Plasticity of process-bearing glial cell cultures from neonatal rat cerebral cortical tissue

The factors and cellular interactions that influence the commitment of cells to specific neural lineages are not well understood. We have used cultured non-neuronal process-bearing (PB) cells from neonatal rat cerebral cortices as a model to assess the influence of various culture conditions on the determination of cells as either astroglia or oligodendroglia. Increasing postseparation plating density was significantly associated (p less than 0.001) with decreasing percentages of glial fibrillary acidic protein (GFAP+) cells, increasing percentages of galactocerebroside (GC+) cells, and increasing percentages of nonstained cells. As the fetal calf serum content of growth medium was increased, the percentage of GFAP+ cells increased, and as the serum content was decreased, the percentage of GC+ cells increased. Evidence of minimal cell proliferation and the observation of PB cells that coexpressed GFAP and GC supported the conclusion that PB cells switched their phenotypic expression from GFAP+ in serum to GC+ in serum-free medium. PB cells exhibited plasticity in their phenotypic expression as cells grown for 9 d in serum-free medium were still responsive to the effects of serum, while cells grown for 6 d in serum were refractory to serum withdrawal. This research has demonstrated the plasticity of PB cells separated from polygonal astroglia as they expressed GFAP in the presence of serum and GC in serum-free medium.     

Development of O4+/O1− immunopanned pro-oligodendroglia in vitro

In this study, O4+/O1− pro-oligodendroglia isolated by immunopanning from cerebral hemispheres of P3–P5 rats were evaluated during their maturation in culture. Immunopanning yielded 3–4×10⁵ cells/cerebrum, with 98% O4+ and 6% O1+. There was heterogeneity in the morphologies of immunopanned cells ranging from simple bipolar cells to more complex multipolar cells. As a first step in determining potential differentiative responses of mature oligodendroglia, we examined glial fibrillary acidic protein (GFAP) expression in response to fetal bovine serum (FBS) by cultures established from O4+/O1− immunopanned cells grown for 1, 14, or 21 days, exposed to 20% FBS for 6–7 days and fixed and immunostained on days 7, 21 or 28 in culture (DIC). When immunopanned cells were exposed to FBS following 1 day in serum-free medium, 88% expressed GFAP and when immunopanned cells were cultured for 14 days prior to FBS exposure, 78% expressed GFAP. By contrast, when cells were cultured for 21 days prior to FBS exposure (when a majority of the cells expressed O1 and myelin basic protein (MBP)), only 19% of the cells expressed GFAP (p<0.001). Cells that were O4+/GFAP− even in the presence of FBS often exhibited a mature oligodendroglial morphology. Among immunopanned cells that responded to FBS by expression of GFAP, both process-bearing (similar to type 2 astroglia) and flattened, polygonal (similar to type 1 astroglia) GFAP+ cells were observed. These results confirm the utility of immunopanning for the isolation of pro-oligodendroglia and demonstrate that oligodendroglia that develop in vitro from O4+/O1− immunopanned cells become resistant to GFAP induction by FBS.     

Astrocytes cultured from mature brain derive from glial precursor cells

We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Cerebral white matter contains PDGF-responsive precursors to O2A cells

Cells dissociated from the cerebral white matter of immature rats were maintained in monolayer culture. Treatment with platelet-derived growth factor (PDGF) caused a large increase in the numbers of "O2A" oligodendroglial precursor cells (which bind the monoclonal antibody A2B5) and subsequently in the numbers of galactocerebroside (galC)-positive oligodendroglia. A2B5-negative "pre-O2A cells" in cerebral white matter cultures in which O2A cells and oligodendroglia had been killed by antibody-dependent complement-mediated cytolysis were induced by PDGF to proliferate and to differentiate into O2A cells and subsequently into oligodendroglia and type 2 astroglia. The most mature pre-O2A phenotype in these cultures was a small, round, process-bearing cell which expressed vimentin but not glial fibrillary acidic protein or galC. Cells of this phenotype were not observed upon PDGF treatment of immature rat optic nerve monolayer cultures from which O2A cells and oligodendrocytes had been depleted, and PDGF also failed to elicit the accumulation of O2A cells and oligodendroglia in such cultures.     

