Human Glioma-derived Cell Research Articles

Potent Antitumour Effects of Novel Pentabromobenzylisothioureas Studied on Human Glial-derived Tumour Cell Lines.

Tumours of astroglial origin are the most common primary brain malignancy characterized by infiltrative growth and resistance to standard antitumour therapy. Glioma progression is thought to be related to various intracellular signal transduction pathways that involve the activation of protein kinases. Protein kinases play important roles in cell differentiation, proliferation, and survival. Recently, novel, specific inhibitors of constitutively active serine/threonine kinases and structurally similar isothiourea derivatives were suggested to induce apoptosis and inhibit proliferation in several types of human cancer cells. In this study, we examined the cytotoxic and proapoptotic activities of selected modified pentabromobenzyl isothioureas (ZKKs) in an adult human glioblastoma (T98G) and a subependymal giant cell astrocytoma cell (SEGA) line. We evaluated cell proliferation, viability, and apoptosis. Two pentabromobenzyl isothiourea bromide derivatives, ZKK-13 and N,N,N'-trimethyl-ZKK1 (TRIM), exhibited the most potent cytotoxic and proapoptotic efficacies against human glioma-derived cells, even at a very low concentration (1 μM). ZKK-13 (25-50 μM) inhibited cell growth by approximately 80-90% in 24 and 48 h of treatment. We showed that selected ZKKs exerted antiproliferative activity against astroglial neoplastic cells of both low- and high-grade tumour malignancy classes. No synergistic effects were detected when ZKKs were combined with serine/threonine kinase inhibitors. Our findings indicated that modified ZKKs show promise for the treatment of glioma-derived brain tumours.

Heat shock protein-90 inhibitors enhance antigen expression on melanomas and increase T cell recognition of tumor cells.

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer.

The expression of Wnt-inhibitor DKK1 (Dickkopf 1) is determined by intercellular crosstalk and hypoxia in human malignant gliomas.

Wnt signalling pathways regulate proliferation, motility and survival in a variety of human cell types. Dickkopf 1 (DKK1) gene codes for a secreted Wnt inhibitory factor. It functions as tumour suppressor gene in breast cancer and as a pro-apoptotic factor in glioma cells. In this study, we aimed to demonstrate whether the different expression of DKK1 in human glioma-derived cells is dependent on microenvironmental factors like hypoxia and regulated by the intercellular crosstalk with bone-marrow-derived mesenchymal stem cells (bmMSCs). Glioma cell line U87-MG, three cell lines from human glioblastoma grade IV (glioma-derived mesenchymal stem cells) and three bmMSCs were selected for the experiment. The expression of DKK1 in cell lines under normoxic/hypoxic environment or co-culture condition was measured using real-time PCR and enzyme-linked immunoadsorbent assay. The effect of DKK1 on cell migration and proliferation was evaluated by in vitro wound healing assays and sulphorhodamine assays, respectively. Glioma-derived cells U87-MG displayed lower DKK1 expression compared with bmMSCs. Hypoxia led to an overexpression of DKK1 in bmMSCs and U87-MG when compared to normoxic environment, whereas co-culture of U87-MG with bmMSCs induced the expression of DKK1 in both cell lines. Exogenous recombinant DKK1 inhibited cell migration on all cell lines, but did not have a significant effect on cell proliferation of bmMSCs and glioma cell lines. In this study, we showed for the first time that the expression of DKK1 was hypoxia dependent in human malignant glioma cell lines. The induction of DKK1 by intracellular crosstalk or hypoxia stimuli sheds light on the intense adaption of glial tumour cells to environmental alterations.

