Meningioma Cultures Research Articles

Autocrine growth stimulation of human meningioma cells by platelet-derived growth factor.

The authors have previously shown that meningioma-derived conditioned medium profoundly stimulates the in vitro proliferation of meningioma cells. In this paper, self mitogenic agents found in the conditioned medium-autocrine growth-stimulatory factors actually secreted by human meningioma cells-are characterized as proteins related to the B chain of platelet-derived growth factor (PDGF) and possibly to the A chain of PDGF as well. The addition to conditioned medium of a neutralizing antibody against PDGF-BB caused a significant inhibition of the conditioned medium-stimulated DNA synthesis in all three meningioma cultures studied. A similar neutralizing effect was observed with an anti-PDGF-AA antibody in one meningioma culture studied. Gel filtration chromatography of concentrated conditioned medium from two different meningiomas using a Sephadex G-100 column revealed similar profiles from both conditioned media with a major peak of mitogenic activity against meningioma cells at a molecular weight (M(r)) of approximately 32 to 36 kD, accompanied by a minor peak at approximately 22 kD. The major peak mitogenic activity was significantly reduced by addition of an anti-PDGF-BB antibody. Western blot analysis of protein extracts from five meningioma specimens was performed using a monoclonal antibody against the B chain of PGDF, and a major band of PDGF-B immunoreactivity was detected at an M(r) of approximately 19 kD in all five meningiomas under both reducing and nonreducing conditions. Exogenous human and porcine PDGFs both exhibited a significant dose-dependent stimulation of DNA synthesis in two of three and three of five meningioma cultures examined, respectively. Although not all meningiomas investigated proved to share the biological activity associated with PDGF and these results may be preliminary, it seems that the autocrine growth-stimulatory loop established by PDGF-B-related molecules plays an important functional role in meningioma cell proliferation.

RT-PCR DETECTION OF CYTOKINE TRANSCRIPTS IN A SERIES OF CULTURED HUMAN MENINGIOMAS

The expression of cytokine transcripts has been investigated in a series of cultured human meningiomas using reverse transcriptase linked polymerase chain reaction (RT-PCR), which allowed simultaneous analysis of a range of cytokines. The main histological subgroups of meningioma were investigated; these included transitional, fibroblastic, and syncytial as well as atypical meningiomas. Meningiomas from each of the different histological subgroups were subjected to a standard tissue culture regime. Total RNA was extracted from representative cultures and reverse-transcribed to yield cDNA. PCR was performed using oligonucleotide primers designed to detect interleukin (IL)-1 alpha/beta to IL-8, transforming growth factor (TGF)beta 1-3, tumour necrosis factor (TNF)alpha/beta, and interferon (IFN)gamma. Transcripts for IL-3, IL-6, IL-8, and TGF beta 3 were detected in all cultures. Transcripts for the three isomers of TGF beta were expressed in the transitional and fibroblastic meningioma cells. TGF beta 2 and TGF beta 3 transcripts were expressed in the syncytial and TGF beta 1 and TGF beta 3 in the atypical meningioma cells. IL-1 beta transcripts were expressed in fibroblastic and atypical cultures and TNF beta transcripts were expressed in syncytial and transitional cultures only. Transcripts for IL-1 alpha, IL-2, IL-4, IL-5, IL-7, TNF alpha, or IFN gamma were not detected in any of the meningioma cultures. This investigation using cells cultured from a small number of tumours from each of the classic histological subtypes suggests that there is a distinct pattern of cytokine mRNA expression linked with histological classification.

Inhibition of In Vitro Meningioma Proliferation after Growth Factor Stimulation by Calcium Channel Antagonists

We have previously reported that calcium channel antagonists can block both the growth of meningiomas in culture and the potent growth stimulation of meningioma cells by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study further defines the nature of this growth inhibition. Primary meningioma cultures were established, and cells were characterized. Fibroblast growth factor or insulin-like growth factor-I growth stimulation in the presence of calcium channel antagonists was examined. In addition, the effects of ethylene glycol-bis-(aminoethylether) N,N,N',N"-tetraacetic acid and Bay K 8644, a calcium channel agonist, on the growth factors were analyzed. Growth factor receptor immunohistochemistry was performed on the original tumors and the in vitro meningioma cells. Twelve of 17 (71%) meningiomas in this study were positive for the EGF receptor, and 14 of 17 (82%) were positive for the PDGF receptor. Five of six (83%) of the culture cells were positive for the EGF receptor, and four of five (80%) were positive for the PDGF receptor. Intracellular calcium changes were quantified using the intracellular calcium-chelating, fluorescent dye, Fura-2. The growth stimulation of fibroblast growth factor and insulin-like growth factor-I on meningioma cells in culture was decreased in a dose-dependent manner by calcium channel antagonists. The growth stimulation of fibroblast growth factor and insulin-like growth factor-I was not affected by a reduction of extracellular calcium, whereas the growth stimulation of EGF and PDGF was. Interestingly, intracellular calcium was not increased after exposure to growth factors but was increased after serum stimulation. This increase could be blocked by preincubation with verapamil. Calcium channel antagonists can inhibit proliferation of meningioma cells in culture after stimulation with a number of growth factors. These drugs might disrupt intracellular calcium homeostasis or interfere with key elements of the growth factor signal transduction pathways. These mechanisms as well as the potential clinical relevance of these findings are discussed.

