epidermal-growth-factor and Glioma

Glial cells in the central nervous system of adult mammals outnumber neurons 10-fold. Their number remains stationary throughout adulthood, controlled by the concomitant presence of mitogens and mitogen inhibitors. The most abundant inhibitor, neurostatin, is ganglioside GD1b O-acetylated on hydroxyl 9 of its outermost sialic acid. Neurostatin inhibited the proliferation of primary microglia and astroblasts in culture (cytostatic) as well as both rodent and human glioma cells (cytotoxic) at nanomolar concentrations. At those concentrations neurostatin had no effect on non-glial lineage cells or differentiated glia. Neurostatin shows direct antimitotic activity on tumoral cells, interfering with multiple signals regulating cell cycle progression. But it also promotes indirectly total destruction of experimental rat brain glioma, presumably by making it visible to the host immune system and activating CD4+ and CD8+ lymphocytes. Neurostatin could be a new anti-inflammatory agent, with multiple convergent direct and indirect actions on glioma growth, a pathology without satisfactory clinical treatment. Neurostatin is produced by neurons but its expression is up-regulated by neuron-astrocyte contact. The action of neurostatin could be mediated by a number of receptor proteins, including integrins, Toll-like receptors and siglecs.. Glicolipidos neuronales regulan negativamente la division glial durante el desarrollo y tras una lesion.. En el sistema nervioso central de los mamiferos, las celulas gliales superan diez veces en numero a las neuronas. Su numero permanente estacionario durante la edad adulta, controlado por la presencia simultanea de mitogenos gliales e inhibidores de esos mitogenos. El inhibidor mas abundante, la neurostatina, es el gangliosido GD1b O-acetilado en el grupo 9 del acido sialico mas externo. La neurostatina y los oligosacaridos sinteticos inhiben la proliferacion de astroblastos en cultivo primario (citostaticos) y de celulas de gliomas (citotoxicos), tanto de roedores como de humanos, en concentracion nanomolar. A esas concentraciones, la neurostatina no tuvo efecto sobre celulas de linaje no glial ni sobre glia madura. La neurostatina y sus analogos mostraron actividad antimitotica directa sobre las celulas tumorales, interfiriendo con la progresion del ciclo celular en multiples sitios, pero tambien actuaron indirectamente, haciendo visibles las celulas tumorales al sistema inmune del huesped y activando linfocitos CD4+ y CD8+. Analogos de neurostatina podrian generar nuevos farmacos antiinflamatorios, con multiples acciones directas e indirectas contra el crecimiento de gliomas, una patologia todavia sin tratamiento clinico satisfactorio. La neurostatina es producida por las neuronas, pero el contacto de estas con astrocitos estimula notablemente su expresion. La accion de la neurostatina puede estar mediada por numerosas proteinas receptoras, incluyendo integrinas, siglecs y receptores Toll-like.

Topics: Animals; Brain Injuries; Carbohydrate Conformation; Carbohydrate Sequence; Cell Division; Cicatrix; Epidermal Growth Factor; Gangliosides; Glioma; Glycolipids; Glycosphingolipids; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Macrophages; Mammals; Mice; Neural Stem Cells; Neurogenesis; Neuroglia; Neurons; Spinal Cord Injuries; Toll-Like Receptors; Xenograft Model Antitumor Assays

Numerous studies have investigated the risk of cancer associated with the polymorphism of epidermal growth factor (EGF) 61A>G, but results have been inconsistent. We performed this meta-analysis to drive a more precise estimation of the association between this polymorphism and risk of glioma. A comprehensive search was conducted to identify all case-control studies on the EGF +61A>G polymorphism and glioma risk. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to assess the strength of the association. Statistical analysis was performed with the software program Stata (version 12.0). A total of ten eligible studies, including 1,888 cases and 2,836 controls were included in this work. Overall, there was a significant association between EGF +61A>G polymorphism and glioma risk in the allele model (OR = 1.419, 95% CI = 1.144-1.759, P = 0.001). In the subgroup analysis by ethnicity, significant associations were also found in Asian populations under all different genetic models (homozygote model: OR = 1.727, 95% CI = 1.310-2.275, P = 0.000; heterozygote model: OR = 1.202, 95% CI = 1.023-1.413, P = 0.025; dominant model: OR = 1.279, 95% CI = 1.096-1.491, P = 0.002; recessive model: OR = 1.590, 95% CI = 1.221-2.070, P = 0.001; and A-allele versus G-allele OR = 1.600, 95% CI = 1.145-2.236, P = 0.006). However, no significant associations were found among Caucasians in all comparison models. In conclusion, the results suggest that there is a significant association between EGF +61A>G polymorphism and glioma risk among Asians.

Topics: Alleles; Brain Neoplasms; Case-Control Studies; Epidermal Growth Factor; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Glioma; Humans; Odds Ratio; Polymorphism, Single Nucleotide; Publication Bias

Gliomas account for almost 80% of primary malignant brain tumors. Epidermal growth factor (EGF) is an interesting research candidate in which to look for genetic polymorphisms because of its role in mitogenesis and proliferation. Extensive studies have found that a single nucleotide polymorphism (SNP) +61 G/A (rs4444903) in the EGF gene is associated with the susceptibility of glioma, however, the results have been controversial. Furthermore, the association between EGF +61 G/A polymorphism with the development and grade progress of glioma has not been established.. We examined the association of EGF +61 G/A polymorphism and glioma by performing a meta-analysis. Nine studies testing the associations between EGF +61 G/A polymorphism and risk of glioma with 1758 cases and 2823 controls were retrieved. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. The pooled ORs were performed for the allele model, codominant model, dominant model, and recessive model, respectively.. Overall, this meta-analysis showed significant associations between the EGF +61 G/A polymorphism and glioma susceptibility in all four genetic models. However, in the stratified analysis by the grade of glioma, we only found this association existed in patients with Grade IV glioblastoma, but not in patients with Grade I-III glioma. We further compared EGF +61 G/A polymorphism in patients with glioblastoma and Grade I-III glioma accordingly, the stronger association between the EGF +61 G/A polymorphism and the malignancy of glioma was found.. The results of this meta-analysis suggested that the EGF +61 G/A polymorphism is associated with both the susceptibility of glioma and the malignance of glioma.

Topics: Alleles; Brain Neoplasms; Case-Control Studies; Epidermal Growth Factor; Gene Frequency; Genetic Predisposition to Disease; Glioma; Humans; Models, Genetic; Neoplasm Grading; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors

The development of glioma is a complex process which may be influenced by many factors including the epidermal growth factor (EGF) gene polymorphism. Previous studies showed that EGF rs4444903 polymorphism could result in increased risk of tumorigenesis in multiple human cancers, but published data regarding the association between EGF rs4444903 polymorphism and glioma risk were inconsistent. To derive a more precise estimation of the association between EGF rs4444903 polymorphism and glioma risk, we performed a systematic review and meta-analysis of previous published studies. PubMed, Embase, and the Wanfang databases were systematically searched to identify relevant studies. Odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were calculated to assess the strength of the association. Ten published studies with 1,891 glioma cases and 2,836 controls were finally included into the study. Overall, there was a significant association between EGF rs4444903 polymorphism and glioma risk in all four genetic models (the allele model: OR=1.25, 95 % CI 1.15-1.37, P<0.001; the codominant model: OR=1.65, 95 % CI 1.36-1.99, P<0.001; the dominant model: OR=1.27, 95 % CI 1.12-1.44, P<0.001; the recessive model: OR=1.48, 95 % CI 1.25-1.75, P<0.001). Subgroup analyses by ethnicity showed that EGF rs4444903 polymorphism resulted in a higher risk of glioma among both Asians and Caucasians. In conclusion, the results suggest that there is a significant association between EGF rs4444903 polymorphism and glioma risk, and genotypes of EGF rs4444903 mutation contribute to increased host susceptibility to glioma.

Topics: Case-Control Studies; Epidermal Growth Factor; Genetic Predisposition to Disease; Glioma; Humans; Polymorphism, Genetic; Risk Factors

Epidermal growth factor (EGF) plays a key role in survival of neural and glial precursor cells. The +61A/G polymorphism of the EGF gene is located in the 5'-untranslated region of EGF mRNA and may affect DNA folding or gene transcription, leading to the increase in EGF protein expression. The association between the +61G allele and glioma risk has been widely reported; however, in general the data from published studies with individually low statistical power were controversial and underpowered. We conducted a search in the PubMed database without a language limitation, covering all papers published by the end of October 2010. Overall, 6 case-control studies with 1453 glioma cases and 1947 controls were retrieved based on the search criteria for glioma susceptibility related to the +61A/G polymorphism. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. We found that EGF +61G allele is associated with the low glioma risk in Chinese population [G-allele vs. A-allele, OR = 0.93, 95%CI (0.89-0.97), P(heterogeneity) = 0.318, I² = 0.0], but with the high glioma risk in European population [G-allele vs. A-allele, OR = 1.14, 95%CI (1.04-1.24), P(heterogeneity) = 0.310, I² = 14.6]. In the stratified analysis by source of control, significant association was observed between hospital-based control and glioma risk [homozygote comparison, OR = 1.14, 95%CI (1.02-1.27), P(heterogeneity) = 0.179, I² = 71.8]. In conclusion, EGF +61G allele represents a risk factor for glioma in European population and conversely a protective factor in Chinese population.

Topics: Alleles; Asian People; Databases, Genetic; Epidermal Growth Factor; Ethnicity; Genetic Heterogeneity; Genetic Predisposition to Disease; Glioma; Humans; Polymorphism, Single Nucleotide; Publication Bias; White People

The epidermal growth factor receptor (EGFR) and its ligands figure prominently in the biology of gliomas, the most common tumors of the central nervous system (CNS). Although their histologic classification seems to be straightforward, these tumors constitute a heterogeneous class of related neoplasms. They are associated with a variety of molecular abnormalities affecting signal transduction, transcription factors, apoptosis, angiogensesis, and the extracellular matrix. Under normal conditions, these same interacting factors drive CNS growth and development. We are now recognizing the diverse molecular genetic heterogeneity that underlies tumors classified histologically into three distinct grades. This recognition is leading to new therapeutic strategies targeted directly at specific molecular subtypes. In this article, we will review the role of EGFR and related molecular pathways in the genesis of the normal CNS and their relationship to glial tumorigenesis. We will discuss barriers to effective treatment as they relate to anatomic specialization of the CNS. We will also consider the ways in which specific EGFR alterations common to glioma reflect outcomes following treatment with targeted therapies, all with an eye towards applying this understanding to improved patient outcomes.

Topics: Brain; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Models, Biological; Multigene Family; Mutation; Protein Binding; Signal Transduction

Topics: Brain Neoplasms; Cell Differentiation; Cell Lineage; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Glioma; Humans; Neurons; Patched Receptors; Receptors, Cell Surface; Signal Transduction; Stem Cells; Transcription Factors; Zinc Finger Protein GLI1

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Amino Acid Motifs; Animals; Antineoplastic Agents; Brain Neoplasms; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; HSP90 Heat-Shock Proteins; Humans; Neuregulins; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured

Topics: Antibiotics, Antineoplastic; Brain Neoplasms; Cell Cycle; Clinical Trials as Topic; Epidermal Growth Factor; Glioma; Humans; Middle Aged; Molecular Biology; Receptors, Vascular Endothelial Growth Factor; Tumor Suppressor Protein p53

Despite optimal clinical treatment, the prognosis for gliomas remains poor, and little progress has been observed during the last few years. Meanwhile, understanding of glioma oncogenesis has improved greatly. This review focuses on recent advances in molecular biology of glial tumors, with particular emphasis on lineage markers, genetic mechanisms underlying tumor progression, new diagnostic and prognostic markers, and potential therapeutic targets.. The question of the cell of origin, illustrated by the evidence of tumor-derived multipotent progenitors, by the animal models of gliomas, and by lineage markers such as Olig1/2 markers, remains unsolved. Genotype/phenotype correlation studies have identified early and late genetic alterations related either to astrocytic or oligodendroglial phenotype. They complement the existing World Health Organization morphologic classification and provide additional prognostic markers such as 1p/19q deletion in oligodendrogliomas. Most of these genetic alterations result in the disruption of three main cellular systems: RB1, P53, and tyrosine kinase receptor pathways. New gene alterations have also been identified in glioma, promoting mitotic signal transduction, cell cycle regulation, apoptosis, angiogenesis, or invasion. Gene and protein profiling has been correlated with outcome.. Management of gliomas, especially oligodendrogliomas with 1p19q deletion, benefits from advances in molecular genetics. A better understanding of the molecular pathogenesis and cellular lineage of gliomas will improve tumor classification and define more reliable prognostic markers. There is a hope that it will also lead to novel targets for therapy.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Central Nervous System Neoplasms; Epidermal Growth Factor; Gefitinib; Glioma; Humans; Neovascularization, Pathologic; Quinazolines

Gliomas are primary central nervous system tumors that arise from astrocytes, oligodendrocytes, or their precursors. Gliomas can be classified into several groups according to histological features. A number of genetic alterations have been identified in human gliomas; these generally affect either signal transduction pathways activated by receptor tyrosine kinases or cell cycle growth arrest pathways. These observed genetic alterations are now being used to complement histopathological diagnosis. The aim of the present review is to give a broad overview of the receptor tyrosine kinase signaling machinery involved in gliomagenesis, with an emphasis on the cooperative interaction between receptor tyrosine kinase signaling and the cell cycle-regulatory machinery. Understanding molecular features of primary glial tumors will eventually allow for target-selective intervention in distinct glioma subsets and a more rational approach to adjuvant therapies for these refractory diseases.

Topics: Adult; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; MAP Kinase Signaling System; Platelet-Derived Growth Factor; Receptor Protein-Tyrosine Kinases; Receptors, Platelet-Derived Growth Factor

We review the role of peptide growth factors in angiogenesis and progression of low grade glial tumors to higher grade glioblastoma multiforme (GBM).. Vascular pathology is a key feature of glioblastoma multiforme characterized by hypervascularity, vascular permeability, and hypercoagulability.. Vascular endothelial growth factor (VEGF) can mediate all of these effects, but by itself does not promote malignant growth. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta (TGF-beta) are implicated in the angiogenesis of a number of tumors including those of glial origin.. These growth factors are suggested to play a role in autocrine and/or paracrine mediated tumorogenesis of astrocytic tumors. VEGF secretion might be the product of induction by physiologic concentrations of other growth factors with VEGF being the common pathway of neovascularization and progression to GBM.

Topics: Animals; Disease Progression; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Growth Substances; Humans; Lymphokines; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cyclohexanes; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Glioma; Humans; Lymphokines; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Prognosis; Sesquiterpenes; Suramin; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Brain Neoplasms; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Growth Substances; Humans; Lymphokines; Nerve Growth Factors; Nerve Tissue Proteins; Platelet-Derived Growth Factor; Somatomedins; Transforming Growth Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Specific and recurring chromosomal and genetic alterations have been identified in gliomas and could described a model of tumoral progression from benin glioma to glioblastoma multiforme. However, the heterogeneity of profiles of molecular alterations that have been observed in gliomas seems to reflect the variety of clinical evolutions which characterise those tumors. Loss of genetic material on chromosomes 17, 9 and 19, then of chromosome 10 have been associated to pathogenesis of glioma and a pejorative prognostic value have been attributed to the alteration of chromosome 10. Gliomas also express growth factors and growth factors receptors that may be important in promoting tumor growth, like Epidermal growth factor (EGF), fibroblast growth factors (FGF) and vascular endothelial growth factor (VEGF). Tumoral invasion which characterise also gliomas, may involve proteases like plasminogen activators (PA) and metalloproteases, under the regulation of specific receptors and inhibitors. PA inhibitor type 1 (PAI1), associated to the most aggressive form of gliomas, may also participate to tumoral neoangiogenesis. Description and understanding of these alterations may contribute to develop new treatment modalities in gliomas.

Topics: Chromosome Aberrations; Chromosome Disorders; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Neoplasm Invasiveness; Protein Kinases; Serine Endopeptidases; Tumor Suppressor Protein p53

The understanding of the signal transduction cascade involving growth factors and their receptors is one major key for diagnostic and therapeutic improvements in human neoplasms. Using receptor autoradiography, an inverse relationship for the incidence of somatostatin receptors (SSR) and epidermal growth factor receptors (EGFR) was found in gliomas [1]. In the majority of low grade gliomas, SSR were present but EGFR were absent. In contrast, EGFR were present in most glioblastomas, but no SSR were detected. Recently, the amplification of the EGFR gene and its overexpression was demonstrated to be associated with the development of glioblastomas. Several independent reports revealed that 40-50% of tumors show amplified EGFR [2-4]. The frequency of EGFR amplification was directly associated with tumor malignancy. In addition, amplified EGFR levels indicate a bad prognosis and shorter overall survival [5]. Recent analysis of the EGFR gene in tumors has shown that regions of this gene frequently undergo alteration. Hence, not only amplification but also mutation may be the cause of the increased malignancy in EGFR overexpressing cells [6].

Topics: Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glioblastoma; Glioma; Growth Substances; Humans; Receptors, Growth Factor; Receptors, Somatostatin; Second Messenger Systems; Signal Transduction

The failure of most available treatment modalities to improve the survival time and the quality of survival in patients with gliomas calls for the intense biological analysis of glial neoplasia. The untimely activation of cellular protooncogenes is often related to the process of neoplasia. Several protein growth factors as well as their cellular receptors have been identified as products of proto-oncogenes. Furthermore, these growth factors have been identified as glial mitogens in normal glial cell cultures as well as in tumors. The analysis of growth factor biology gains additional weight by the demonstration of autocrine growth factor secretion by tumor cells. The role of epidermal growth factor (EGF) and its receptor system in glial cell proliferation and differentiation is presented, with the speculation that the EGF receptor system may integrate the biological actions of many different factors, of which EGF itself may be least important. The literature on platelet-derived growth factor and insulin-like growth factors is reviewed, and the biology of fibroblast growth factor is presented in perspective with such phenomena as neovascularization and cell-matrix interactions.

Topics: Epidermal Growth Factor; ErbB Receptors; Glioma; Growth Substances; Humans; Proto-Oncogene Proteins

Normal cell replication is regulated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) that act through binding to specific surface receptors on target cells. Oncogenes may exert their transforming activity by encoding proteins that mimic the function of the normal regulatory factors along the mitogenic pathway, growth factors, their receptors or elements along the postreceptor signaling system. This may be exemplified by the human malignant glioma, in which the sis gene (encoding a growth factor homologous to PDGF) and the erb B gene (encoding a membrane protein homologous to the EGF receptor) have been implicated.

Topics: Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioblastoma; Glioma; Growth Substances; Humans; Middle Aged; Mitosis; Oncogenes; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Brain Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunotherapy; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Suramin; Treatment Outcome; Tumor Cells, Cultured

Tumor cells respond actively to the extracellular microenvironment via biomechanical and biochemical stimulation. Microchips provide a flexible platform to integrate multiple factors for cell research. In this work, we developed a subtle microfluidic chip that can generate a controllable stiffness gradient and orthogonal chemical stimulation to study the behaviors of glioma cells. Fibronectin-conjugated polyacrylamide (PAA) hydrogel with a longitudinal stiffness gradient ranging from about 1 kPa to 40 kPa is integrated within the cell culture chamber while lateral diffusion-based chemical stimulation is generated through circumambient microchannel arrays. The synergistic effect of epidermal growth factor (EGF) stimulation and hydrogel stiffness gradient on U87-MG cell migration was studied. By tracing cell migration, we discovered that hydrogel stiffness can promote cell chemotaxis, while the EGF gradient can accelerate cell migration. In addition, cell morphology showed typical cell spreading, increased aspect ratios, and decreased circularity in response to a stiffer substrate and plateaued at a certain stiffness level. Meanwhile, the content of intracellular reactive oxygen species (ROS) on the hydrogel soft end is enhanced by approximately 2 fold compared with that on the hydrogel stiff end. The enhancement of substrate stiffness on cell chemotaxis is very significant for in vitro model simulation and tissue engineering.

Topics: Acrylic Resins; Biocompatible Materials; Biomechanical Phenomena; Biosensing Techniques; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Elasticity; Epidermal Growth Factor; Equipment Design; Fibronectins; Glioma; Humans; Hydrogels; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Tumor Microenvironment

Tumor-associated microglial cells promote glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Milk fat globule EGF factor 8 protein (MFG-E8), a secreted glycoprotein, is closely related to tissue homeostasis and anti-inflammation. In the present study, we investigated the role of MFG-E8 in microglial polarization and glioma progression in vitro and in vivo. We found that glioma cells secrete comparable amounts of MFG-E8 in culture media to astrocytes. Recombinant MFG-E8 triggered microglia to express the M2 polarization markers, such as arginase-1 (ARG-1), macrophage galactose-type C-type lectin-2 (MGL-2), and macrophage mannose receptor (CD206). Forced expression of MFG-E8 in BV-2 microglia cells not only promoted IL-4-induced M2 polarization but also inhibited lipopolysaccharide (LPS)-induced M1 microglial polarization. Mechanistic studies demonstrated that recombinant MFG-E8 markedly induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the STAT3 inhibitor stattic significantly blocked MFG-E8-induced ARG-1 expression. Administration of antibody against MFG-E8 and knockdown of its receptor, integrin β3, significantly attenuated MFG-E8-induced ARG-1 expression. Similarly, knockdown of MFG-E8 also markedly reduced IL-4-induced M2 marker expression and increased LPS-induced M1 marker expression in microglia cells. Moreover, the knockdown of MFG-E8 in GL261 glioma cells inhibited cell proliferation and enhanced chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which was likely associated with the downregulation of FAK/AKT activation and STAT3/cyclin D1 signaling. The murine GL261 glioma experimental model demonstrated that knockdown of MFG-E8 significantly reduced tumor size and extended survival times. Additionally, attenuated CD11b

Topics: Animals; Antigens, Surface; Astrocytes; Cell Proliferation; Epidermal Growth Factor; Glioma; Humans; Lectins, C-Type; Mannose Receptor; Mannose-Binding Lectins; Mice, Inbred C57BL; Microglia; Milk Proteins; Receptors, Cell Surface; STAT3 Transcription Factor; Tumor Microenvironment

Glioma, the most frequent primary tumor of the central nervous system, has poor prognosis. The epidermal growth factor receptor (EGFR) pathway and angiogenesis play important roles in glioma growth, invasion, and recurrence. The present study aimed to use proteomic methods to probe into the role of the EGF-EGFR-angiogenesis axis in the tumorigenesis of glioma and access the therapeutic efficacy of selumetinib on glioma.. Proteomic profiling was used to characterize 200 paired EGFR-positive and EGFR-negative glioma tissues of all pathological types. The quantitative mass spectrometry data were used for systematic analysis of the proteomic profiles of 10 EGFR-positive and 10 EGFR-negative glioma cases. Consensus-clustering analysis was used to screen target proteins. Immunofluorescence analysis, cell growth assay, and intracranial xenograft experiments were used to verify and test the therapeutic effect of selumetinib on glioma.. Advanced proteomic screening demonstrated that the expression of EGF-like domain multiple 7 (EGFL7) was higher in EGFR-positive tumor tissues than in EGFR-negative tumor tissues. In addition, EGFL7 could act as an activator in vitro and in vivo to promote glioma cell proliferation. EGFL7 was associated strongly with EGFR and prognosis. EGFL7 knockdown effectively suppressed glioma cell proliferation. Selumetinib treatment showed tumor reduction effect in EGFR-positive glioblastoma xenograft mouse model.. EGFL7 is a potential diagnostic biomarker and therapeutic target of glioma. Selumetinib could target the EGFR pathway and possibly improve the prognosis of EGFR-positive glioma.

Topics: Adult; Animals; Benzimidazoles; Calcium-Binding Proteins; Cell Movement; EGF Family of Proteins; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; Humans; Male; Mice; Neoplasm Recurrence, Local; Proteomics; Xenograft Model Antitumor Assays

Annexin A2 (ANXA2) acts as a calcium‑dependent phospholipid‑binding protein that is widely expressed in vertebrate cells and has abnormally high expression in various tumor cells. However, the detailed molecular mechanism underlying the effects of ANXA2 on glioma cells remains unclear. The present study aimed to investigate the role and underlying molecular mechanisms of ANXA2 in glioma cell proliferation. The results revealed that knockdown of ANXA2 inhibited the proliferation of U251 and U87 glioma cell lines and decreased phosphorylated (p) signal transducer and activator of transcription 3 (STAT3)(Y705) and cyclin D1 expression, leading to impedance of the G1‑to‑S phase transition. Furthermore, it was suggested that ANXA2 may regulate pSTAT3(Y705) levels through direct binding with STAT3, thereby affecting STAT3‑cyclin D1 pathway‑mediated cell proliferation. When ANXA2 was re‑expressed in ANXA2‑knockdown cells, the expression of pSTAT3(Y705) and cyclin D1 was restored. Furthermore, overexpression of ANXA2 significantly promoted the proliferation of U251 cells, as determined by an MTT assay and a tumor formation assay in nude mice, but had no statistically significant effect on colony formation rate, cell cycle progression or the STAT3‑cyclin D1 pathway, suggesting that endogenous ANXA2 may be redundant. Additionally, the present study provided evidence that the overexpression of ANXA2 enhanced the expression of pSTAT3(Y705) in the presence of epidermal growth factor (EGF), indicating that the proliferation‑promoting effect of ANXA2 may be due to the accumulation and synergistic effect of paracrine EGF. Taken together, the present results indicated that ANXA2 may affect the proliferation of human glioma cells through the STAT3‑cyclin D1 pathway via direct interaction with STAT3 in U251 and U87 glioma cells. ANXA2 was redundant in this pathway, but positive synergy was revealed to exist between ANXA2 and EGF.

