CD44 mediates human glioma cell adhesion and invasion in vitro.

Human gliomas are characterized by their invasion of normal brain structures irrespective of their grade of malignancy. Factors involved in the control of this invasive behavior are poorly documented. Human gliomas have also been found to express CD44 adhesion molecules. Expression of splice variants of CD44 has been correlated to metastasis in nonglial solid tumors. In this study, 8-microns porosity polycarbonate filters incorporated in modified Boyden chambers and coated with the extracellular matrix composite Matrigel were used to investigate the role of CD44 in invasion of eight human glioma cell lines in vitro. Invasion of Matrigel was found to be inhibited to different extents by a CD44 monoclonal antibody. Moreover, this invasion was highly inhibited in two cell lines and completely arrested in five other glioma cell lines by a CD44-specific antisense oligonucleotide which inhibited CD44 expression. In addition, adhesion of glioma cells to fibronectin, laminin, vitronectin, and collagen I was inhibited by the CD44 monoclonal antibody. These results strongly suggest that CD44 is involved in human glioma cell invasion in vitro, probably through its role in cell interactions with extracellular matrix proteins. Interference with glioma invasion, by targeting CD44 expression, may be envisaged in animal models.

Cooperation between the H-ras oncogene and a truncated derivative of the v-myb oncogene in transformation of hamster embryo fibroblasts.

The ras oncogenes alone fully transform established (immortalized) rodent fibroblasts in a few days, but generally transform early-passage fibroblasts only partially, unless their action is complemented by that of a nuclear, immortalizing, oncogene. Here we show that transfection of second-passage Syrian hamster embryo fibroblasts (HEFs) by the EJ-H-ras oncogene coupled to the neo gene, followed by selection with G418, gives rise to apparently normal, or only slightly transformed, clonal colonies, only a few of which become established. The study of two established clonal lines showed that they acquired only after some weeks, and stepwise, the main characteristics of full neoplastic transformation, i.e. anchorage independence, reduced requirement for serum growth factors and tumorigenicity. Later both clonal lines became increasingly tumorigenic and completely independent of exogenous growth and attachment factors, without increase in the expression of the H-ras oncogene. Transfection of one of the clones, early after its isolation, with a truncated derivative of the nuclear v-myb oncogene devoid of its transcriptional negative regulatory domain and able to partially transform chicken embryo fibroblasts [(myb(KXANM)] gave rise to more transformed cells, expressing both EJ-H-ras and myb(KXANM), which became tumorigenic earlier than the controls and remained more tumorigenic later on. With more efficient transfection techniques, numerous foci of fully transformed cells were subsequently obtained, in a few days, in cultures transfected sequentially with EJ-H-ras(neo) and myb(KXANM) and in cultures co-transfected with the two oncogenes. Highly tumorigenic, serum-independent and immortalized clones expressing both oncogenes were obtained from these cultures. Hence, the truncated myb(KXANM) oncogene accelerate the stepwise transformation of unestablished HEFs by the EJ-HH-ras oncogene and, together with this oncogene, fully transforms these same cells in a single step. The two oncogenes acting in cooperation also induce cell immortalization, but myb(KXANM), by itself, is not an immortalizing oncogene. No cooperation was observed between EJ-H-ras(neo) and the unaltered v-myb oncogene.

Transforming potential of truncated v-myb and stimulation of replication by gag-myb fusion products.

We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.

Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines.

The influence of an artificial basement membrane (BM), Matrigel, and four individual extracellular matrix proteins, fibronectin, laminin, collagen I and vitronectin, on cell proliferation, morphology and migration was assessed in four glioma cell lines. Matrigel and individual BM proteins differentially inhibited cell proliferation of all cell lines studied. In addition, Matrigel was found to induce extensive morphological changes in glioma cells. Polycarbonate filters, of 8-microns porosity in modified Boyden chambers, were used to assess the chemoattraction activity of Matrigel and the individual proteins on glioma cells. All these components were found to stimulate cell migration, albeit to different extents but laminin proved to be the most effective chemoattractant for glioma cells in vitro. These data suggest that basement membrane proteins may inhibit proliferation and stimulate migration in order to facilitate invasion.