Control of meningioma cell growth by platelet-derived growth factor (PDGF)

We have examined the possible involvement of PDGF and PDGF receptors in the growth control of five meningiomas, analyzing the biopsy specimens and the primary cultures derived from the same tumors. Light and electron microscopy demonstrated that MAbs against PDGF β-receptors immunodecorate meningioma cells in vivo and in vitro, while those against α-receptors gave negative results. The effects of PDGF isoforms AA, AB, BB and of PDGF neutralizing antibodies on meningioma cultures were examined using [ 3H]thymidine incorporation analysis. Only with PDGF-AB and -BB a mitogenic effect was observed, while PDGF-neutralizing antibodies produced a reduction of [ 3H]thymidine incorporation. The production of PDGF-like growth factors by meningioma cells was tested analyzing the effects of meningioma culture-conditioned media on the growth of Swiss 3T3 cells. In all cases meningioma conditioned media stimulated the in vitro growth of 3T3 fibroblasts and this stimulatory effect was strongly reduced by PDGF-neutralizing antibodies. Furthermore, Northern blot analysis demonstrated expression of c-sis/PDGF-B and PDGF β-receptors mRNA in all meningioma biopsies and in all the derived cultures. Our results provide strong evidence that PDGF-B chain and PDGF β-receptors are involved in growth control mechanisms of human meningiomas through autocrine and/or paracrine mechanisms.

Involvement of protein kinase C in growth regulation of human meningioma cells.

In order to investigate the possible role of protein kinase C (PKC)-mediated signal pathways in growth regulation of meningiomas, we examined the effect of two PKC-activating phorbol esters, 12-O-tetradecanoyl-13-phorbol acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu), and PKC inhibitor, staurosporine, on cell proliferation using low-passage human meningioma cells in culture. TPA (0.1 to 100 ng/ml) caused a dose-dependent stimulation of cell proliferation in six of eight meningioma cultures. At optimal concentrations of TPA, the cell growth ranged from 113% to 251% versus control. In contrast, PDBu (0.1 to 100 ng/ml) caused a significant inhibition of cell proliferation in three of five meningioma cultures. At optimal concentrations of PDBu, the cell growth ranged from 52% to 79% of control. Staurosporine exhibited a stimulation of cell proliferation (135% to 178%) in three of four meningioma cultures studied at a concentration of 10(-10) to 10(-9)M, although a tendency of growth inhibition was observed at a lower concentration. A time course of DNA synthesis in response to TPA, assessed by [3H] thymidine incorporation studies, revealed a time- and dose-dependent stimulation and/or inhibition which further depended on the serum concentration of the growth medium used. The overall results indicate that PKC-mediated signal pathways are closely involved in growth regulation of human meningioma cells. The results further suggest that the signalling processes consist of complex mechanisms which await to be elucidated.

Secretion of interleukin-6 by human meningioma cells: possible autocrine inhibitory regulation of neoplastic cell growth.

Using cell culture techniques, the authors have previously shown that human meningioma cells secrete an autocrine growth stimulator related to platelet-derived growth factor. Here, they further demonstrate potential autocrine inhibitory regulation of meningioma cell growth by interleukin (IL)-6. Constitutive IL-6 production was detected in all meningiomas studied, in the form of protein as well as IL-6-specific messenger ribonucleic acid. The IL-6 immunoreactivity in conditioned medium from three different meningioma cultures eluted from a Sephadex G-100 column was evidenced by a single peak corresponding to a molecular weight of about 32 kD. Interleukin-6 secretion was remarkably stimulated by tumor necrosis factor-alpha, IL-1 beta, and IL-4, and was also influenced by a combination of epidermal growth factor and bromocriptine. Recombinant IL-6 exhibited a significant dose-dependent inhibitory effect on meningioma cell proliferation. The maximum effect was observed at concentrations of 10 to 100 pg/ml, with the decrease in thymidine incorporation ranging from 21% to 35% versus control. Addition of an anti-IL-6 antibody enhanced the growth-stimulating effect of meningioma-derived conditioned medium. The rate of IL-6 secretion tended to show an inverse correlation with meningioma growth rate. The results presented here and the previous results suggest that the regulation of meningioma cell proliferation is defined by a complex network of autocrine stimulation, autocrine inhibition, and influences from multiple exogenous factors.

