transforming-growth-factor-alpha and Glioma

Gliomas, the most frequent primitive CNS tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. TGFα, an EGF family member, is frequently over-expressed in the early stages of glioma progression. We questioned whether prolonged TGFα exposure affects the stability of the normal mature astrocyte phenotype and, eventually, their propensity to cancerous transformation. Using mouse astrocyte cultures devoid of residual neural stem cells or progenitors, we demonstrate that several days of TGFα-treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors, without any accompanying sign of cancerous transformation. In contrast, when astrocytes de-differentiated with TGFα were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties, forming high-grade glioma-like tumors after brain grafting. Gamma irradiation was without effect on astrocytes which were not treated with TGFα. These results suggested that most gliomas should contain tumor cells with stem-like properties (TSCs). Our study of 55 pediatric brain tumors show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a majority of gliomas. Survival analysis showed an association between isolation of TSCs with extended self-renewal capabilities and a patient's higher mortality rate.

Topics: Animals; Astrocytes; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Gamma Rays; Glioma; Humans; Phenotype; Stem Cells; Transforming Growth Factor alpha

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. Despite treatment with surgery, radiation therapy, and chemotherapy, the prognosis remains poor, particularly for glioblastoma, which has a median survival of 12 to 15 months. An important impediment to finding effective treatments for malignant gliomas is the presence of the blood brain barrier, which serves to prevent delivery of potentially active therapeutic compounds. Multiple efforts are focused on developing strategies to effectively deliver active drugs to brain tumor cells. Blood brain barrier disruption and convection-enhanced delivery have emerged as leading investigational delivery techniques for the treatment of malignant brain tumors. Clinical trials using these methods have been completed, with mixed results, and several more are being initiated. In this review, we describe the clinically available methods used to circumvent the blood brain barrier and summarize the results to date of ongoing and completed clinical trials.

Topics: Animals; Antineoplastic Agents; Blood-Brain Barrier; Brain; Brain Neoplasms; Catheterization; Drug Delivery Systems; Drug Implants; Exotoxins; Genetic Vectors; Glioblastoma; Glioma; Humans; Immunotoxins; Interleukins; Transferrin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Ultrasonic Therapy

Despite advances in our knowledge about the genesis, molecular biology, and natural history of malignant gliomas and the use of a multi-disciplinary approach to their treatment, patients harboring this diagnosis continue to face a grim prognosis. At the time of diagnosis, patients typically undergo surgery for the establishment of a histologic diagnosis, the reduction of tumor burden, and the relief of mass effect, with the maintenance of the patient's neurological function in mind. This is followed by the administration of adjuvant therapeutics, including radiation therapy and chemotherapy. Many investigational agents with laboratory evidence of efficacy against malignant gliomas have not met their promise in the clinical setting, largely due to the barriers that they must overcome to reach the tumor at a therapeutically meaningful concentration for a durable period of time. The relevant aspects of the blood-brain barrier, blood-tumor barrier, and blood-cerebrospinal fluid barrier, as they pertain to the delivery of agents to the tumor, will be discussed along with the strategies devised to circumvent them. This discussion will be followed by a description of agents currently in preclinical and clinical development, many of which are the result of intense ongoing research into the molecular biology of gliomas.

Topics: Animals; Antineoplastic Agents; Bacterial Toxins; Biological Transport; Blood-Brain Barrier; Brain Neoplasms; Convection; Diphtheria Toxin; Drug Delivery Systems; Exotoxins; Glioma; Humans; Interleukin-13; Interleukin-4; Recombinant Fusion Proteins; Transferrin; Transforming Growth Factor alpha