Cell Patterning on Photolithographically Defined Parylene-C: SiO<sub>2</sub> Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined Parylene-C: SiO<sub>2</sub> Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

March 2014: This Month in JoVE - Food-Related Motivation, Pathogens in the Deli, Diagnosing ALS, and Bioengineering Cells into Patterns

Here's a look at what's coming up in the March 2014 issue of JoVE: The Journal of Visualized Experiments. This month, we have a special culinary focus with food-related articles from various JoVE sections. In JoVE Environment, we have a method for tracking the spread of the bacterium Listeria monocytogenes. This foodborne pathogen contaminates all kinds of foods in the retail environment - including dairy, eggs, produce, meat, and fish. According to the Centers for Disease Control and Prevention (CDC), Listeria sickens around 1,600 people in the United States every year, and is the 3rd leading cause of death related to food poisoning. Many cases of Listeria-related illness can be traced to contaminated deli meats, especially those sliced by retailers. To study how this potentially deadly pathogen spreads in retail environments, Sirsat et al. simulate bacterial contamination of deli meat using a fluorescent compound that glows under black light, focusing on the deli slicer itself as a critical point of Listeria cross contamination. This method can be adapted to study other microbial pathogens in retail and industrial environments, and facilitate sanitary practices in multiple settings. In a culinary article from JoVE's Behavior section, we examine what drives us to eat. Besides the obvious impulse of hunger, the rewarding aspects of food can drive certain behaviors, especially overeating. Ziauddeen et al. use several methods to examine food-related motivation. In one experiment, subjects have to physically work to win food or non-food rewards. In another experiment, they test whether subliminal images of a food reward can affect an individual's willingness to work for it. In a third experiment, subjects assign monetary values on various foods, which yields quantitative measures of food-related rewards. These methods can be used to study the effectiveness of anti-obesity treatments that alter the reward value of food. In JoVE Clinical and Translational Medicine, we feature an article on amyotrophic lateral sclerosis (ALS, commonly known as Lou Gehrig's disease). In this degenerative neuromuscular disease, muscles become paralyzed when motor neurons disconnect from muscle fibers at the neuromuscular junction (NMJ). An early symptom that is specific to ALS is the split hand phenomenon, or the preferential weakness of a specific group of hand muscles, while other hand muscles are spared. Menon and Vucic demonstrate a novel method of obtaining split hand index scores. To do this, they record maximal motor responses from three hand muscles. By comparing the responses of various patients, our authors demonstrate how this index can distinguish ALS from other neuromuscular diseases. In JoVE Bioengineering, many applications involve growing cells on synthetic surfaces. Therefore, a common bioengineering research goal is to understand the mechanisms of cell adhesion and patterning on synthetic materials. Hughes et al. have developed a method for photolithographically printing silicon dioxide chips with paralene-C. Cells specifically adhere to the paralene-C while being repelled by the silicon dioxide. This technique has allowed patterning of a broad range of cell types, including HEK 293 cells, primary human glioma-derived stem-like cells, and 3T3 L1 cells You've just had a sneak peek of the March 2014 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Engineering neuronal networks on photolithographically defined, biologically activated silicon substrates

BackgroundTo interface neurons with silicon semiconductors is a bioengineering challenge. Such constructs provide a platform for understanding neuronal coding and learning, an arena for modelling disease, and ultimately a step towards creating intelligent neuroprostheses. A prerequisite is the ability to dictate the spatial organisation of neuronal cells, in a context in which electrical and synaptic behaviour can be assessed. Our aim was to pattern cells on silicon, leading the way to the eventual incorporation of a wide range of biomicroelectromechanical systems for cellular interrogation. MethodsA predetermined design of the chlorine-containing polmer parylene-C was photolithographically printed onto oxidised silicon wafers (in a microelectronics cleanroom facility). Parylene-patterned chips were activated by acid etching and then incubated in fetal calf serum. The resulting substrate became differentially cell-adhesive (parylene) or repulsive (SiO2) for certain cell types. A human glioma-derived stem-like cell line that had been isolated during debulking surgery was cultured on-chip, growing selectively on parylene. Neurons (Lund Human Mesencephalic [LUHMES] cell line) were then plated, adhering to the glial template and extending neurites to connect with adjacent cells to form a network. The effect of parylene design on neuronal network configuration was assessed by use of a pattern of circular nodes with four orthogonal spokes printed in arrays of three different configurations (node diameter 50 μm, spoke length 125 μm; node 100 μm, spoke 100 μm; node 250 μm, spoke 100 μm). FindingsTen different cell lines were assessed on the parylene–silicon cell-patterning platform. Cell adhesion behaviour was heterogeneous, with only select cell lines patterning at high resolution. LUHMES neurons in isolation failed to pattern. However, in co-culture with glioma-derived stem-like cells (which pattern accurately), the neurons adhered and differentiated to form networks. Neurite directionality differed significantly according to parylene design (Kolmogorov-Smirnov 250 μm vs 100 μm nodes D=0·204, p=0·001; 100 μm vs 50 μm nodes D=0·147, p=0·028), allowing creation of reticular neuronal networks. InterpretationThe cell adhesion molecule profile of different cell lines affects their behaviour on the parylene–silicon platform. By consolidating DNA microarray data from cell types with contrasting patterning behaviour, we hope to correlate gene expression profiles with a cell's tendency to adhere to, or be repulsed by, the two contrasting substrates. The co-culture approach we describe has allowed creation of defined neuronal networks on silicon. The next steps include assessing long-term viability and functionality of patterned networks, while also improving control over their spatial organisation. FundingWellcome Trust.