Autoradiographic quantitation of beta-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether beta-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). beta-Adrenergic receptors were labeled with [125I]pindolol or [125I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of beta-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of 125I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of beta-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of beta-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the beta-adrenergic receptor is lost from this population of astroglia during development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic divergence of oligodendrocyte and astrocyte lineages in developing rat cerebrum

Oligodendrocyte and astrocyte lineages were traced in rat forebrain sections using single- and double-label immunoperoxidase and indirect immunofluorescent techniques. Antibodies were directed against antigenic markers, the expressions of which overlapped in time: GD3 ganglioside in immature neuroectodermal cells; vimentin in radial glia; glial fibrillary acidic protein (GFAP) in astrocytes; and carbonic anhydrase (CA) and galactocerebroside (GC) in oligodendrocytes. A histochemical stain for iron was also used as a marker of oligodendrocytes. Small cells of the subventricular zone (SVZ) were stained with anti-GD3 but not with the other antibodies. By 16 d of gestation (E16), the SVZ generated large, round cells and thick, process-bearing cells that were GD3+/CA+/iron+. These cells then appeared in the cingulum and, with time, increased in numbers and extended thick processes as they filled the subcortical white matter. These cells eventually lost their reactivity to anti-GD3 but became GC+/CA+ with processes extending to myelin sheaths. At E15 radial glia were stained with the anti-vimentin antibody but were negative for GFAP. At birth, only the vimentin+ radial glia midline between the 2 ventricles were GFAP+, but with time more vimentin+ cells became GFAP+. By 7 d of postnatal age all the vimentin+ cells were GFAP+ and had converged predominately on the cingulum. With time these cells condensed and took on characteristic shapes of astrocytes. The embryonic separation of the oligodendrocyte and the astrocyte lineage is supported by four pieces of evidence: (1) GD3+ cells were double labeled with anti-CA, and then went on to become GC+; (2) vimentin+ and GFAP+ cells were not also GD3+; (3) ultrastructural localization of anti-GD3 was confined to cells with characteristics consistent with developing oligodendrocytes; and (4) the shapes of GD3+, CA+, GC+, or iron+ cells did not resemble those of the vimentin+ or GFAP+ cells.     

Autoradiographic quantitation of β-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether β-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). β-Adrenergic receptors were labeled with [¹²⁵I]pindolol or [¹²⁵I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of β-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of¹²⁵I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of β-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of β-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the β-adrenergic receptor is lost from this population of astroglia during development. This method provides a novel approach to the problem of measuring cell-type specific, membrane-associated receptors on individual, well-characterized cells in mixed primary neural cultures.     

Co-expression of glial fibrillary acidic protein- and vimentin-type intermediate filaments in human astrocytomas

Summary The expression of intermediate filament (IF) proteins was studied in 71 cases of malignant human astrocytoma and in 17 cases of reactive gliosis, using immunocytochemical techniques with polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and vimentin. In all cases of astrocytoma, varying in degree of malignancy from grade I to grade IV, co-expression of GFAP and vimentin was found. No change in vimentin- or GFAP-IF expression with increasing anaplasia was seen. In addition astrocytic cells in reactive gliosis showed simultaneous expression of GFAP and vimentin. The intracellular distribution of these IF proteins differed. Vimentin was found to be located in a more juxta-nuclear position, whereas GFAP immunoreactivity showed a more intense staining of the cellular processes. Astrocytes in reactive gliosis behaved more or less like neoplastic cells. However, thin cell processes of reactive astrocytes in the cortex and superficial white matter only contained GFAP immunoreactivity. Simultaneous expression of GFAP and vimentin and their proportion in malignant and reactive glial cells are discussed in the light of earlier reports on the IF content of glial cells during development and maturation, in which vimentin precedes GFAP-expression. The existence of two separate (functional) IF systems in astroglia is suggested.     

Establishment of a permanent rat brain-derived glial cell line as a source of purified oligodendrocyte-type 2 astrocyte lineage cell populations

A permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process-bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process-bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum-containing culture medium, most of the process-bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1+ or O4+ cells were rarely GFAP-, and GFAP+ cells were rarely LB1- or O4-. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process-bearing cells accumulated the neurotransmitter gamma-aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the O-2A (oligodendrocyte-type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of O-2A lineage cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vimentin isoform expression in the human retina characterized with the monoclonal antibody 3CB2

The antigen recognized by the monoclonal antibody 3CB2 (3CB2-Ag and 3CB2 mAb) is expressed by radial glia and astrocytes in the developing and adult vertebrate central nervous system (CNS) of vertebrates as well as in neural stem cells. Here we identified the 3CB2-Ag as vimentin by proteomic analysis of human glial cell line U-87 extracts (derived from a malignant astrocytoma). Indeed, the 3CB2 mAb recognized three vimentin isoforms in glial cell lines. In the human retina, 3CB2-Ag was expressed in Müller cells, astrocytes, some blood vessels, and cells in the horizontal cell layer, as determined by immunoprecipitation and immunofluorescence. Three populations of astrocytes were distinguishable by double-labeling immunohistochemistry: vimentin+/GFAP+, vimentin-/GFAP+, and vimentin+/GFAP-. Hence, we conclude that 1) the 3CB2-Ag is vimentin; 2) vimentin isoforms are differentially expressed in normal and transformed astrocytes; 3) human retinal astrocytes display molecular heterogeneity; and 4) the 3CB2 mAb is a valuable tool to study vimentin expression and its function in the human retina.     