Topics: Animals; Annexin A2; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Glioma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Signal Transduction; STAT3 Transcription Factor

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

Topics: Amino Acid Transport System y+; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Disease Progression; Dizocilpine Maleate; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Glioma; Glutamic Acid; Humans; Mice; Neoplasm Transplantation; Phosphorylation; Protein Domains; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Sulfasalazine

Shc family signalling adaptors connect activated transmembrane receptors to proximal effectors, and most also contain a sequence involved in clathrin-mediated receptor endocytosis. Notably, this AP2 adaptin-binding motif (AD) is absent from the ShcD (also known as Shc4) homolog, which also uniquely promotes ligand-independent phosphorylation of the epidermal growth factor receptor (EGFR). We now report that cultured cells expressing ShcD exhibit reduced EGF uptake, commensurate with a decrease in EGFR surface presentation. Under basal conditions, ShcD colocalises with the EGFR and facilitates its phosphorylation, ubiquitylation and accumulation in juxtanuclear vesicles identified as Rab11-positive endocytic recycling compartments. Accordingly, ShcD also functions as a constitutive binding partner for the E3 ubiquitin ligase Cbl. EGFR phosphorylation and focal accumulation likewise occur upon ShcD co-expression in U87 glioma cells. Loss of ShcD phosphotyrosine-binding function or insertion of the ShcA AD sequence each restore ligand acquisition through distinct mechanisms. The AD region also contains a nuclear export signal, indicating its multifunctionality. Overall, ShcD appears to possess several molecular permutations that actively govern the EGFR, which may have implications in development and disease.

Topics: Adaptor Protein Complex Subunits; Amino Acid Motifs; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Shape; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Ligands; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Domains; Protein Transport; Proto-Oncogene Proteins c-cbl; rab GTP-Binding Proteins; Shc Signaling Adaptor Proteins; Subcellular Fractions; Transport Vesicles; Ubiquitination

The epidermal growth factor (EGF) receptor EGFR is a major receptor tyrosine kinase whose role in gliomagenesis is well established. We have recently identified EHD3 [Eps15 homology (EH) domain-containing protein 3], an endocytic trafficking regulatory protein, as a putative brain tumor suppressor. Here, we investigate the underlying mechanisms, by establishing a novel mechanistic and functional connection between EHD3 and the EGFR signaling pathway. We show that, in response to stimulation with the EGF ligand, EHD3 accelerates the rate of EGFR degradation by dramatically increasing its ubiquitination. As part of this process, EHD3 also regulates EGFR endosomal trafficking by diverting it away from the recycling route into the degradative pathway. Moreover, we found that upon EGF activation, rather than affecting the total MAPK and AKT downstream signaling, EHD3 decreases endosome-based signaling of these two pathways, thus suggesting the contribution of EHD3 in the spatial regulation of EGFR signaling. This function explains the higher sensitivity of EHD3-expressing cells to the growth-inhibitory effects of EGF. In summary, this is the first report supporting a mechanism of EHD3-mediated tumor suppression that involves the attenuation of endosomal signaling of the EGFR oncogene.

Topics: Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Endosomes; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; Mutation; Protein Transport; Proteolysis; Proto-Oncogene Proteins c-akt; Signal Transduction; Ubiquitination

Activation of epidermal growth factor receptor (EGFR), which occurs in many types of tumour, promotes tumour progression. However, no extracellular antagonist of human EGFR has been identified. We found that human macrophage migration inhibitory factor (MIF) is O-GlcNAcylated at Ser 112/Thr 113 at its carboxy terminus. The naturally secreted and O-GlcNAcylated MIF binds to EGFR, thereby inhibiting the binding of EGF to EGFR and EGF-induced EGFR activation, phosphorylation of ERK and c-Jun, cell invasion, proliferation and brain tumour formation. Activation of EGFR through mutation or its ligand binding enhances the secretion of MMP13, which degrades extracellular MIF, and results in abrogation of the negative regulation of MIF on EGFR. The finding that EGFR activation downregulates its antagonist in the tumour microenvironment represents an important feedforward mechanism for human tumour cells to enhance EGFR signalling and promote tumorigenesis.

Topics: Acetylglucosamine; Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Macrophage Migration-Inhibitory Factors; Matrix Metalloproteinase 13; Mice, Nude; Microscopy, Fluorescence; Mutation; Phosphorylation; Protein Binding; RNA Interference; Serine; Survival Analysis; Threonine; Transplantation, Heterologous

The aim of the present study was to investigate the extensive invasion of tumor cells into normal brain tissue, a life‑threatening feature of malignant gliomas. How invasive tumor cells migrate into normal brain tissue and form a secondary tumor structure remains to be elucidated. In the present study, the morphological and phenotypic changes of glioma cells during invasion in a C6 glioma model were investigated. C6 glioma cells were stereotactically injected into the right putamen region of adult Sprague‑Dawley rats. The brain tissue sections were then subjected to hematoxylin and eosin, immunohistochemical or immunofluorescent staining. High magnification views of the tissue sections revealed that C6 cells formed tumor spheroids following implantation and marked invasion was observed shortly after spheroid formation. In the later stages of invasion, certain tumor cells invaded the perivascular space and formed small tumor clusters. These small tumor clusters exhibited certain common features, including tumor cell multilayers surrounding an arteriole, which occurred up to several millimeters away from the primary tumor mass; a high proliferation rate; and similar gene expression profiles to the primary tumor. In conclusion, the present study revealed that invading tumor cells are capable of forming highly proliferative cell clusters along arterioles near the tumor margin, which may be a possible cause of the recurrence of malignant glioma.

Topics: Animals; Arterioles; Biomarkers, Tumor; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Eosine Yellowish-(YS); Epidermal Growth Factor; Gene Expression; Glioma; Hematoxylin; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Neoplasm Transplantation; Nerve Growth Factor; Putamen; Rats; Rats, Sprague-Dawley; Spheroids, Cellular; Staining and Labeling; Stereotaxic Techniques; Twist-Related Protein 1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Host malignant stromal cells induced by glioma stem/progenitor cells were revealed to be more radiation-resistant than the glioma stem/progenitor cells themselves after malignant transformation in nude mice. However, the mechanism underlying this phenomenon remains unclear.. Malignant stromal cells induced by glioma stem/progenitor cell 2 (GSC-induced host brain tumor cells, ihBTC2) were isolated and identified from the double color-coded orthotopic glioma nude mouse model. The survival fraction at 2 Gy (SF2) was used to evaluate the radiation resistance of ihBTC2, the human glioma stem/progenitor cell line SU3 and its radiation-resistant sub-strain SU3-5R and the rat C6 glioma cell line. The mRNA of Notch 1 and Hes1 from ihBTC2 cells were detected using qPCR before and after 4 Gy radiation. The expression of the Notch 1, pAkt and Bcl-2 proteins were investigated by Western blot. To confirm the role of the Notch pathway in the radiation resistance of ihBTC2, Notch signaling blocker gamma secretase inhibitors (GSIs) were used.. The ihBTC2 cells had malignant phenotypes, such as infinite proliferation, hyperpentaploid karyotype, tumorigenesis in nude mice and expression of protein markers of oligodendroglia cells. The SF2 of ihBTC2 cells was significantly higher than that of any other cell line (P<0.05, n = 3). The expression of Notch 1 and Hes1 mRNAs from ihBTC2 cells was significantly increased after radiation. Moreover, the Notch 1, pAkt and Bcl-2 proteins were significantly increased after radiation (P<0.05, n = 3). Inhibition of Notch signaling markedly enhanced the radiosensitivity of ihBTC2 cells.. In an orthotopic glioma model, the malignant transformation of host stromal cells was induced by glioma stem/progenitor cells. IhBTC2 cells are more radiation-resistant than the glioma stem/progenitor cells, which may be mediated by activation of the Notch signaling pathway.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Glioma; Humans; Karyotyping; Mice; Mice, Nude; Neoplastic Stem Cells; Phenotype; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Rats; Receptor, Notch1; Signal Transduction

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION-EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION-EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION-EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION-EGF conjugates in animals provided receptor-mediated targeted delivery across the blood-brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION-EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Dextrans; Epidermal Growth Factor; Glioma; Magnetite Nanoparticles; Nanocapsules; Rats; Recombinant Proteins; Treatment Outcome

We have characterised, using both in vivo and in vitro methods, the effects of the metabotropic glutamate receptor subtype 3 (mGlu3) antagonist (LY341495) and agonist (LY379268) on the proliferation and differentiation of glioma stem cells (GSC). For in vitro studies, a CCK-8 assay was used to determine the cell proliferation, flow cytometry was performed to determine cell cycle phases, and immunohistochemistry and laser confocal microscopy were employed to detect CD133 expression. For in vivo studies, GSCs were injected into nude mice treated with either LY379268 or LY341495 and the growth of the tumours was measured after 3 weeks. When compared with controls, the proliferation rates and proportion of cells in S phase within the LY341495 treated group decreased in a time-dependent manner. In the presence of differentiation medium containing LY341495, GSC differentiation was diverted into an astrocyte rather than neuronal phenotype. The growth rate and volume of tumours injected into nude mice was reduced in LY341495 treated mice compared with controls. Thus pharmacological blockade of mGlu3 receptor signalling pathway significantly inhibits the growth and proliferation of GSCs both in vitro and in vivo while promoting differentiation to astrocytes. These results further implicate mGlu3 in the biology of glioma and as a target for continued research.

Topics: AC133 Antigen; Amino Acids; Animals; Antigens, CD; Astrocytes; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Glycoproteins; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Peptides; Phenotype; Receptors, Metabotropic Glutamate; Recombinant Proteins; Transplantation, Heterologous; Xanthenes

G19 is a novel homogeneous sulfated oligosaccharide, prepared from Grateloupia filicina. In the present study, we first reported that oligosaccharide G19 exhibited a dose- and time-dependent anti-proliferation effect against U-87 malignant gliomas (MG) human glioma cells. Further studies indicated that G19 strongly bound to epidermal growth factor (EGF), suppressed EGF receptor phosphorylation and interrupted the phosphatidylinositol-3 kinase/Akt pathway in the cancer cells. Moreover, G19 elevated intracellular reactive oxygen species levels and caused endogenous DNA damage. These actions were associated with activation of ataxia-telangiectasia-mutated/checkpoint kinase 2 pathway. The downregulation of MDM2 with stabilizing p53 and the nuclear location of p21 were induced by G19 to cause cell cycle arrest and apoptosis to some extent. Meanwhile, intrinsic mitochondrial pathway and extrinsic death receptor pathway were involved in G19-mediated apoptosis. Pretreatment with free radical scavenger N-acetyl-l-cysteine nearly completely inversed G19-induced cell growth inhibition, cell cycle arrest and apoptosis in U-87 MG cells. Importantly, G19 could inhibit the growth of U-87 MG tumor cells xenograft in nude mice. The results suggested that G19 could be served as a new targeting drug candidate for human glioma treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Glioma; Humans; Molecular Targeted Therapy; Oligosaccharides; Rhodophyta; Signal Transduction; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1α (HIF-1α) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1α was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1α protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1α and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1α and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1α and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Phosphatidic Acids; Phospholipase D; Signal Transduction; Transfection; Vascular Endothelial Growth Factor A

Cyclooxygenase-2 (COX-2) is linked to poor prognosis in patients with malignant gliomas. Amplification/overexpression of epidermal growth factor receptor (EGFR) is commonly seen in these tumors. EGFR signaling, through activation of the p38-MAPK/PKC-δ/Sp1 cascade, plays an essential role in the regulation of COX-2 expression in glioma cells. Here, we report that Src kinase contributes upstream to this signaling cascade. In addition, more detailed analysis revealed the involvement of FOXM1, a member of the forkhead box family of transcriptional activators, in EGF-dependent COX-2 induction. FOXM1 protein increased after stimulation with EGF, although its role in modulating COX-2 expression does not depend on this increase. While a conventional FOXM1 responsive element resides in a distal region (-2872/-2539 relative to the transcriptional start site) of the COX-2 promoter, this is not required for EGF-dependent induction of COX-2. Instead, FOXM1 forms a cooperative interaction with Sp1 at the Sp1-binding site (-245/-240 relative to the start site) of the COX-2 promoter to mediate EGF-induced COX-2 expression. Definition of this novel interaction provides a clearer understanding of the mechanistic basis for EGF induction of COX-2.. These data provide a guide for the evaluation of potential newer therapeutic targets that have relevance in this disease.

Topics: Cell Line, Tumor; Cyclooxygenase 2; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Response Elements; Signal Transduction; Sp1 Transcription Factor; src-Family Kinases

Glioma malignancy greatly depends on its aggressive invasion. The establishment of cell polarity is an important initial step for cell migration, which is essential for cell-directional translocation. However, our understanding of the molecular mechanisms underlying cell polarity formation in glioma cell invasion remains limited. Glycogen synthase kinase-3 (GSK-3) has a critical role in the formation of cell polarity. We therefore investigated whether localized GSK-3β, a subtype of GSK-3, is important for glioma cell invasion. We reported here that the localized phosphorylation of GSK-3β at the Ser9 (pSer9-GSK-3β) was critical for glioma cell invasion. Scratching glioma cell monolayer up-regulated pSer9-GSK-3β specifically at the wound edge. Inhibition of GSK-3 impaired the cell polarity and reduced the directional persistence of cell migration. Consistently, down-regulation of GSK-3α and 3β by specific small interfering RNAs inhibited glioma cell invasion. Over-expressing wild-type or constitutively active forms of GSK-3β also inhibited the cell invasion. These results indicated the polarized localization of GSK-3 regulation in cell migration might be also important for glioma cell migration. Further, EGF regulated both GSK-3α and 3β, but only pSer9-GSK-3β was enriched at the leading edge of scratched glioma cells. Up- or down-regulation of GSK-3β inhibited EGF-stimulated cell invasion. Moreover, EGF specifically regulated GSK-3β, but not GSK-3α, through atypical PKC pathways. Our results indicated that GSK-3 was important for glioma cell invasion and localized inhibition of GSK-3β was critical for cell polarity formation.

Topics: Cell Line, Tumor; Cell Polarity; Enzyme Inhibitors; Epidermal Growth Factor; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Protein Kinase C; Protein Kinase Inhibitors

Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma.. Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown.. EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α.. EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

Topics: Animals; Anthracenes; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Autocrine Communication; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cetuximab; Endoribonucleases; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Gene Expression; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Mice; Protein Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Epidermal growth factor (EGF) plays a key role in survival of neural and glial precursor cells. A single nucleotide polymorphism of the EGF gene +61G/A in the 5'-untranslated region has been reported to be associated with susceptibility to glioma. The purpose of this study was to investigate the potential association between EGF +61G/A and brain glioma in a Chinese population. A case-control study involving 180 patients with glioma and 360 controls was done. Polymerase chain reaction restriction fragment-length polymorphism assay was used to analyze the EGF +61G/A genotypes. Patients with glioma had a significantly higher frequency of AA genotype (odds ratio, 2.25; 95% confidence interval, 1.20, 4.21; p=0.01] than controls. When stratified by histologic features and World Health Organization grade of glioma, distribution of each genotype did not significantly differ. Our data suggested that the EGF +61G AA genotype was associated with a higher glioma risk in a Chinese population. This finding is in contrast with previous studies that reported the G allele as a risk factor for glioma in white patients.

Topics: Adult; Asian People; Brain Neoplasms; Case-Control Studies; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Glioma; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Factors

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

Topics: Brain Neoplasms; Cell Adhesion; Cell Migration Assays; Cell Movement; Epidermal Growth Factor; Epithelial Cells; Glioma; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; Molecular Chaperones; Osteonectin; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Binding; Protein Folding; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Deletion; Tumor Cells, Cultured

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Neoplasm Invasiveness; Nonmuscle Myosin Type IIA; Phosphorylation; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Signal Transduction

Malignant gliomas are associated with alteration in EGF/EGFR signaling. Functional EGF+61A>G polymorphism is implicated with risk, recurrence, and progression of glioma. This study aimed to establish a putative association of EGF+61A>G with risk of glioma development, production of angiogenic growth factor EGF, and the response to perillyl alcohol administered by intranasal route.. The study included 83 patients with recurrent glioma enrolled in Phase I/II trial for intranasal perillyl alcohol therapy and subjects without cancer (n = 196) as control group. DNA was extracted from blood samples, EGF genotype performed with PCR-RFLP assay, and EGF circulating levels by enzyme immunoassay. Adequate statistical tests were performed to verify associations between polymorphism and glioma risk, and genotype correlation with EGF circulating levels. The log-rank test was also used to evaluate differences on patient survival.. Patients with primary glioblastoma had high frequency of AA genotype (p = 0.037) and A allele (p = 0.037). Increased EGF circulating levels were observed in glioma patients with AA (p = 0.042), AG (p = 0.006), and AA + AG (p = 0.008) genotypes compared with GG. Patients with GG genotype showed increased but not significant (p > 0.05) survival rate, and EGF levels lower than 250 pg/mL was consistently (p = 0.0374) associated with increased survival.. Presence of EGF+61A>G polymorphism in Brazilian subjects was associated with glioma risk and increased circulating EGF levels. Better response to perillyl alcohol-based therapy was observed in a group of adult Brazilian subjects with lower EGF levels.

Topics: Administration, Intranasal; Adolescent; Adult; Aged; Aged, 80 and over; Alleles; Brain Neoplasms; Brazil; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Epidermal Growth Factor; Female; Gene Frequency; Genotype; Glioma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Monoterpenes; Polymorphism, Single Nucleotide; Prospective Studies; Survival Analysis; Treatment Outcome; Young Adult

Studies of genetic mutations that have been used in predicting glioma prognosis have revealed a complex relationship between clinical and genetic factors. Epidermal growth factor (EGF) and the NAT2 gene play a central role in carcinogenesis. An adenine (A) to guanine (G) single nucleotide polymorphism at position 61 in the 5'-untranslated region (5'-UTR) of the EGF gene has been found to be associated with levels of EGF production, and the mutations in the NAT2 gene have been postulated as a risk factor for cancer. We investigated EGF and the NAT2 gene in 13 glioma tissue samples and 12 normal controls. In the EGF 5'-UTR 61G polymorphism, the heterozygote GA was the most common genotype in the glioma patients. In the NAT2 polymorphism at nucleotide position 857G/A, the G allele and the GG genotype were the most prevalent forms in both the glioma and normal samples. We did not find any homozygous AA genotypes in the glioma patients. Based on this preliminary evidence, the EGF 5'-UTR at position 61 and the NAT2 SNP at position 857 polymorphisms are associated with increased risk for glioma.

Topics: 5' Untranslated Regions; Alleles; Arylamine N-Acetyltransferase; Brain Neoplasms; Case-Control Studies; Electrophoresis, Agar Gel; Epidermal Growth Factor; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Glioma; Humans; Malaysia; Nucleotides; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide

Epidermal growth factor (EGF), a potent mitogenic protein, plays an important role in the development of cancers, including glioma. Previous studies showed that the EGF +61G/A polymorphism (rs4444903) may lead to an alteration in EGF production and/or activity, which can result in individual susceptibility to glioma. However, published data regarding the association between the +61G/A polymorphism and glioma risk was contradictory.. The aim of this study was to perform a meta-analysis of eligible studies to derive precise estimation of the association of EGF +61G/A with glioma risk.. We performed a pooled analysis of seven published studies that included 1,613 glioma cases and 2,267 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. The pooled ORs were performed for codominant model, dominant model, and recessive model, respectively.. Overall, no significant associations between the EGF +61G/A polymorphism and glioma cancer risk were found for AA versus GG (OR=0.95, 95% CI=0.62-1.45), GA versus GG (OR=0.94, 95% CI=0.72-1.22), AA/GA versus GG (OR=0.93, 95% CI=0.70-1.23), and AA versus GA/GG (OR=1.04, 95% CI=0.77-1.39). However, in the stratified analysis by ethnicity, the EGF +61G/A polymorphism had a higher risk of glioma development among Asians, but a lower risk among Caucasians.. Taken together, the results suggest that the EGF +61G/A polymorphism may contribute to the susceptibility of glioma in different ethnic groups.

Topics: Asian People; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Glioma; Humans; Male; Polymorphism, Single Nucleotide

Gap junctions (GJs) are sites of direct cell-to-cell communication formed by the connexin (Cx) family of ion channel proteins. The aberrant intercellular communication mediated by GJs is associated with a variety of hereditary and acquired human diseases. GJs utilize a highly interconnected network that is indispensible for synthesis, trafficking and degradation of their constituent proteins. By unbiased proteomic examination and network enrichment, we identified interacting components of the ubiquitin proteasome system associated with Cx43. LC-MS/MS identification and quantification of tryptic peptides from IP materials revealed a variety of interacting candidates, including the E3 ligase TRIM21 and ubiquitin. The interaction of Cx43 with TRIM21 was confirmed by confocal microscopy and coimmunoprecipitation of these proteins from C6 rat glioma and mouse primary astrocyte cultures. To gain a better understanding of this interaction, complexes isolated by high-resolution size-exclusion chromatography revealed signal integration by phosphorylation, ubiquitylation and proteolytic turnover within complexes of Cx43/TRIM21. Cx43/TRIM21 is also responsive to E1 UBE1 and E2 UbcH5a, with the interruption of this activity being an effective inhibitor of in vitro ubiquitin-conjugation. Mathematical models of these complexes demonstrated a mechanism for the switch-like degradation of GJs that were validated in EGF-stimulated cell cultures. Our finding of the interaction of Cx43 with TRIM21 provides mechanisms for the down-regulation of GJ intercellular communication that are known to impact a variety of physiological processes.

Topics: Animals; Astrocytes; Cell Communication; Cell Membrane; Chromatography, Gel; Connexin 43; Epidermal Growth Factor; Gap Junctions; Glioma; Immunoprecipitation; Mice; Microscopy, Confocal; Models, Biological; Phosphorylation; Primary Cell Culture; Proteasome Endopeptidase Complex; Protein Binding; Protein Interaction Mapping; Protein Interaction Maps; Protein Transport; Proteolysis; Proteomics; Rats; Tandem Mass Spectrometry; Transfection; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ubiquitination

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; Cholesterol; Epidermal Growth Factor; Glioma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Membrane Microdomains; Membrane Proteins; Neoplasm Metastasis; Neoplasm Proteins

Glioblastoma multiforme (GBM) is an aggressive cancer with a poor survival rate. A key component that contributes to the poor prognosis is the capacity of glioma cells to invade local brain tissue in a diffuse manner. Among various proteases that aid in the process of invasion, matrix metalloproteinase-1 (MMP-1) has been identified as an important contributory factor in various cancers. Apart from its traditional role in cleaving its primary extracellular matrix (ECM) substrates, and like other members of the matrix metalloproteinase family, MMP-1 can activate latent forms of bio-active molecules initiating downstream pro-invasive and pro-oncogenic signaling mechanisms. MMP-1 expression is regulated by several growth factors including epidermal growth factor (EGF). Due to the fact that the epidermal growth factor receptor (EGFR) is aberrantly overexpressed in GBM, we wanted to examine in greater detail the signaling mechanisms by which MMP-1 expression and invasion is driven by EGF in GBM cells. T98G cells treated with EGF resulted in an induction of MMP-1 expression following EGFR activation. Inhibition of EGFR by both pharmacologic and genetic approaches abrogated this induction. Repression of the mitogen activated protein kinase (MAPK) signaling led to the inhibition of EGF-induced MMP-1 whereas the PI3-kinase/AKT signaling was not associated with EGFR-mediated MMP-1 induction. Inhibition of EGFR signaling also led to a decrease in T98G invasion. These data suggest that EGFR mediated MMP-1 regulation is mainly via the MAPK pathway in T98G cells and inhibition of EGFR and MMP-1 results in a decrease in T98G cell invasion.

