuroepithelial tumors, 3 craniopharyngiomas, 2 ependymomas, 2 metastatic carcinomas, 3 arachnoid cysts, 1 glial ependymal cyst, and 1 inflammatory cyst. RESULTS: We detected no expression of TN in the cyst fluids of the ependymomas, craniopharyngiomas, and nonpilocytic low-grade astrocytoma. By contrast, TN was detected in the cyst fluids of all the other tumors. Results of quantitative immunoblotting using a PhosphorImager unit (Molecular Dynamics, Sunnyvale, CA) revealed that, on average, a 5-fold higher signal was observed in the glioblastoma multiforme tumors as compared with the anaplastic astrocytoma, and a 10-fold higher signal as compared with the mixed glioma, juvenile pilocytic astrocytomas, and dysembryoplastic neuroepithelial tumors. Results of TN immunohistochemistry in the astrocytomas correlated with glioma grade, with stronger staining of the hyperplastic vessels and tumor cells being observed in higher grade gliomas. No TN immunoreactivity was detected in the walls of the ependymomas, arachnoid cysts, and glial ependymal cyst that lack hyperplastic vessels, and minimal TN immunoreactivity was observed in the perivascular gliotic rim of the craniopharyngiomas. No TN was detected in the cyst fluid of these cystic processes. CONCLUSION: The presence of TN in and around the hyperplastic vessels and tumor cells present in the cyst walls of astrocytomas and its deposition in the intratumoral cyst fluid in which angiogenic factors have been detected further suggests a role for TN as an angiogenic modulator. These preliminary results suggest that immunodetection of TN in the tumor cyst fluid may indicate tumor type and grade.

Migration of brain tumor cells on extracellular matrix proteins in vitro correlates with tumor type and grade and involves alphaV and beta1 integrins.

An important contributor to the malignancy of brain tumors is their ability to infiltrate the brain. Extracellular matrix molecules and cell adhesion molecules on cell surfaces play key roles in cell migration. In the present study, we used reaggregates of dissociated cells from freshly excised human brain tumors to analyze the migration of cells from human brain tumors of different types and grades on many different adhesion proteins adsorbed to glass substrates. Proteins were chosen based on their presence in normal or neoplastic nervous tissue, and included the extra-cellular matrix molecules fibronectin, collagens, fibrinogen, laminin, tenascin-C, thrombospondin, and the neuron-glia cell adhesion molecule, Ng-CAM. Cells from astrocytomas (n = 24) migrated on a variety of substrates, in contrast to cells from primitive neuroectodermal tumors cells (n=6), which only migrated well on laminin, fibronectin, or type IV collagen but not on the other substrates. Typically, migrating cells from astrocytomas of all grades had long, slender processes, were usually bipolar, and their cell bodies did not spread well on any substrate. Although there was variability in the migration of cells from astrocytomas of the same grade, cells from high-grade astrocytomas tended to migrate more extensively (42.3 +/- 4.7 micrometers/16 h: n = 16) than cells from lower grade astrocytomas (28.9 +/- 3.9 micrometers/16 h; P = 0.07; n = 8); the most striking differences were observed for collagen substrates, on which cells from lower grade astrocytomas migrated at very low levels (7.6 +/- 2 .6 micrometers/16 h) and cells from high-grade astrocytomas at higher levels (24.4 +/- 5.2 micrometers;P = 0.01). In contrast to primary cells from glioblastomas (n = 13), glioblastoma cell lines (n = 10) consistently spread on various substrates and migrated at high levels (69.5 +/- 7.6 versus 46.4 +/-5.7 micrometers/16 h; P = 0.03), in particular, on collagens (108.4 +/- 20.2 versus 28.0 +/- 6.1 micrometers/16 h; P= 0.001). Specific monoclonal antibodies to alphaV and beta1 integrin monomers completely inhibited the migration of astrocytoma cells on most substrates, suggesting that alphaV and beta1 integrins play a crucial role in brain tumor infiltration. These studies also suggest that although a large number of extracellular matrix molecules may promote tumor cell migration, disrupting the function of only a few tumor cell receptors may be critical for tumor infiltration in the brain.

Expression of polypeptide variants of receptor-type protein tyrosine phosphatase beta: the secreted form, phosphacan, increases dramatically during embryonic development and modulates glial cell behavior in vitro.

Glial cells express three splicing variants of a receptor-type protein tyrosine phosphatase called RPTP beta. Two are receptor forms that differ in a large extracellular domain. The third is a secreted proteoglycan called phosphacan that lacks the cytoplasmic phosphatase domains. We have now identified, by immunoblotting, proteins corresponding to these three forms of RPTP beta in rat C6 glioma cells and brain. The short receptor form is much more prevalent than the full-length receptor in C6 glioma cells. Phosphacan is much more abundant than either of the receptor forms in rat brain, and its expression increases progressively during embryonic development, while the receptor forms show only moderate changes. In contrast to the long form and phosphacan that were detected as proteoglycans, the short receptor form, lacking the large alternatively spliced domain, was not detected as a chondroitin sulfate proteoglycan. We recently showed that phosphacan binds to the neuron-glia cell adhesion molecule, Ng-CAM, and we now report that glia expressing RPTP beta adhere and extend processes on substrates coated with Ng-CAM. After one day in culture, however, the glia retract their processes and often lift off the substrate. Conditioned medium from glial cells, which contains large amounts of phosphacan, inhibits glial adhesion to Ng-CAM, and depletion of phosphacan from the conditioned medium by immunoadsorption reduces the inhibitory activity. The results show that phosphacan increases dramatically during development, and indicate that secreted forms of RPTP beta can modulate glial cell adhesion and behavior.

