Expression of proto-oncogenes in neural tissues

Several proto-oncogenes have been shown to be expressed in tissues of neural origin. In most cases, their expression is developmentally regulated and they encode proteins similar in their sequence to a variety of known proteins involved in transferring information from the cell surface to the nucleus. Some of the proto-oncogenes, including src and yes, are expressed preferentially in neural tissues and one of them, src⁺, is expressed there exclusively. Many of neurally expressed proto-oncogenes, including src, yes, ras andmyc, are also found in organs containing epithelial cells involved in ion transport. It is possible that proteins encoded by these proto-oncogenes are themselves involved in some aspects of ion transport. Among defined categories of neurons expressing proto-oncogenes, cerebellar Purkinje cells are most frequently mentioned. They express at least 3 protooncogenes, src, yes, myc, as well as protein kinase C. Purkinje cells make an attractive model for functional studies of these proteins.Although an integrated picture-illuminating cooperative action of proto-oncogenes in neural or other tissues is missing, it is hoped that discovery of new classes of proto-oncogenesand functional interactions among them, may help us to underst and not only oncogenesis but also biology of the nervous system.     

Expression of neuroectodermal antigens common to melanomas,gliomas, and neuroblastomas

Summary The reactivity spectrum of five different monoclonal anti-melanoma antibodies cross-reacting with gliomas and neuroblastomas and one monoclonal anti-glioma antibody cross-reacting with melanomas and neuroblastomas was investigated. Comparison of the binding activity of these monoclonal antibodies for 11 melanoma, seven glioma, and three neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Since all these tumors are known to derive from cells originating embryologically from the neural crest, it can be assumed that the antigens recognized by our monoclonal antibodies are neuroectodermal differentiation antigens.However, absorption with fetal brain homogenates abolished only the binding of monoclonal anti-glioma antibody, but did not modify the binding of monoclonal anti-melanoma antibodies.Abbreviations RIA radioimmunoassay - HAT medium containing 10^–4 M hypoxanthine, 5×10^–7 M aminopterin, 1.6×10^–5 M thymidin     

Mouse fetal brain specific protein "GP68" is expressed in human tumor cells]

It is well known that some fetal antigens are expressed in malignant tumor cells. Likewise, brain tumors, especially histologically malignant cases, may have any antigenic relationships with fetal brain. So, we investigated the relationship by immunohistochemical technique, utilizing a polyclonal antibody to mouse fetal stage-specific polypeptide "GP68". We prepared GP68 from homogenate of head part of embryos at the 14th day of gestation mice by RCA-1 agarose column chromatography. And immunized it to Japanese white rabbits and the titer was measured by enzyme-linked immunosorbent assay. We analyzed operatively resected brain tumors and autopsy brain tissues. Frozen tissues were fixed in cold acetone and immunostained with anti-GP68 serum according to biotin-streptavidin peroxidase method. Remained tissues were homogenized in Laemmli's sample buffer and electrophoresed. The proteins were transferred to nitrocellulose membrane and immunostained with anti-GP68. Normal brain tissues were not positively stained, except for capillary endothelium which showed a weak staining. On the other hand, brain tumors of neuroectodermal origin were positively stained in varying degrees, and other tumors were negative. It is especially noteworthy that, in astrocytoma cases, there exists a definite correlation between the intensity of stain and the degree of histological malignancy. Immunoblot studies demonstrated a very weak band at 68 KD in normal brain and meningioma. In contrast, very strong band at the same position was seen in malignant astrocytomas. These results suggested that in brain tumors, especially those of neuroectodermal origin, GP68 antigen is expressed and the degree of expression is related to their histological malignancy. So this fetal antigen may be useful for evaluation of biological malignancy of gliomas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnosis and management of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.     

Polycomb genes expression as a predictor of poor clinical outcome in children with medulloblastoma

Introduction  

Medulloblastoma is the most frequent type of embryonal tumor in the pediatric population, accounting for 20–25% of all brain tumors in children. Recently, the suspected contribution of the Polycomb group (PcG) genes in medulloblastoma development was described. PcG genes play an important role in developmental processes; they are also involved in the self-renewal of hematopoietic and neural stem cells as well as in malignant transformation.     