Topics: Benzamides; Cell Line, Tumor; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioma; Humans; Laminin; Matrix Metalloproteinase 1; Mitogen-Activated Protein Kinase Kinases; Proteoglycans; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection

The majority of malignant primary brain tumors are gliomas, derived from glial cells. Grade IV gliomas, Glioblastoma multiforme, are extremely invasive and the clinical prognosis for patients is dismal. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. In this study, we investigated the localization and functional relevance of transient receptor potential canonical (TRPC) channels in glioma migration. We show that gliomas are attracted in a chemotactic manner to epidermal growth factor (EGF). Stimulation with EGF results in TRPC1 channel localization to the leading edge of migrating D54MG glioma cells. Additionally, TRPC1 channels co-localize with the lipid raft proteins, caveolin-1 and β-cholera toxin, and biochemical assays show TRPC1 in the caveolar raft fraction of the membrane. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knockdown of TRPC1 channels, yet without affecting unstimulated cell motility. Moreover, lipid raft integrity was required for gliomas chemotaxis. Disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents in whole cell recordings and decreased store-operated calcium entry as revealed by ratiomeric calcium imaging. These data indicated that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF.

Topics: Brain Neoplasms; Calcium Signaling; Caveolin 1; Cell Line, Tumor; Chemotaxis; Cholera Toxin; Cholesterol; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Membrane Microdomains; Membrane Potentials; Membrane Transport Modulators; Neoplasm Invasiveness; Patch-Clamp Techniques; RNA Interference; TRPC Cation Channels

Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.. STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.. STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.. Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

Topics: Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Databases, Factual; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Glioma; Humans; Neoplasm Invasiveness; Phosphorylation; RNA, Small Interfering; STAT6 Transcription Factor; Survival Analysis; Tyrosine

EGF-modified Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide-targeted Au NPs. Compared to the untargeted conjugates, EGF-Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor. This delivery system holds promise for future delivery of a wider range of hydrophobic therapeutic drugs for the treatment of hard-to-reach cancers.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Delayed-Action Preparations; Epidermal Growth Factor; ErbB Receptors; Glioma; Gold; Humans; Hydrophobic and Hydrophilic Interactions; Indoles; Isoindoles; Metal Nanoparticles; Mice; Nanocapsules; Nanoconjugates; Photochemotherapy; Photosensitizing Agents; Treatment Outcome

Stem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors.. Primary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots.. SLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe.. Our results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53-deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumor cells.

Topics: Apoptosis; Brain Neoplasms; Cell Proliferation; Cell Survival; DNA Damage; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gamma Rays; Glioma; Histones; Humans; Mitosis; Neoplastic Stem Cells; Radiation Tolerance; Radiation, Ionizing

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

Topics: AC133 Antigen; Antigens, CD; Cell Division; Cell Lineage; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Glycoproteins; Humans; Laminin; Mitosis; Neoplastic Stem Cells; Peptides

For the treatment of cancer, the therapeutic potential of short-range, low-energy Auger-electron emitters, such as (125)I, is getting progressively wider recognition. The potency of Auger-electron emitters is strongly dependent on their location in close vicinity to DNA. We have developed a new two-step targeting strategy to transport (125)I into cancer-cell nuclei using PEG-stabilized tumour-cell targeting liposomes named "Nuclisome-particles".. In the present study, epidermal growth factor (EGF) was used as a tumour-cell-specific agent to target the EGF-receptor (EGFR) and the liposomes were loaded with (125)I-Comp1, a recently synthesized daunorubicin derivative.. As analysed with cryo-TEM, the derivative precipitates inside liposomes at a drug-to-lipid molar ratio of 0.05:1. Receptor-specific uptake in cultured U-343MGaCl2:6 tumour cells of EGFR-targeting liposomes increased with time while non-specific and receptor-blocked uptake remained low. Nuclisome-particles were able to target single U-343MGaCl2:6 cells circulating in human blood during 4 h, with low uptake in white blood cells, as demonstrated in an ex vivo system using a Chandler loop. Autoradiography of targeted cells indicates that the grains from the radiolabelled drug are mainly co-localized with the cell nuclei. The successful targeting of the nucleus is shown to provide high-potency cell killing of cultured U-343MGaCl2:6 cells. At the concentration used, Nuclisome-particles were up to five orders of magnitude more effective in cell killing than EGFR-targeting liposomes loaded with doxorubicin.. The results thus provide encouraging evidence that our two-step targeting strategy for tumour cell DNA has the potential to become an effective therapy against metastasizing cancer cells in the bloodstream.

Topics: Cell Line, Tumor; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Liposomes; Polyethylene Glycols; Radiopharmaceuticals

Uncontrolled growth and diffused invasion are major causes of mortality in patients with malignant gliomas. Nodal has been shown to have a central role in the tumorigenic signaling pathways of malignant melanoma. In this study, we show that grade IV human glioma cell lines expressed different levels of Nodal, paralleled to the potential for cell invasiveness. Treatment of glioma cell lines with recombinant Nodal (rNodal) increased matrix metalloproteinase 2 (MMP-2) secretion and cell invasiveness. The ectopic expression of Nodal in GBM glioma cells that expressed Nodal at low level resulted in increased MMP-2 secretion, enhanced cell invasiveness, raised cell proliferation rates in vitro, increased tumor growth in vivo, and was associated with poor survival in a mice xenograft model. In contrast, the knockdown of Nodal expression in U87MG glioma cells with high Nodal expression level had reduced MMP-2 secretion, less cell invasiveness, lower tumor growth in vivo and longer lifespan in mice with U87MG/shNodal cell xenografts. In addition, Nodal knockdown promoted the reversion of malignant glioma cells toward a differentiated astrocytic phenotype. Furthermore, our data support the notion that Nodal may regulate glioma progression through the induction of the leukemia inhibitory factor (LIF) and Cripto-1 through activated Smad.

Topics: Animals; Cell Division; Cell Line, Tumor; Central Nervous System Neoplasms; Disease Progression; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Glioma; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Leukemia Inhibitory Factor; Matrix Metalloproteinase 2; Membrane Glycoproteins; Membrane Proteins; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Recombinant Proteins; Smad Proteins; Tissue Inhibitor of Metalloproteinase-2; Transfection

To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas.. Paraffin sections of the original surgical specimens, primary glioma spheroids, and U87 derived spheroids were stained immunohistochemically with the stem cell markers CD133, podoplanin, Sox2, Bmi-1, and nestin; the endothelial cell markers CD31, CD34, and Von Willebrand Factor (VWF); the chemosensitivity markers P-glycoprotein and tissue inhibitor of metalloproteinases-1 (TIMP-1); and glial fibrillary acidic protein, neural cell adhesion molecule CD56, and the proliferation marker Ki67.. Scoring of the immunohistochemical stainings showed that the expression of CD133 and all other markers included was preserved in primary spheroids, confirming the in vivo-like nature of these spheroids. Spheroids in SFM better mimicked the in vivo phenotype with significantly more CD133, CD34, VWF, P-glycoprotein, TIMP-1, and Ki67 compared with SCM.. In this first study of the influence of SFM on primary glioma spheroids, the conditions favored an in vivo-like phenotype with increased expression of CD133. More vascular structures were found in SFM, suggesting that the close relationship between blood vessels and tumor stem-like cells was better preserved in this medium.

Topics: Adult; Aged; Biomarkers, Tumor; Brain Neoplasms; Cell Culture Techniques; Culture Media; Culture Media, Serum-Free; Endothelial Cells; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Phenotype; Spheroids, Cellular; Stem Cells; Tumor Cells, Cultured

The antiproliferative effect of tandem somatostatin receptor (SSTR) activation, epidermal growth factor receptor (EGFR) inhibition, and induction of DNA damage was analyzed using octreotide (OCT), a SSTR agonist, the clinical DNA methylating agent temozolomide (TMZ), Iressa, an EGFR inhibitor, and dual EGFR-DNA targeting agents termed "combi-molecules". Using SSTR-expressing glioma cells harbouring low levels of EGFR (U87MG) or transfected to overexpress EGFR (U87/EGFR) or a variant (U87/EGFRvIII), we showed that Iressa, alone or in combination with the DNA damaging agent TMZ, and combi-molecules RA2 and RA5 inhibited EGF-induced phosphorylation of EGFR in U87MG and more moderately in U87/EGFR and U87/EGFRvIII transfected cells. This translated into equivalent levels of Erk 1/2 inhibition. Activation of SSTRs with OCT did not modulate the effects of the various treatments on Erk 1/2 phosphorylation. Likewise, SSTR activation did not alter TMZ- or DNA-damaging combi-molecules, RA2 and RA5, induced p53 activation nor upregulation. However, SSTR activation significantly shifted TMZ-, RA2- and RA5-induced cell-cycle arrest to earlier phases (i.e., G2/M to late S, late S to S, S to G1). Further analysis showed that apoptosis was not induced. This was in agreement with the fact that p53 activation did not induce Bax upregulation nor did EGFR inhibition promote Bad dephosphorylation. Moreover, enhancement of survivin, an anti-apoptotic protein, expression was observed. The results in toto suggest that the combination of SSTR activation with EGFR inhibition and DNA damage affects cell-cycle progression but a disconnection between the targeted signalling pathways in these brain tumour cells precludes synergistic cell-killing by the triple growth inhibitory events.

Topics: Analysis of Variance; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Line, Tumor; Dacarbazine; DNA Damage; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gefitinib; Gene Expression Regulation, Neoplastic; Glioma; Humans; Models, Biological; Mutation; Octreotide; Phosphorylation; Quinazolines; Signal Transduction; Temozolomide; Time Factors; Transfection

Epidermal growth factor (EGF) is critical in cancer process. EGF and EGF receptor (EGFR) interaction plays a pivotal role in cell proliferation, differentiation, and tumorigenesis of epithelial tissues. Variations of the EGF +61G/A (rs4444903) may lead to an alteration in EGF production and/or activity, which can result in individual susceptibility to brain glioma. The purpose of this study was to investigate the potential association between EGF +61G/A and brain glioma in a Chinese population.. In this study, we analyzed single nucleotide polymorphism of EGF +61G/A in 677 patients with glioma and 698 gender- and age-matched controls. Genotyping was performed by polymerase chain reaction-ligation detection reaction (PCR-LDR) method.. The A allele (minor Allele) was 33.0% in cases and 27.3% in controls. The additive model was more powerful to reveal the association in our study than that of recessive and dominant model. Our data showed the genotype G/A and A/A was associated with increased risk for glioma (adjusted OR = 1.48, 95%CI: 1.17-1.87, p = 0.001 for G/A, adjusted OR = 1.81, 95%CI: 1.20-2.72, p = 0.005 for A/A, respectively), and for glioblastoma (adjusted OR = 1.51, 95%CI: 1.06-2.17, p = 0.024 and adjusted OR = 2.35, 95%CI: 1.34-4.15, p = 0.003, respectively). The A allele significantly increased glioma risk (OR = 1.31, 95%CI: 1.11-1.55, p = 0.001). The additive model (G/G vs G/A vs A/A) showed that both G/A and A/A genotype increased glioma risk (adjusted OR = 1.40, 95% CI: 1.17-1.66, p = 0.0002).G/A and A/A genotypes or EGF +61 A allele increased risk in both low and high WHO grade glioma. Non-smokers with G/A and A/A genotype showed increased glioma risk compared with G/G genotype (adjusted OR = 1.72, 95%CI: 1.29-2.30, p = 0.0002 and adjusted OR = 1.81, 95%CI: 1.10-2.99, p = 0.020, respectively). This association was not found in ever- or current-smokers.. Our study indicated that G/A and A/A genotypes or EGF +61 A allele were associated with higher glioma risk in Chinese. This is in contrast with previous studies which reported G allele as a risk factor of glioma in Caucasian. The role of EGF +61 A/G polymorphism in glioma susceptibility needs further investigation.

Topics: Adolescent; Adult; Aged; Asian People; Brain Neoplasms; Case-Control Studies; Chi-Square Distribution; Child; Child, Preschool; China; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Glioma; Humans; Logistic Models; Male; Middle Aged; Odds Ratio; Phenotype; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Risk Assessment; Risk Factors; Smoking; Young Adult

Overexpression and/or amplification of the epidermal growth factor receptor (EGFR) is present in 35-45% of primary glioblastoma multiforme tumors and has been correlated with a poor prognosis. In this study, we investigated the effect of cetuximab and intracellular signaling pathways downstream of EGFR, important for cell survival and proliferation. We show insufficient EGFR downregulation and competition with endogenous EGFR ligands upon cetuximab treatment. Dose-response experiments showed inhibition of EGFR phosphorylation without affecting two of the prominent downstream signaling pathways. Our results indicate that amplification and/or overexpression of EGFR is an unsatisfactory predictor for response to cetuximab.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cell Survival; Cetuximab; Cycloheximide; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioma; Head and Neck Neoplasms; Humans; Phosphorylation; Signal Transduction

Numerous factors that contribute to malignant glioma invasion have been identified, but the upstream genes coordinating this process are poorly known.. To identify genes controlling glioma invasion, we used genome-wide mRNA expression profiles of primary human glioblastomas to develop an expression-based rank ordering of 30 transcription factors that have previously been implicated in the regulation of invasion and metastasis in cancer.. Using this approach, we identified the oncogenic transcriptional repressor, SNAI2/Slug, among the upper tenth percentile of invasion-related transcription factors overexpressed in glioblastomas. SNAI2 mRNA expression correlated with histologic grade and invasive phenotype in primary human glioma specimens, and was induced by EGF receptor activation in human glioblastoma cells. Overexpression of SNAI2/Slug increased glioblastoma cell proliferation and invasion in vitro and promoted angiogenesis and glioblastoma growth in vivo. Importantly, knockdown of endogenous SNAI2/Slug in glioblastoma cells decreased invasion and increased survival in a mouse intracranial human glioblastoma transplantation model.. This genome-scale approach has thus identified SNAI2/Slug as a regulator of growth and invasion in human gliomas.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Staging; Neoplasm Transplantation; Neovascularization, Pathologic; Phenotype; RNA Interference; RNA, Messenger; Snail Family Transcription Factors; Time Factors; Transcription Factors; Transfection; Tumor Burden

Calixarenes are synthetic macrocyclic compounds that may serve as scaffolds for biologically active molecules and have been proposed as potential anticancer agents. We synthesized a ureido-calix[8]arene carrying N-acetyl-D-glucosamine residue (compound 1) and had previously demonstrated that it inhibits C6 glioma cell migration and proliferation, with divergent mechanisms. In the present work we explored in more detail the antiproliferative effect of compound 1, comparing it to related compounds lacking either the sugar moieties (compound 2), the multiple ureido groups (compound 3) or both (compound 4). The results show that the action of compound 1 is independent of the N-acetyl-D-glucosamine residues, requires the presence of multiple ureido groups and does not seem to involve focal adhesion kinase signaling. Inhibition of proliferation is reduced by preincubation with epidermal growth factor (EGF) and vascular endothelial growth factor (20 ng/ml) with compound 1, and extracellular-related kinase phosphorylation is reduced by treatment with compound 1 in both basal and EGF-stimulated conditions, suggesting that the observed effect depends on a direct interference with growth factor signaling.

Topics: Acetylglucosamine; Animals; Antineoplastic Agents; Calixarenes; Cell Division; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Focal Adhesion Kinase 1; Glioma; Phosphorylation; Rats; Signal Transduction; Vascular Endothelial Growth Factor A

A common pathobiological feature of malignant gliomas is the insidious infiltration of single tumor cells into the brain parenchyma, rendering these deadly tumors virtually incurable with available therapies. In this study, we report that ADP-ribosylation factor 6 (ARF6), a Ras superfamily small GTPase, is abundantly expressed in invasive human glioma cells. Cellular depletion of ARF6 by small interfering RNA decreased Rac1 activation, impaired HGF-stimulated and serum-stimulated glioma cell migration in vitro, and markedly decreased the invasive capacity of invasive glioma in the brain. Furthermore, ectopic expression of ARF6 in glioma cells promoted cell migration via the activation of Rac1. Upon stimulation of glioma cells with HGF, we show that IQ-domain GTPase-activating protein 1 (IQGAP1) is recruited and overlaps with ARF6 at the leading edge of migrating cells. However, cellular depletion of ARF6 abrogated this recruitment of IQGAP1 and attenuated the formation of surface protrusions. ARF6 forms complexes with Rac1 and IQGAP1 in glioma cells upon HGF stimulation, and knockdown of IQGAP1 significantly inhibits ARF6-induced Rac1 activation and cell migration. Taken together, these data suggest that ARF6-mediated Rac1 activation is essential for glioma cell invasion via a signaling pathway that requires IQGAP1.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Animals; Brain Neoplasms; Cell Movement; Epidermal Growth Factor; Glioma; Hepatocyte Growth Factor; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; rac1 GTP-Binding Protein; ras GTPase-Activating Proteins; RNA, Small Interfering; Signal Transduction

Earlier, we showed that epidermal growth factor receptor (EGFR) signaling in human glioma cells increased cyclooxygenase-2 (COX-2) expression through p38-mitogen-activated protein kinase (MAPK)-dependent activation of the Sp family of transcription factors. Further mechanistic details of EGFR-dependent induction of COX-2 expression in glioma cells remained elusive. Protein kinase Cs (PKCs) comprise a family of serine-threonine kinases that are major mediators of signaling from receptor tyrosine kinases. Here, we report that PKC-delta, a novel PKC isoform, plays a role in EGF-dependent COX-2 induction in human glioma cells. Pharmacological inhibition and genetic silencing (through siRNA or dominant-negative expression) of PKC-delta confirm a role for this PKC isoform in EGF-dependent COX-2 induction. Overexpression of a functional PKC-delta enhanced COX-2 expression indicating that PKC-delta is not only necessary but also sufficient to regulate COX-2 levels. Inhibition of p38-MAPK pharmacologically or with siRNA further shows that p38-MAPK is required for activation of PKC-delta by EGF while inhibition of PKC-delta had no discernible effects on p38-MAPK activation. Finally, EGF stimulation promotes physical interactions between PKC-delta and Sp1 resulting in phosphorylation and nuclear localization of this transcription factor. These data provide the first evidence that PKC-delta is a critical link between p38-MAPK and Sp1-dependent COX-2 expression in human glioma cells.

Topics: Active Transport, Cell Nucleus; Brain Neoplasms; Cell Line; Cyclooxygenase 2; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C-delta; Sp Transcription Factors; Transcription, Genetic

Despite surgical advances and recent progress in adjuvant therapies, the prognosis for patients with malignant brain tumors such as glioblastoma multiforme has remained poor, and the neurological deterioration suffered by most patients as a consequence of tumor progression is dramatic and severe. In addition, malignant brain tumors have >>95% recurrence close to the primary site of initial resection. Unfortunately, standard imaging techniques do not permit the intraoperative identification of individual or small clusters of residual tumor cells, precluding their selective removal while sparing the surrounding normal brain tissue. In this report, we show that quantum dots (QDs) coupled to epidermal growth factor (EGF) or anti-EGF receptor receptor (EGFR, Her1) specifically and sensitively label glial tumor cells in cell culture, glioma mouse models, and human brain-tumor biopsies. A clear demarcation between brain and tumor tissue at the macroscopic as well as the cellular level is provided by the fluorescence emission of the QDs.

Topics: Animals; Antibodies, Monoclonal; Brain Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Glioma; Humans; Image Enhancement; Mice; Microscopy, Fluorescence; Molecular Probe Techniques; Quantum Dots; Sensitivity and Specificity

Enrichment of cancer stem cells for studies of carcinogenesis remains a difficult issue. We hypothesized that the unique features of cancer stem cells (CSCs) may allow formation of their colonies in vitro with distinct morphology. We therefore investigated the possibility to use morphological diversity of colonies to identify and enrich CSCs from cultured malignant human glioma cells. We found that a small proportion of the cells from a human glioma cell line U251 formed tight and round-shaped colonies in culture. Most cells in such colonies were capable of self-renewal, generating tumor spheres and differentiating into lineages with markers for neurons, astrocytes and oligodendrocytes. In addition, several neural stem cell-related genes were highly expressed by tumor cells in those tight colonies. Our results thus demonstrate a novel approach to the identification and enrichment of CSCs based on unique morphology of their colonies formed in vitro.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Separation; Clone Cells; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glial Fibrillary Acidic Protein; Glioma; Humans; Hyaluronan Receptors; Intermediate Filament Proteins; Neoplasm Proteins; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Spheroids, Cellular; Vimentin

In the present study, we have evaluated a boronated dendrimer-epidermal growth factor (BD-EGF) bioconjugate as a molecular targeting agent for boron neutron capture therapy (BNCT) of the human EGFR gene-transfected F98 rat glioma, designated F98(EGFR). EGF was chemically linked to a heavily boronated polyamidoamine dendrimer (BD) by means of the heterobifunctional reagent, mMBS. Biodistribution studies were carried out at 6 h and 24 h following intratumoral (i.t.) injection or intracerebral (i.c.) convection enhanced delivery (CED) of (125)I-labeled or unlabeled BD-EGF (40 microg (10)B/10 microg EGF) to F98 glioma bearing rats. At 24 h. there was 43% more radioactivity in EGFR(+) tumors following CED compared to i.t. injection, and a doubling of the tumor boron concentration (22.3 microg/g vs. 11.7 microg/g). CED of BD-EGF resulted in a 7.2x increase in the volume of distribution within the infused cerebral hemisphere and a 1.9x increase in tumor uptake of BD-EGF compared with i.t. injection. Based on these favorable biodistribution data, BNCT was carried out at the Massachusetts Institute of Technology nuclear reactor 14 days following i.c. tumor implantation and 24 h. after CED of BD-EGF. These animals had a MST of 54.1 +/- 4.7 days compared to 43.0 +/- 2.8 days following i.t. injection. Rats that received BD-EGF by CED in combination with i.v. boronophenylalanine (BPA), which has been used in both experimental and clinical studies, had a MST of 86.0 +/- 28.1 days compared to 39.8 +/- 1.6 days for i.v. BPA alone (P < 0.01), 30.9 +/- 1.4 days for irradiated controls and 25.1 +/- 1.0 days for untreated controls (overall P < 0.0001). These data have demonstrated that the efficacy of BNCT was significantly increased (P < 0.006), following i.c CED of BD-EGF compared to i.t injection, and that the survival data were equivalent to those previously reported by us using the boronated anti-human-EGF mAb, C225 (cetuximab).

Topics: Animals; Boron Compounds; Boron Neutron Capture Therapy; Brain Neoplasms; Cell Line, Tumor; Convection; Dendrimers; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glioma; Iodine Radioisotopes; Kaplan-Meier Estimate; Magnetic Resonance Imaging; Neoplasm Transplantation; Polyamines; Proportional Hazards Models; Radiometry; Rats; Rats, Inbred F344; Transfection

The aim of the present study was to investigate if treatment with lysosomotropic weak bases could increase the intracellular retention of radiohalogens and thereby increase the therapeutic effect of radionuclide tumor targeting.. Four different lysosomotropic bases, chloroquine, ammonium chloride, amantadine, and thioridazine, were investigated for their ability to increase radiohalogen retention in vitro. The two most promising substances, chloroquine and ammonium chloride, were studied in several cell lines (A431, U343MGaCl2:6, SKOV-3, and SKBR-3) in combination with radiolabeled epidermal growth factor (EGF) or the HER2 binding affibody (Z(HER2:4))(2).. The uptake and retention of radionuclides was found to be substantially increased by simultaneous treatment with the lysosomotropic bases. The effect was, however, more pronounced in the epidermal growth factor:epidermal growth factor receptor (EGF:EGFR) system than in the (Z(HER2:4))(2):HER2 system. The therapeutic effect of ammonium chloride treatment combined with (211)At-EGF was also studied. The effect obtained after combined treatment was found to be much better than after (211)At-EGF treatment alone.. The encouraging results from the present study indicate that the use of lysosomotropic weak bases is a promising approach for increasing the therapeutic effect of radionuclide targeting with radiohalogens.

Topics: Amantadine; Ammonium Chloride; Antimalarials; Antipsychotic Agents; Antiviral Agents; Astatine; Carcinoma, Squamous Cell; Cell Line, Tumor; Chloroquine; Epidermal Growth Factor; Glioma; Humans; Iodine Radioisotopes; Radioimmunotherapy; Radioisotopes; Recombinant Fusion Proteins; Thioridazine

Epidermal growth factor (EGF) plays a critical role in cancer. A polymorphism in the EGF gene (EGF+61) may influence its expression and contribute to cancer predisposition and aggressiveness. In the present study, we aimed to elucidate the role of EGF+61 in glioma susceptibility and prognosis.. A case-control study involving 197 glioma patients and 570 controls was done. Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95% confidence intervals (95% CI). False-positive report probability was also assessed. The luciferase reporter gene assay was used to ascertain the functional consequences of this polymorphism.. Corroborating the univariate analysis, the multivariate model showed that the G allele conferred higher risks for gliomas (OR, 1.32; 95% CI, 1.04-1.67), glioblastomas (OR, 1.47; 95% CI, 1.02-2.10), and oligodendrogliomas (OR, 1.55; 95% CI, 1.07-2.23). The GG genotypes were associated with increased risk for gliomas (OR, 1.71; 95% CI, 1.07-2.73), glioblastomas (OR, 2.03; 95% CI, 1.02-4.05), and oligodendrogliomas (OR, 2.72; 95% CI, 1.18-6.28). In addition, the AG+GG genotypes were associated with higher risk for gliomas (OR, 1.52; 95% CI, 1.03-2.23) and oligodendrogliomas (OR, 2.80; 95% CI, 1.35-5.79). No significant association was observed between the EGF+61 polymorphism and glioblastoma or oligodendroglioma patients' overall survival. The luciferase reporter gene assay exhibited a significant increased promoter activity for the G variant compared with the reference A allele.. These findings support the role of the EGF+61 polymorphism as a susceptibility factor for development of gliomas and show its implication on EGF promoter activity.