Targeted toxins represent a new class of agents with high specificity for tumor cells. Toxins in current clinical use for the treatment of brain tumors are mostly recombinant polypeptides consisting of a tumor-selective ligand coupled to a peptide toxin of bacterial origin. Targeted toxins are highly potent - one single molecule of toxin is enough to cause cell death. Toxins are able to kill tumor cells independent of any malignancy-associated genetic alterations and/or mutations. The blood-brain barrier has been a major obstacle for using targeted toxins for treatment of malignant glioma. Convection-enhanced delivery (CED), a method for delivery of large molecules to brain tissue via continuous interstitial microinfusion, has permitted direct administration of toxins to brain tumors or to surrounding brain tissue infiltrated by tumor cells. Four targeted toxins advanced to at least phase II clinical trials and are being used for treatment of adult or pediatric patients with recurrent or progressive malignant glioma. These are IL4-P. aeruginosa exotoxin (IL4-PE, NBI-3001), tumor growth factor (TGF)alpha-P. aeruginosa exotoxin (TP-38), IL13-P. aeruginosa exotoxin (IL13-PE38), and transferrin-C. diphtheriae toxin (TransMID(trade mark), Tf-CRM107). All of these toxins have shown an acceptable profile of toxicity and safety in phase I and II clinical studies and have demonstrated some evidence for tumor response. Current phase I and II clinical protocols are exploring several parameters, such as placement of catheters for CED either intratumorally or in the brain tissue surrounding a tumor, surgical resection of tumor before or after toxin infusion, and single vs. repeated infusion. Two large randomized and controlled phase III multicenter studies using IL13-PE38 or TransMID(trade mark) are currently enrolling patients. This review summarizes the study protocols and key findings of all previously completed and currently ongoing clinical studies with targeted toxins for malignant glioma. It offers in addition an outlook into future areas of development of targeted toxins, such as improved delivery modes and non-invasive in vivo imaging of intracerebral and intratumoral distribution of toxin in patients.

Topics: Animals; Bacterial Toxins; Brain Neoplasms; Clinical Trials as Topic; Drug Delivery Systems; Exotoxins; Glioma; Humans; Immunologic Factors; Immunotoxins; Interleukin-13; Interleukin-4; Recombinant Fusion Proteins; Transferrin; Transforming Growth Factor alpha

Antisense strategy using synthetic oligodeoxynucleotides has been applied to the suppression of specific gene expression, the modulation of various gene expression, and its biological activity. Antisense strategy is applicable for the growth suppression of glioma cells. Several genes, including transforming growth factor-alpha, basic fibroblast growth factor, fibroblast growth factor receptor 1, vascular endothelial growth factor, telomerase, topoisomerase II alpha-subunit, protein kinase C-alpha, and microtubule-associated protein 1A, have been targeted by antisense strategy in glioma cells. These antisense strategies provide a potential novel antitumor therapy for gliomas.

Topics: Brain Neoplasms; Cell Division; Glioma; Humans; Oligonucleotides, Antisense; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; Telomerase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Advances in our understanding of the molecular changes and resultant cellular effects in malignant glioma are expanding the opportunities for novel targeted therapies. At present, chemotherapy and radiation followed by chemotherapy with nonselective cytotoxic agents is emerging as a standard treatment option for patients with malignant glioma. Unfortunately significant improvements in response and survival have not occurred for the majority of patients. The continued improvement in patient outcomes will require the incorporation of glioma-specific therapies either in the form of compounds specifically targeted to glioma-specific receptors or that inhibit signaling pathways promoting glioma survival and progression.

Topics: Glioma; Humans; Interleukin-13; Interleukin-4; Scorpion Venoms; Transferrin; Transforming Growth Factor alpha

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: Base Sequence; Brain Neoplasms; Genetic Therapy; Glioma; Humans; Interleukin-8; Molecular Sequence Data; Oligonucleotides, Antisense; Transforming Growth Factor alpha