The effect of exogenous gangliosides on matrix metalloproteinase secretion by human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are zinc-dependent peptidases and are amongst those enzymes responsible for extracellular matrix (ECM) degradation during tumour-cell migration. Gangliosides are a family of acidic membrane glycolipids thought to play a role during cell development, differentiation and oncogenic transformation. In this descriptive study, we investigated the effects of six exogenous gangliosides (GM1, GM3, GD1a, GD1b, GD3 and GT1b) on the secretion of MMP-2 (72 kDa gelatinase or gelatinase-A) and MMP-9 (92 kDa gelatinase or gelatinase-B). Cell-conditioned media from eight human glioma-derived cell-lines served as the source of MMPs and were investigated using SDS-PAGE zymography. Six of the cell lines showed upregulation of secretion of both enzymes by all six gangliosides. Of the remaining two cell lines, one showed inhibition of MMP secretion by all gangliosides and the other had a small but differential response to the range of gangliosides investigated. These results suggest that gangliosides may stimulate glioma cell invasiveness by promoting MMP expression.

Vascular endothelial growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells in vitro.

Vascular endothelial growth factor (VEGF) is an angiogenic factor which is known to be expressed in several malignancies including glioma. The effect of transforming growth factor-beta (TGF-beta) isoforms as well as gangliosides on VEGF production was investigated in human glioma cell lines. TGF-beta isoforms and gangliosides were found to differentially stimulate VEGF production by these cells. The ganglioside GD3 enhanced this release to the greatest extent and the stimulation was more marked in a glioblastoma cell line than in the two other anaplastic astrocytoma cell lines. These results suggest that both TGF-betas and gangliosides may act as indirect angiogenic factors by stimulating VEGF secretion.

Synergism between growth-factors in the control of glioma cell-proliferation, migration and invasion in-vitro.

Gliomas constitute more than 50% of primary brain tumours in man. Perhaps the most important hallmark of these tumours is their diffuse invasion of the normal brain structures. The biological factors involved in the control of both their proliferation and invasion are, however, not well known. We studied the expression of receptors for epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and transforming growth factor beta-1 (TGF-beta 1) in low grade astrocytoma (IPNT-H)-, grade III astrocytoma (IPSB-18)-, and glioblastoma (IPRM-5)- derived cell lines. The effects of EGF, bFGF, PDGF, and TGF-beta 1 on proliferation, migration, and invasion in vitro were also investigated. When tested individually, EGF, bFGF and PDGF, were found to differentially stimulate proliferation, motility and invasion of the cell lines examined. When combined, these three growth factors acted synergistically to stimulate these biological properties. In addition, TGF-beta 1 exhibited positive and negative effects on the mitogenic action of the other growth factors in IPNT-H cells but inhibited their activity in IPSB-18 and IPRM-5 cells. Moreover, TGF-beta 1 was found to modulate negatively and positively the migration and invasion promoting action of the other growth factors in IPNT-H and IPSB-18 cells, while it strongly potentiated this action in IPRM-5 cells. These results suggest that all the growth factors examined may play key roles in the control of the biological properties of human glioma cells in vitro. Together with our findings that TGF-beta 1 is overexpressed in human glioblastoma in vivo, these results also suggest that co-operation between growth factors and TGF-beta 1 may be of central importance in tumour progression of gliomas.

Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration.