Topics: Alleles; Brain Neoplasms; Case-Control Studies; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Glioma; Humans; Male; Polymorphism, Genetic; Risk

Expression of cyclooxygenase-2 (COX-2) has been linked to many cancers and may contribute to malignant phenotypes, including enhanced proliferation, angiogenesis, and resistance to cytotoxic therapies. Malignant gliomas are highly aggressive brain tumors that display many of these characteristics. One prominent molecular abnormality discovered in these astrocytic brain tumors is alteration of epidermal growth factor (EGF) receptor (EGFR) through gene amplification and/or mutation resulting in excessive signaling from this receptor. We found that EGF-mediated stimulation of EGFR tyrosine kinase in human glioma cell lines induces expression of both COX-2 mRNA and protein. The p38 mitogen-activated protein kinase (p38-MAPK) pathway was a strong downstream factor in this activation with inhibition of this pathway leading to strong suppression of COX-2 induction. The p38-MAPK pathway can activate the Sp1/Sp3 transcription factors and this seems necessary for EGFR-dependent transactivation of the COX-2 promoter. Analysis of COX-2 promoter/luciferase constructs revealed that transcriptional activation of the COX-2 promoter by EGFR requires the Sp1 binding site located at -245/-240. Furthermore, Sp1/Sp3 binding to this site in the promoter is enhanced by EGFR activation both in vitro and in vivo. Enhanced DNA binding by Sp1/Sp3 requires p38-MAPK activity and correlates with increased phosphorylation of the Sp1 transcription factor. Thus, EGFR activation in malignant gliomas can transcriptionally activate COX-2 expression in a process that requires p38-MAPK and Sp1/Sp3. Finally, treatment of glioma cell lines with prostaglandin E2, the predominant product of COX-2 activity, results in increased vascular endothelial growth factor expression, thus potentially linking elevations in COX-2 expression with tumor angiogenesis in malignant gliomas.

Topics: Brain Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Models, Genetic; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Ribonucleases; Sp1 Transcription Factor; Sp3 Transcription Factor

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

Topics: Adenovirus E1A Proteins; Base Sequence; Cell Line, Tumor; Cell Movement; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioma; Humans; Molecular Sequence Data; Neoplasm Invasiveness; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Sp1 Transcription Factor; Transcriptional Activation

Osteoporosis pseudoglioma syndrome (OPPG) is an autosomal recessive disorder due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we report two novel missense mutations found in a southern Chinese family of a non-consanguineous marriage. Three out of four children had blindness, low bone mineral density (BMD) and multiple fractures in their childhood. Genotyping by DNA sequencing demonstrated 2 new mutations in exon 7 of the LRP5 gene. Tryptophans at amino acid residue positions 478 and 504 were replaced by arginine (W478R) and cysteine (W504C), respectively. While the parents that possessed either heterozygous W478R or W504C were apparently normal, all affected subjects were compound heterozygotes for the W478R and W504C mutations in the LRP5 gene. W478R is located immediately C-terminal to the third YWTD repeat of the second YWTD/EGF domain in LRP5, while W504C is located between the third and the fourth YWTD repeats of the second YWTD/EGF domain in LRP5. Using LRP5-related proteins, such as the low-density lipoprotein receptor (LDLR) and nidogen as reference models, a homology model of LRP5 suggested that the observed mutations may affect the molecular interactions of LRP5 and so lead to the observed OPPG phenotypes.

Topics: Adolescent; Base Sequence; Child; Epidermal Growth Factor; Exons; Female; Glioma; Heterozygote; Humans; Introns; LDL-Receptor Related Proteins; Low Density Lipoprotein Receptor-Related Protein-5; Male; Middle Aged; Models, Molecular; Mutation; Osteoporosis; Pedigree; Polymorphism, Genetic; Protein Structure, Quaternary; Syndrome

The effects of epidermal growth factor (EGF) on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry (FCM), and laser scan confocal microscope (LSCM) was used to measure the cytoplasmic free calcium. The results showed that GFAP was diffusedly expressed in GL15 cells and EGFR was over-expressed. EGF at doses of < or =1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100 ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas: low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.

Topics: Apoptosis; Brain Neoplasms; Calcium; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Glial Fibrillary Acidic Protein; Glioma; Humans

Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation. EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells. Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells. Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells.. EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR. EGF-E4orf4 fragment was cloned into the yeast expression vector. Recombinant plasmid DNA was transformed into the yeast Pichia pastoris. Fusion proteins were purified by SP Sepharose ion exchange chromatography. Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry.. The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4. Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes. Apparent molecular weight of fusion protein was 20 ku. Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody. EGF-E4orf4 in high concentrations (5, and 0.5 microg/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls. Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 microg/3 x 10(5) cells.. Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.

Topics: Adenovirus E4 Proteins; Apoptosis; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Female; Glioma; Humans; Open Reading Frames; Pichia; Recombinant Fusion Proteins; Recombination, Genetic; Transformation, Genetic

The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma.. Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached approximately 100 mm3 (approximately 6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 microg in 50 microL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers.. The maximal tolerated dose (MTD) of DAB389EGF was 3 microg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF.. DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor-positive glioblastoma multiforme.

Topics: Animals; Blood Urea Nitrogen; Cell Line, Tumor; Diphtheria Toxin; Epidermal Growth Factor; Female; Glioblastoma; Glioma; Humans; Inhibitory Concentration 50; Kidney; Liver; Maximum Tolerated Dose; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Neoplasm Transplantation; Nitrogen; Protein Structure, Tertiary; Recombinant Fusion Proteins; Time Factors; Treatment Outcome

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-alpha to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.

Topics: Actins; Animals; Bone Marrow Cells; Brain Neoplasms; Cell Line; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Genetic Therapy; Glioma; Mice; Phosphatidylinositol 3-Kinases; Protein Kinase C; Stromal Cells; Transfection

Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer.. DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts.. A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors.. (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Drug Evaluation, Preclinical; Epidermal Growth Factor; ErbB Receptors; Feasibility Studies; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Isotope Labeling; Metabolic Clearance Rate; Mice; Mice, Inbred BALB C; Mice, Nude; Organ Specificity; Organometallic Compounds; Radionuclide Imaging; Radiopharmaceuticals; Tissue Distribution

To isolate and clone the differentially expressed genes induced by epithelial growth factor (EGF) with inhibiting dosage in cultured glioma BT325 cells and understand the molecular mechanism that inhibits glioma cells growth.. Using differential display reversed transcription polymerase chain reaction (DDRT-PCR) method to analyze the differentially expressed cDNA in BT325 cells induced by EGF with inhibiting dosage. After sequencing and homology research, the differentially expressed cDNA fragments were further confirmed by Dot blot analysis and one of them by Northern blot.. Up-regulated genes cDNA fragments were isolated in growth inhibited BT325 cells. It was found that five cDNA fragments were highly homologous to the known human genes, while one was a fragment of a novel genes. Among these genes, one has coding sequence homology with transaldolase (TAL), which has been proved to be associated with apoptosis in recently research.. High-dose EGF could change the expression of many genes in BT325 cells. EGF can inhibit the growth of BT325 cell growth, which may be resulted from its potential role in promoting TAL gene expression and thus inducing cell apoptosis.

Topics: Base Sequence; Brain Neoplasms; Cell Division; Cloning, Molecular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glioma; Humans; Molecular Sequence Data; Sequence Analysis, DNA; Tumor Cells, Cultured

Increased expression of membrane-type matrix metalloproteinases (MT-MMPs) has previously been reported to correlate with increasing grade of malignancy in gliomas, a relationship shared with alterations in epidermal growth factor receptor (EGFR) signaling. To investigate the possibility of a causative role for EGFR signaling in increasing MT-MMP expression and subsequent peritumoral proteolysis, we characterized glioma cell lines for expression of MT1-MMP, MT2-MMP, MT3-MMP, and MT5-MMP by Western blotting and by quantitative real-time polymerase chain reaction analysis, and for MMP-2 activity following epidermal growth factor (EGF) stimulation. EGF stimulation of glioma cell lines resulted in a 2- to 4-fold increase in MT1-MMP mRNA levels. Although there were slight differences in MT2-, MT3-, and MT5-MMP mRNA expression following EGF stimulation, none of these demonstrated an increase similar to that of MT1-MMP expression. Treatment of high-grade glioma cell lines U251MG and IPSB-18 with EGF for 24 h resulted in a several-fold increase in MT1-MMP protein (2.5- and 5.1-fold, respectively) and in cyclin D1 (2.9-fold), as compared to untreated controls. No significant increase was detected in other MT-MMPs at the protein level. Although there was no detectable increase in proMMP-2 protein, there was an increase in MMP-2 activity. Furthermore, the MT1-MMP induction by EGF was prevented by pretreatment with the EGFR-specific tyrphostin inhibitor AG1478. Similarly, treatment with the phosphatidylinositol 3-kinase inhibitor LY294002 prevented the induction of MT1-MMP protein by EGF stimulation. These compounds additionally inhibited EGF-stimulated invasion in Matrigel Transwell assays. Our results indicate that one mechanism of EGFR-mediated invasiveness in gliomas may involve the induction of MT1-MMP.

Topics: Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Matrix Metalloproteinase 15; Matrix Metalloproteinase 16; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Signal Transduction

Cetuximab (IMC-C225) is a monoclonal antibody directed against both the wild-type and mutant vIII isoform of the epidermal growth factor receptor (EGFR). The purpose of the present study was to evaluate the monoclonal antibody (MoAb), cetuximab, as a boron delivery agent for neutron capture therapy (NCT) of brain tumors. Twenty-four hours following intratumoral (i.t.) administration of boronated cetuximab (C225-G5-B(1100)), the mean boron concentration in rats bearing either F98(EGFR) or F98(WT) gliomas were 92.3+/-23.3 microg/g and 36.5+/-18.8 microg/g, respectively. In contrast, the uptake of boronated dendrimer (G5-B(1000)) was 6.7+/-3.6 microg/g. Based on its favorable in vivo uptake, C225-G5-B(1100) was evaluated as a delivery agent for BNCT in F98(EGFR) glioma bearing rats. The mean survival time (MST) of rats that received C225-G5-B(1100), administered by convection enhanced delivery (CED), was 45+/-3d compared to 25+/-3d for untreated control animals. A further enhancement in MST to >59d was obtained by administering C225-G5-B(1100) in combination with i.v. boronophenylalanine (BPA). These data are the first to demonstrate the efficacy of a boronated MoAb for BNCT of an intracerebral (i.c.) glioma and are paradigmatic for future studies using a combination of boronated MoAbs and low molecular weight delivery agents.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Boron; Boron Neutron Capture Therapy; Brain Neoplasms; Cetuximab; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glioma; Iodine Radioisotopes; Mutation; Rats; Rats, Inbred F344

The purpose of the present study was to further evaluate a boronated dendrimer (BD)-epidermal growth factor bioconjugate (BD-EGF), administered by means of convection enhanced delivery (CED), as a molecular targeting agent for boron neutron capture therapy (BNCT) of the F98(EGFR) glioma. Twenty-four hours following CED of (125)I-labeled BD-EGF 47.4% of the injected dose (ID) was retained in F98(EGFR) gliomas compared to 12.3% in F98(WT) (wildtype) receptor negative tumors. Normal brain values were in the range of 5.9-10.1% ID in the tumor bearing cerebral hemisphere. Boron concentrations in F98(EGFR) gliomas were 22.3 and 11.7 microg/g following CED and i.t. injection, respectively. Based on these results, BNCT studies were initiated at the Massachusetts Institute of Technology nuclear reactor (MITRII). The mean survival time (MST) of rats that received BD-EGF either alone or in combination with boronophenylalanine (BPA), injected i.v., were 53+/-13 d and >61+/-14 d, respectively, compared to 40+/-5 d for BPA alone and 31+/-4 d for irradiated controls. These data show that CED improved the radiobiological effectiveness of BD-EGF and lay the groundwork for future studies using combinations of boron delivery agents for NCT of EGFR(+) gliomas.

Topics: Animals; Boron Compounds; Boron Neutron Capture Therapy; Brain Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Injections, Intravenous; Phenylalanine; Radiotherapy Dosage; Rats; Rats, Inbred F344; Recombinant Proteins; Tissue Distribution; Transfection

Epidermal growth factor (EGF)-mediated Ca2+ signaling in multiple cell lines derived from human gliomas and in the A431 epidermoid carcinoma cell line was observed using fluorescence videomicroscopy. Bath application of EGF evoked an oscillatory increase in [Ca2+]i in 4 different human glioma cell lines as well as the A431 cell line. This effect was blocked by the EGF receptor tyrosine kinase inhibitors gefitinib and erlotinib, as well as by the EGFR antibody cetuximab. In addition to this acute Ca2+ signaling response, transient exposure to EGF also potentiated subsequent Ca2+ signaling responses to other stimuli. Tumor cells transiently exposed to EGF (5 minutes), showed a sustained increase in propagation of intercellular Ca2+ waves, which have been previously shown to involve release of ATP and activation of purinergic receptors. Cells transiently exposed to EGF also showed a sustained potentiation of the Ca2+ signaling response to ATP. In contrast to the acute Ca2+ signaling response to EGF, this sustained potentiation of purinergic intercellular signaling was not blocked by gefitinib or erlotinib, while it was blocked by cetuximab. These results indicate that while the acute Ca2+ signaling response requires tyrosine kinase activation, the sustained potentiation of intercellular signaling occurs via a distinct pathway. Distinct intra- and intercellular Ca2+ signaling pathways may be mechanisms by which EGF modulates the growth and migration of tumor cells.

Topics: Adenosine Triphosphate; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Calcium; Calcium Signaling; Carcinoma, Squamous Cell; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Glioma; Humans; Protein Kinase Inhibitors; Quinazolines; Receptors, Purinergic; Tumor Cells, Cultured

The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ("Iressa", ZD1839), a reversible growth inhibitor of EGFR-expressing tumour cells, has been shown to enhance the antitumour effect of ionising radiation, and also to increase the uptake of radioiodinated EGF. Thus, combination of gefitinib treatment and radionuclide targeting is an interesting option for therapy of brain tumours that are difficult to treat with conventional methods. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF ((211)At-EGF) to cultured glioma U343 cells, which express high levels of EGFR. The growth of U343 cells in the presence of gefitinib was investigated, and it was found that gefitinib does not significantly inhibit the growth of these cells. Nevertheless, the uptake of (211)At-EGF in U343 cells was markedly increased (up to 3.5 times) in cells pre-treated with gefitinib (1 microM). This indicates that a combination of gefitinib treatment and radionuclide targeting to EGFR might be a useful therapeutic modality, even for patients who do not respond to treatment with gefitinib alone.

Topics: Astatine; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioma; Humans; Metabolic Clearance Rate; Protein-Tyrosine Kinases; Quinazolines; Radioisotopes; Radiopharmaceuticals

Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with (125)I on different parts: [(125)I]-EGF-dextran-[(125)I], [(125)I]-EGF-dextran and EGF-dextran-[(125)I]. Comparisons were made with [(125)I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that (131)I, instead of (125)I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/microg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose.

Topics: Animals; Dextrans; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Glioma; Half-Life; Mice; Tumor Cells, Cultured

Liposomes are, when coupled to receptor ligands, candidates for receptor mediated delivery of boron for tumour therapy since they have capacity to deliver large amounts of boron per receptor interaction. With EGF-liposomes we present a pegylated ligand liposome delivery vehicle, containing water soluble boronated phenanthridine, WSP1, or water soluble boronated acridine, WSA1, for EGFR targeting. In the case of WSA1 a ligand dependent uptake was obtained and the boron uptake was as good as if free WSA1 was given. No ligand dependent boron uptake was seen for WSP1 containing liposomes. Thus, WSA1 is a candidate for further studies. Approximately 10(5) boron atoms were in each liposome. A critical assessment indicates that after optimization up to 10(6) boron atoms can be loaded. Since it is known that, for therapeutic effect, approximately 10(8)-10(9) boron atoms are needed in a single tumour cell it is realized that 10(2)-10(3) receptor interactions are needed to meet the demand. Tests applying cultured glioma cells indicate, without optimization of the delivery conditions, a boron uptake in the ppm range, which is necessary for successful BNCT. Thus, it seems possible to kill micro-invasive tumour cells with targeted liposomes if the delivery conditions are optimal.

Topics: Acridines; Boron Compounds; Boron Neutron Capture Therapy; Brain Neoplasms; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Ligands; Liposomes; Polyethylene Glycols; Tumor Cells, Cultured

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 microM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.

Topics: Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Glioma; Humans; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Tetradecanoylphorbol Acetate; Tyrphostins; Vascular Endothelial Growth Factor A

Dextran-conjugated EGF (EGF-dextran) has a potential use for targeted radionuclide therapy of tumors that overexpress the epidermal growth factor receptor (EGFR). There are plans to treat both bladder carcinomas and malignant gliomas with local injections of radiolabeled EGF-dextran since these tumors often express high levels of EGFR. In this report we show that EGF and EGF-dextran differentially activate the EGFR. In the human glioma cell line U-343, activation of the serine/threonine kinases Erk and Akt is identical upon stimulation with EGF or EGF-dextran. However, the effect on phospholipase Cgamma1 (PLCgamma1) phosphorylation differs. In cells stimulated with EGF-dextran, the PLCgamma1 phosphorylation is lower than in cells stimulated with EGF. This observation could be explained by the fact that the PLCgamma1 association sites in the EGFR, tyrosine residues 992 and 1173, were phosphorylated to a lower degree when the receptor was stimulated with EGF-dextran as compared to with EGF.

Topics: Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Isoenzymes; Phospholipase C gamma; Phosphorylation; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine

Convection enhanced delivery (CED) is potentially a powerful method to improvethe targeting of macromolecules to the central nervous system by applying a pressure gradient to establish bulk flow through the brain interstitium during infusion. The purpose of the present study was to evaluate CED as a means to improve the intracerebral and intratumoral (i.t.) uptake of a heavily boronated macromolecule (dendrimer; BD) linked to epidermal growth factor (EGF) for neutron capture therapy in rats bearing a syngeneic epidermal growth factor receptor (EGFR) + glioma. Boronated EGF was radiolabeled with 125I and administered by CED at a rate of 0.33 micro l/min for 15, 30, and 60 min [infusion volumes (V(I)) of 5, 10, and 20 micro l, respectively], using a syringe pump connected to an indwelling cannula implanted into the right caudate nucleus of normal rats or i.t. in rats bearing either F98(EGFR) or F98 wild-type (F98(WT)) gliomas. After infusion, rats were euthanized, and their brains were removed and serially sectioned. The uptake and biodistribution of (125)I-boronated EGF in tumor or brain was studied by quantitative autoradiography and gamma-scintillation counting. The volume of distribution (V(d)) in brain was assessed using a computer interfaced image analysis system. After CED, the V(d) increased from 34.4 to 123.5 micro l with corresponding V(i) ranging from 5 to 20 micro l. The V(d) of BD-EGF in the brain was 64.8 +/- 13.4 micro l with CED (V(i) 10 micro ), and the V(d):V(i) ratio was 6.5 compared with a V(d) of 9.4 +/- 1.6 micro l and a V(d):V(i) ratio of 0.9 after direct intracerebral injection. As determined by quantitative autoradiography and gamma-scintillation counting at 24 h after CED, 47.4% of the injected dose per gram tissue (%ID/g) was localized in F98(EGFR) gliomas compared with 33.2%ID/g after direct i.t. injection and 12.3%ID/g in F98(WT) gliomas. On the basis of these observations, we have concluded that CED is more effective than i.t. injection as a way to deliver boronated EGF to EGFR (+) gliomas for boron neutron capture therapy.

Topics: Animals; Autoradiography; Boranes; Boron Neutron Capture Therapy; Brain; Brain Neoplasms; Dendrimers; Epidermal Growth Factor; ErbB Receptors; Glioma; Iodine Radioisotopes; Polyamines; Radionuclide Imaging; Rats; Rats, Inbred F344; Tissue Distribution

The promoter of the early growth response gene (Egr-1) has been described to be activated by ionizing radiation, and it seems to be clear that this process involves different mitogen activated protein (MAP) kinases, dependent on the specific cell type examined. However, early steps leading to activation of the corresponding pathways and thus to overexpression of Egr-1 are not well understood. In this study, deletion mutants of the 5' upstream region of the Egr-1 gene were generated which allowed us to correlate the radiation-induction of the Egr-1 promoter in U87 glioma cells to five serum response elements. Based on the data shown, a possible role of two cAMP responsive elements for radiation-dependent promoter regulation could be ruled out. On the basis of activator/inhibitor studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and potentially SAPK/JNK, but not the p38MAPK/SAPK2, pathway contribute to the radiation-induction of Egr-1 promoter. In addition, it was observed that irradiated cells secrete a diffusible factor into the culture media which accounts for the radiation-induced promoter upregulation. By blocking growth factor receptor activation with suramin, this effect could be completely abolished.

Topics: Androstadienes; Anisomycin; Blotting, Western; Colforsin; DNA-Binding Proteins; Early Growth Response Protein 1; Epidermal Growth Factor; Flavonoids; Glioma; Humans; Imidazoles; Immediate-Early Proteins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Pyridines; Suramin; Transcription Factors; Wortmannin

Chinese hamster ovary (CHO) cells transfected with the wild-type gene for the human epidermal growth factor-receptor (EGFR) and expressing the receptor in their cell membrane are, together with the receptor negative parent CHO cells, an interesting model system for experimental EGFR-targeting tumour therapy. Comparisons of effects on nearly identical cells with and without receptors can be made. The main purpose of this work was to compare the internalisation and retention of the radioactivity delivered as 125I-EGF or 125I-EGF-dextran in transfected cells (called CHO-EGFR), and human glioma cells U-343 which naturally express wild-type EGFR. We found that radioactivity delivered as 125I-EGF-dextran was retained intracellularly by both cell types to a higher degree than radioactivity delivered as 125I-EGF. Prolonging the cellular exposure time for 125I-EGF-dextran considerably increased postincubation intracellular retention in both cell types. No major differences between the two EGFR expressing cell lines were found and, based on the results in this work, CHO-EGFR cells seem an adequate model for experiments with agents targeting the EGF-receptor.

Topics: Animals; Cell Membrane; CHO Cells; Cricetinae; Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Mice; Mutation; Protein Binding; Time Factors; Transfection; Tumor Cells, Cultured

Success of boron neutron capture therapy (BNCT) is dependent on cellular and molecular targeting of sufficient amounts of boron-10 to sustain a lethal (10)B (n, alpha) (7)Li capture reaction. The purpose of the present study was to determine the efficacy of boronated epidermal growth factor (EGF) either alone or in combination with boronophenylalanine (BPA) as delivery agents for an epidermal growth factor receptor (EGFR) -positive glioma, designated F98(EGFR). A heavily boronated precision macromolecule [boronated starburst dendrimer (BSD)] was chemically linked to EGF by heterobifunctional reagents. Either F98 wild-type (F98(WT)) receptor (-) or EGFR gene-transfected F98(EGFR) cells, which expressed 5 x 10(5) receptor sites/cell, were stereotactically implanted into the brains of Fischer rats, and 2 weeks later biodistribution studies were initiated. For biodistribution studies rats received an intratumoral (i.t.) injection of (125)I-labeled BSD-EGF and were euthanized either 6 or 24 h later. At 6 h, equivalent amounts of BSD-EGF were detected in F98(EGFR) and F98(WT) tumors. Persistence of the bioconjugate in F98(EGFR) tumors was specifically determined by EGFR expression. By 24 h 33.2% of injected dose/g of EGF-BSD was retained by F98(EGFR) gliomas compared with 9.4% % of injected dose/g in F98(WT) gliomas, and the corresponding boron concentrations were 21.1 microg/g and 9.2 microg/g, respectively. Boron concentrations in normal brain, blood, liver, kidneys, and spleen all were at nondetectable levels (<0.5 microg/g). On the basis of these results, BNCT was initiated at the Brookhaven National Laboratory Medical Research Reactor. Two weeks after implantation of 10(3) F98(EGFR) or F98(WT) tumor cells, rats received an i.t. injection of BSD-EGF (approximately 60 microg (10)B/approximately 15 microg EGF) either alone or in combination with i.v. BPA (500 mg/kg). Rats were irradiated at the Brookhaven Medical Research Reactor 24 h after i.t. injection, which was timed to coincide with 2.5 h after i.v. injection of BPA for those animals that received both capture agents. Untreated control rats had a mean survival time (MST) +/- SE of 27 +/- 1 day, and irradiated controls had a MST of 31 +/- 1 day. Animals bearing F98(EGFR) gliomas, which had received i.t. BSD-EGF and BNCT, had a MST of 45 +/- 5 days compared with 33 +/- 2 days for animals bearing F98(WT) tumors (P = 0.0032), and rats that received i.t. BSD-EGF in combination with i.v. BPA had a MST of 57 +

Topics: Animals; Boron Compounds; Boron Neutron Capture Therapy; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Rats; Rats, Inbred F344; Tissue Distribution; Transfection

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the "glial" serotonin transporter in astrocytes was consistent with that reported for the "neuronal" serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [3H]serotonin uptake in astrocytes in a time (1-4 days)- and concentration (20-100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Topics: Astrocytes; Carrier Proteins; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Glioma; Growth Substances; Humans; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Neuroglia; RNA, Messenger; Serotonin; Serotonin Plasma Membrane Transport Proteins; Time Factors; Tumor Cells, Cultured

In various cell types, the neuro- and endocrine peptide somatostatin induces inhibitory and anti-secretory effects. Since somatostatin receptors, especially of the subtype sst2A, are constantly over-expressed in gliomas, we investigated the influence of somatostatin and the receptor subtype-selective peptide/non-peptide agonists octreotide and L-054,522 on the secretion of the most important angiogenesis factor produced by gliomas, vascular endothelial growth factor (VEGF). Cultivated cells from solid human gliomas of different stages and glioma cell lines secreted variable amounts of VEGF, which could be lowered to 25% to 80% by co-incubation with somatostatin or sst2-selective agonists (octreotide and L-054,522). These effects were dose-dependent at nanomolar concentrations. Stimulation with different growth factors (EGF, bFGF) or hypoxia considerably increased VEGF production over basal levels. Growth factor-induced VEGF synthesis could be suppressed to <50% by co-incubation with somatostatin or an sst2-selective agonist; this was less pronounced in hypoxia-induced VEGF synthesis. The effects were detected at the protein and mRNA levels. These experiments indicate a potent anti-secretory action of somatostatin or sst2 agonists on human glioma cells that may be useful for inhibiting angiogenesis in these tumors.