Biological aspects of brain tumors in infancy and childhood

Notwithstanding the definitive systematization of theclinical features of childhood brain tumors, manybiological laws governing this vast area of pathology still escape us. There are no sure explanations for the fickleness of supratentorial/subtentorial distribution in fetal life and in the first 15 years of life — something not found in later years. Another focus of discussion is the tendency of brain tumors in infancy to concentrate along the midline due to the fact that most infantile neuroectodermal tumors arise from the phylogenetically older structures of the CNS (periventricular regions, brainstem, cerebellum), in contrast to tumors of adulthood. Neuroepithelial tumors, much more frequent in childhood than in later years, exhibit substantial histological differences in infancy: mixed gliomas and primitive neuroectodermal tumors (not otherwise specified, or with astrocytic, ependymal, oligodendrocytic ... cells) are the best examples. As to the question of whether a given oncotype exhibits different biological behavior according to patient age, there is no single answer: some malignant tumors (medulloblastomas, ependymomas, neuroblastomas) are more aggressive in infancy, in line with Collins' law, while others (optic gliomas) offer a better prognosis in younger patients. The most peculiar and disquieting aspect of brain tumors in infancy/childhood, however, is what emerged from a recent epidemiological survey. The survey was conducted on the close relatives of a boy with a brain tumor from point of view of a possible second malignancy in this boy. Since a brain tumor is a rare occurrence in the very young, it may signal heightened susceptibility to malignancy in the individual, extending to other tumors in the patient himself and even to other members of his family.This paper was presented in part at the XIth Congress of the European Society for Paediatric Neurosurgery, Naples, 1988     

c- and N-<Emphasis Type="Italic">myc</Emphasis> Regulate Neural Precursor Cell Fate,Cell Cycle,and Metabolism to Direct Cerebellar Development

Separate murine knockout (KO) of either c- or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes microcephaly in Feingold Syndrome. We created a double KO (DKO) of c- and N-myc using nestin-cre, which strongly impairs brain growth, particularly that of the cerebellum. Granule neurons were almost absent from the Myc DKO cerebellum, and other cell types were relatively overrepresented, including astroglia, oligodendrocytes, and Purkinje neurons. These findings are indicative of a profound disruption of cell fate of cerebellar stem and precursors. DKO Purkinje neurons were strikingly lacking in normal arborization. Inhibitory neurons were ectopic and exhibited very abnormal GAD67 staining patterns. Also consistent with altered cell fate, the adult DKO cerebellum still retained a residual external germinal layer (EGL). CGNP in the DKO EGL were almost uniformly NeuN and p27KIP1 positive as well as negative for Math1 and BrdU at the peak of normal cerebellar proliferation at P6. The presence of some mitotic CGNP in the absence of S phase cells suggests a possible arrest in M phase. CGNP and NSC metabolism also was affected by loss of Myc as DKO cells exhibited weak nucleolin staining. Together these findings indicate that c- and N-Myc direct cerebellar development by maintaining CGNP and NSC populations through inhibiting differentiation as well as directing rapid cell cycling and active cellular metabolism.     

β-Endorphin modulates T-cell intracellular calcium flux and c-myc expression via a potassium channel

To characterize the effect of β-endorphin on T-lymphocyte activation, we examined its influence on membrane currents, intracellular calcium flux, and c-myc mRNA levels during mitogenic stimulation of Jurkat cells. While β-endorphin weakly enhanced voltage-activated K⁺ currents of Jurkat cells by itself, it suppressed these currents in the presence of mitogen. Naloxone, by itself, also enhanced K⁺ current amplitude, but in the presence of mitogen partially reversed the suppressive effect of β-endorphin. A 5–30 min exposure to β-endorphin resulted in an increase in the rate of mitogen-stimulated intracellular calcium release and an increase in c-myc mRNA levels relative to controls. Longer exposure (1–2 h) to β-endorphin retarded intracellular calcium release, and suppressed c-myc expression. The suppressive effects were reversed by naloxone and mimicked by the K⁺ channel blocker, tetraethylammonium ion. These data suggest that opiate receptors and K⁺ channels of Jurkat cells are functionally coupled in a way that modulates intracellular calcium release and c-myc expression — two key processes in T-cell mitogenesis.     

Reductions in the plating efficiency of the fetal neural precursor cells following maternal alcohol consumption

Maternal alcohol abuse has been associated with reduced neural cell number and abnormal cell differentiation and organization in many regions of the developing mammalian brain. Experiments were conducted to determine whether changes in the neural precursor cells could be detected in primary culture following maternal alcohol consumption during the early proliferative period of the fetal brain growth spurt. Alcohol was administered to pregnant mice in the drinking water from days 11 to 19 of gestation. There was no alcohol-related increase in the incidence of fetal mortality or malformation, but fetal body and brain weights were reduced. When disaggregated cells from the day 19 fetal neopallium were grown in culture, there was a reduction in the number of astroglial colonies yielded in 42% of alcohol-exposed brains. This effect was expressed as a reduction in the absolute plating efficiency (APE) of the neural precursor cells. The APE was reduced as much as 80% in severely affected brains. There was no alcoholrelated difference in the in vitro morphogenesis of the astroglial colonies. Observations of the proliferative neural cells in situ suggest that there is an impaired recruitment of all neural cell types, but that the reduced APE reflects primarily a proportionate increase in the number of immature neurons among the cells obtained from the fetal neopallium. It appears that a prolongation of mitosis may be resulting in a general developmental delay in the fetal neocortex.     