Expression of seven serotonin or 5-hydroxytryptamine (5-HT) receptors (5-HT1D alpha, 5-HT1E, 5-HT2, 5-HT1A, 5-HT1C, 5-HT1D beta, and 5-HT6) was investigated in human normal fetal astrocytes and eight glioma cell lines by reverse transcription and polymerase chain reaction (RT-PCR). No expression of 5-HT1D beta and 5-HT6 was observed in any of the cell lines studied. The 5-HT1D alpha receptor was found to be expressed in two human glioma cell lines but not in normal astrocytes. In addition, only three glioma cell lines expressed the 5-HT1E receptor. The 5-HT1C receptor was expressed in six glioma cell lines but not in normal astrocytes while the 5-HT1A was found to be expressed in normal astrocytes from the left hemisphere and in six glioma cell lines but not in normal astrocytes from the cerebellum. Interestingly, the 5-HT2 receptor was expressed in all cells studied but very weakly in normal astrocytes. The effect of 5-HT on glioma cell proliferation, migration, and invasion was also investigated. Serotonin was found to positively modulate these three processes in vitro. These results suggest that 5-HT may play an important role in the control of the biological properties of human glioma cells.

Growth factors and gangliosides stimulate laminin production by human glioma cells in vitro.

Neoplastic cells from intrinsic, neuroectodermal tumours may migrate up to several millimeters away from the original tumour mass into normal nervous tissue. The biological mechanisms underlying this local invasive behaviour of gliomas are poorly understood. We have demonstrated recently that growth factors and cell surface gangliosides are positively involved in human glioma cell adhesion, migration and invasion in vitro. In order to study the mechanism of action of gangliosides and growth factors in this process, their role in the production of laminin, the major component of glioma vascular basal lamina, was investigated. Both growth factors and gangliosides stimulated laminin production in vitro suggesting that these factors increase laminin production in order to enable glioma cells to adhere and then migrate and invade in vivo.

Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro.

The involvement of cell surface gangliosides in glioma cell invasion in vitro was examined using the ganglioside-specific antibody A2B5 and Matrigel-coated 8-microns porosity polycarbonate filters. Invasion of six cell lines derived from glial tumours of different histological grades was found to be markedly inhibited by A2B5 (50-96% inhibition) in a dose-dependent manner. Furthermore, exogenous gangliosides were found to prevent cell invasion when they were incubated with cells during the invasion assay. These results suggest that cell surface gangliosides are involved in glioma cell invasion in vitro, probably because of their adhesion-promoting action to basement membrane components.

Human wild type p53 inhibits cell proliferation and elicits dramatic morphological changes in human glioma cell lines in vitro.

A human pilocytic astrocytoma-derived cell line, a grade III astrocytoma-derived cell line, and a glioblastoma-derived cell line were transfected with the human wild-type p53 gene, in order to demonstrate the possible suppressor role of this gene in low grade as well as in high grade human astrocytomas. p53 exhibited a strong growth suppressor effect on the three cell lines studied, irrespective of the grade of malignancy of the tumours from which they originate. Furthermore, the p53 gene elicited important morphological changes in these cell lines. p53-Transfected cells displayed a flat morphology, a large cell body, and a stellate shape with long processes, characteristic of differentiated astrocytes. In addition, the growth inhibitory effect of p53 was found not to be due to induction of apoptosis. These results indicate that p53 plays a tumour suppressor role in low grade and high grade human astrocytomas and raise the possibility of the involvement of p53 in glioma cell differentiation in vitro.

Vascular endothelial growth-factor production is stimulated in response to growth-factors in human glioma-cells.

Neovascularization is essential for tumour growth and is mediated by physiological substances produced by tumours. Vascular endothelial growth factor (VEGF) is one such potent angiogenic factor. Human gliomas, the most important class of intrinsic brain tumours, express VEGF both in vivo and in vitro. Factors involved in the control of VEGF production by glioma cells are not well known. In this study, we investigated the role of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet derived growth factors (PDGF) on VEGF production by four different glioma cell lines in vitro. With the exception of PDGF A/A and B/B in one cell line, all growth factors differentially stimulated VEGF production in all cell lines investigated. These data suggest that VEGF production in human glioma may be regulated by other growth factors which are also known to be expressed in such tumours.