Topics: Antineoplastic Agents, Hormonal; Benzimidazoles; Blotting, Northern; Brain Neoplasms; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Hormones; Humans; Hypoxia; Indoles; Lymphokines; Octreotide; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatostatin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Amplification and/or mutations of the epidermal growth factor (EGF) receptor have been frequently reported in human malignant gliomas, the most common primary tumor of the adult central nervous system. We have analyzed a panel of established human glioma cell lines for EGF receptor expression. The EGF receptor was expressed in all of the glioma cell lines tested, with highest levels found in the cell line U343MG-a. In addition, various amounts of a truncated form of the EGF receptor were detected. The platelet-derived growth factor (PDGF) alpha receptor, analyzed for comparison, was expressed at low levels in human glioma cells, with the exception of U-118MG and U-373MG cells. The truncated form of the EGF receptor has been discussed as a constitutively active variant of the receptor. Using antibodies directed against the active form of the EGF receptor, we show here that the truncated variant of the EGF receptor in U343MG-a cells is not in the active conformation. However, the full-length EGF receptor, highly expressed in U343MG-a cells, was very rapidly activated following EGF treatment. In line with this, phosphorylation and activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) in U343MG-a cells required administration of EGF. Moreover, using highly specific riboprobes we observed that EGF signaling increased the Egr-1 mRNA concentration in human glioma cells within 30 min. The increase in the Egr-1 mRNA concentration was followed by a transient synthesis of the Egr-1 protein. Likewise, Egr-1 mRNA and protein concentrations were increased in U-118MG and U-373MG cells treated with PDGF. The synthesis of Egr-1 in human glioma cells as a result of EGF or PDGF stimulation indicates that Egr-1 may be an important "late" part of the EGF and PDGF-initiated signaling cascades suggesting that Egr-1 functions as a "third messenger" in glioma cells connecting growth factor stimulation with changes in gene transcription.

Topics: Astrocytoma; Brain Neoplasms; DNA-Binding Proteins; Early Growth Response Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Immediate-Early Proteins; MAP Kinase Signaling System; Neoplasm Proteins; Peptide Fragments; Platelet-Derived Growth Factor; Protein Conformation; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

We have previously reported a method for labeling epidermal growth factor (EGF) with technetium-99m and have shown that 99mTc-EGF localized in EGF receptor (R) positive intracerebral C6EGFR rat gliomas following intratumoral (i.t.) injection of the radioligand. In the present study, we have evaluated the potential use of 99mTc-EGF as a tumor targeting agent after systemic administration to Fischer rats bearing intracerebral implants of C6EGFRgliomas. Radiolocalization was determined following intravenous (i.v.) or intracarotid (i.c.) injection with or without hyperosmotic mannitol induced disruption of the blood-brain barrier (BBB-D). As determined by gamma-scintillation counting, 4 h after i.c. injection of 99mTc-EGF, 0.34% of the injected dose per gram (% ID/g) was localized in C6EGFR tumors. which expressed 10(5)-10(6) EGFR sites per cell, compared to 0.07% ID/g in animals bearing C6 wildtype gliomas, which do not express EGFR. The corresponding tumor to brain ratios were 5.6 and 1.6, respectively. Tumors could be visualized by external gamma-scintigraphy in rats bearing C6EGFR but not C6 wildtype gliomas, thereby establishing that radiolocalization was dependent upon receptor expression. Intracarotid administration of 99mTc-EGF significantly increased tumor uptake compared to i.v. injection (0.34 vs 0.14% ID/g, p < 0.04). BBB-D disruption, followed by i.c. injection of 99mTc-EGF, however, did not significantly enhance tumor uptake compared to i.c. injection without BBB-D (0.45% vs 0.34% ID/g, p > 0.1). The uptake of 99mTc-EGF was approximately 4-9% ID/g in the liver and 12-20% ID/g in the kidneys after i.c. or i.v. administration. External gamma-scintigraphy of regions of interest over the liver and kidneys revealed that approximately 70-80% of the whole body radioactivity accumulated in these organs, and only 0.47-0.83% in the tumor following i.v. or i.c. administration of 99m9Tc-EGF. Our study has demonstrated that EGF can be used as a specific targeting agent for EGFR (+) rat brain tumors. However, it is unlikely that systemic injection of EGF-based bioconjugates can deliver sufficient amounts of the ligand to brain tumors for therapeutic purposes and direct delivery by means of either intratumoral injection or a variant of it such as convection enhanced delivery will be required.

Topics: Animals; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Glioma; Injections, Intralesional; Injections, Intravenous; Organotechnetium Compounds; Radionuclide Imaging; Rats; Rats, Inbred F344; Tissue Distribution

The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.

Topics: Bradykinin; Brain Neoplasms; Cell Adhesion; Culture Media, Serum-Free; Cyclin-Dependent Kinase Inhibitor p16; Ecdysone; Ecdysterone; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, p16; Glioma; GTP-Binding Proteins; Humans; Insulin; Integrins; Neoplasm Proteins; Promoter Regions, Genetic; Pseudopodia; Recombinant Fusion Proteins; Signal Transduction; Tumor Cells, Cultured

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-beta1,2) and vascular endothelial growth factor (VEGF)].on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-beta1 and TGF-beta2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.

Topics: Brain Neoplasms; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Growth Substances; Humans; Lymphokines; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Meningeal Neoplasms; Meningioma; Neoplasm Proteins; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A number of epidermal growth factor receptor (EGFR) deletion mutants have been identified in gliomas, in which the EGFR gene is frequently amplified and rearranged. We have previously characterized the structure of a gene in A-172 human glioma cells that encodes a 190-kDa EGFR mutant with tandem duplication of the tyrosine kinase (TK) and calcium-mediated internalization (CAIN) domains. Here we describe a 185-kDa tandem duplication mutant (TDM) that is expressed in KE and A-1235 glioma cells, along with certain functional characteristics of the mutants. The corresponding transcripts in KE and A-1235 cells contain 1053 additional nucleotides representing an in-frame duplication of exons 18 through 25 which encode the entire TK region and a portion of the CAIN domain. As with duplication of the entire TK/CAIN region (exons 18-26) in A-172 cells, duplication of exons 18-25 is associated with a specific genomic rearrangement between flanking introns. Involved introns contain homology to recombination signal sequence (RSS) heptamers present in the V(D)J region of the T lymphocyte receptor gene. In defined medium, both oncogenic TDM are constitutively autophosphorylated and inefficiently downregulated. High-affinity binding is reduced in EGFR.TDM/18-26, although the t1/2 of receptor internalization is not prolonged.

Topics: Animals; Biopsy; Brain Neoplasms; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Exons; Fibroblasts; Gene Duplication; Gene Expression Regulation, Neoplastic; Glioma; Humans; Introns; Mice; Molecular Weight; Phosphorylation; Protein Processing, Post-Translational; Protein Structure, Tertiary; Radioligand Assay; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

We have previously documented that the vast majority of high-grade gliomas over-express binding sites for interleukin 13 (IL13) in situ. We now extend this analysis to evaluate the distribution of the binding of IL13 among other brain tumors. Tumor specimens from patients with low-grade gliomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, gliosarcomas, medulloblastomas, meningiomas, and metastases to the brain were analyzed and compared to a new series of glioblastoma multiforme (GBM) samples. Serial tumor tissue sections were incubated with 125I-labeled (i) IL13, (ii) antibody against transferrin (Tf) receptor, and (iii) epidermal growth factor (EGF). Most (17/18) GBMs stained specifically for IL13 binding sites while sections from 3/11 low-grade gliomas, 5/5 high-grade gliomas (grade III), 3/5 oligodendrogliomas (all three were anaplastic), and 1/2 gliosarcomas also showed specific binding for IL13. We did not detect IL13 binding sites in medulloblastomas (0/4) and found them only in 2/20 meningiomas. Metastases to the brain (4/12, i.e., lung adenocarcinomas and renal cell carcinoma) showed some binding of 125I-IL13. The presence of receptors for Tf was ubiquitous among all studied tumors while EGF receptor expression was much more variable. Since it appears that primarily the least differentiated forms of gliomas possess IL13 binding sites in abundance, it is plausible that IL 13 receptor expressed in low-grade gliomas might be a prognostically significant marker associated with their progression to high-grade gliomas. Finally, we demonstrate that the glioma-associated IL13 receptor is truly more restrictive in nature also due to its selective representation among brain tumors of glial origin.

Topics: Adenocarcinoma; Biomarkers, Tumor; Brain Neoplasms; Carcinoma; Cell Transformation, Neoplastic; Disease Progression; Ependymoma; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Gliosarcoma; Humans; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Interleukin-4; Medulloblastoma; Meningeal Neoplasms; Meningioma; Neoplasm Proteins; Oligodendroglioma; Receptors, Interleukin; Receptors, Interleukin-13; Receptors, Transferrin; Recombinant Proteins; Substrate Specificity

The early incidence of p53 mutation in astrocytomas suggests that it plays an important role in astrocyte transformation. Astrocytes isolated from homozygous p53 knockout mice grow rapidly, lack contact inhibition, and are immortal. Here we tested whether the loss of p53 is sufficient for progression to tumorigenicity of astrocytes. We grew primary astrocytes under three conditions for over 120 population doublings and assessed their antigenic phenotype, chromosome number, and expression of glioma-associated genes as well as their ability to form colonies in soft agarose and tumors s.c. and intracranially in nude mice. Under two conditions (10% FCS and 0.5% FCS plus 20 ng/ml EGF), cells acquired the ability to form colonies in soft agarose and tumors in nude mice, and this was accompanied by the expression of genes, including epidermal growth factor receptor, platelet-derived growth factor receptor alpha and beta, protein kinase Cdelta, and vascular endothelial growth factor, which are known to be aberrantly regulated in human astrocytomas. Under the third condition (0.5% FCS plus 10 ng/ml basic fibroblast growth factor), astrocytes gained the ability to form colonies in soft agarose and had abnormal chromosome numbers similar to cells in the first two conditions but did not form tumors in nude mice or overexpress glioma-associated genes. These data provide experimental evidence for the idea that the malignant progression initiated by the loss of p53 may be subject to modulation by extracellular environmental influences.

Topics: Aneuploidy; Animals; Astrocytes; Blotting, Northern; Brain; Brain Neoplasms; Carcinogenicity Tests; Cell Culture Techniques; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Glioma; Humans; Mice; Mice, Nude; Receptors, Platelet-Derived Growth Factor; Time Factors; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

The goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors.

Topics: Angiogenesis Inducing Agents; Animals; Becaplermin; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glioma; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Magnetic Resonance Imaging; Mice; Mice, Nude; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Spheroids, Cellular; Swine; Wounds and Injuries

The purpose of this study was to compare the differential susceptibility to photodynamic therapy (PDT) mediated damage in human U-105MG glioma cells and CH-157MN meningioma cells in vitro using 5-amino-levulinic acid (ALA) as photosensitizer, and to determine if growth factors would enhance PDT-mediated damage of these cells.. U-105MG or CH-157MN cells were irradiated with polychromatic light in the presence of ALA. A Xenon lamp (150 W) was used as the light source. For the study on the effect of growth factor on ALA-PDT, cells were cultured in serum free medium for 24 hours. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet derived growth factor BB (PDGF-BB) was added to achieve a final concentration of 50 ng/ml. 30 minutes later, cells were incubated with ALA (100 microg/ml) for 24 hours, washed, and irradiated with light (11 J/cm2). MTT tetrazolium assays were performed 24 hours after light irradiation.. The inhibition of metabolic cellular function in U-105MG cells by ALA depended on both light energy density and ALA concentration. The susceptibility to ALA-PDT was profoundly lower for CH-157MN meningioma cells than U-105MG glioma cells. When incubated with ALA (100 microg/ml), U-105MG cells exhibited an LD50 around 8 J/cm2 of light irradiation, whereas that of CH-157MN cells was more than 25 J/cm2. EGF, bFGF, or PDGF-BB did not have any effects on the susceptibility of these two cell lines to ALA-PDT.. ALA-PDT was more effective in killing U-105MG glioma cells than CH-157MN meningioma cells. The differential susceptibility was likely due to differential accumulation of PpIX in these cells. EGF, bFGF, or PDGF-BB did not have stimulatory or inhibitory effect on the efficiency of ALA-PDT.

Topics: Aminolevulinic Acid; Analysis of Variance; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Glioma; Humans; Meningeal Neoplasms; Meningioma; Photochemotherapy; Photosensitizing Agents; Tumor Cells, Cultured

Hepatocyte growth factor/scatter factor (HGF/SF) contributes to the malignant progression of human gliomas. We investigated the effect of HGF/SF on matrix metalloproteinase-2 (MMP-2), membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitors of metalloproteinases (TIMPs), expressions of c-Met/HGF receptor-positive human glioblastoma cells. Treatment of U251 human glioblastoma cells with HGF/SF resulted in enhanced secretion of MMP-2 with an increased level of the active form. This was accompanied by enhanced expression (2.5-fold) of mRNA specific for MMP-2. The stimulatory effect of HGF/SF on MMP-2 expression did not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. MT1 -MMP, a cell-surface activator of proMMP-2, was also up-regulated by HGF/SF in a dose-dependent manner. By contrast, the level of TIMP- 1 mRNAs was not altered significantly and that of TIMP-2 was reduced mildly by the HGF/SF treatment, suggesting that HGF/SF may eventually modulate a balance between MMP-2 and TIMPs in favor of the proteinase activity in the glioma cell microenvironment. HGF/SF also stimulated MMP-2 expression of other glioblastoma cell lines. Since glioblastomas frequently co-express HGF/SF and its receptor, our results suggest that HGF/SF might contribute to the invasiveness of glioblastoma cells through autocrine induction of MMP-2 expression and activation.

Topics: Benzoquinones; Brain Neoplasms; Disease Progression; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Gelatinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Hepatocyte Growth Factor; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.

Topics: Cell Adhesion; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Kinetics; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-crk; Proto-Oncogenes; src Homology Domains; Tumor Cells, Cultured

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.

Topics: Amphiregulin; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glioma; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mitosis; Recombinant Proteins; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

A conjugate with specific binding to the epidermal growth factor receptor, EGFR, and of interest for clinical tests was prepared using mouse epidermal growth factor, mEGF, and dextran. The mEGF was first coupled to dextran by reductive amination in which the free amino group on the N-terminal of mEGF was reacted with the aldehyde group on the reductive end of the dextran chain. The end-end coupled intermediate was further activated by the cyanopyridinium agent CDAP and tyrosines introduced to the dextran part of the conjugate. The mEGF-dextran-tyrosine conjugate was, with high efficiency, iodinated with the chloramine-T method. Approximately 25-35% of the radioactivity could be removed from the conjugate after exposure to protease K while 65-75% of the radioactivity could be removed after exposure to dextranase. Thus, the largest amount of the iodine was on the dextran part of the conjugate. The iodinated mEGF-dextran-tyrosine had EGFR specific binding since the binding to an EGFR rich human glioma cell line could be displaced by an excess of non-radioactive mEGF. The conjugate was to a large extent internalized in these cells and the administrated radioactivity was thereby retained inside the cells for at least up to 50 h.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Iodine Radioisotopes; Mice; Tumor Cells, Cultured; Tyrosine

N-linked oligosaccharides appear to be important for the function of the epidermal growth factor (EGF) receptor. In a previous study (Rebbaa, A., Yamamoto, H., Moskal, J. R., and Bremer, E. G. (1996) J. Neurochem. 67, 2265-2272), we showed that binding of the erythroagglutinating phytohemagglutin lectin from Phaseolus vulgaris to the bisecting structures on the EGF receptor from U373 MG glioma cells blocked EGF binding and receptor autophosphorylation. In this study we examined the consequences of overexpression of the bisecting structure on the EGF receptor by gene transfection of U373 MG cells with the N-acetylglucosaminyltransferase III (GnT-III). This modification leads to a significant decrease in EGF binding and EGF receptor autophosphorylation. In addition, the cellular response to EGF was found to be altered. Proliferation of U373 MG cells in serum-free medium is inhibited by EGF. In contrast, proliferation of the GnT-III-transfected cells was stimulated by EGF. These data demonstrate that changes in EGF receptor glycosylation by GnT-III transfection reduces the number of the active receptors in U373 MG cells and that this change results in change in the cellular response to EGF.

Topics: Acetylglucosamine; Epidermal Growth Factor; ErbB Receptors; Glioma; Glycosylation; Humans; N-Acetylglucosaminyltransferases; Oligosaccharides; Phosphorylation; Phytohemagglutinins; RNA, Messenger; Transfection; Tumor Cells, Cultured

The gene for epidermal growth factor receptor (EGFR) is amplified or overexpressed in high-grade gliomas but is low or undetectable in normal brain. Recently, there has been increasing interest in using epidermal growth factor (EGF)-based bioconjugates as targeting agents for brain tumors. In the present study, we have investigated the potential use of boronated EGF as a delivery agent for boron neutron capture therapy, which is based on the capture reaction that occurs when 10B, a stable isotope, is irradiated with low-energy thermal neutrons. A fourth generation starburst dendrimer was boronated and linked to EGF using heterobifunctional reagents. Either wild-type or EGFR gene transduced C6 glioma cells (C6EGFR), which expressed 10(5)-10(6) receptor sites/cell, were stereotactically implanted into the right cerebral hemisphere of Fischer rats. Four weeks later, the rats received either i.v. or intratumoral (i.t.) injection of 131I-labeled boronated starburst dendrimer (BSD) or BSD-EGF. The biodistribution of 131I-BSD-EGF and 131I-BSD was studied by means of whole-body scintigraphy, autoradiography, and gamma scintillation counting. Following i.t. injection of 131I-BSD-EGF, 21.8% of the injected dose per gram tissue (% ID/g) was localized in C6EGFR tumors at 24 h and 16.3% at 48 h compared to 5 and 1.3% ID/g in C6 wild-type tumors, respectively, and 0.01 and 0.006% ID/g, respectively, for i.v. injected animals at the corresponding times. In contrast, following i.t. injection of BSD-EGF, only 0.01-0.1% ID/g was localized in the liver and spleen at 24 and 48 h compared to 5-12% ID/g following i.v. injection. Our data indicate that direct i.t. injection can selectively deliver BSD-EGF to EGFR-positive gliomas and suggest that intracerebral administration may be the most effective way for delivering EGF-based bioconjugates to EGFR-positive brain tumors.

Topics: Animals; Boron; Boron Neutron Capture Therapy; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Glioma; Injections, Intralesional; Injections, Intravenous; Isotopes; Neoplasm Proteins; Rats; Tumor Cells, Cultured

The development of primary human brain tumors, particularly glioblastoma multiforme (GBM), has been associated with a number of molecular and chromosomal abnormalities. In this study, a novel tumor suppressor locus was identified and localized after the transfer of a human chromosome 4 into U251 human GBM cells. Hybrid clones containing a transferred neomycin-resistance tagged chromosome 4 revealed an inability to form tumors in nude mice and a greatly decreased efficiency of soft agarose colony formation. As a control, clones containing a transferred chromosome 2 were generated, and these retained the tumorigenic phenotype of the parental U251 cells. The presence of the transferred chromosomes was demonstrated by gain of polymorphic loci and FISH analyses. Several suppressed hybrid clones were shown to contain spontaneously reduced versions of the transferred chromosome 4. A common region of the fragmented chromosome 4 was retained among these clones that included the epidermal growth factor locus at 4q24-26 and several adjacent markers. The identification of a common fragment in the suppressed clones suggests the presence of a tumor suppressor gene or genes in this region, involved in glioma oncogenesis.

Topics: Animals; Blotting, Southern; Chromosome Mapping; Chromosomes, Human, Pair 4; DNA; Epidermal Growth Factor; Genes, Tumor Suppressor; Glioma; Humans; Hybrid Cells; Mice; Microsatellite Repeats; Phenotype; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

Topics: 3T3 Cells; Animals; Brain Neoplasms; Cell Survival; Epidermal Growth Factor; Female; Glioma; Growth Inhibitors; Humans; Isoxazoles; Leflunomide; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Ovarian Neoplasms; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; Recombinant Proteins; Signal Transduction; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Gliomas are characterized by their extensive invasion into the brain parenchyma. Recently it has been shown that normal brain cells can produce laminin, fibronectin and collagen type IV when confronted by invading glioma cells. Laminin stimulates cell migration of several human glioma cell lines in vitro. This migration can be inhibited by adding blocking monoclonal antibodies (MAbs) against the most expressed integrin subunits, alpha3 and beta1. Previous studies have shown that glioma cell migration, invasion and growth are stimulated by epidermal growth factor (EGF). However, MAb directed against the EGF receptor (EGFR) did only partly inhibit the invasive process in vitro. Since laminin has regional peptide homology with EGF (EGF-like repeats), the present work was aimed at studying how two human glioma cell lines exposed to antibodies to the EGFR, reacted to laminin stimulated migration. Furthermore, we wanted to study which role the EGFR and the laminin receptor integrin subunits alpha3 and beta1 play during glioma cell invasion. EGFR expression of two glioma cell lines, AN1/lacZ and U-251/lacZ was studied by flow cytometry and immunofluorescence microscopy. A cell migration assay was used to study effects of MAbs against EGFR on migration from laminin-stimulated tumor spheroids. Tumor cell invasion was evaluated by using an in vitro co-culture model, where normal fetal brain cell aggregates were confronted with multicellular tumor spheroids. The results show that both cell lines expressed EGFR, AN1/lacZ 4-fold more than U-251/lacZ. MAb against EGFR inhibited the laminin-stimulated migration only from AN1/lacZ spheroids. MAbs against alpha3 and beta1 integrin subunits inhibited glioma cell invasion in vitro. The present work indicates possible connections between laminin-stimulated cell migration and the EGFR expression on glioma cells. These elements contribute to the characteristic features of glioma cells and may be an important part of the complex relationships between growth factors, integrins and extracellular matrix during glioma cell invasion.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Cell Division; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Glioma; Humans; Integrin alpha3; Integrin beta1; Integrins; Microscopy, Fluorescence; Neoplasm Invasiveness; Rats; Receptors, Laminin; Spheroids, Cellular; Tumor Cells, Cultured

To explore the relationship between the proliferative activities, oncoprotein expression, cell differentiation and prognosis of gliomas.. Immunohistochemistry and image analysis were used to study the expression of proliferating cell nuclear antigen (PCNA) and several oncoproteins both qualitatively and quantitatively in 124 brain gliomas.. It was found that the intensities of PCNA reaction were significantly related to both the grade and prognosis of gliomas. Overexpression of c-erbB-2 protein was slightly stronger in well than in poorly-differenciated gliomas. Moreover, the reactions in patients who survived over 5 years were stronger than in those under 5 years. EGF (40.0%), EGFR (91.4%) and p21ras (53.3%) expression levels were related to neigher the grading nor prognosis of this tumor. The positive ratios of the three antibodies to p53 protein were higher in grades II-IV than in grade I gliomas. The intensity of p53 reaction was correlated to that of PCNA.. It is suggested that the aberration of c-erbB-2, p21ras, EGF and EGFR might be the early events in the initiation and progression of gliomas, whereas p53 is involved in all stages of these tumors. PCNA could reflect the degree of malignancy to a certain extent.