Tumor-associated macrophages (TAMs) are enriched in gliomas and help create a tumor-immunosuppressive microenvironment. A distinct M2-skewed type of macrophages makes up the majority of glioma TAMs, and these cells exhibit pro-tumor functions. Gliomas contain large hypoxic areas, and the presence of a correlation between the density of M2-polarized TAMs and hypoxic areas suggests that hypoxia plays a supportive role during TAM recruitment and induction. Here, we investigated the effects of hypoxia on human macrophage recruitment and M2 polarization. We also investigated the influence of the HIF inhibitor acriflavine (ACF) on M2 TAM infiltration and tumor progression in vivo. We found that hypoxia increased periostin (POSTN) expression in glioma cells and promoted the recruitment of macrophages. Hypoxia-inducible POSTN expression was increased by TGF-α via the RTK/PI3K pathway, and this effect was blocked by treating hypoxic cells with ACF. We also demonstrated that both a hypoxic environment and hypoxia-treated glioma cell supernatants were capable of polarizing macrophages toward a M2 phenotype. ACF partially reversed the M2 polarization of macrophages by inhibiting the upregulation of M-CSFR in macrophages and TGF-β in glioma cells under hypoxic conditions. Administering ACF also ablated tumor progression in vivo. Our findings reveal a mechanism that underlies hypoxia-induced TAM enrichment and M2 polarization and suggest that pharmacologically inhibiting HIFs may reduce M2-polarized TAM infiltration and glioma progression.

Topics: Acriflavine; Animals; Antineoplastic Agents; Brain Neoplasms; Cell Adhesion Molecules; Cell Communication; Cell Plasticity; Cell Proliferation; Chemotaxis; Cytokines; ErbB Receptors; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinase; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; RNA Interference; Signal Transduction; THP-1 Cells; Time Factors; Transfection; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Burden; Tumor Hypoxia; Tumor Microenvironment; Up-Regulation

Inappropriate expression of Ets-1 is observed in a variety of human cancers, and its forced expression in cultured cells results in transformation, autonomous proliferation, and tumor formation. The basis by which Ets-1 confers autonomous growth, one of the primary hallmarks of cancer cells and a critical component of persistent proliferation, has yet to be fully explained. Using a variety of cancer cell lines, we show that inhibition of Ets-1 blocks tumor formation and cell proliferation in vivo and autonomous growth in culture. A screen of multiple diffusible growth factors revealed that inhibition of Ets-1 results in the specific downregulation of transforming growth factor alpha (TGFalpha), the proximal promoter region of which contains multiple ETS family DNA binding sites that can be directly bound and regulated by Ets-1. Notably, rescuing TGFalpha expression in Ets-1-silenced cells was sufficient to restore tumor cell proliferation in vivo and autonomous growth in culture. These results reveal a previously unrecognized mechanism by which Ets-1 oncogenic activity can be explained in human cancer through its ability to regulate the important cellular mitogen TGFalpha.

Topics: Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Glioma; Humans; Kidney Neoplasms; Male; Neoplasms; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; Proto-Oncogene Protein c-ets-1; Transfection; Transforming Growth Factor alpha

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min(-1) and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor alpha (TGF-alpha) and transforming growth factor beta1 (TGF-beta1). Monoclonal antibodies for TGF-alpha (mAb TGF-alpha) and TGF-beta1 (mAb TGF-beta1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-alpha was not present in the ICCM and addition of mAb TGF-alpha to the ICCM had no effect on bystander cell survival. No active TGF-beta1 was present in the ICCM; however, addition of mAb TGF-beta1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-beta1 plays a downstream role in this response.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Bystander Effect; Cell Death; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Dose-Response Relationship, Radiation; Electrons; Glioma; Humans; Transforming Growth Factor alpha; Transforming Growth Factor beta1

Gliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress.