Abstract 2314: Erlotinib response in lung- and glioma-derived EGFR alleles: Correlating kinase site occupancy with efficacy

Abstract INTRODUCTION: Mutational activation of the Epidermal Growth Factor Receptor (EGFR) gene features prominently in malignant glioma (MG) and non-small-cell lung cancer (NSCLC). However, while NCSLC-derived mutants of EGFR exhibit high sensitivity to small molecule tyrosine kinase inhibitors, patients with MG-derived EGFR alleles respond poorly to TKIs. We tested MG- and NSCLC-derived alleles of EGFR to determine whether differences in occupancy within the mutant kinase domains determine response to therapy. METHODS: Human malignant glioma-derived cell lines expressing wild-type and mutant alleles of EGFR (derived from MG (EGFRvIII) or NSCLC (EGFR L858R and EGFRdel746-750)) were generated by viral transduction. Cell cycle analyses and viability assays were performed to confirm the in vitro recapitulation of the range of erlotinib's clinical efficacy in cells expressing MG- or NSCLC-specific alleles of EGFR. A novel affinity probe selective for the kinase site of EGFR was used to quantify the level of erlotinib binding in this panel of EGFR-allele expressing cell lines. Cell-signaling was analyzed through western-blotting and quantified with densitometry. RESULTS: Using the EGFR-specific affinity probe, erlotinib's kinase site occupancy was measured at clinically relevant doses. In cells expressing MG-derived EGFRvIII, significantly lower levels of kinase site occupancy were reached as compared to NSCLC-derived EGFR L858R and EGFRdel746-750. Surprisingly, these data did not correlate with levels of phospho-EGFR. Across all alleles, kinase site occupancy rather than levels of p-EGFR correlated with downstream signaling through Akt and mTOR, and with growth inhibition and cell cycle arrest. CONCLUSION: Erlotinib, a small molecule tyrosine kinase inhibitor, achieves higher levels of kinase site occupancy in NSCLC-derived mutations of EGFR, as compared with MG-derived mutations. Kinase site occupancy correlated directly with efficacy. In contrast, levels of p-EGFR correlated poorly with efficacy for these agents. These data suggest kinase site occupancy as a potential biomarker for efficacy of EGFR inhibitors. This also raises the intriguing possibility that the reason for the differential efficacy of erlotinib in lung- and MG-derived activating mutations in EGFR is due to differences in ATP binding affinity: the lung-derived activating mutations in EGFR have lower ATP affinity as compared to the wild-type kinase, resulting in higher relative erlotinib affinity, while MG-derived activating mutations in EGFR have a higher affinity for ATP, which outcompetes erlotinib and results in poor drug affinity for the kinase site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2011-2314

Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in the irradiated cells was analyzed by Western blotting. When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21(WAF1/CIP1) was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1α