Topics: Brain Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Oligodendroglioma; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins p21(ras); Receptor, ErbB-2; Survival Rate; Tumor Suppressor Protein p53

Epidermal growth factor (EGF) and platelet-derived growth (PDGF) are suggested to be involved in the proliferation of human gliomas. We examined the effects of these growth factors on two human malignant glioma cell lines. Treatment of the A172 glioblastoma and the Hs683 glioma cell line with EGF and PDGF resulted in the tyrosine autophosphorylation, and hence activation, of the respective growth factor receptors. In addition, both cell lines responded to EGF and PDGF with increased deoxyribonucleic acid (DNA) synthesis. Because the intrinsic protein tyrosine kinase activity of this class of growth factor receptors is indispensable for their functioning, we tested the effects of specific protein tyrosine kinase inhibitors on growth factor-induced DNA synthesis and glioma cell proliferation. Genistein inhibited both EGF- and PDGF-stimulated autophosphorylation of the receptors and induction of DNA synthesis. However, genistein seemed to be cytotoxic to the cells. The tyrphostins RG 50875 and RG 13022 dose-dependently inhibited DNA synthesis induced by EGF, PDGF, and serum. RG 13022 completely blocked the EGF- and PDGF-induced DNA synthesis at a concentration of 50 mumol/L. The tyrphostins showed no selectivity in blocking either EGF or PDGF signaling. With concentrations up to mumol/L, no cytotoxic side effects of the tyrphostins were observed. Both tyrphostins also inhibit serum-driven cell growth in a dose-dependent manner. These results support the hypothesis that activated protein tyrosine kinase receptors are involved in the proliferation of A172 and Hs683 glioma cells. Selective inhibitors of protein tyrosine kinases, therefore, might have the potential to contribute to the treatment of growth factor-dependent gliomas.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Division; Cell Survival; DNA Replication; Dose-Response Relationship, Drug; Epidermal Growth Factor; Genistein; Glioblastoma; Glioma; Humans; Isoflavones; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Tumor Cells, Cultured

The expression of vascular endothelial growth factor (VEGF) has been implicated in brain tumor angiogenesis, and the promoter region for the VEGF gene contains several SP-1 and AP-1 (c-Fos and c-Jun) binding motifs. Among eight human glioma cell lines, cellular mRNA levels of transcription factors SP-1 and AP-1 (c-Fos and c-Jun) were found to be closely correlated with those of VEGF. VEGF expression appears to be highly susceptible to hypoxia or exogenous cytokines and growth factors. Of various cytokines and growth factors, basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 most potently enhanced VEGF mRNA levels of a glioma cell line, U251. Incubation of the glioma cells with bFGF or TNF-alpha increased both VEGF and SP-1 mRNA at 30 min and c-Fos mRNA at 1-3 h, over 5-fold. Nuclear run-on assays showed an apparent increase of the transcription of the VEGF gene as well as the SP-1 gene by bFGF or TNF-alpha. Gel mobility shift assays demonstrated that only SP-1 binding activity was increased 1 h after exposure to bFGF or TNF-alpha, and also that AP-1, but not SP-1, activity was significantly activated by hypoxia. Mithramycin, an inhibitor of SP-1, at 1-10 nM inhibited activation of the VEGF gene by bFGF or TNF-alpha but not that by hypoxia. Western blot analysis also demonstrated an increase in cellular amounts of VEGF by TNF-alpha and a decrease by co-administration with mithramycin. The promoter activity of the VEGF gene, which contains five SP-1 binding sites and one AP-1 binding site but not hypoxia regulatory elements, was enhanced by bFGF or TNF-alpha but not by hypoxia. The chloramphenicol acetyltransferase assay with VEGF promoter deletion constructs demonstrated that four clusterized SP-1 binding sites in the proximal promoter were essential for the basal transcription and the TNF-alpha-dependent activation. These data indicated that the expression of the VEGF gene enhanced by bFGF or TNF-alpha appeared to be mediated in part through the transcription factor SP-1, suggesting a different mechanism from that for hypoxia-induced activation of the VEGF gene.

Topics: Antineoplastic Agents; DNA-Binding Proteins; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Glioma; Humans; Lymphokines; Oxygen; Platelet-Derived Growth Factor; Plicamycin; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Transcription Factor AP-1; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Morphological and immunocytological changes of intermediate filaments of cultured human malignant glioma cells were studied by adding various growth factors or cytokines using stereoscopic high voltage electron microscopy operated at 1,000 kV. The gold-colloid immuno-cytochemical method was used to stain GFAP and vimentin. Growth rate of tumor cells increased when EGF, TGF-alpha, and PDGF administered and decreased when FGF, TNF, and CLN-IgG administered. Morphological changes of cells were not remarkable when EGF, PDGF, IL-1, and FGF were administered. The cytoplalsmic organellaes were damaged after administrating TNF and CLN-IgG to cells.

Topics: Brain Neoplasms; Cell Division; Cytokines; Epidermal Growth Factor; Glial Fibrillary Acidic Protein; Glioma; Growth Substances; Humans; Immunohistochemistry; Intermediate Filaments; Microscopy, Immunoelectron; Organelles; Platelet-Derived Growth Factor; Transforming Growth Factor alpha; Tumor Cells, Cultured; Vimentin

The discovery of EGFR gene amplification in glioblastoma multiforme has prompted interest in experimental therapies to target the receptor on brain tumor cells. To develop an animal model for in vivo study of such strategies, we transfected C6 glioma cells with a plasmid containing the neomycin resistance gene and the human EGFR gene under the control of the glucocorticoid-inducible MMTV promoter. Following selection with G418, individual clones that expressed EGFR at high levels were selected. Kinetics of EGF binding fit a dual site model indicating the presence of both high (KA = 2.5 x 10(9) M-1) and low (KA = 3.3 x 10(7) M-1) affinity receptors. To assess growth in vivo, graded numbers of either wild-type or transfected cells were implanted into the brains of CD Fischer 344 rats. No differences in survival were observed between groups of animals injected with either wild-type or transfected cells at inocula of 10(3) or 10(4) respectively. In addition, one-third of animals (7/21) challenged with 10(5) or 10(6) transfected cells survived > 50 days compared to 0% of animals (0/12) challenged with 10(5) or 10(6) wild-type cells. Such an effect suggests greater immunogenicity of transfected cells, but only at the larger inocula. Since C6 glioma cells will grow in both outbred and inbred strains, our model should have a number of applications including the in vivo study of EGFR targeting for glioma therapy.

Topics: Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Complementary; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Male; Rats; Rats, Inbred F344; Recombinant Proteins; Time Factors; Transfection; Tumor Cells, Cultured

Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5'-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

Topics: Antibodies; Cell Division; Cell Line; Cell Nucleus; Cross-Linking Reagents; Deoxyadenosines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioblastoma; Glioma; Humans; Immunoblotting; Iodine Radioisotopes; Methyltransferases; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Precipitin Tests; Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Thionucleosides; Tyrosine

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.

Topics: Brain Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gangliosides; Glioma; Growth Substances; Humans; Laminin; Recombinant Proteins; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured

In 75 gliomas and 31 meningiomas, mutations at the epidermal growth factor receptor (EGFR) gene locus were restricted to gliomas. The ligands of this receptor, epidermal growth factor and transforming growth factor alpha, lacked quantitative changes at their loci in gliomas and meningiomas. EGFR gene amplification occurred in astrocytomas, oligodendrogliomas, ependymomas and glioblastomas. The frequency of this mutation significantly increased with the malignancy grade and the patient's age. Especially in glioblastomas of individuals aged over 64 years, EGFR gene mutations were observed without chromosome-10-specific allele losses. This finding contradicts the hypothesis that deletion of one entire chromosome 10 regularly precedes EGFR gene amplification in primary glioblastomas of patients aged over 50 years. It was found that most individuals whose gliomas carry an EGFR gene mutation have a poor prognosis, comparable to that of glioblastoma patients even when the tumour is graded as benign.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Child; Child, Preschool; Chromosome Mapping; Epidermal Growth Factor; ErbB Receptors; Female; Gene Amplification; Glioblastoma; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma; Middle Aged; Mutation; Prognosis; Remission Induction; Survival Rate; Transforming Growth Factor alpha

Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy.. EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made.. The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF.. The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Topics: Adenocarcinoma; Animals; Boron Compounds; Boron Neutron Capture Therapy; Carcinoma; Colonic Neoplasms; Dextrans; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Male; Mammary Neoplasms, Experimental; Models, Biological; Neoplasms; Neoplasms, Experimental; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha; Tumor Cells, Cultured

Loss of genetic material on chromosome 10 is regarded as a prominent feature in the genesis of glioblastomas. To use chromosome 10 deletions as diagnostic markers for glioblastomas we investigated, if the loss of chromosome 10 material could be restricted on the region 10q21-26. By PCR microsatellite analysis on frozen tissue and paraffin material from the ZULCH brain tumor collection we found (1) loss of heterozygosity in 10q21-26 in 75% of the investigated DNA from frozen tissue and (2) an interstitial loss in the region of the microsatellite marker D10S186. The combined immunohistochemical analysis of overexpression of EGFR, EGF and TGF alpha with LOH on chromosome 10 showed that chromosome 10 deletions are not exclusively bound to EGFR overexpression.

Topics: Brain Neoplasms; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 10; DNA, Neoplasm; DNA, Satellite; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glioblastoma; Glioma; Humans; Immunohistochemistry; Paraffin; Polymerase Chain Reaction; Transforming Growth Factor alpha

When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.

Topics: Animals; Antibodies; Cell Division; Cell Line; Cell Line, Transformed; Cells, Cultured; DNA Replication; Embryo, Mammalian; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Glioma; Kinetics; Mice; Plasmids; Rats; Receptor, IGF Type 1; Tumor Cells, Cultured

In addition to its powerful vasoconstrictive activity, endothelin-1 (ET-1) has been recognized to stimulate DNA synthesis in some cell lines. In this study, we confirmed the existence of ET-1 receptor in YKG-1 human glioma cells, and investigated its effect on DNA synthesis in YKG-1 for 6 consecutive days, comparing it with that of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Scatchard analysis of the binding data revealed the presence of a single class of high-affinity binding molecule. The apparent dissociation constant (Kd) was 5.2 x 10(-9) M and the maximal binding capacity (B max) was 4.7 x 10(4) sites/cell. The percentage of non-cycling cells was initially more than 85%, and decreased to 55.40%, 24.22%, 11.50%, and 7.51% on days 1, 2, 4, and 6, respectively, after ET-1 stimulation. Although ET-1 reduces the fraction of non-cycling cells more slowly than other growth factors such as EGF, PDGF and IGF-I, it reaches the same level as the others by day 6. These results indicate that YKG-1 human glioma cells have ET-1 receptors and that ET-1 initiates a peculiar slow induction of DNA synthesis in these cells, suggesting that secondary factors might exist to accelerate the DNA synthesis in response to ET-1.

Topics: Cell Cycle; Cell Division; Cell Line; DNA Replication; DNA, Neoplasm; Endothelins; Epidermal Growth Factor; Glioma; Growth Substances; Humans; Insulin-Like Growth Factor I; Kinetics; Platelet-Derived Growth Factor; Receptors, Endothelin; Time Factors; Tumor Cells, Cultured

The effects of tyrphostin, a selective protein tyrosine kinase inhibitor, on epidermal growth factor (EGF)-stimulated cell growth and EGF-receptor tyrosine kinase activity were studied in four human glioma cell lines. Stimulation by EGF induced variable enhancements of cell growth as well as tyrosine phosphorylation of EGF receptor and intracellular target proteins in all glioma cell lines. The level of immunoreactive EGF receptor detected with antibodies against extra- and intracellular domains was moderate in all four glioma cell lines, but markedly decreased with the latter antibody in two glioma cell lines. This variation was associated with considerable reduction of the EGF-stimulated tyrosine autophosphorylation level. Tyrphostin inhibited dose-dependently the EGF-stimulated cell growth and tyrosine autophosphorylation in all glioma cell lines, and the optimum time for the maximum inhibitory effect on tyrosine autophosphorylation was 12 to 18 hours after treatment with tyrphostin. The antiproliferative activity of tyrphostin nearly correlated quantitatively with its potency as an inhibitor of the EGF-stimulated EGF receptor tyrosine kinase activity. Tyrphostin had no significant effect on the immunoreactive EGF receptor levels, on the affinity constants and numbers of EGF receptor, or on the down-regulation and specific internalization of EGF receptor in any glioma cell line, suggesting that the effects of tyrphostin are not likely to be the results of reduction in EGF receptor and EGF binding capacity. In addition, the serum-stimulated cell growth was also inhibited dose-dependently by higher concentrations of tyrphostin in all glioma cell lines. It might be suggested, therefore, that tyrphostin inhibits EGF-stimulated cell growth by a specific suppression of EGF receptor tyrosine kinase activity, and at higher concentrations there appears to be some degree of either nonspecific inhibition or inhibition of serum-stimulated protein tyrosine kinase activity to induce the cell growth inhibition of gliomas.

Topics: Catechols; Cell Division; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Kinetics; Nitriles; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrphostins

Autocrine growth due to dysregulation of growth factor production may have a role in the development of neoplasia. We demonstrated that U251MG, a well characterized human malignant glioma cell line, had high affinity receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) by enzyme-linked immunosorbent assay. We assessed the inhibitory effect of anti-EGF receptor (EGFR), anti-FGF, and anti-PDGF monoclonal antibodies (MoAbs) on the growth of U251MG cells using the MTT assay and 3H thymidine uptake. At 50 micrograms/ml, the EGFR, FGF, and PDGF MoAbs significantly decreased cell numbers by 31.0%, 31.2%, and 31.0%, respectively, when compared to control cultures in the MTT assay. At the same concentration, the EGFR, FGF, and PDGF MoAbs reduced 3H thymidine uptake by 45.2%, 41.1%, and 40.0%, respectively, when compared to control cultures. At 50 micrograms/ml, a combination of the 3 MoAbs (16.6 micrograms/ml each) caused a 13.7% greater decrease in cell numbers in the MTT assay and an 11.9% greater decrease of 3H thymidine uptake. These findings suggest that the antigrowth factor MoAbs interrupted the autocrine loop at the growth factor receptor level. In conclusion, the demonstration that MoAbs directed against EGFR, FGF, and PDGF inhibit the growth of malignant glioma cells in vitro raises the possibility that these antibodies could be used clinically to treat malignant glioma either alone or conjugated to other agents.

Topics: Antibodies, Monoclonal; Cell Division; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Humans; Platelet-Derived Growth Factor; Tumor Cells, Cultured

The effects of 5 different growth factors [EGF, PDGF(bb), TGF-alpha, bFGF and IL-2] were studied on tumour spheroids obtained from 5 different human glioma cell lines (U-251MG, D-263MG, D-37MG, D-54MG, GaMG). The expression of EGF and PDGF receptors as well as the endogenous production of TGF-alpha and PDGF were studied by Northern blot analyses. After growth-factor-exposure, tumour spheroid volume growth, and directional cell migration from the spheroids were studied. In addition, tumour-cell invasion was studied in vitro, where foetal rat-brain aggregates were used as a target for the tumour cells. In all the assays a common stimulator for most of the cell lines was EGF. The other growth factors had a more heterogeneous stimulatory effect. Tumour-cell invasion, cell growth and cell migration are biological properties which are not necessarily related to each other. This may explain why the tumours often responded differently to the growth factors in the various assay systems. Two of the cell lines studied were non-invasive (U-251MG, D-263MG). It is shown that these were stimulated both in the directional migration assay and in the spheroid-volume-growth assay. However, their non-invasive behaviour was not influenced by the growth factors studied.

Topics: Animals; Blotting, Northern; Brain; Brain Neoplasms; Cell Aggregation; Cell Division; Cell Movement; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Growth Substances; Interleukin-2; Neoplasm Invasiveness; Phenotype; Platelet-Derived Growth Factor; Rats; Receptors, Growth Factor; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Targeting with toxic EGF-based conjugates against tumour cells with amplified EGF-receptors might be a possible approach towards improved therapy of certain malignancies such as gliomas and squamous carcinomas. In this study, the penetration and binding of 125I delivered by EGF-dextran conjugates were analysed in cultured spheroids applied as a tumour nodule model. The spheroids consisted of human glioma cells, U-343MGaCl2:6, with large amounts of EGF-receptors. The penetration and binding patterns of 125I delivered by 125I-EGF and 125I-dextran were analysed for comparison. The EGF-dextran associated 125I-activity showed a rather slow penetration but after some hours significant amounts of radioactivity had reached the deeper regions and good penetration was obtained within 5 hours. The penetration seemed somewhat faster when the 125I-activity was delivered with EGF possibly dependent on the lower molecular weight allowing for faster diffusion. Furthermore, EGF-dextran associated 125I seemed to penetrate somewhat faster after the EGF-receptors were blocked with non-radioactive EGF, probably due to the lack of binding preventing free diffusion. After administration of 125I-EGF-dextran or 125I-EGF, the binding patterns were superimposed on the penetration patterns. In the penetration studies, the superimposed accumulations due to binding were removed by presaturation of the receptors with non-radioactive EGF. After a 1 hour incubation, binding of EGF-dextran associated 125I-activity could be seen only in an outer region, with an approximative thickness of 50 microns, of the viable cell layer. Extensive receptor specific binding in the deeper regions, at a depth of 100-200 microns, was seen after several hours incubation. In addition, low levels of non-specific binding in the central regions were seen when the 125I-activity was delivered with dextran without EGF. A similar low background binding was seen also in the centre of spheroids incubated with 125I-EGF-dextran or 125I-EGF after saturation of the receptors with non-radioactive EGF. However, the major amount of radioactivity delivered as 125I-EGF-dextran or 125I-EGF had a receptor specific binding and, also in inner regions, it could be displaced by non-radioactive EGF. Thus, EGF-dextran, which is a candidate compound for targeted therapy, allowed penetration of the applied radioactivity and binding could be observed, after some hours, also in the inner regions of the spheroids.

Topics: Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Tumor Cells, Cultured

Conjugates based on transforming growth factor alpha, TGF alpha, or epidermal growth factor, EGF, are candidates for targeted radiotherapy against EGF-receptor rich tumours such as gliomas or squamous carcinomas. In this study, binding, internalization and excretion of radiolabelled TGF alpha and TGF alpha-dextran conjugates was analysed in an EGF-receptor rich human glioma cell line. The binding of 125I-TGF alpha was EGF-receptor specific and the binding pattern was similar to that of 125I-EGF. The TGF alpha-dextran conjugate also bound specifically but gave maximum binding for a longer time during continuous incubation compared to when only TGF alpha was used. The excretion pattern of internalized radioactivity was somewhat slower for 125I-TGF alpha-dextran, with 125I-labelling on the TGF alpha part, as compared to 125I-TGF alpha although most of the radioactivity in both cases was excreted within 4 hours. The fate of the dextran part of the conjugate, as followed by means of 125I-labelling of the dextran, was different since all radioactivity in that case remained cell-associated for at least up to 22 hours. Furthermore, by comparison with previously published results, it was seen that the radioactivity delivered through the TGF alpha part of TGF alpha-dextran was retained for a shorter period of time by the cells than when delivered by EGF in EGF-dextran conjugates. However, when the radioactivity was delivered by the dextran part of the conjugates, the radioactivity seemed to be retained equally well or even better when TGF alpha-dextran was applied. It is concluded that TGF alpha-dextran, as well as EGF-dextran, have interesting properties for targeting against EGF-receptors and that the dextran part is well retained in the cells and therefore might be a suitable carrier for toxic agents such as radionuclides. It is of high interest to continue with toxicological and pharmacological in vivo studies of the conjugates.

Topics: Binding Sites; Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Transforming Growth Factor alpha; Tumor Cells, Cultured

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

Topics: Brain Neoplasms; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glioma; Humans; Immunohistochemistry; Lymphokines; Models, Biological; Neovascularization, Pathologic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Epidermal growth factor (EGF) content in urine from patients with glial tumors was examined by radioimmunoassay techniques with labeled human EGF and its rabbit EGF polyclonal antibody. There was no cross-reaction with transforming growth factor-alpha, which has a common receptor with EGF. Forty glial tumors were divided into three groups according to the clinical stage: Samples from Group A patients were obtained before therapy and/or after biopsy; in these patients a large volume of tumor was apparent on computerized tomography (CT). Group B samples were obtained after gross total removal of the tumor and/or chemo- and radiation therapy; these patients showed a small volume of residual tumor on CT. Samples from Group C patients were obtained after gross tumor total removal and/or chemo- and radiation therapy; no tumor was detected on CT scans in these patients. Urinary EGF levels in Group A samples were statistically significantly higher than in samples from healthy individuals (p < 0.001), Group B patients (p < 0.10), and Group C patients (p < 0.02). In addition, high-grade glial tumors in Group A cases showed a significantly higher level of urinary EGF than low-grade tumors in Group A patients (p < 0.05), or patients with meningioma (p < 0.02), metastatic brain tumor (p < 0.05), and cerebral infarction (p < 0.001). Longitudinal changes of urinary EGF levels in glioma patients mostly synchronized with the clinical course and therapeutic interventions. Therefore, urinary EGF, as a glial tumor marker, may be of practical value for diagnosing a malignant glioma and evaluating for the efficacy of chemo- and radiation therapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Brain Neoplasms; Epidermal Growth Factor; Female; Glioma; Humans; Male; Middle Aged; Radioimmunoassay

Spheroids initiated directly from human primary gliomas were used to investigate the effects of EGF, bFGF, NGF and PDGF(bb) on cell proliferation, migration and invasion into foetal rat brain tissue. EGF increased tumour spheroid volume in 10 of 13 glioblastomas studied, whereas 5 of 11 tumours responded to bFGF. NGF increased the spheroid volume in 2 of 5 tumours. In 8 tumours, PDGF(bb) had no effect on tumour spheroid volume. An increase in BUdR-labelling indices confirmed that cell proliferation was responsible for the volume increase observed in stimulated spheroids. EGF stimulated cell migration in 5 and bFGF in 3 of 8 tumours studied. NGF stimulated cell migration in 1 of 5 glioblastomas, whereas 1 of 3 glioblastomas responded to PDGF(bb). The effects of growth factors on the invasion of spheroids prepared from the glioblastoma biopsy specimens were also studied in vitro using foetal rat brain aggregates as target tissue. EGF stimulated invasion in 7 of 8 glioblastomas studied, whereas bFGF stimulated invasion in 2 of these tumours. NGF or PDGF(bb) did not increase the invasiveness of the glioblastoma tissue. Our results represent the net effect of the growth factors on a complex tumour-cell population. We conclude that exogenously administered growth factors, EGF in particular, increase the cell proliferation as well as migratory and invasive capacities of cultured primary brain tumour biopsies in vitro.

Topics: Animals; Brain; Brain Neoplasms; Cell Division; Cell Movement; Epidermal Growth Factor; Fetus; Fibroblast Growth Factor 2; Glioblastoma; Glioma; Growth Substances; Humans; Neoplasm Invasiveness; Nerve Growth Factors; Platelet-Derived Growth Factor; Rats; Tumor Cells, Cultured

The insulin-like growth factors are postulated to play a role during brain development. Because they are believed to act in a paracrine/autocrine manner, the production of insulin-like growth factor-I (IGF-I) by cultured astroglial cells was examined. Quantities of IGF-I in conditioned media were determined by RIA after separation of IGFs from IGF-binding proteins by high-pressure liquid chromatography. Astrocytes from 1-day-old rats and the rat glioma cell line (C6) both secreted 7.5-kDa IGF-I. A peak of immunoreactivity with an apparent mol wt of 12,000 was additionally present in media conditioned by C6 cells. Exposure to epidermal growth factor (EGF) increased media content of immunoreactive IGF-I slightly (60%) in C6 cells but more than 2-fold in normal astrocytes. Fibroblast growth factor also increased the amount of IGF-I contained in media conditioned by normal astrocytes. To determine whether the secreted IGF-I was biologically active, media IGFs were immunoneutralized with a monoclonal antibody (Sm 1.25). In the presence of the antibody, EGF-stimulated astrocyte replication was blocked. These data indicate that IGF-I secretion by rodent astrocytes is stimulated by factors thought to be important for brain growth and development and that the IGFs are likely intimate participants in EGF-induced astrocyte growth.

Topics: Animals; Antibodies, Monoclonal; Astrocytes; Brain Neoplasms; Cell Division; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Indicators and Reagents; Insulin; Insulin-Like Growth Factor I; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

Cloned neoplastic astrocytes from a human glioma-derived cell line (IPSB-18) were grown in fetal calf serum (FCS)-supplemented culture medium in the presence of three growth factors. Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF) induced an increase in the number of cells positive for the ganglioside-recognizing monoclonal antibody, A2B5. No such growth factor-mediated induction could be detected in cells maintained in plasma-derived serum (PDS)-supplemented medium. Small molecules, removed from PDS during dialysis, may, therefore, act synergistically with growth factors in the control of ganglioside synthesis.