Topics: Animals; Astrocytes; Brain Neoplasms; Cell Dedifferentiation; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media, Serum-Free; Gamma Rays; Glioma; Mice; Mice, Inbred C57BL; Mice, Nude; Stem Cell Transplantation; Stem Cells; Stress, Physiological; Transforming Growth Factor alpha

Both the Notch-signaling pathway and extracellular signal regulated kinase (ERK) cascade are involved in a wide variety of biological processes, such as proliferation, differentiation, survival, and tumorigenesis. Their dysregulation in recent studies have been shown to be associated with glioma formation. Here, we show that transforming growth factor-alpha (TGF-alpha) stimulated glioma cell line U251 growth and can partly compensate for the inhibitory effect of Notch-signaling inhibitor DAPT. The effect of TGF-alpha on ERK1/2 phosphorylation was prompt and transient and could be inhibited by mitogen-activated/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) specific inhibitor PD98059. Moreover, TGF-alpha was capable of up-regulating Hairy-enhancer of split1 (Hes1) expression which was independent of Notch1 activation, and of introducing Hes1 nuclear import in the presence of ERK1/2 activation. Collectively, our data suggest a potential linkage between ERK activation and the Notch-signaling pathway.

Topics: Active Transport, Cell Nucleus; Amyloid Precursor Protein Secretases; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Dipeptides; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation, Neoplastic; Glioma; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Phosphorylation; Protease Inhibitors; Protein Kinase Inhibitors; Protein Transport; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor HES-1; Transforming Growth Factor alpha; Up-Regulation

Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.

Topics: Aging; Animals; Astrocytes; Cell Death; Cell Survival; Cells, Cultured; Cerebellar Cortex; Down-Regulation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; MAP Kinase Signaling System; Mice; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Receptor, ErbB-2; Time Factors; Transforming Growth Factor alpha

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Cell Division; DNA, Complementary; Enzyme Inhibitors; ErbB Receptors; Glioma; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; RNA; RNA, Messenger; Tetracycline; Time Factors; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine; Tyrphostins

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

Glioblastoma multiforme is a highly malignant tumor that is extremely refractory to therapy. One reason is its highly invasive nature into brain tissue. Metalloproteinases and their inhibitors, plasminogen activators (PA) and their inhibitors and cathepsins are thought to be involved in invasion by tumor cells. In this study, we determined if the urokinase-type plasminogen activator (uPA) and/or the urokinase-type plasminogen activator receptor (uPAR) were responsible for the invasion activity of a human glioma cell line. We determined the invasion activity of a human glioma U251 cell line using an in vitro invasion assay system. A 2.4- to 5.8-fold increase in invasion activity was observed in the presence of basic fibroblast growth factor (bFGF) or transforming growth factor (TGF)-alpha. Northern blot analysis showed that bFCF and TGF-alpha treatment was associated with increases in cellular mRNA levels of uPA and uPAR. Zymographic activity correlated to mRNA levels of uPA and uPAR. Addition of an anti-uPAR monoclonal antibody significantly inhibited the invasion activity induced by bFGF- and TGF-alpha. Irsogladine, an inhibitor of uPA synthesis, also blocked the invasion activity. These observations suggest that uPA and its receptor have a role in the invasion process of human gliomas.

Topics: Fibroblast Growth Factor 2; Glioma; Humans; Neoplasm Invasiveness; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Tissue Plasminogen Activator; Transforming Growth Factor alpha; Triazines; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

The effects of transforming growth factor-alpha (TGF-alpha) on cell growth were studied in human glioma U251 cells transfected with antisense TGF-alpha vectors (pcDNAI.neo). Several antisense clones showed a marked decrease in growth rate in serum-free medium but not in medium containing 10% FBS, compared with those of parental cells and clones from sense or vector transfectants. Antisense clones also produced fewer and smaller colonies in anchorage-independent growth assays. Moreover, there was a reduction in TGF-alpha expression in these antisense clones at both the protein and mRNA levels, as determined by enzyme linked immuno-sorbent assay and reverse transcriptase polymerase chain reaction analysis. A U251 clone transfected by TGF-alpha antisense in a different vector (pMT/Ep) also showed a marked suppression in cell growth and TGF-alpha mRNA level. Finally, transfected clones with either vector system, showed decreased tumorigenicity in nude mice. In summary, a strong correlation between the inhibition of glioma cell growth and TGF-alpha expression was obtained from two different plasmid vectors, indicating that the expression of TGF-alpha could be specifically and effectively down-regulated by TGF-alpha antisense vector, which in turn led to growth inhibition. These studies suggests that TGF-alpha plays an essential role in controlling human glioma cell proliferation and may serve as a potential target for treatment of malignant glioma.