Expression of vascular endothelial growth-factor in the cyst fluid of human cerebral gliomas.

Vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF) has been shown to play a key role in angiogenesis in several solid tumours including human brain neoplasms. Its expression has also been found to be correlated to malignancy in the major class of these tumours, gliomas. Moreover, it has been suggested that cyst fluids (CFs) associated with human gliomas may contain a permeability factor responsible for the formation of brain edema and disruption of the blood-brain barrier generally observed in these tumours. We demonstrate that VEGF is present in low and high grade gliomas of seven patients. We also show that VEGF concentration increases with increasing malignancy of the tumours. Although further cases should be investigated, these results suggest that the amount of CF-VEGF may be of value in the diagnosis of human gliomas.

Establishment and characterization of two paediatric brain tumour cell lines in vitro.

Well-characterised, established cell lines derived from paediatric brain tumours are rare. The paucity of medulloblastoma cell lines may reflect--at least in part--the differences in cell adhesion molecule expression between the cells of primitive neoplasms of childhood and their better-differentiated adult counterpart neoplasms. This frequently results in primitive neoplastic cells failing to adhere to the culture dish substrate and leads to suspension, rather than monolayer, growth. Furthermore, low grade astrocytic neoplasms of infancy and early adulthood often have low mitotic indices and, accordingly, fail to establish themselves in vitro. We report here the establishment and characterisation of a surface adherent medulloblastoma-derived cell line (IPNN-8) from a 13 year old boy and a pilocytic astrocytoma of the hypothalamus-derived cell line (IPNT-H) from a 6 month old child which have undergone 39 and 36 serial passages respectively. Growth curves and PCNA staining results show that these cell lines have similar population doubling times (24 hours and 27 hours respectively) and high proliferative indices. Immunocytochemical analysis shows that the IPNN-8 cell line is weakly positive for NFP and S-100 protein and negative for both NSE and CD44 but positive for A2B5 and GFAP, indicating glial differentiation; whereas, the IPNT-H cell line is positive for GFAP, glutamine synthetase, A2B5 and CD44 but only weakly positive for vimentin.

Control of human glioma cell growth, migration and invasion in vitro by transforming growth factor beta 1.

Factors involved in the control of the biological properties of gliomas, the major form of brain tumour in man, are poorly documented. We investigated the role of transforming growth factor beta 1 (TGF-beta 1) in the control of proliferation of human glioma cell lines as well as normal human fetal brain cells. The data presented show that TGF-beta 1 exerts a growth-inhibitory action on both human fetal brain cells and three cell lines derived from human glioma of different grades of malignancy. In addition, this growth-inhibitory effect is dose dependent and serum independent. Since TGF-beta 1 is known to be involved in the control of cell migration during ontogenesis and oncogenesis, we investigated the role of this factor in the motile and invasive behaviour that characterises human gliomas in vivo. TGF-beta 1 was found to elicit a strong stimulation of migration and invasiveness of glioma cells in vitro. In combination with recent data showing an inverse correlation between TGF-beta 1 expression in human gliomas and survival, these findings may suggest that TGF-beta 1 plays an important role in the malignant progression of gliomas in man. A study of the molecular mechanisms involved in the antiproliferative action and the invasion-promoting action of TGF-beta 1 may help to identify new targets in therapy for brain tumours. A combined antiproliferative and anti-invasive therapy could be envisaged.

Hyaluronic acid/CD44H interaction induces cell detachment and stimulates migration and invasion of human glioma cells in vitro.