Functions of brain chondroitin sulfate proteoglycans during developments: interactions with adhesion molecules.

Chondroitin sulfate proteoglycans (CSPGs), including neurocan and phosphocan, are believed to be major components of brain extracellular matrix that interact with other matrix proteins and cell surface receptors. In addition, several brain CSPGs such as receptor protein tyrosine phosphatase beta are expressed as cell surface receptors that interact with proteins in the extracellular matrix and with receptors on neural cells. Recent in vitro studies demonstrate that, although the brain CSPGs neurocan and phosphocan can promote transient adhesion of neuronal cells, they inhibit stable cell adhesion and neurite growth promoted by the cell adhesion molecule Ng-CAM/L1. Neurocan and phosphocan bind with high affinity to Ng-CAM/L1 and N-CAM which may be their major receptors on neurons. These CSPGs also bind to other adhesion molecules, such as tenascin-C, and can differentially modulate adhesion of glia of tenascin-C. Both the glycosaminoglycan and the core glycoproteins contribute to the function of the brain CSPGs. When expressed in regions containing low levels of adhesion molecules, various CSPGs including phosphocan, neurocan, versican, aggrecan, and NG2 proteoglycan may act as barriers to cell migration and axonal growth. In regions containing high levels of adhesion proteins, brain CSPGs may still act to maintain certain boundaries while allowing selective axonal extension to proceed. There are numerous regions of overlap in the expression patterns of CSPGs and adhesion molecules in vivo, and the relative levels of these molecules as well as the organization of the extracellular matrix may be important factors that regulate the rate of axonal growth locally. Differential expression of CSPGs may be important for modulating cell adhesion as well as axonal growth and guidance during neural development, and continued expression may prevent these processes in the normal nature nervous system as well as following brain injury.

Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro.

The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis.

Tenascin expression in astrocytomas correlates with angiogenesis.

We investigated the expression and distribution of the extracellular matrix protein tenascin (TN) in 59 astrocytomas and 11 samples of normal brain by Western blot analysis and immunohistochemistry using antibodies against human TN. The tumors included 14 juvenile pilocytic astrocytomas (grade 1), 13 low grade fibrillary astrocytomas (grade II), 8 anaplastic astrocytomas (grade III), and 24 glioblastomas multiforme (grade IV). Proliferation indices were calculated by computer-based image analysis after immunostaining with the MIB-1 antibody against the Ki-67 proliferation-associated antigen. Western blot analysis for TN on fresh frozen tumor tissue from 23 of the 59 astrocytomas indicated up to 4-fold higher TN expression in glioblastomas multiforme than in nontumorous control tissues. Enhanced intercellular expression of TN was observed by immunohistochemistry in glioblastomas multiforme. More-over, TN immunostaining was consistently greater within and around the walls of hyperplastic blood vessels than nonhyperplastic vessels of both high grade tumors and juvenile pilocytic astrocytomas. Juvenile pilocytic astrocytomas with increased TN expression by Western blot analysis had vascular hyperplasia by light microscopy. Proliferation indices moderately correlated with tumor grade. Enhanced immunohistochemical expression of TN was associated with higher tumor grade with higher proliferation indices. The strong association of TN and vascular hyperplasia, regardless of tumor grade, suggests that TN may play a crucial role in angiogenesis.

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules.

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth.

We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

Evidence for the binding of Ng-CAM to laminin.

Ng-CAM is a cell adhesion molecule mediating neuron-glia and neuron-neuron adhesion via different binding mechanisms. While its binding can be homophilic as demonstrated by the self-aggregation of Ng-CAM coated beads (Covaspheres), Ng-CAM has also been shown to bind to glia by a heterophilic mechanism. In the present study, we found that the extent of Ng-CAM Covasphere aggregation was strongly diminished in the presence of the extracellular matrix glycoprotein laminin. When proteolytic fragments of laminin were tested, the P1' fragment (obtained from the short arms by pepsin treatment) was found to inhibit aggregation of Ng-CAM-Covaspheres while the elastase fragments E3 and E8 (from the long arm) were ineffective. To provide other means of analyzing interactions between laminin and Ng-CAM, the two proteins were covalently linked to differently fluorescing Covaspheres and tested for coaggregation. Laminin-Covaspheres coaggregated with Ng-CAM-Covaspheres, and this binding was inhibited both by anti-Ng-CAM and by anti-laminin antibodies. Covaspheres coated with other proteins including BSA and fibronectin did not coaggregate with Ng-CAM-Covaspheres. Moreover, using a solid phase binding assay, we found that 125I-labeled Ng-CAM bound to laminin and to Ng-CAM but not to fibronectin. The results suggest that regions in the short arms of laminin can bind to Ng-CAM. To test whether Ng-CAM present on neurons could be involved in binding to laminin, adhesion of neurons to substrates coated with various proteins was tested in the presence of specific antibodies. Anti-Ng-CAM Fab' fragments inhibited neuronal binding to laminin but not binding to fibronectin. The combined results open the possibility that Ng-CAM on the surface of neurons may mediate binding to laminin in vivo, and that interactions with laminin can modulate homophilic Ng-CAM binding.

Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development.

Evidence is accumulating that molecules involved in cell-cell and cell-substratum interactions are important in the establishment and maintenance of borders between cell groups during development. In this report, we review evidence supporting this conclusion, particularly in regard to the role of adhesion molecules in the formation of cell collectives and in the modulation of cell and neurite movements.

Differential effects of the cytoplasmic domains of cell adhesion molecules on cell aggregation and sorting-out.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that play important roles in morphogenesis and histogenesis, particularly in defining discrete borders between cell populations. Previous studies have suggested that the cytoplasmic domains of CAMs play a significant role in their adhesion properties. These domains may also be involved in regulating other cellular interactions, such as those involved in the sorting-out of cells to form tissues. In the present studies, we have compared the effects of replacing the cytoplasmic domain of one CAM with that of another CAM of different homophilic binding specificity on cell adhesion and cell sorting-out. The molecules studied were liver CAM (L-CAM) and the neural CAM (N-CAM) sd polypeptide. One cDNA was constructed that encodes a chimeric molecule composed of the extracellular domain of L-CAM and the cytoplasmic plus transmembrane domains of the sd polypeptide of chicken N-CAM (called L/N-CAM). Another was constructed encoding a truncated L-CAM missing the last 50 residues of the cytoplasmic domain. Permanently transfected lines of mouse L cells were obtained expressing the truncated L-CAM ("L-L-50 cells") or the chimeric L/N-CAM ("L-L/N cells") and were compared with cells expressing intact L-CAM ("L-L cells"). Immunoblotting and ELISA analyses demonstrated that these various cell lines expressed similar amounts of CAMs at the cell surface. Aggregation of L-L and L-L/N cells occurred at similar rates in short-term aggregation assays and was inhibited by antibodies to the extracellular L-CAM binding domain. In contrast, L-L-50 cells did not aggregate. Incubation of transfected cells with cytochalasin D, which disrupts microfilaments, markedly inhibited aggregation of L-L cells but had no effect on L-L/N cell aggregation. Mixed L-L and L-L/N cells co-aggregated in short-term assays; in the longer-term sorting-out assays, however, they behaved differently: L-L cells sorted out from both L-L/N and untransfected cells, whereas L-L/N cells did not sort out from untransfected cells. These studies not only suggest that interactions of cytoplasmic domains of different CAMs with the cytoskeleton can modulate cell adhesion but also suggest that specific interactions with certain cytoskeletal components are required for events such as cell sorting and cell patterning.

cDNAs of cell adhesion molecules of different specificity induce changes in cell shape and border formation in cultured S180 cells.

The liver cell adhesion molecule (L-CAM) and N-cadherin or adherens junction-specific CAM (A-CAM) are structurally related cell surface glycoproteins that mediate calcium-dependent adhesion in different tissues. We have isolated and characterized a full-length cDNA clone for chicken N-cadherin and used this clone to transfect S180 mouse sarcoma cells that do not normally express N-cadherin. The transfected cells (S180cadN cells) expressed N-cadherin on their surfaces and resembled S180 cells transfected with L-CAM (S180L cells) in that at confluence they formed an epithelioid sheet and displayed a large increase in the number of adherens and gap junctions. In addition, N-cadherin in S180cadN cells, like L-CAM in S180L cells, accumulated at cellular boundaries where it was colocalized with cortical actin. In S180L cells and S180cadN cells, L-CAM and N-cadherin were seen at sites of adherens junctions but were not restricted to these areas. Adhesion mediated by either CAM was inhibited by treatment with cytochalasin D that disrupted the actin network of the transfected cells. Despite their known structural similarities, there was no evidence of interaction between L-CAM and N-cadherin. Doubly transfected cells (S180L/cadN) also formed epithelioid sheets. In these cells, both N-cadherin and L-CAM colocalized at areas of cell contact and the presence of antibodies to both CAMs was required to disrupt the sheets of cells. Studies using divalent antibodies to localize each CAM at the cell surface or to perturb their distributions indicated that in the same cell there were no interactions between L-CAM and N-cadherin molecules. These data suggest that the Ca(++)-dependent CAMs are likely to play a critical role in the maintenance of epithelial structures and support a model for the segregation of CAM mediated binding. They also provide further support for the so-called precedence hypothesis that proposes that expression and homophilic binding of CAMs are necessary for formation of junctional structures in epithelia.