Topics: Animals; Brain Neoplasms; Cell Division; Clone Cells; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Mice, Nude; Oligodeoxyribonucleotides, Antisense; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Transplantation, Heterologous; 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

Nitric oxide (NO) is thought to play an important role in neurotransmission, inflammation, and regulation of cell death in the mammalian brain. Here, we examined the synthesis and biological effects of NO in human malignant glioma cells. Exposure to cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta and lipopolysaccharide (LPS) induced NO synthesis in rat C6 and A172 human glioma cells, but not in LN-229, T98G or LN-18 human malignant glioma cells. Induced release of NO involved enhanced expression of inducible NO synthase (iNOS). Failure to detect NO release in the latter cell lines was not overcome by neutralization of endogenous TGF-beta or by coexposure to cytokines, LPS, and antioxidants. Apoptosis induced by CD95 ligand (CD95L) did not involve NO formation. Neither NOS inhibitors nor NO donators modulated CD95L-induced apoptosis. Dexamethasone (DEX)-mediated protection of glioma cells from CD95L-induced apoptosis was also independent of DEX effects on NO metabolism. DEX inhibited not only cytokine/LPS-evoked NO release but also attenuated the toxicity of NO in three of five cell lines. Forced expression of temperature-sensitive p53 val135 in C6 cells in either mutant or wild-type conformation inhibited cytokine/LPS-induced NO synthesis. Further, accumulation of p53 in both mutant or wild-type conformation protected glioma cells from the toxicity of exogenous NO, consistent with a gain of p53 function associated with p53 accumulation. We conclude that resistance to NO-dependent immune defense mechanisms may contribute to the malignant progression of human cancers with p53 alterations, notably those associated with the accumulation of mutant p53 protein.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Cycloheximide; Cytokines; Dexamethasone; fas Receptor; Genes, p53; Glioma; Humans; Interleukin-1; Lipopolysaccharides; Neoplasm Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Synthesis Inhibitors; Rats; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Alterations in the epidermal growth factor receptor (EGFR) and its main ligand, transforming growth factor-alpha (TGF alpha), were investigated for a possible prognostic relevance in 125 astrocytic gliomas (44 World Health Organization (WHO) Grade II, 19 WHO Grade III, and 62 WHO Grade IV tumors). The TGF alpha and EGFR proteins were detected immunohistochemically using monoclonal antibodies. A positive immunoreaction to TGF alpha was detected in 33 (75%) of 44 WHO Grade II astrocytomas, 18 (95%) of 19 WHO Grade III astrocytoma, and 50 (81%) of 62 WHO Grade IV glioblastomas. No correlation between TGF alpha immunoreaction and duration of survival could be found. A positive EGFR immunoreaction was detected in seven (16%) of 44 WHO Grade II astrocytomas, five (26%) of 19 WHO Grade III astrocytomas, and 32 (52%) of 62 WHO Grade IV glioblastomas. Of these gliomas, 97 (26 WHO Grade II, 17 WHO Grade III, and 54 WHO Grade IV gliomas) were examined for EGFR gene amplification using a differential polymerase chain reaction assay. Amplification of the EGFR gene was detected in none of the WHO Grade II astrocytomas, one (6%) of 17 WHO Grade III astrocytomas, and 18 (33%) of 54 WHO Grade IV glioblastomas. Twenty-two of the tumors investigated showed a positive EGFR immunoreaction without detectable gene amplification (five WHO Grade II, four WHO Grade III, and 13 WHO Grade IV tumors). Gene amplification was invariably associated with a positive EGFR immunoreaction. For the entire study group, a strong correlation between EGFR alterations (gene amplification and positive immunoreaction) and survival could be found. However, this correlation only reflected the higher percentages of cases with EGFR alterations in malignant gliomas and was not an independent prognostic factor as determined by multifactorial analysis. These data demonstrate that EGFR alterations are frequent events in astrocytic gliomas and are largely restricted to glioblastomas. However, within one tumor grade they do not provide prognostic information.