The mechanisms underlying the invasive properties of gliomas, the major form of intrinsic brain tumours in humans, are poorly understood. We have reported that CD44 plays an important role in this behaviour in vitro. In the present work, we investigated the role of its ligand, hyaluronic acid (HA), in invasion in 8 human glioma cell lines. We found that HA mediates cell detachment via its interaction with its high affinity receptor, CD44H. Using 8 microns porosity polycarbonate filter transwells, we demonstrate that HA strongly stimulates migration in all 8 cell lines. This effect was found to be partially counteracted by a CD44H monoclonal antibody (MAb), suggesting the involvement of CD44H, as well as other HA receptors, in this process. Furthermore, incorporation of increasing concentrations of HA in Matrigel in an in vitro invasion assay resulted in a substantial increase in the invasive propensity of the glioma cell lines. Moreover, blocking experiments with the CD44H MAb suggest that CD44H and other receptors interact with HA to promote cell invasion in vitro. Our results show that HA induces cell detachment, stimulates migration and promotes invasion via its interaction with CD44H and other HA receptors in vitro. These effects could be prevented by use of specific HA receptor antibodies.

Adhesion of human glioma cell lines to fibronectin, laminin, vitronectin and collagen I is modulated by gangliosides in vitro.

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.

Transforming growth factor beta stimulates mitogenically mouse NIH3T3 fibroblasts and those cells transformed by the EJ-H ras oncogene.

TGF-beta 1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-beta in NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-beta 1 is as active, or more active than other mitogens (TGF-alpha, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S-) medium, is stimulated by only 10 picograms/ml of TGF-beta 1, and that of TR15 cells, in S- medium, by only 1 picogram/ml. In contrast, TGF-beta 1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S- medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-beta activated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of the c-myc proto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activated c-myc RNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.

Gangliosides modulate proliferation, migration, and invasiveness of human brain tumor cells in vitro.

Gliomas, the most common form of intrinsic brain tumor, are characterized by diffuse local invasion of the normal brain structures, irrespective of their histological grade of malignancy; a feature that is a major obstacle to successful therapy. They generally infiltrate the central nervous system (CNS) as individual tumor cells several centimeters beyond the macroscopic tumor margin and consequently often recur, after subtotal surgical resection. Factors involved in the control of both their proliferation and invasiveness are poorly documented. In this work, the role of gangliosides on proliferation of both human fetal human brain cells and five cell lines derived from human gliomas with different grades of malignancy was investigated. In addition, 8 microns-porosity polycarbonate filters were used to study cell motility. In addition, these filters were coated with the reconstituted extracellular matrix (ECM) composite, Matrigel, to assess invasiveness. The results presented show that gangliosides generally exert a proliferation inhibitory effect on fetal brain cells and glioma cell lines in vitro and play an important role in promoting glioma cell motility and invasiveness. The molecular mechanisms involved in the action of gangliosides may prove useful in identifying new targets for an anti-invasion therapy.

Overexpression of the 18A2/mts1 gene and down-regulation of the TIMP-2 gene in invasive human glioma cell lines in vitro.

Invasion of the reconstituted extracellular matrix composite, Matrigel, by eight human glioma-derived cell lines and human fetal brain cells was assessed in vitro using 8 microns polycarbonate filters in a modified Boyden migration chamber. With the exception of one low grade glioma derived cell line, all lines studied proved to be invasive while normal fetal brain cells failed to invade. This invasive potential was independent of the histological grade of the tumour from which the cell lines originated. In addition, the expression of the metastasis-associated gene 18A2/mts1 as well as the tissue inhibitor of metalloproteinases-2 (TIMP-2) was analysed in each of the glioma-derived cell lines. The 18A2/mts1 was expressed in all the cells studied with the exception of fetal brain cells and the low grade non-invasive glioma derived IPRK-7 cell line. The 18A2/mts1 related genes coding for the S100 subfamily of calcium binding proteins were found to be differentially and overexpressed in invasive cell lines. TIMP-2 was expressed only in non-invasive cell lines. These results suggest that the 18A2/mts1 and TIMP-2 genes could play an important role in the invasive behaviour of human glioma cells in vitro.