The influence of ImuVert, a biological response modifier, on the growth and ganglioside composition of murine neural tumors

ImuVert is a biological response modifier (BRM) that has antitumor effects in humans and rats. The influence of ImuVert on the ganglioside composition of two experimental brain tumors, ependymoblastoma and CT-2A, was studied in C57BL/6J mice. Gangliosides are expressed on plasma membranes and can serve as markers to distinguish neural cells from nonneural cells in mouse brain tumors.N- acetylneuraminic (NeuAc) is the predominant sialic acid in mouse neural cells, whereasN-glycolylneuraminic (NeuGc) is a major sialic acid in nonneural cells, e.g., macrophages and lymphocytes. ImuVert treatment increased the NeuGc ganglioside concentration in the ependymoblastoma, but had no effect on the sialic acid concentration in the CT-2A brain tumor. ImuVert also had a slight inhibitory effect on the growth of both brain tumors.     

In Vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53-deficient immortalized neuroectodermal progenitor cell line, NE-4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res. 47, 405–416]. Rearrangement of nestin filaments was an early marker of neuron-formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4–5 days compared to the appearence of neurons.Future neurons were sorted out from the substrate-attached population of apparently non-induced cells. The sorting out of future neurons resembled the separation of neural precursorsin vivo. The continuous changes in the shape and also in the position of the cells resulted in the formation of characteristic morphological patterns. On the basis of morphological changes, five characteristic stages of in vitro neural differentiation were distinguished.The analysis of the morphological changes revealed that cell-to-cell interactions played an essential role in the cell fate decision made by induced precursors. Our observations indicate that the NE-4C cell line can serve as an in vitro model to investigate some early steps of neurogenesis.     

Platelet-derived growth factor in human glioma

Platelet-derived growth factor (PDGF) is a 30 kDa protein consisting of disulfide-bonded dimers of A- and B-chains. PDGF receptors are of two types, α- and β-receptors, which are members of the protein-tyrosine kinase family of receptors. The receptors are activated by ligand-induced dimerization, whereby the receptors become phosphorylated on tyrosine residues. These form attachment sites for signalling molecules, which inter alia activate the Ras · Raf pathway. PDGF has important functions in development and is required for a proper timing of oligodendrocyte differentiation. The v-sis oncogene of simian sarcoma virus (SSV) is a retroviral homolog of the B-chain gene, and induces transformation by an autocrine activation of PDGF receptors at the cell surface. SSV induces malignant glioma in experimental animals, suggesting a role for autocrine PDGF in glioma development. PDGF and PDGF receptors are frequently coexpressed in human glioma cell lines. Specific and nonspecific PDGF antagonists block the growth of some glioma cell lines in vitro and in vivo, suggesting that autocrine PDGF is involved in transformation and tumorigenesis. In situ studies of human gliomas show overexpression of α-receptors in glioma cells of high-grade tumors. In a few cases, overexpression is caused by receptor amplification. Since high-grade glioma cells also express the PDGF A-chain, an autocrine activation of the α-receptor may drive the proliferation of glioma cells in vivo. © 1995 Wiley-Liss, Inc.     

SK-ER3 Neuroblastoma Cells as a Model for the Study of Estrogen Influence on Neural Cells

The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.     

Human fetal brain antigen expression common to tumors of neuroectodermal tissue origin

The antigenic relationship between human tumors of neuroectodermal origin and fetal brain were further investigated by characterization of two hybridoma antibodies derived from a fusion of P3-NS1/1-Ag 4-1 (NSI) myeloma cells and splenocytes hyperimmunized to second trimester human fetal brain homogenate. Monoclonal antibodies (MAs) 1H8cl 2 and 1H8cl 3 were analyzed by cell surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-antiperoxidase (PAP) immunohistology. MA 1H8cl 3 is the more broadly reactive, binding to 9/14 glioblastoma (GBM), 2/3 neuroblastoma, 1/2 melanoma, and 1 medulloblastoma cell line(s) by CS-RIA analysis, and to 12/15 GBM, fetal brain, spleen, and liver, and adult spleen by PAP analysis. MA 1H8cl 2 is more restricted, binding to 7/14 GBM, 2/3 neuroblastoma, 1 medulloblastoma, and 2/3 fetal skin fibroblast cell line(s) by CS-RIA, and to 9/15 GBM and fetal brain and spleen by PAP analysis. Control non-central nervous system tumors and normal adult tissue including brain, thymus, lymph node, liver, kidney, lung, skin, and pancreas, were unreactive with both 1H8cl 2 and 1H8cl 3 by CS-RIA, PAP, and absorption analysis. The data presented here establish the unique nature of the detected antigenic specificities as compared to previously described oncofetal and onconeural antigens, and define two immune reagents which are operationally specific for tumors of neuroectodermal origin within the adult central nervous system.     