Topics: Adult; Brain Neoplasms; ErbB Receptors; Female; Glioma; Humans; Immunohistochemistry; Male; Polymerase Chain Reaction; Prognosis; Transforming Growth Factor alpha

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

To examine which growth factors correlate with neovascularization in human brain tumors, the mRNA levels of transforming growth factor alpha, transforming growth factor beta, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand factor. We normalized the growth factor mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and 16 meningiomas examined, the mRNA of transforming growth factors alpha and beta, basic fibroblast growth factor, and VEGF were expressed at various levels. Among those 4 growth factors, the mRNA levels of VEGF, but not those of transforming growth factors alpha and beta and basic fibroblast growth factor, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive factor in tumor angiogenesis in both human gliomas and meningiomas.

Topics: Brain Neoplasms; Endothelial Growth Factors; Fibroblast Growth Factor 2; Glioma; Humans; Lymphokines; Meningioma; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Physical and chemical agents can damage the genome. Part of the protective response to this damage is the increased expression of p53. p53, a transcription factor, controls the expression of genes, leading to cell cycle arrest and apoptosis. Another protective mechanism is the proliferative response required to replace the damaged cells. This proliferation is likely to be signaled by growth factors. In this communication, we show that the transforming growth factor alpha (TGF-alpha) gene is a direct target for p53-mediated transcriptional activation. In a stable cell line containing an inducible p53 construct, p53 induction leads to a threefold accumulation of the native TGF-alpha mRNA. IN cotransfection assays using a TGF-alpha promoter reporter construct, we show that expression of wild-type but not mutant p53 increases transcriptional activity of the TGF-alpha promoter by approximately 2.5-fold. In vitro, wild-type p53 binds to a consensus binding site found in the proximal portion of the promoter, and this sequence is necessary for the p53 transcriptional response. Furthermore, this element confers p53 induction to the otherwise nonresponsive adenovirus major late promoter. In addition to these results, we found that the TGF-alpha promoter contains a nonconsensus but functional TATA box-binding protein-binding site approximately 30 bp upstream of the transcription start site. Although p53 can repress transcription from promoters containing a TATA box, the nonconsensus TGF-alpha TATA motif is resistant to this effect. On the basis of these results, we propose that p53 may play a dual role, which includes both the elimination of irreparably genetically damage cells and the proliferative response necessary for their replacement, in the response to physical-chemical damage.

Topics: Adenoviridae; Base Sequence; Blotting, Northern; Blotting, Western; Cell Line; DNA Damage; DNA Mutational Analysis; Fluorescent Antibody Technique; Gene Expression Regulation; Glioma; Humans; Molecular Sequence Data; Precipitin Tests; Promoter Regions, Genetic; Protein Binding; Sequence Deletion; TATA Box; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Tumor Suppressor Protein p53; Viral Proteins

The effects of specific antibodies against growth factors and receptors on deoxyribonucleic acid (DNA) synthesis in two established human glioma cell lines, A172 and TM-1, were examined. Anti-platelet-derived growth factor (PDGF), anti-basic fibroblast growth factor (bFGF), and anti-epidermal growth factor receptor (EGF-R) antibodies inhibited thymidine incorporation by both cell lines in serum-free medium. Antibody specific to transforming growth factor-alpha only slightly suppressed DNA synthesis by both cell lines. Although the antiproliferative effects of anti-PDGF and anti-bFGF antibodies decreased in serum-supplemented medium, the effect of anti-EGF-R antibody was little changed. The combination of anti-PDGF, anti-bFGF, and anti-EGF-R antibodies significantly inhibited thymidine incorporation by the two cell lines even in serum-supplemented medium. This preliminary study suggests that simultaneous blockade of multiple autocrine loops may provide a new approach to the treatment of human malignant gliomas.