Topics: Astrocytes; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fluorescent Antibody Technique; Gangliosides; Glioma; Histocytochemistry; Humans; Platelet-Derived Growth Factor; Tumor Cells, Cultured

Epidermal growth factor, EGF, and 131I or 125I-labelled tyrosine were conjugated to the sugar polymer dextran. The conjugates bound to EGF-receptor rich human glioma cells in culture and the binding was mainly receptor specific because cells presaturated with nonradioactive EGF gave strongly reduced binding. The 131I labelled conjugates were used in tests on cellular retention and therapeutical effects. 131I activity delivered to the cells as EGF-dextran-tyrosine-131I remained cell-associated for much longer periods of time than 131I activity delivered by only EGF. The amount of cell-associated 131I activity was nearly constant for up to 25 hours. The 131I labelled conjugate gave, after a one hour incubation period for binding followed by a 25 hour incubation in nonradioactive medium, a good therapeutical effect. This effect, which corresponded to about 3.0 Gy of external 60Co radiation, was due to the specifically bound 131I. The comparatively small effects of nonbound 131I in the culture medium, present only during the first incubation hour, were measured in control experiments with presaturated receptors and were corrected for in the evaluation of the EGF-receptor mediated effects. Control experiments showed that neither nonradioactive EGF nor non-radioactive EGF-dextran conjugates gave measurable effects on clonogenic growth. The results obtained were promising and the possibilities to use EGF-dextran conjugates for therapy should be further examined.

Topics: Brain Neoplasms; Cell Survival; Culture Media; Dextrans; Drug Carriers; Epidermal Growth Factor; Glioma; Humans; Iodine Radioisotopes; Time Factors; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrosine

Analysis of genomic organization and expression of platelet-derived growth factor receptors (PDGFR) and epidermal growth factor receptor (EGFR) in human malignant gliomas showed amplification and overexpression of both receptors in distinct subsets of tumors. Amplification of the alpha PDGFR was detected in 4 of 50 glioblastomas (8%). EGFR was amplified in 9 of the 50 tumors (18%). Western blot analysis showed elevated expression of alpha PDGFR and EGFR proteins in 4 (24%) and 3 (18%), respectively, of 17 tumor specimens analyzed. Increased production of alpha PDGFR as well as EGFR proteins was observed in the presence or absence of gene amplification. Three of the 4 tumors with elevated levels of alpha PDGFR also overexpressed the beta PDGFR, which was present as a single copy gene in all 50 tumors analyzed. Our findings suggest that the amplification and/or overexpression either of EGFR or of the alpha PDGFR along with the coordinate overexpression of the beta PDGFR can contribute to the malignant phenotype of distinct subsets of human glioblastoma.

Topics: Blotting, Western; Brain Neoplasms; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioma; Humans; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor

The proliferation rates of gliomas may be modulated by the protein kinase C (PKC) signal transduction system. The present study was undertaken to further examine the role of PKC system in growth regulation of gliomas in vitro by measurement of PKC activity over various phases of tumor growth and by assessing its potential role as a signal transduction system induced by serum mitogens and the known glioma mitogens epidermal growth factor and fibroblast growth factor. All human glioma lines examined, and the rat glioma C6, displayed high PKC activity relative to nonmalignant glial cells, which correlated with their proliferation rates over their respective growth phase. Frozen surgical human malignant glioma specimens also displayed high PKC activity. The relatively selective PKC inhibitor staurosporine (SP) reduced PKC activity and corresponding growth rates in a dose-related manner. Stimulation of PKC with phorbol esters under different concentrations of serum in the growth medium indicated that the high PKC activity, which correlated with their rapid growth rates, is highly susceptible to down-regulation by these agents. Epidermal growth factor and fibroblast growth factor increased both PKC activity and the growth rate of glioma line A172; addition of SP reduced the growth rate to levels observed in SP-treated control tumors, indicating that PKC may be a common signal transduction system induced by these mitogens. These results implicate PKC as an important signal transduction system regulating glioma growth, and offers a potential target for tumor inhibition.

Topics: Animals; Brain Neoplasms; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Growth Substances; Humans; Protein Kinase C; Rats; Signal Transduction; Tumor Cells, Cultured

Cultures of fetal rat brain cell aggregates and tumor spheroids from the human glioma cell line GaMG were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF) or isoforms of platelet-derived growth factor (PDGF AA or BB). Radioreceptor binding studies displayed a high binding capacity for EGF and FGF, but not binding of PDGF isoforms in the glioma cells. In serum-free culture, 10 ng/ml of both EGF and FGF caused increased growth and cell shedding in the tumor spheroids, whereas PDGF produced no such effect. Similarly, EGF and FGF stimulated tumor cell migration. EGF increased the proliferation and outgrowth of glial fibrillary acidic protein (GFAP)-positive cells in brain cell aggregates, while PDGF AA and BB both stimulated the outgrowth of oligodendrocyte-like cells which were negative for GFAP and neuron-specific enolase. FGF stimulated GFAP+ as well as GFAP- cell types. In co-culture experiments using brain aggregates and tumor spheroids, both EGF and FGF treatment caused increased tumor cell invasion. PDGF had no effect on the tumor cells, but instead stimulated the proliferation of oligodendrocyte-like cells in the brain aggregates. The present results indicate that growth factors may facilitate glioma growth as well as invasiveness, and cause reactive changes in the surrounding normal tissue.

Topics: Animals; Brain; Brain Neoplasms; Cell Migration Inhibition; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glial Fibrillary Acidic Protein; Glioma; Growth Substances; Humans; Immunoenzyme Techniques; Immunohistochemistry; Platelet-Derived Growth Factor; Radioligand Assay; Rats; Tumor Cells, Cultured

The expression of epidermal growth factor receptor (EGF-R) was examined in 27 primary human brain tumors (7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas, 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependymoma, 1 histiocytic lymphoma), in 6 brain metastases from lung carcinomas and in 20 meningiomas. Peritumoral tissues histologically normal excised surgically along with a large tumor were used as control. All plasma membranes from brain tissues tested showed specific EGF binding. The EGF receptor is expressed at low levels in the control human brain and at very high levels in 60% of the total intracranial tumors studied. When the various histological types of tumors were analyzed, the higher percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71%), while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). A good correlation between binding and total amount of EGF-R protein detected by Western Blot was also observed.

Topics: Adult; Aged; Astrocytoma; Blotting, Western; Brain Neoplasms; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; Humans; Iodine Radioisotopes; Male; Meningioma; Middle Aged; Neurilemmoma; Oligodendroglioma; Radioligand Assay

Using an in vitro monolayer natural killer (NK) cytolysis assay, the authors examined the effects of serum concentration and epidermal growth factor (EGF) on sensitivity to NK cytolysis. It was found that target cells cultured in high concentrations of serum (10% fetal bovine serum (FBS)) had higher cytotoxicity levels than those in low serum concentrations (0% to 0.5% FBS). Exposure of target cells to EGF had no effect on their sensitivity to NK cytolysis. Both glioma cell lines showed decreased NK cell sensitivity with longer times in culture. The results of cytofluorometric studies on these cell lines indicate that the differences in NK cell sensitivity may reflect the growth fraction of the target population and that a population with a higher proportion of cycling cells is more susceptible to lysis by NK cells. Whether it is possible to separate the proliferative rate of these cells from their NK cell sensitivity is unknown, but worthy of consideration.

Topics: Blood; Brain; Brain Neoplasms; Cell Cycle; Culture Media; Epidermal Growth Factor; Glioma; Humans; Immunotherapy; Killer Cells, Natural; Tumor Cells, Cultured

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Chromosome Deletion; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Rearrangement; Glioma; Humans; Kinetics; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogenes; Receptor, ErbB-2; Transforming Growth Factor alpha

Targeting of toxic substances to the epidermal growth factor, EGF, receptor might be an attractive therapeutic approach because of the increased receptor-expression in some human tumours such as, for example, malignant gliomas and squamous lung carcinomas. Radiation effects of [131I]EGF on human malignant glioma cells growing as monolayers were analysed in this study. The cells were, in all cases, incubated for 25 min with about 350 kBq/ml [131I]EGF, which gave a total binding of 3.2-3.5 kBq/10(5) cells. The rapid release of activity from the cells caused by the normal degradation of EGF was inhibited by incubation with 30 microM chloroquine or 5 mM lidocaine added to the cell culture medium. These substances are, at these concentrations, known to inhibit proteolytic processes in lysosomes. No effects of the inhibitors alone were observed on cell growth and clonogenic survival. Inhibition of EGF degradation by chloroquine or lidocaine resulted in a significantly prolonged association of 131I with the test cells. About 70% of the initially bound radioactivity remained in the cells giving, after 6 h, a binding of 2.1-2.5 kBq/10(5) cells. A 6 h exposure to the radiation from 131I decays, mediated mainly by specifically bound EGF, gave a survival value of about 50%. Such an effect corresponds to a treatment of 2.5 Gy 60Co gamma-radiation. This is promising considering that, when monolayers are applied, only a very small fraction of the released energy from the 131I decays is deposited in the cells. Effects from non-receptor bound [131I]EGF were analysed after presaturation of the receptors with non-radioactive EGF, and gave no or very small changes in survival.

Topics: Cells, Cultured; Chloroquine; Epidermal Growth Factor; Glioma; Humans; Iodine Radioisotopes; Lidocaine

Some gliomas, melanomas and squamous carcinomas have large numbers of EGF receptors which could, in these cases, be used for targeting with toxic agents. We investigated whether EGF could be conjugated to dextran, which is a suitable carrier for toxic agents, without losing its ability to bind to the receptor. Dextran of 20 kDa molecular weight was activated with I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) yielding highly active pyridinium-isourea derivatives. EGF was coupled to the activated dextran through the amino terminus and glycine was added to block residual activity. The EGF-dextran conjugate was, after purification on Sephadex G25 and Sephacryl 200 columns, tested for its receptor binding properties on human malignant glioma, U343MGaC12:6, cells. The conjugate inhibited binding of 125I-EGF in a competitive assay, showing that the binding was receptor-specific. Dextran conjugated with glycine, without EGF, had no inhibitory effect. The conjugate was radio-labelled either on the EGF part with 125I or on the dextran part with 3H-glycine, and the internalization patterns were compared to the internalization of 125I-EGF. The radioactivity of the conjugates remained cell-associated for more than 20 hr, regardless of whether the radioactivity was on the EGF or on the dextran part, while the radioactivity of unconjugated EGF rapidly disappeared from the cells. Most of the cell-associated radioactivity was, at all analysed time intervals, located intracellularly. Thus, it seems promising to use dextran, conjugated with EGF, as a carrier of, for example, toxic radioactive nuclides.

Topics: Dextrans; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Kinetics; Nitriles; Protein Binding; Pyridinium Compounds; Temperature; Time Factors; Tritium; Tumor Cells, Cultured

Low-grade astrocytomas, anaplastic astrocytomas and glioblastomas in vitro were found to ubiquitously produce the mRNA of transforming growth factor-beta (TGF beta). TGF beta 1 and TGF beta 2 mRNA were expressed to a lesser degree among the hyperdiploid malignant gliomas. By radioreceptor assay of conditioned medium, TGF beta was secreted predominantly in latent form, in both latent and active form, or only in active form within a panel of low-grade and malignant gliomas. The TGF beta receptor (types I, II, and III) was evident among the glioma lines. Many near-diploid gliomas were growth-inhibited by TGF beta 1 and TGF beta 2 in vitro. Most hyperdiploid glioblastomas showed a positive mitogenic response to exogenous TGF beta 1 and TGF beta 2. A synergistic or additive mitogenic interaction with epidermal growth factor and insulin was observed among some. Under serum-free conditions, anti-TGF beta antibody neutralized the expected growth-regulatory effect of endogenous TGF beta, thus establishing the specificity of the response in vitro. TGF beta 1 also enhanced the clonogenicity of certain gliomas which had been growth-stimulated in monolayer. Thus, basic elements in support of an autocrine hypothesis have been demonstrated: TGF beta mRNA was expressed among low-grade and malignant gliomas, TGF beta was secreted in latent and/or active form into conditioned media and appeared to serve as an endogenous regulator of glioma proliferation in vitro. The mitogenic response, either positive or negative, correlated with the degree of anaplasia and karyotypic divergence.

Topics: Blotting, Northern; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; Gene Expression; Glioma; Humans; In Vitro Techniques; Insulin; Radioligand Assay; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

We have studied binding of 125I-EGF to the human malignant glioma cell line U-343 MG aCl2:6, which is planned to be used as a model system in studies of toxic effects of EGF conjugates. Special care has been taken to fulfil the requirements for a correct Scatchard analysis of binding parameters. Binding as a function of time, temperature and pH was investigated as well as dissociation and internalization of bound EGF. The stability of EGF during incubation was also determined. After binding to the receptor, EGF is rapidly internalized and degraded at physiological temperature. We found that binding experiments should be performed at 4 degrees C, since at this temperature practically no internalization took place, whereas dissociation occurred. From displacement experiments using increasing concentrations of unlabelled EGF competing with 125I-EGF for binding, binding parameters were calculated using a computerized, nonlinear, least-squares regression analysis of binding data. We found that EGF bound to a class of high affinity receptors with an apparent dissociation constant KD of about 4 x 10(-10) M. The mean number of receptors was 25,000 per cell. In experiments where receptors were saturated with 125I-EGF an additional class of low affinity receptors was detected. This had an apparent KD of 1 x 10(-8) M with a mean receptor number per cell of 780,000. We also noticed enhanced dilution-induced dissociation of bound 125I-EGF in the presence of excess unlabelled EGF, suggesting negative cooperativity.

Topics: Cell Count; Cell Line; Chemical Precipitation; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Hydrogen-Ion Concentration; Iodine Radioisotopes; Kinetics; Radioligand Assay; Temperature; Trichloroacetic Acid

Growths factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in culture and probably also for cells in vivo. Evidence has been derived for autocrine system in which the cell produces its own growth factor. Several growth factors as well as their cellular receptors have been identified as productions of proto-oncogenes. Furthermore, these growth factors have been identified as mitogens in tumors of the central nervous system. The roles of growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and its receptor. Insulin-like growth factors (IGFs), transforming growth factors (TGFs) and fibroblast growth factor (FGF) on the proliferation of brain tumors, especially glioma were reviewed. The activation of cellular proto-oncogenes resulting in the autocrine system of growth factors and their receptors offers the opportunity for therapeutic interference. Therapeutic efforts will be based on the concepts of neutralization of growth factors, antagonizing growth factors at their receptors, irreversibly blocking receptors, and interference with oncogene product synthesis. Specific antibody for growth factors or receptors will be able to inhibit the proliferation. Trapidil, an antagonist for PDGF, can inhibit the proliferation of a PDGF-producing glioma cell. We can assume that the further analysis of growth regulatory mechanism will allow the design of new therapeutic approaches.

Topics: Brain Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Humans; Oncogenes; Platelet-Derived Growth Factor; Transforming Growth Factors; Trapidil

Anomalies of the epidermal growth factor receptor (EGFR) gene, including amplification, rearrangement, and overexpression, have been reported in malignant human gliomas in vivo. In vitro glioma cell lines coexpress EGFR and at least one of its ligands, transforming growth factor alpha, suggesting the existence of an autocrine growth stimulatory loop. We have studied the tumor tissue from 62 human glioma patients and examined the structure and quantity of the EGFR gene and its transcripts, as well as the quantity of the receptor protein. In addition we have examined the genes and transcripts coding for the pre-pro forms of epidermal growth factor and transforming growth factor alpha, the two endogenous EGFR ligands. EGFR gene amplification was detected in 16 of the 32 malignancy grade IV gliomas (glioblastoma) studied (50%), but only in 1 of 30 gliomas of lesser malignancy grade (I-III). All tumors with an amplified gene overexpressed EGFR mRNA. More than one-half (62.5%) of the glioblastomas with amplified EGFR genes also showed coamplification of rearranged EGFR genes and concomitant expression of aberrant mRNA species. Overexpression, without gene amplification, was observed in some of the low grade gliomas, and aberrant EGFR transcripts were also seen in some cases without gene amplification or detected gene rearrangements. mRNA expression for one or both of the pre-pro forms of the ligands was detected in every tumor studied. Thus, several mechanisms for the activation of the EGFR-mediated growth stimulating pathway are possible in human gliomas in vivo: expression of a structurally altered receptor that may have escaped normal control mechanisms; and/or auto-, juxta-, or paracrine stimulating mechanisms involving coexpression of receptor and ligands, with or without overexpression of the receptor.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Middle Aged; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

The expression of epidermal growth factor receptor (EGFR) was determined in cryosections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and two monoclonal antibodies (mAb) against EGFR. All gliomas were found to express EGFR when examined with B-EGF, whereas 33 expressed EGFR when examined with the two mAbs. The highly malignant gliomas (glioblastomas and anaplastic astrocytomas) had a more heterogeneous staining strongly with B-EGF than did the low-grade gliomas (astrocytomas, oligodendrogliomas, mixed gliomas, and ependymomas). This indicates that high-grade gliomas contain more tumour cells rich in EGFR than do the low-grade gliomas. Reactive astrocytes, ependymal cells, and many types of nerve cells (cerebral cortical pyramidal cells, pyramidal and granular hippocampal cells, Purkinje cells, cerebellar granular cells and neurons in the molecular layer of the cerebellum) expressed EGFR, whereas small neurons and normal glial cells were not found to express EGFR.

Topics: Adult; Aged; Antibodies, Monoclonal; Brain Chemistry; Brain Neoplasms; Child; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescence; Glioma; Histocytochemistry; Humans; Male; Middle Aged; Neurons

Malignant glioma cells often have more epidermal growth factor (EGF) receptors than normal cells and targeting of toxic substances to the receptor might therefore be an attractive therapeutical approach. Radiation effects were analysed on human glioma cells growing as monolayers after exposure to 131I-EGF. Unspecific effects were analysed with 131I-BSA or after presaturation with nonradioactive EGF. The radiation effects were compared to the effects obtained by external 60Co gamma irradiation. Administration of the highest radioactive concentrations, 0.2-0.5 MBq/ml in the culture medium, corresponded, after 20 min incubation, to a binding of about 1.0-2.5 dpm/cell. Such an exposure to 131I decays gave effects on cell survival corresponding to about 2.5 Gy of external gamma irradiation. Somewhat less than half of this effect came from the specific bound radioactivity and the rest from nonbound radioactivity. When administrating lower concentrations of radioactivity both the binding and the radiation effects were smaller. The observations showed that it is possible to inactivate cell-proliferation of glioma cells with specific bound 131I-EGF. The possibilities to fractionate the treatments and of binding also other toxic agents than 131I to the EGF receptor are discussed.

Topics: Cell Line; Cell Survival; Clone Cells; Cobalt Radioisotopes; Epidermal Growth Factor; ErbB Receptors; Gamma Rays; Glioma; Humans; Iodine Radioisotopes; Kinetics

Stimulation of primary cultures of rat astrocytes with appropriate agents results in the mobilization of arachidonic acid from intracellular lipid pools and the synthesis of eicosanoids. Thromboxane A2 is one of the major prostanoids released upon stimulation with calcium ionophore, phorbol esters, and ATP; but a number of other predicted effectors are inactive. In an attempt to understand the pathophysiological significance of eicosanoid release from astrocytes, primary cultures have been derived from human astrocytic glioma biopsies. The majority of cells in the cultures expressed glial fibrillary acidic protein (GFAP), frequently in conjunction with vimentin and fibronectin. Cell sorting revealed that a significant proportion of cells in the cultures from the high-grade (malignant) tumors expressed epidermal growth factor receptor, indicative of neoplastic cells. Both effective and ineffective agents in rat cultures were tested for their ability to stimulate release of thromboxane from these gliomas, and also from cultures of medulloblastoma and ependymoma which contained significant numbers of GFAP-positive cells. Only cells from the high-grade tumors released thromboxane in response to the known effective stimuli. While the muscarinic agonist carbachol was ineffective, norepinephrine evoked thromboxane release from malignant astrocytomas. These data show that cells derived from malignant human gliomas retain the ability to release thromboxane upon stimulation and suggest that a transformation in receptor coupling might accompany neoplasia, such that the cells now respond to a previously ineffective agonist.

Topics: Astrocytes; Carbachol; Epidermal Growth Factor; Flow Cytometry; Glial Fibrillary Acidic Protein; Glioma; Humans; Norepinephrine; Thromboxanes; Tumor Cells, Cultured

To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and C6), and two low-passage human glioma lines (obtained from surgical specimens) were studied. With the exception of the C6 line, all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma. At optimal concentrations (5 to 10 ng/ml), TPA produced a two- to five-fold increase in the rate of DNA synthesis (p less than 0.05) as assessed by incorporation of 3H-thymidine. However, TPA had no additive effect on the mitogenic response produced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). Inhibition of PKC using the antibiotic polymyxin B (20 micrograms/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.

Topics: Animals; Brain; Brain Neoplasms; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; Glioma; Humans; Platelet-Derived Growth Factor; Polymyxin B; Polymyxins; Protein Kinase C; Rats; Thymidine; Tumor Cells, Cultured

The gene amplification and expression of transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R) in human gliomas was determined by Southern blot analysis and a receptor binding study. Amplification of the EGF-R gene was demonstrated in 3 of 11 tumors examined. All were glioblastomas, whereas the TGF-alpha gene was amplified in 7 of 11 tumors, and 6 of the 7 were recurrent glioblastomas and anaplastic astrocytomas. A competitive binding study with iodinated EGF showed a TGF-alpha-like activity ranging from 1.6-31.5 ng of EGF/microgram protein. These results suggest a differential expression of EGF-R and TGF-alpha genes among untreated and recurrent malignant gliomas.

Topics: Binding, Competitive; Blotting, Southern; Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioblastoma; Glioma; Humans; Iodine Radioisotopes; Tumor Necrosis Factor-alpha

Both permanent cultured cell lines and athymic mouse xenografts were established from two human glioblastomas. Biopsies from D-245 MG and D-270 MG contained amplified and rearranged epidermal growth factor receptor (EGFR) genes. Although the gene amplification and rearrangement seen originally was maintained in the xenografts, cultured cell lines established from these biopsies lost the amplified rearranged genes in vitro. Analysis of these cell lines and 11 additional permanent human glioma cell lines with normal EGFR gene copy number showed from 2.7 x 10(3) to 4.1 x 10(5) high affinity EGFRs/cell by radioreceptor assay. The RNase A protection assay showed minimal differences in the quantity of EGFR mRNA among the 13 glioma lines, while the D-245 MG and D-270 MG xenografts expressed approximately 10-20 times as much EGFR mRNA as the corresponding cell lines. Immunoprecipitation of EGFR from these lines, including D-245 MG and D-270 MG, demonstrated only the intact Mr 170,000 Da form, while truncated Mr 145,000 Da and 100,000 Da EGFR proteins were immunoprecipitated from the D-270 MG and D-245 MG xenografts, respectively. These studies demonstrate that gliomas with amplification of the EGFR gene are capable of establishing in culture but that the amplified rearranged genes are not maintained. Possible explanations are that the abnormal genes are lost during serial passage or that the cells with amplified rearranged genes only represent a minor subpopulation of cells, which are unable to grow in culture. In either case, these observations suggest that high expression and structural abnormalities of EGFR proteins generated by amplification and rearrangement of the EGFR gene provide a growth advantage for gliomas in vivo but not in vitro.

Topics: Adult; Aged; Animals; Cell Line; Chromosome Mapping; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioma; Humans; Karyotyping; Kinetics; Male; Mice; Mice, Nude; Neoplasm Transplantation; Nucleic Acid Hybridization; Transplantation, Heterologous

The use of a serum-free culture system for assessing the growth factor responsiveness of malignant glial cells is described. The mitogenic properties of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were examined in three human malignant glioma cell lines (T98G, U87, and U138). Each of the three had high-affinity EGF receptors and all responded in a dose-dependent fashion to physiological concentrations of EGF. These cell lines also showed a pronounced mitogenic response to PDGF which equaled or exceeded that achieved with EGF. Simultaneous stimulation with both factors produced an additive response, which approximated that obtained in medium supplemented with 10% fetal calf serum. The authors conclude that functional EGF and PDGF receptors were present in the human malignant glial tumors studied. The response of the human glioma lines to these growth factors in many respects parallels the response seen in fetal astrocytes tested under similar conditions. In contrast, the behavior of two chemically induced rat gliomas (9L and C6) differed significantly from that seen in the human lines, suggesting that the rat lines may not be entirely acceptable as models for studying the growth characteristics of human malignant glial tumors.