Interleukin-6 (IL-6) production and cell growth of cultured human ameningiomas:- Interactions with interleukin-1β (IL-1β) and interleukin-4 (IL-4) in vitro

We examined the production of interleukin (IL)-6 by human meningioma cells in vitro, and the effects of IL-1β and IL-4 on IL-6 production and meningioma cell growth. The histological classification of the tumours studied included transitional, syncytial, fibroblastic and atypical. All 10 meningiomas studied produced IL-6 (range 0.22–7.6ng/ml/10 6 cells/24 h). Separate addition of IL-1β or IL-4 to cultures increased IL-6 production up to ten fold, and two to three fold, respectively. Growth studies with IL-6 indicated that this cytokine significantly increased terminal cell density at a concentration greater than 1 ng/ml in 60% of the meningioma cultures studied. IL-1β caused a significant decrease in the terminal cell density in 25% of the meningioma cultures studied whereas IL-4 had a tendency to significantly inhibit growth in 16.6% of the cultures. These data suggest that IL-6 production by meningiomas can be modified by other cytokines and secondly, that IL-6, IL-1β and IL-4 can modify growth in vitro and may act as autocrine factors in vivo. By further determining the cytokine profiles within meningiomas and their effects, a better understanding of meningioma growth characteristics may be obtained.

An Ultrastructural and Immunocytochemical Analysis of Leptomeningeal and Meningioma Cultures

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Modulation of meningioma cell growth by sex steroid hormones in vitro.

Meningiomas were removed from four patients and estradiol binding was measured in the tumor tissue. Cell cultures were established and an in vitro system was developed to test the biological activity of physiologically relevant concentrations (10(-7) M and 10(-9) M) of estradiol-17 beta, progesterone, and the anti-estrogen, tamoxifen, on the growth of meningioma cells in early culture (passages 3 to 5). Assays of the frozen surgical specimens demonstrated cytosolic estradiol binding, with levels of 0.3 to 26.7 femtomoles (fM)/mg protein, in all four tumors. Nuclear estradiol binding was detected in three tumors, with levels of 16.8 to 39.5 fM/mg protein. In cell culture, estradiol at either 10(-7) M or 10(-9) M consistently stimulated cell growth in all four cultures. When tested alone, progesterone stimulated the growth of all four tumors and tamoxifen stimulated the growth of three of the four tumors, but the levels of stimulation produced by either of these compounds were less pronounced than the level produced by estradiol. When tested in combination with estradiol, progesterone significantly inhibited the growth stimulation produced by estradiol in all four meningioma cultures and tamoxifen significantly inhibited estradiol-induced growth stimulation in three of four cultures. It is not known if these effects are mediated by a hormone receptor or by a hormone binder different from a true receptor, or if they are caused by alterations in cellular metabolism that are independent of specific hormone binding. However, the authors conclude that this in vitro technique can be used to further study the biological activity of hormones on human meningiomas in order to answer these questions.

The role of acrocentric chromosomes in nucleolar organization. I. Correlation between the loss of acrocentric chromosomes and a decrease in the number of nucleoli in meningioma cell cultures.

Only few human meningiomas show a normal karyotype whereas in most of them a G-Chromosome loss is the primary cytogenetic event, often followed by the loss of further, mostly acrocentric, chromosomes. As the karyotype of each meningioma is normally uniform, these tumors seemed to us expecially suitable to investigate which chromosomes are involved in nucleolar organisation. We compared the number of nucleoli in cells of meningioma cultures which have lost none, one, two or more acrocentric chromosomes. Between tumours with normal karyotype and those showing a G monosomy only a slight difference was found. However there was a highly significant decrease in the nucleolar count after the loss of two or more acrocentric chromosomes (p<0.0l). The loss of non-acrocentric chromosomes did not influence the number of nucleoli. Furthermore we observed that meningioma cells being cultured only for 2–3 days, contained less nucleoli than cells of cultures from the 7th to 9th day (p<0.01). Already in this early stage of proliferation a strongly diminished number of nucleoli appeared in tumours having lost more than one acrocentric chromosome. These results prove acrocentric chromosomes play the most important role in nucleolar organisation.

Study on the effect of actinomycins in tissue cultures from human brain tumours.

The authors prepared tissue cultures from the following human brain tumours-glioblastoma multiforme, astrocytoma malignum and meningeoma endotheliomatosum. The cytostatic effects of two antibiotics, Actinomycin C (Sanamycin) and Actinomycin D were studied on these cultures. Quantitatively, the results were rated by the determination of the growth inhibition rate and the mitotic index of the explants; qualitatively, by evaluating the alteration of cellular morphology. As compared with the meningiomas, the malignant gliomas reacted more sensitively to both drugs. In the glioma cultures quantitative differences were observed between the responses to both drugs: a higher concentration of AD was required to obtain the same effect as with AC. In the meningioma cultures the identical concentration of both drugs brought about the same effect. The authors discuss the problem of thein vitro testing andin vivo application of chemotherapeutic drugs. They raise the idea of performing a sensitivity test in tissue culture prior to chemotherapy.