Topics: Antibodies, Monoclonal; Cell Movement; Cells, Cultured; Fibroblast Growth Factor 2; Glioma; Glucose-6-Phosphate Isomerase; Humans; In Vitro Techniques; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Thymidine; Transforming Growth Factor alpha

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

We have previously established a human malignant glioma cell line, TM-1. TM-1 cells could proliferate in the serum-free medium. In the present study, immunochemical analysis demonstrated that platelet-derived growth factor (PDGF), transforming growth factor (TGF)-alpha, and TGF-beta are present in the serum-free medium conditioned by growing TM-1 cells. While the cells appeared to possess a single type of binding sites for epidermal growth factor (EGF) with properties comparable to those determined for other tumor cells, the conditioned medium did not contain EGF.PDGF, TGF-alpha, and EGF added exogenously to serum-free media stimulated thymidine incorporation into DNA of TM-1 cells. In addition, antibodies specific for PDGF and TGF-alpha suppressed this activity. These results indicate autocrine and stimulatory roles of PDGF and TGF-alpha for the proliferation of TM-1 cells. As observed for other tumor cells, TGF-beta by itself weakly suppressed thymidine incorporation by TM-1 cells. However, TGF-beta employed in combination with TGF-alpha or EGF appeared to stimulate thymidine incorporation, suggesting that a cooperative action of TGF-beta with different growth factors may be involved in the stimulatory growth regulation at least for TM-1 cells.

Topics: Cell Division; DNA; ErbB Receptors; Glioma; Humans; Platelet-Derived Growth Factor; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain. In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38. In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor. Treatment with i.p. TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c. xenografts produced tumor regression and growth delay. For intracranial xenograft implants treated with i.p. TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression. However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested. These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression. For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p. administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38. Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; ErbB Receptors; Exotoxins; Female; Glioma; Humans; Medulloblastoma; Mice; Mice, Nude; Neoplasm Transplantation; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; 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

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

We have developed two different models of tumor angiogenesis by human brain tumors: one being tube formation by bovine aortic endothelial (BAE) cells cocultured with tumor cells in vitro, and other being in vivo angiogenesis in mice when tumor cells are transplanted into the dorsal sac. We investigated whether tube formation could be induced in BAE cells in type I collagen gel when these cells were cocultured with seven human glioma cell lines. Four of the seven glioma cell lines, which had high levels of basic fibroblast growth factor (bFGF) mRNA, induced tube formation by BAE cells. The tube formation was blocked by coadministration of anti-bFGF antibody. In in vivo model system of tumor angiogenesis in mice, these four cell lines were highly angiogenic. In contrast, with the other three glioma cell lines, which had poor expression of bFGF, BAE cells showed no apparent tube formation. These three cell lines did not efficiently develop capillary networks in mice. The results demonstrated a correlative relationship in the tubulogenesis of BAE cells, bFGF mRNA levels and angiogenesis in mice. The present study with two model systems of tumor angiogenesis suggests that the angiogenesis of some human glioma cell lines is mediated by bFGF, possibly via paracrine control.

Topics: Animals; Endothelium, Vascular; Fibroblast Growth Factor 2; Gene Expression; Glioma; Growth Substances; Humans; In Vitro Techniques; Mice; Morphogenesis; Neoplasm Transplantation; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