Hypoxia contributes to the progression of a variety of cancers by activating adaptive transcriptional programs that promote cell survival, motility and tumor angiogenesis. Although the importance of hypoxia and subsequent hypoxia-inducible factor-1alpha (HIF-1alpha) activation in tumor angiogenesis is well known, their role in the regulation of glioma-derived stem cells is unclear. In this study, we show that hypoxia (1% oxygen) promotes the self-renewal capacity of CD133-positive human glioma-derived cancer stem cells (CSCs). Propagation of the glioma-derived CSCs in a hypoxic environment also led to the expansion of cells bearing CXCR4 (CD184), CD44(low) and A2B5 surface markers. The enhanced self-renewal activity of the CD133-positive CSCs in hypoxia was preceded by upregulation of HIF-1alpha. Knockdown of HIF-1alpha abrogated the hypoxia-mediated CD133-positive CSC expansion. Inhibition of the phosphatidylinositol 3-kinase(PI3K)-Akt or ERK1/2 pathway reduced the hypoxia-driven CD133 expansion, suggesting that these signaling cascades may modulate the hypoxic response. Finally, CSCs propagated at hypoxia robustly retained the undifferentiated phenotype, whereas CSCs cultured at normoxia did not. These results suggest that response to hypoxia by CSCs involves the activation of HIF-1alpha to enhance the self-renewal activity of CD133-positive cells and to inhibit the induction of CSC differentiation. This study illustrates the importance of the tumor microenvironment in determining cellular behavior.

TWIST is Expressed in Human Gliomas, Promotes Invasion

TWIST is a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, promotes tumor cell metastasis, and, in response to cytotoxic stress, enhances cell survival. Our screen for bHLH gene expression in rat C6 glioma revealed TWIST. To delineate a possible oncogenic role for TWIST in the human central nervous system (CNS), we analyzed TWIST message, protein expression in gliomas, normal brain. TWIST was detected in the large majority of human glioma-derived cell lines, human gliomas examined. Increased TWIST mRNA levels were associated with the highest grade gliomas, increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis, subsequent malignant progression. TWIST was also expressed in embryonic, fetal human brain, in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.

Human autologous in vitro models of glioma immunogene therapy using B7-2, GM-CSF, and IL12.

Cancer immunogene therapy is based on vaccination with radiated, autologous tumor cells transduced with immunostimulatory genes. To help determine an optimal glioma immunogene therapy strategy, we stimulated lymphocytes with autologous human glioma cells transduced with B7-2 (CD86), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin-12 (IL12). A human glioma-derived cell culture (Ed147.BT) was transduced with B7-2, GM-CSF, and/or IL12 using retroviral vectors. Autologous peripheral blood mononuclear cells (PBMC) were co-cultured with irradiated gene-transduced tumor alone or a combination of radiated wild type and gene-transduced cells. Peripheral blood mononuclear cells proliferation was determined by serial cell counts. Peripheral blood mononuclear cells phenotype was assessed by flow cytometry for CD4, CD8, and CD16. Anti-tumor cytotoxicity was determined by chromium-51 (51Cr) release assay. Peripheral blood mononuclear cells cell numbers all decreased during primary stimulation but tumor cells expressing B7-2 or GM-CSF consistently caused secondary proliferation. Tumors expressing B7-2 and GM-CSF or B7-2, GM-CSF, and IL12 consistently increased PBMC CD8+ (cytotoxic T) and CD16+ (natural killer) percentages. Interestingly, anti-tumor cytotoxicity only exceeded that of PBMC stimulated with wild type tumor alone when peripheral blood mononuclear cells were stimulated with both wild type tumor and B7-2/GM-CSF- (but not IL12) transduced cells. PBMC proliferation and phenotype is altered as expected by exposure to immunostimulatory gene-transduced tumor. However, transduced tumor cells alone do not stimulate greater anti-tumor cytotoxicity than wild type tumor. Only B7-2/GM-CSF-transduced cells combined with wild type produced increased cytotoxicity. This may reflect selection of tumor subclones with limited antigenic spectra during retrovirus-mediated gene transfer.

Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU.