GFAP promoter driven transgenic expression of PDGFB in the mouse brain leads to glioblastoma in a Trp53 null background

Glioblastomas are the most common and malignant astrocytic brain tumors in human adults. The tumor suppressor gene TP53 is commonly mutated and/or lost in astrocytic brain tumors and the TP53 alterations are often found in combination with excessive growth factor signaling via PDGF/PDGFRα. Here, we have generated transgenic mice over‐expressing human PDGFB in brain, under control of the human GFAP promoter. These mice showed no phenotype, but on a Trp53 null background a majority of them developed brain tumors. This occurred at 2–6 months of age and tumors displayed human glioblastoma‐like features with integrated development of Pdgfrα⁺ tumor cells and Pdgfrβ⁺/Nestin⁺ vasculature. The transgene was expressed in subependymal astrocytic cells, in glia limitans, and in astrocytes throughout the brain substance, and subsequently, microscopic tumor lesions were initiated equally in all these areas. With tumor size, there was an increase in Nestin positivity and variability in lineage markers. These results indicate an unexpected plasticity of all astrocytic cells in the adult brain, not only of SVZ cells. The results also indicate a contribution of widely distributed Pdgfrα⁺ precursor cells in the tumorigenic process. © 2008 Wiley‐Liss, Inc.     

MAP-2e,a novel MAP-2 isoform,is expressed in gliomas and delineates tumor architecture and patterns of infiltration

The MAP-2 isoform containing exon 13 (MAP-2e) is expressed in human fetal development as early as 15 gestational weeks and parallels oligodendrocyte maturation. MAP-2e is down-regulated following myelination and is expressed in few cells in the adult central nervous system (CNS). To determine whether CNS tumors express MAP-2e, we screened 122 archival, paraffin-embedded adult and pediatric tumors of the CNS and non-CNS. All oligodendrogliomas were positive and extensive staining was observed in glioblastomas, various malignant gliomas and dysembryoplastic neuroepithelial tumors. MAP-2e was not expressed in non-CNS tumors or neuroblastomas. Thus. neuroectodermal tumors that have glial characteristics express this developmental marker of immature glia. Analysis of oligodendrogliomas demonstrated numerous cell morphologies from round cells with no processes to cells with single or multiple processes. MAP-2e immunostaining also delineated tumor invasion into adjacent gray and white matter, indicating that MAP-2e appears to be a useful marker for examining the infiltration of malignant cells into surrounding tissue.     

Human fetal brain antigen expression common to tumors of neuroectodermal tissue origin : Gliomas, neuroblastomas, and melanomas

The antigenic relationship between human tumors of neuroectodermal origin and fetal brain were further investigated by characterization of two hybridoma antibodies derived from a fusion of P3-NS1/1-Ag 4-1 (NSI) myeloma cells and splenocytes hyperimmunized to second trimester human fetal brain homogenate. Monoclonal antibodies (MAs) 1H8cl 2 and 1H8cl 3 were analyzed by cel; surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-antiperoxidase (PAP) immunohistology. MA 1H8cl 3 is the more broadly reactive, binding to 9/14 glioblastoma (GBM), 2/3 neuroblastoma, 1/2 melanoma, and 1 medulloblastoma cell line(s) by CS-RIA analysis, and to 12/15 GMB, fetal brain, spleen, and liver, and adult spleen by PAP analysis. MA 1H8cl 2 is more restricted, binding to 7/14 GBM, 2/3 neuroblastoma, 1 medulloblstoma, and 2/3 fetal skin fibroblast cell line(s) by CS-RIA, and to 9/15 GBM and fetal brain and spleen by PAP analysis. Control non-central nervous system tumors and normal adult tissue including brain, thymus, lymph node, liver, kidney, lung, skin, and pancreas, were unreactive with both 1H8cl 2 and 1H8cl 3 by CS-RIA, PAP, and potential interest in terms of cell lineage evolution and investigations of cell-cell interactions from immunologic, developmental, and neuronal communication viiewpoints. The most critical observation remains, however, that the operational specificity of MAs 1H8cl 2 and 1H8cl 3 within the adult central nervous system makes them useful reagents for the purification and biochemical characterization of the family of human neuroectodermal tumor-associated antigens.