Topics: Brain Neoplasms; Cell Division; Culture Media; Epidermal Growth Factor; Glioma; Humans; Iodine Radioisotopes; Neurons; Platelet-Derived Growth Factor; Thymidine; Tumor Cells, Cultured

Effects of epidermal growth factor (EGF) and an antibody (Ab-528) reactive against the binding site for EGF on human EGF receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EGF receptor expression. The D-247 MG and D-263 MG spheroids grew slowly or not at all in the absence of EGF, while in the presence of EGF they were growth stimulated. Tumor cell migration, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of EGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 micrograms/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to EGF receptor expression; the two lines with medium to high receptor expression (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an in vitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both D-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal-sized Mr 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-alpha may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor.

Topics: Cell Division; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Immunohistochemistry; Neoplasm Invasiveness; Tumor Cells, Cultured

Growth factors contained in cultured medium of rat glioma C6 cells (C6 cells) were examined mainly for the activity of transforming growth factors (TGFs). Cultured medium of C6 cells was dialyzed against acetic acid, lyophilized and chromatographed by gel-permeation method, the fractions were assayed by soft agar colony formation, iodine 125 (125I)-epidermal growth factor (EGF)-binding competition and incorporation of tritium-thymidine. Two nontransformed cell lines, clonal NRK49F and BALB/3T3 A 31-1-1 (3T3) cells, were used as indicator cells for the soft agar colony assay. 3T3 cells were also used for the incorporation of tritium-thymidine. EGF receptor-rich A 431 cells were used for 125I-EGF-binding competition assay. The activity of alpha-type TGFs was examined by soft agar colony formation of NRK49F cells and inhibition of EGF-binding to A 431 cells since TGF alpha has sequence homology with EGF and binds to EGF receptors on the cell membrane, while the activity of beta-type TGFs was examined by soft agar colony formation of 3T3 cells and NRK 49 F cells with the addition of EGF. High level of activities of both TGF alpha and TGF beta were detected in 14,000 to 45,000 daltons, and also high level of the activity of DNA synthesis was detected at the same molecular weight. These results suggest that C6 cells produce TGF alpha and TGF beta as well as platelet-derived growth factors (PDGFs)-analogue. Since amplification of EGF receptor gene has been demonstrated in glioma, TGF alpha released by glioma may provide autocrine stimulation through the binding to the amplified EGF receptors. TGF beta is known to increase EGF receptors on the cell membrane. TGF beta has been demonstrated not only to stimulate but also inhibit cell proliferation under certain circumstances.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Brain Neoplasms; Colony-Forming Units Assay; DNA; Epidermal Growth Factor; Glioma; Growth Substances; Molecular Weight; Platelet-Derived Growth Factor; Rats; Transforming Growth Factors; Tumor Cells, Cultured

Cells from gliomas induced by N-ethyl-N-nitrosourea have a high basal level of plasminogen activator activity compared with cells from normal tissue. Plasminogen activator activity is known to be affected by many substances but whether inhibition or stimulation occurs depends on the cell and agent involved. It is not clear whether tumour and control cells from the same type of tissue respond similarly. A comparison has been made of the effect of several factors on both cell associated and secreted enzyme activity of cloned lines from a glioma and normal tissue. The effect of two cAMP elevating compounds was stimulatory while that of the steroid, dexamethasone, was generally inhibitory for both cells. However, the polypeptide hormone, epidermal growth factor, had a differential effect. It caused an increase in secreted enzyme activity in the tumour line but had no such effect on the control clone. The precise mechanism by which this occurs is unknown. Co-operative effects of the enzyme and growth hormone could result in more aggressive behaviour of the tumour cells.

Topics: Animals; Brain Neoplasms; Cell Line; Cholera Toxin; Colforsin; Dexamethasone; Epidermal Growth Factor; Ethylnitrosourea; Glioma; Plasminogen Activators; Rats

Formal proof for an involvement of autocrine stimulation in the disturbed growth of malignant cells has been difficult to obtain, in part due to lack of precise methods of assessing growth factor production and receptor occurrence. In this study we have analyzed the mRNA levels for two growth factors and the corresponding receptors in a number of established human malignant glioma cell lines. Twenty-one tested lines all contained transcripts for the platelet-derived growth factor (PDGF) A chain while 16-17 of 21 expressed the c-sis/PDGF B chain gene; these two genes were expressed independently of each other. PDGF receptor transcripts were present in 15-16 of the 21 lines. Transcripts for the epidermal growth factor receptor were found in all 15 tested lines, in 2 of them at high levels, and the corresponding ligand transforming growth factor-alpha was found in 11 of 15 lines. No amplification or structural rearrangements of the genes, as analyzed by Southern blot hybridization, could explain the varying expression of PDGF A and B chain transcripts or the elevated levels of epidermal growth factor receptor mRNA. A correlation was found between cell morphology and expression of growth factor and receptor mRNA in these lines. The highest amount of PDGF receptor transcripts was found in cells with fibroblast-like morphology, and c-sis/B chain transcripts were found in small cell types and in cells with astrocyte-like morphology, while no clear relationship was found between PDGF receptor and A chain transcript levels or between morphology and A chain transcripts. It is possible that the findings reflect a coordinated expression of these genes in the progenitor cells. In conclusion, the data imply the existence of two possible autocrine loops in human malignant glioma lines, affecting the PDGF and epidermal growth factor receptor pathways.

Topics: Cell Line; DNA; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Peptides; Platelet-Derived Growth Factor; Poly A; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA; RNA, Messenger; Transforming Growth Factors

Primary cell cultures were initiated from surgically obtained specimens from human gliomas. After 4-10 passages in vitro the cells from 18 individual cases were seeded onto cluster dishes and left to attach for 24 h in medium containing fetal calf serum. Thereafter the medium was changed to serumfree, defined conditions and the following growth factors were added: EGF 1.5 x 10-8 M, PDGF 35 ng/ml, FGF 80 pg/ml. The growth factors were added fresh every 2 days. After 9 or 11 days in culture, the cells on one multiwell-plate were counted. In 8 cases another set which was cultured also in a multiwell-plate but on glass cover slides was immunostained for glial fibrillary acidic protein (GFAP), fibronectin (FN), A2B5 and galactocerebroside (galC). The proliferative response pattern in consequence to the growth factor addition varied greatly between different cases. In all cases a pronounced proliferative response was accompanied by marked changes on the culture morphology. Usually the best proliferative response was obtained with PDGF (10 cases) or FGF (8 cases) whereas EGF was most effective only in one case. Two cases showed only minimal response, one of which was an oligodendroglioma D according to Ludwig and another a gliosarcoma which was re-evaluated after 1 year in culture. It could be noted, however, that FGF had a tendency to be less effective in the group of malignant astrocytomas. The staining pattern of the cultures with the afore mentioned markers was not affected by the growth factor treatment. The proliferative response usually resulted in increased staining for fibronectin and never an induction of GFAP. A2B5 staining was positive only in one case of gliosarcoma and galC staining was regularly negative.

Topics: Cell Division; Epidermal Growth Factor; Fibroblast Growth Factors; Fibronectins; Glial Fibrillary Acidic Protein; Glioma; Humans; Platelet-Derived Growth Factor; Tumor Cells, Cultured

Xenografts from eight malignant human gliomas were established in athymic mice and were used to study amplification and expression of the epidermal growth factor receptor (EGFR) gene. Tissue identity between biopsy and xenografts was confirmed by karyotypic profiles, which showed that each glioma xenograft retained structural abnormalities, including double minute chromosomes, present in the parent glioma. EGFR gene amplification was found in six of the eight glioma biopsies and their corresponding xenografts. Expression of the EGFR gene was measured by Scatchard analysis, affinity reactions, immunoprecipitations, Western immunoblots, and immunocytochemistry; significant expression of the EGFR gene was only detectable in xenografts with EGFR gene amplification. Moreover, five of the six xenografts with EGFR gene amplification demonstrated structural alterations of the EGFR gene, which was associated with low-molecular-weight EGFR proteins. These xenografts represent an excellent tissue source and in vivo model system for characterizing the epidermal growth factor receptor in malignant human gliomas.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Female; Gene Amplification; Glioma; Glycosylation; Humans; Immunohistochemistry; Iodine Radioisotopes; Karyotyping; Male; Mice; Mice, Inbred BALB C; Molecular Weight; Neoplasm Transplantation; Transplantation, Heterologous

Urine specimens obtained from 19 patients with primary brain tumors were examined for the activity of transforming growth factors (TGF's). Urine was assayed for TGF's by soft agar colony formation and iodine-125 (125I)-epidermal growth factor (EGF)-binding competition. Two nontransformed cell lines, clonal NRK49F and BALB/3T3 A31-1-1 cells, were used as indicator cells for the soft agar colony assay, while EGF receptor-rich A431 cells were used for 125I-EGF-binding competition assay. Urine samples were dialyzed against acetic acid, then lyophilized, prepared with gel-permeation chromatography, and assayed. All 19 patients and a control group of healthy individuals showed high levels of alpha-type TGF's with low molecular weight (4 to 8 kD) in all urine samples. In addition, alpha-type TGF's of high molecular weight (20 to 50 kD) were detected at high levels in urine from all 10 patients with high-grade astrocytoma; at intermediate levels in urine from one of two patients with low-grade astrocytoma and from two of four patients with meningioma; and at low levels in urine from one of two patients with low-grade astrocytoma, from two of four patients with meningioma, from one patient with oligodendroglioma, from two patients with neurinoma, and from all healthy control individuals. The high level of alpha-type TGF's with high molecular weight detected in urine from patients with high-grade astrocytoma could be useful as a tumor marker.

Topics: Adult; Aged; Brain Neoplasms; Epidermal Growth Factor; Female; Glioma; Humans; Male; Meningioma; Middle Aged; Peptides; Transforming Growth Factors

The gene encoding the receptor for epidermal growth factor was amplified two- to fivefold in the human glioblastoma cell line SF268. The amplified gene gave rise to abundant quantities of receptor that bound EGF with a high affinity (Kd, 0.35 nM). The binding of ligand failed to elicit cellular DNA synthesis, however, and the receptor was enzymatically inactive. We presume that the amplified receptor gene carries a mutation(s) that affects several aspects of the receptor's function. Characterization of the mutation(s) may illuminate how structure dictates function in the receptor protein.

Topics: Cell Cycle; DNA; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Glioma; Humans; Mutation; Protein-Tyrosine Kinases; Structure-Activity Relationship; Tumor Cells, Cultured

A monoclonal antibody (Amersham) to epidermal growth factor receptor (EGFR) was used for immunohistochemical study to confirm the presence of EGFR in the glioma tissue. Fresh surgical material was stored at -80 degrees C, and frozen sections were stained. Positive staining was demonstrated in 8 of 9 gliomas. In 2 of 8, more than 50% of tumor cells were positively stained. In 4 of 8, positive cells were seen scattered in the tissue. In the remaining 2 gliomas, few tumor cells were positively stained. The most representative staining was seen on the cytoplasmic membrane. But there were also nuclear and cytoplasmic stainings. The distribution of EGFR in the glioma cells suggests possible role of the epidermal growth factor in the proliferation of gliomas.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; Humans; Immunohistochemistry; Male; Middle Aged

Type beta transforming growth factor (B-TGF) is a potent growth inhibitor to many human tumor cell lines. Very little is known about the mechanism for this growth inhibitory action of B-TGF. We here report the effect of B-TGF on proliferation and epidermal growth factor receptor (R-EGF) expression in a human glioblastoma cell line named T-MG1. B-TGF inhibit the soft agar growth of T-MG1 cells. Maximum inhibition was 70%, achieved with 0.5 units B-TGF. B-TGF had no effect on monolayer growth of T-MG1 cells. T-MG1 cells contained abundant R-EGF, which could be divided into two subpopulations, one high affinity and one low affinity population of R-EGF. Treatment with B-TGF caused an initial decrease (0-6 h) in EGF-binding, followed by an increase in EGF-binding which reached maximum after 24 h exposure to B-TGF. Since addition of EGF to agar cultures gave no additional increase in inhibition by B-TGF and EGF alone had no inhibitory effect, we believe that binding of EGF to its receptor is not part of the pathway mediating the inhibitory effect of B-TGF. All neoplastic cells have lost some measure of growth control and the cellular elements involved are growth factors, growth factor receptors and oncogenes. T-MG1 cells contain abundant R-EGF and this may partly explain their malignant nature (malignant nature is here defined as ability to proliferate in agarose). Type alpha transforming growth factors, which in some cancer cells act as uncontrolled autocrine growth factors, were not found in protein extracts from T-MG1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Cell Division; Cell Line; Epidermal Growth Factor; Glioma; Humans; Transforming Growth Factors; Tumor Cells, Cultured

In the human glioblastoma cell line, T-MG1, plasminogen activator activity (PA-activity) was demonstrated by using the chromogenic substrate S-2251. Using monoclonal antibodies against human urokinase type PA (u-PA) and human tissue type PA (t-PA), only u-PA activity was found in T-MG1 cell extracts. The u-PA activity in T-MG1 cells was suppressed in a dose-dependent manner by B-TGF and EGF after 24 hours of exposure to these growth factors. Twenty units of B-TGF caused a decrease in PA-activity of 80%, while 10 ng/ml EGF gave a decrease in PA-activity of 60%. The suppressive effects of B-TGF and EGF were observed after 2 hours and 4 hours of incubation, respectively and sustained for at least 24 hours. The effects of B-TGF and EGF were not antienzymatic, but rather mediated through regulatory mechanisms. In view of the capacity of invasive growth of gliomas and the potential role of PA in invasive growth, the suppression of PA-activity in gliomas by B-TGF and EGF may be of importance.

Topics: Aged; Antibodies, Monoclonal; Epidermal Growth Factor; Glioma; Humans; Plasminogen Activators; Transforming Growth Factors; Tumor Cells, Cultured

Using the adhesive tumor cell culture system, we studied the effect of epidermal growth factor (EGF) on 16 primary and 7 metastatic fresh human tumors of the nervous system cultured in serum-free and serum-supplemented media at low cell density. In serum-free conditions, EGF significantly enhanced the growth of glial tumor cells. This positive effect was less pronounced for metastases to the brain. No significant enhancement was observed for the other tumor types (primitive neuroectodermal tumors and various tumors of neuroepithelial/mesenchymal origin). The addition of serum obscured this effect of EGF, even at the lowest cell densities. In 7 tumors, the simultaneous addition of platelet-derived growth factor did not enhance the EGF response. Subtypes of brain tumors respond to EGF in vitro under serum-free conditions.

Topics: Brain Neoplasms; Cell Division; Child; Child, Preschool; Epidermal Growth Factor; Glioma; Humans; Meningeal Neoplasms; Meningioma; Tumor Cells, Cultured

Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 2:6, and D-37 MG, with 2.9 x 10(4), 1.5 x 10(5), 8.6 x 10(5) and 1.59 x 10(6) EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 2:6 by 39%. U-343 MGa Cl 2:6 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MHG, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.

Topics: Antibodies, Monoclonal; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Immunotherapy; Tumor Cells, Cultured

The binding capacity for epidermal growth factor (EGF) was determined in 34 intracranial neoplasms (14 glioblastoma, seven low-grade gliomas, six meningiomas, and seven others) and four specimens of normal brain by using [I125]EGF. EGF binding and binding affinity of the sites in the tumour and brain samples were compared to placenta and rat liver. All specimens of normal brain were negative. Ten of 14 glioblastoma specimens contained EGF binding (level range 10-39,660 fmol/mg protein), however, ligand binding affinity was high in only three tumours. Only one of nine low-grade gliomas contained EGF binding activity. Five of six meningiomas contained EGF binding sites (level range 49-776 fmol/mg protein) and binding affinity was high in two. When present EGF binding activity was found in all cellular fractions except the cytosol. There were no clinical or histopathological features within major tumour groups that were predictive of either high or specific EGF binding activity. These preliminary studies have confirmed that EGF receptor-like activity is present in the particulate fractions of intracranial neoplasms of both mesenchymal and neuroctodermal origin. In a large proportion of these tumours the EGF binding affinity is low, suggesting either a less specific or truncated EGF binding site.

Topics: Brain Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma

Using acid-ethanol extraction, two proteins with Mr=8 and 12 kD were extracted from rat glioma tissue induced with ethylnitrosourea. These proteins were shown to complete for the receptor with [125I]EGF (epidermal growth factor) on A431 cells. The 8 kD protein exhibited a marked mitogenic effect by stimulating DNA synthesis in resting NIH 3T3 cells. Stepwise chromatography of the acid-ethanol extract on Biogels P-60 and P-10 resulted in preparative amounts of the protein and allowed for its partial characterization. It was found that the half-maximum stimulation of DNA synthesis in NIH 3T3 cells was achieved at growth factor protein concentration of 5 micrograms/ml. The preparation obtained possessed the EGF-competing activity of 10 ng-equiv. EGF per 1 microgram of protein and stimulated protein phosphorylation of the 170 kD protein in NRK cell membranes. The data obtained suggest that this factor may be related to the family of the so-called EGF-like growth factors.

Topics: Animals; Brain Neoplasms; Chromatography, Gel; Epidermal Growth Factor; ErbB Receptors; Glioma; Membrane Proteins; Peptides; Phosphorylation; Rats; Transforming Growth Factors

Three cell samples in different passages of the line U-343 MGa, derived from a human malignant glioma biopsy, gave rise to clones with different amounts of platelet-derived growth factor (PDGF)-like activity secreted to extracellular medium, and of 125I-labeled PDGF binding. Sixteen clones were completely karyotyped with the G-banding technique. The unique markers 1p-q+, 16p- found in all clones, as well as in the parallel uncloned line, U-343 MG, provided evidence of their common origin. The deduced early, possibly partly primary, deviations had the formula 44, XY, 1p-q+, -14, 16p-, -22, where loss of one chromosome 22 is in accordance with previous reports on early chromosomal deviations in gliomas. Two clones, the hypodiploid 26L and 5H, represented early progressional changes. The other clones followed two patterns of late progressional changes, probably starting from the karyotype of 5H, with additional markers and doubling of the stemlines. In late progressional line I 12q+ and in II +7 were the most characteristic findings. Northern blot analysis using complementary DNA clones for the A and B chains of PDGF showed that both PDGF chains were expressed in 26L and 5H indicating that activation of the PDGF genes could have been an early event in the development of this glioma. Clones with late progression pattern II had been subjected to the highest selective pressure in vitro, and they secreted the highest amount of PDGF-like activity to the extracellular medium. Among them were the most rapidly and tightly growing cells and some clones with high 125I-labeled epidermal growth factor binding. Possibly these findings reflect progressional changes including defective regulation of the growth factor/growth factor receptor genes, selected for in vitro, without involving gross rearrangements or amplifications of the genes. The possible significance of extra chromosomes 7, with the PDGF A chain and epidermal growth factor receptor genes, and of the 12q+ marker, located near the gamma interferon gene is discussed.

Topics: Cell Line; Chromosome Aberrations; Chromosomes, Human, Pair 7; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Karyotyping; Platelet-Derived Growth Factor

Two cell lines (U-343 MG and U-343 MGa) with different phenotypic characteristics were established from the same human glioblastoma multiforme biopsy. Previous studies have shown that a clonal derivative (Cl 2) of the U-343 MGa line produces a PDGF-like growth factor. In the present investigation glioma PDGF production and 125I-PDGF binding were found to be differently expressed in U-343 MG, U-343 MGa, and U-343 MGa Cl 2 cultures, providing evidence for a clonal variation in these properties. In order to investigate this point further, several clones were derived from low (23 clones) and high (30 clones) passage U-343 MGa cultures, as well as from U-343 MGa Cl 2 cells (30 clones). The clones could be divided into 4 groups according to morphology and growth pattern. A determination of the amount of PDGF receptor competing activity in serum-free conditioned media gave evidence for a clonal variation in the production of glioma PDGF, corresponding to 0-87 ng of authentic PDGF per ml. There was also a considerable range in 125I-PDGF binding (0-44 fmol of tracer bound per 10(6) cells). Scatchard plots performed on two clones confirmed the presence of saturable, high affinity PDGF receptors. High passage cultures were found to give rise to a higher number of high producing clones than did low passage cultures. There appeared to be a negative correlation between production of glioma PDGF and binding of 125I-PDGF, probably due to the receptor blocking activity of the endogenous growth factor. However, the presence of clones, apparently devoid of both glioma PDGF production and 125I-PDGF binding, suggests a true clonal variation in these two parameters. The growth rate in serum-free medium was found to correlate fairly well to the extent of glioma PDGF production. Production of glioma PDGF was found to have a morphological correlate and be most prominent among clones of "immature" looking, tightly growing cells. Clones that had large star-shaped cells with some resemblance to normal glia-like cells in culture were found to have a low production and a high 125I-PDGF binding capacity.

Topics: Cell Cycle; Cell Line; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor

The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the alpha-subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated beta-subunit. The functions of the insulin receptor were examined by measuring specific 125I-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down-regulation of the receptor, insulin-stimulated autophosphorylation of the beta-subunit, and phosphorylation of exogenous substrates as well as insulin-stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its alpha-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system.

Topics: Biological Transport; Brain Neoplasms; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Glioma; Glucose; Growth Hormone; Humans; Insulin; Macromolecular Substances; Molecular Weight; Neuraminidase; Receptor, Insulin; Receptors, Cell Surface

In a patient with recurrent grade IV glioma of the brain resistant to conventional treatment an antibody guided isotopic scan showed uptake by the tumour of a monoclonal antibody (9A) that was developed against epidermal growth factor receptor but cross reacted with blood group A antigen. As a therapeutic attempt antibody labelled with 1665 MBq (45.0 mCi) iodine-131 was delivered to the tumour area by infusion into the internal carotid artery. Computed tomography showed regression of the tumour after treatment, and an appreciable and sustained clinical improvement was noted without any toxicity. Delivery of irradiation guided by monoclonal antibody delivered by arterial infusion of the tumour area may be of clinical value in the treatment of brain gliomas resistant to conventional forms of treatment.

Topics: ABO Blood-Group System; Adult; Antibodies, Monoclonal; Brain Neoplasms; Cross Reactions; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Male; Radiation Dosage; Receptors, Cell Surface; Tomography, X-Ray Computed

Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF.

Topics: Affinity Labels; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; DNA Replication; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Photochemistry; Receptors, Cell Surface

The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.

Topics: Brain Neoplasms; Cell Line; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation; Glioma; Humans; Nucleic Acid Hybridization; Oncogenes; Receptors, Cell Surface; RNA, Messenger

We have characterized, in C6 cells, an amiloride-sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation-exchange system can be induced within 24-36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium-dependent step with full activation obtained in the presence of the calcium ionophore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocytochalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride-sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal-dependent step. Since diacylglycerol is the presumed endogenous activator of protein kinase C, we studied the effects of dioleylglycerol. This intermediate of phospholipid turnover was found to increase specifically the amiloride-sensitive sodium pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amiloride; Animals; Biological Transport, Active; Bradykinin; Bucladesine; Cell Line; Colchicine; Diglycerides; Epidermal Growth Factor; Glioma; Protons; Pyrazines; Rats; Sodium; Tetradecanoylphorbol Acetate

Topics: Animals; Brain Neoplasms; Cell Adhesion; Cell Division; Cell Line; Cell Membrane; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Glioma; Humans; Mice; Mice, Nude; Neuroglia

Two clones, designated Cl 2 and Cl 3, were established from the human malignant glioma line U-343 MGa. The astrocytic origin of the cells was proven by the presence in virtually 100% of the cells of the astrocyte marker glial fibrillary acidic protein. The addition of 10 ng epidermal growth factor (EGF) per milliliter to Cl 2 and Cl 3 cells resulted in the rapid appearance of large cell surface ruffles, visualized by scanning electron microscopy. A time course study by phase contrast microscopy showed that the maximal ruffling activity occurred 5 minutes after addition of EGF. Under basic culture conditions (Eagle's MEM, 10% fetal calf serum), Cl 2 and Cl 3 cells were essentially immobile and formed tightly packed, well demarcated colonies. In the presence of 10 ng EGF per milliliter, no defined colonies were formed and the cells seemed to move around freely. The stimulatory effect of EGF on cell migration was confirmed by growing the cells on a deposit of colloidal gold; in the absence of EGF, the cells remained immobile whereas cells grown at 10 ng EGF per ml formed long phagokinetic tracks. The effect of EGF on membrane motility and cell locomotion occurred in the absence of any effect of EGF on growth rate; both clones multiplied at the same rate in the absence as in the presence of EGF. Binding experiments using 125I-labeled EGF demonstrated a single class of high affinity receptors. The number of 180,000 receptors per cell was estimated in both clones. The finding that human glioma cells in culture require EGF for their migration raises the interesting possibility that tumor cells in vivo may respond in a similar fashion, and in that case require a growth factor for migration and for the expression of their infiltrative growth potential. Furthermore, the present findings strengthen the notion that glial cells should be recognized as targets for EGF.

Topics: Cell Division; Cell Membrane; Cell Movement; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Microscopy, Electron, Scanning; Receptors, Cell Surface

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.

Topics: Animals; Blood; Cell Division; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Glioma; Male; Mice; Mitogens; Molecular Weight; Nerve Growth Factors; Rats; Submandibular Gland; Tissue Extracts