Due to minimal treatment success with surgery, radiotherapy, and chemotherapy, the aim of this study was to test the therapeutic potential of gene therapy for the treatment of glioblastoma multiforme (GBM). We have quantitatively analyzed two gene therapy approaches using short-term human glioma cell cultures derived from surgical biopsies (designated IN859, IN1612, IN2045, IN1760, and IN1265) and compared the results of gene therapy with the chemosensitivity of the same cells. All of the glioma cell cultures tested were susceptible to recombinant adenovirus (RAd)-mediated infection. Expression of herpes simplex virus type 1-thymidine kinase (RAd128), followed by ganciclovir treatment, induced apoptosis in all of the glioma cell cultures studied, including three that are resistant to the chemotherapeutic drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Expression of murine Fas ligand (RAdhCMV-mFasL) also induced cell death in four of the five cell cultures studied. One cell culture that was resistant to CCNU was also resistant to apoptosis induced by mFasL expression. These results suggest that sensitivity to chemotherapeutic agents does not necessarily correlate with the sensitivity to gene therapy treatments. RAds expressing therapeutic gene products in human glioma cell cultures are able to induce apoptosis even in some cells that are resistant to a commonly used chemotherapeutic agent. Therefore, RAd-mediated gene transfer could be a good candidate to further develop gene therapy for the treatment of GBM.

Survival of human glioma cells treated with various combination of temozolomide and X-rays

Purpose: To investigate the effect of temozolomide, a 3-methyl derivative of mitozolomide in combination with X-rays in human glioma-derived cell lines. Methods and Materials: Glioma cell lines D384 and U251 were treated with temozolomide for various periods of time in combinations with X-rays. Temozolomide administration was repeated every 24 h for exposures up to 96 h. Cytotoxicity was determined with a clonogenic assay. Results: Incubation of D384 cells with temozolomide during 24 h prior to or following irradiation results in a moderate enhancement of the cytotoxicity. Prolonged treatment with temozolomide, i.e., 48–96 h before X-rays, causes a stronger potentiation. In contrast, no enhancement is observed in irradiated U251 cells in combination with 24–96 h temozolomide treatment. In addition to single-dose irradiation, we investigated the effect of temozolomide in D384 cells with concomitant fractionated irradiation. A 96-h exposure to temozolomide with simultaneous doses of 2 Gy X-rays at 24-h intervals, causes a significant further reduction in cell survival as compared to fractionated irradiation only. Conclusion: Depending on the cell line, treatment of glioma cells with temozolomide and X-rays can have either an additional effect or potentiate cell killing.

SETA: a novel SH3 domain-containing adapter molecule associated with malignancy in astrocytes.

Differential display polymerase chain reaction analysis was used to compare five differentiation states of the O-2A progenitor-like cell line CG4: progenitor cells and cells at 12 h or 4 days after the induction of differentiation into oligodendrocytes or astrocytes. This led to the identification of 52 sequence tags that were expressed differentially with cellular phenotype. One sequence was upregulated during differentiation of CG4 cells and represented a novel gene that we named SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes). This gene encodes an SH3 domain-containing adapter protein with sequence similarity to the CD2AP (CD2 adapter protein) and CMS (Cas ligand with multiple Src homology) genes. SETA mRNA was expressed at high levels in the developing rat brain but was barely detectable in the normal adult rat or human brain. However, SETA mRNA was found in approximately one half of the human gliomas tested, including astrocytomas grades II, III, and IV, as well as oligodendrogliomas, mixed oligoastrocytomas, and human glioma-derived cell lines. A rat glioma generated by treatment with the alkylating carcinogen ethylnitrosourea on postnatal day 1 and a derived cell line also expressed SETA mRNA. Furthermore, in an in vitro model of astrocytoma progression based on p53-/- astrocytes, expression of SETA was restricted to cells that are tumorigenic.