A role for neutral endopeptidase 24.11 (NEP) in growth and development is supported by the demonstration that NEP hydrolyses and inactivates a number of peptide growth factors including atrial natriuretic peptide, endothelins, bombesin-like peptides, and opioid peptides, including the enkephalins. In the present study, suspensions of cells obtained from the ventral mesencephalon or cortex of rat embryos (ED14) were implanted into the striatum of the adult rat brain. Three to 15 weeks after transplantation the relative distribution of NEP-positive cellular elements was visualized histochemically. NEP staining in the transplants consistently appeared before NEP staining in the surrounding host striatum supporting a relative increase in NEP activity in the transplants. The NEP staining richly visualized cells of varying size and morphology which lacked the normal organization of the host striatum. The histochemical staining in the transplants and the surrounding host tissue was completely blocked by a 100 nM concentration of the selective NEP inhibitors phosphoramidon or JHF-26, supporting the exclusive localization of NEP by this method. NEP localization in the embryonic (ED14) cortex and ventral mesencephalon was also confirmed, suggesting one possible origin for the NEP-positive cells visualized in the transplants. Fluorescent double-labeling studies for NEP and glial fibrillary acidic protein (GFAP) or transforming growth factor alpha precursor (TGF alpha p) revealed the presence of rich glial labeling within the transplants for both GFAP and TGFap. NEP-labeled cells in the transplants were closely associated with glial elements, however, only occasional glial elements in the transplants stained for NEP; supporting a non-astrocytic localization for the NEP in the transplants. The marked enhancement of NEP staining in the transplants may have significance for controlling the rate or pattern of growth of the transplanted cells through inactivation of peptide growth factors produced by, or in response to, the transplants.

Topics: Animals; Brain Neoplasms; Brain Tissue Transplantation; Cerebral Cortex; Corpus Striatum; Female; Fetal Tissue Transplantation; Fluorescence; Glial Fibrillary Acidic Protein; Glioma; Immunohistochemistry; Male; Mesencephalon; Neoplasm Transplantation; Neprilysin; Pregnancy; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha

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

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

Surgical specimens from six benign and 16 malignant human gliomas were investigated immunohistochemically to correlate the degree of malignancy, the distribution of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) receptor, and the potential for cell proliferation using monoclonal antibodies to TGF-alpha, EGF receptor, and Ki-67. Fourteen (88%) of the malignant gliomas and one (17%) of the benign gliomas were found to be positive for TGF-alpha, and 14 (88%) of the malignant gliomas and two (33%) of the benign gliomas expressed EGF receptor. The proliferation index with Ki-67 was 18.8% +/- 8.1% (mean +/- standard deviation) in malignant gliomas and 1.9% +/- 1.8% in benign gliomas. In general, cells positive for EGF receptor and Ki-67 were randomly distributed throughout the tumor tissue, and cells positive for TGF-alpha tended to be clustered without obvious relationship to areas of necrosis or blood vessels. In some tumors, cells positive for TGF-alpha, EGF receptor, and Ki-67 were associated in a focal distribution. The more frequent expression of TGF-alpha and EGF receptor in the highly proliferative malignant gliomas is compatible with a role for TGF-alpha and EGF receptor in the induction or stimulation of malignant gliomas.

Topics: Astrocytoma; ErbB Receptors; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Nuclear Proteins; Transforming Growth Factor alpha

Expression of transforming growth factor alpha (TGF alpha) is frequently associated with the development of human and animal tumors. Using a sensitive immunohistochemical assay, which can be applied on formalin-fixed, paraffin-embedded tissue, we have examined the expression of TGF alpha in 71 human gliomas (63 untreated and 8 recurrent tumors). Tumors were graded by a 3-grade-system: grade I = low grade gliomas, grade II = anaplastic gliomas and grade III = glioblastomas. A strong positive correlation between tumor grade and extent of TGF alpha expression was found (P less than 0.0001). Polymerase chain reaction (PCR) was used to amplify the fourth exon of the TGF alpha gene of 8 glioma DNA specimens and increasing amounts of normal human DNA, which served as a standard. No amplification of the TGF alpha gene copy number in tumors could be detected.

Topics: Base Sequence; Brain Neoplasms; DNA, Neoplasm; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunohistochemistry; Molecular Sequence Data; Polymerase Chain Reaction; Transforming Growth Factor alpha