Regulation of glial fibrillary acidic protein (GFAP) expression in CNS development and in pathological states

Studies that have described changes in glial fibrillary acidic protein (GFAP) expression during CNS development and during pathological reactions are summarized. We describe our own studies examining GFAP expression in vitro. In primary cultures established from newborn rat forebrain and maintained in the presence of serum, GFAP expression is first observed in cells from the subventricular germinal zone. Glial precursor cells, at an uncommitted stage, can be induced by serum to begin GFAP synthesis. In astrocytes already established in culture, GFAP synthesis can be further increased by dibutyryl cyclic AMP (dbcAMP). By labelling astrocytes with radioactive methionine, we demonstrate increases of GFAP and vimentin synthesis within 48 h of exposure to dbcAMP. Continuous exposure results in a gradual rise in the cellular level of GFAP.     

Glial fiber pattern in the developing chicken cerebellum: vimentin and glial fibrillary acidic protein (GFAP) immunostaining

The possible relation between glial fibers and the formation of longitudinal granule cell migration patterns that occur in the cerebellar anlage of the chicken was investigated by immunocytochemistry of vimentin (monoclonal antibody) and glial fibrillary acidic protein (polyclonal antibody against GFAP, PGF) on fixed and unfixed brain tissues. In addition, neuronal development was studied with a monoclonal antibody for neurofilament. Vimentin was present in radial and tangential fibers in the cerebellar anlage during granule cell migration in almost all parts of the anlage. However, no specific topographic relation of vimentin and GFAP to the migration pattern of granule cells was observed. In adults, Bergmann fibers and astroglia were stained with vimentin antiserum and not with GFAP antiserum. Conclusions are that radial fibers do not determine the formation of longitudinal cytoarchitectonic patterns in the chick cerebellum and that vimentin is the main cytoskeletal component of Bergmann fibers and astroglial cells in embryonic and adult chicken cerebellum.     

Studies of glial lineage and proliferation in vitro using an early marker for committed oligodendrocytes.

The potential of immature glial cells to differentiate into astrocytes (ASs) or oligodendrocytes (OLs) has been examined using a monoclonal antibody (007) that is specific for OLs in vivo. Cells were dissociated from 2-day postnatal mouse cortex and labeled with the 007 antibody 2 hr after plating. The cells which were labeled during this single, brief exposure to the antibody retained the antibody on their surfaces over the course of the experiments. Cells were double stained at various timepoints for residual 007 antibody and either galactocerebroside (GC) or glial fibrillary acidic protein (GFAP). Shortly after plating, most 007+ cells were GC- and none expressed GFAP. These cells were round, although some had begun to extend very short processes. After 96 hr, greater than 95% of cells with residual 007 on their surfaces also expressed GC. By this time, all the 007+ cells had several processes of varying lengths extending from their cell bodies. Cells expressing both 007 and GFAP were never seen. The 007+/GC+ OLs were not induced to differentiate from 007+ bipotential progenitors since they were grown in fetal calf serum. These results show that under our culture conditions the 007 antibody is OL specific. Immunostaining for bromodeoxyuridine, a marker for dividing cells, revealed that some 007+ cells were proliferating. The majority of these proliferating cells had already extended three or more processes. We therefore conclude that immature, process-bearing cells can be committed to the OL lineage at times before they express detectable amounts of GC. Since these young 007+ OLs are actively proliferating, committed cells can serve as an important source of new OLs.     

Immunocytochemical characteristics of organotypic cerebellar culture using the avidin-biotin complex method (ABC)

The occurrence and cellular localization of some CNS antigenic markers were studied in organotypic cultures of newborn rat cerebellum grown for up to 5 weeks. The avidin-biotin-complex method was employed using polyclonal immune sera against glial fibrillary acidic protein (GFAP), S-100 protein and vimentin, and monoclonal antibodies against neurofilaments. In all cultures GFAP was selectively localized in astrocytes, both in perikarya and in cellular processes. The difference in immunoreactivity between particular cells and culture "zones", and considerable morphological polymorphism of the astrocytes were noted. In majority of cells the immunostaining for vimentin was very intensive, whereas reaction with antiserum against S-100 protein was weak or negative. Neurofilament antigen was localized only in neuronal processes lying in explants of the younger--one- to two-week cultures.     

Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue. A developmental study

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.     

Expression of vimentin and glial fibrillary acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines

Summary The expression of glial fibrillary acidic protein (GFAP) and vimentin was investigated immuno-histochemically in 104 experimental gliomas induced by transplancental application of ethylnitrosourea (ENU) in CDF rats. Immunoreactivity for vimentin was prominent in many astrocytic tumor cells and especially in small glioma cells forming anaplastic medulloblastoma-like foci in many tumors. The majority of tumor cells in oligodendroglial tumors were vimentin negative, except for some of the large polymorphous oligodendrogliomas which contained intermingled vimentin positive glioma cells. GFAP immunoreactivity was detectable only in a low fraction of tumor astrocytes and in a few exceptional cases some oligodendroglial tumor cells stained positive. Immunohistochemistry with antibodies against neurofilaments and cytokeratins revealed no staining in tumor cells of ENU-induced gliomas, while all oligoden-drogliomatous tumors stained positive for HNK-1. Immunocytological and immunoblot investigations of the two rat glioma cell clones RG2 and F98, which are both derived from ENU-induced gliomas, showed a prominent expression of vimentin in monolayer cultures and in syngeneic intracerebral transplantation tumors. F98 additionally demonstrated a fraction of GFAP positive cells especially in confluent cultures and in intracerebral tumors. RG2, on the other hand, exhibited virtually no GFAP immunoreactivity in culture but showed individual GFAP positive tumor cells in intracerebral tumors. Our results revealed a more precise picture of the cellular differentiation in ENU-induced rat gliomas and in two widely used glioma cell lines. They underline the heterogeneity of experimental rat gliomas which may comprise cells at different stages of differentiation towards the oligodendroglial or astroglial phenotype.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin

Possible differentiation mechanisms were investigated in a glioblastoma multiform cell line (GL15) presenting an undifferentiated phenotype with weak glial fibrillary acidic protein (GFAP) and strong vimentin (VIM) expression. Serum-free conditions induced time-dependent increases of GFAP-mRNA and GFAP protein levels, associated with a process-bearing astrocytic morphology. Activation of protein kinase C (PKC) by tumor promoter phorbol 12-myrystate 13-acetate (PMA) induced a rapid morphological differentiation and a decrease in GFAP mRNA, whereas the GFAP level remained unchanged. Such parameters were shown to characterize a physiological differentiation stage in astroglial cultures. Treatment of process-bearing GL15 cells with dibutyryl cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, induced a timedependent decrease in the GFAP mRNA and GFAP protein levels and reverted morphological changes induced by serum-free conditions. Neither PMA nor dbcAMP influenced the VIM mRNA expression. In GL15 cells, PKC and PKA activation have opposite effects. Understanding the role of these kinases in malignant transformation and in the in vitro differentiation process is of both basic and clinical interest. © 1995 Wiley-Liss, Inc.     

Glial fibrillary acidic protein (GFAP) in oligodendrogliomas: a reflection of transient GFAP expression by immature oligodendroglia

Fourteen pure oligodendrogliomas were studied by light- and electronmicroscopy and immunohistochemistry to examine glial fibrillary acidic protein (GFAP) positivity in the tumors. To compare the immunohistochemical staining patterns of neoplastic oligodendroglia and immature oligodendroglia, myelination glia in the white matter of eight normal brains from children under 6 months of age were studied. The tumors possessed light microscopic and ultrastructural features characteristic of oligodendrogliomas. Microtubules were found in the cytoplasm of nine tumors on electronmicroscopy. In one, intermediate filaments and microtubules were observed in occasional tumor cells with polygonal crystalline structures in the cytoplasm. Using the peroxidase-antiperoxidase technique, all specimens were stained for GFAP, vimentin, S-100 and neuron-specific enolase (NSE). In nine tumors, variable numbers of cells with an oligodendroglial morphology reacted positively for GFAP. All tumors were positive for S-100 and negative for vimentin and NSE. The myelination glia in the eight normal brains stained positively for GFAP but not for vimentin. Vimentin is expressed by developing, reactive and neoplastic astrocytes. Thus, GFAP positivity combined with vimentin negativity in both neoplastic and immature oligodendroglia suggests that GFAP positivity in oligodendrogliomas may reflect the transient expression of this intermediate filament by immature oligodendroglia.