Chapter 8 Establishing Human Glioma-Derived Cell Lines

This chapter discusses the establishment of human glioma-derived cell lines. The chapter discusses the primary culture of cells derived from human glioma-tissue specimens and the characterization and handling of the derived cell lines. The developments of new therapeutic and diagnostic strategies in neurooncology rely on cell-culture models in monolayer or multicellular spheroids and in vivo models derived from xenotransplants. Chemoresistance testing, the elucidation of the effects of gene therapy, and the development of new antiproliferative strategies in oncology require appropriate cell-culture models. A major goal in the primary culture of any tumor and brain tumors is the attachment of as many cellular components as possible. The chapter describes the techniques that help obtain permanent cell lines from the tumors taken into tissue culture. Cells need to be characterized into two aspects: (1) they need to be shown to be of glial origin, which is the lineage aspect, and (2) they need to be characterized as truly neoplastic. Lines other than astrocytic or astrocytoma-derived cell lines are difficult to generate and maintain. The chapter discusses the molecular basis of glioma disease.

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins.

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

Reconstituted basement membrane (matrigel) enhances the growth of human glioma cell lines in nude mice

Transplantation of human cancers into immunologically deficient mice is widely used to study potential therapeutic interventions in vivo. For brain tumor research, however, several factors limit more widespread application of this animal model. First, only a minority of human glioma-derived cell lines are tumorigenic in nude mice. In addition, even for tumorigenic cell lines, tumor take is variable and growth is often slow for tumors derived from cell inoculums. Reconstituted components of tumor basement membrane (matrigel) have been found to improve the growth in nude mice of several types of human tumors originating outside the central nervous system when premixed with the tumor cells before subcutaneous inoculation. We investigated the ability of matrigel to enhance the growth in nude mice of tumors derived from the human glioma cell lines U-251 MG, U-373 MG, SNB-78 and SNB-101. Athymic nude mice (NIH Swiss background, nu/nu genotype) were inoculated subcutaneously with 1.0 x 10(6) tumor cells alone or after premixing with an equal volume of liquid matrigel. U-251 and U-373 cells were tumorigenic, with palpable tumors present by about 2 to 3 weeks. Co-injection of these cell lines with matrigel resulted in higher tumor-take rates, from 6/10 to 8/8 animals for U-251 at 60 days, and from 9/12 to 11/11 animals for U-373 at 60 days. Matrigel also enhanced tumor growth, with tumors at 45 days significantly larger than those formed in the absence of matrigel, for both cell lines (p < 0.01). SNB-78 and SNB-101 cells did not give rise to progressively enlarging solid tumors with or without matrigel. Matrigel enhances the growth of tumorigenic human gliomas in athymic nude mice. This technique provides a model with more consistent tumor take and more rapid growth kinetics for human glioma cell lines that are tumorigenic in nude mice.

Hyaluronic acid modulates glioma cell proliferation through its interaction with CD44H in vitro

In a recent study we showed that hyaluronic acid (HA) induces cell detachment and promotes migration and invasion of glioma cells in vitro through its interaction with the cell surface molecule CD44H. In this study, the role of HA in proliferation in eight human glioma-derived cell Lines was investigated. We demonstrate that HA exerts a dose dependent proliferative and anti-proliferative effect on glioma cells. This effect was found to be partially counteracted by a CD44H monoclonal antibody, suggesting the involvement of its high affinity receptor, CD44H and other HA receptors in this process.

Morphologic, immunologic, biochemical, and cytogenetic characteristics of the human glioblastoma-derived cell line, SNB-19

Human glioma-derived cell cultures and lines have proven to be of significant value in the study of the basic properties that contribute to the highly malignant, invasive and angiogenic phenotype of glioblastoma multiforme tumors. It is frequently difficult to establish lines that retain glial tumor properties in long term culture. The SNB-19 cell line has maintained and exhibited properties of transformation, differentiation, autocrine growth response, and tumorigenesis while remaining in culture for over 13 yr and undergoing over 200 passages. This human line has been utilized in a wide range of studies related to the basic properties of human glioblastoma multiforme. In this report, we summarize the immunologic, biochemical, and cytogenetic properties of this versatile cell line and its utility for additional mechanistic investigation into the pathophysiology of the progression of human malignant gliomas.