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     

Glial fibrillary acidic protein and intermediate filaments in human glioma cells

Summary Cultured human glioma cells were studied by double indirect immunofluorescence technique using antisera against intermediate filaments and glial fibrillary acidic protein. With both antisera cytoplasmic fibrillar fluorescence was seen. Perinuclear bundles of intermediate-sized filaments, induced by vinblastine treatment, were strongly stained with both antisera. The degree of codistribution of the two types of antigenic determinants varied considerably from cell to cell. These results suggest that two types of filament-related antigenic determinants can be present in the same cell, and also that glial fibrillary acidic protein-related filaments may possess functional similarities to the intermediate filaments found in other cells. Glial fibrillary acidic protein remains as a useful and specific antigenic marker for the study of glial cells in vitro.     

自身免疫性胶质纤维酸性蛋白星形胶质细胞病

自身免疫性胶质纤维酸性蛋白星形胶质细胞病是一种可治的中枢神经系统自身免疫性炎性疾病,以脑膜、脑、脊髓和视神经等受累为主要表现,磁共振成像可见脑室旁线样放射状强化和(或)脊髓长节段受累伴中央强化病灶,脑活体组织检查提示小血管周围炎症伴小胶质细胞活化,对类固醇激素治疗敏感.胶质纤维酸性蛋白抗体被认为是本病的特异性生物标志物.     

Glial filaments in the subcutaneous tumors of mouse glioma clones differently expressing glial fibrillary acidic protein

Summary Glial filaments contain vimentin and glial fibrillary acidic protein (GFAP). The question of how glial filaments change morphologically according to the expression of vimentin and/or GFAP has remained unclear. In this study, immunohistochemical and ultrastructural examinations were performed on the subcutaneously transplanted tumors of two clones (F6B3 and G10A10) derived from a mouse glioma. F6B3 tumor expressed GFAP and vimentin in large quantities. G10A10 tumor expressed plenty of vimentin but only a little of GFAP. Ultrastructurally, F6B3 tumor contained abundant cytoprocesses in most of which numerous intermediate filaments (IFs) were arranged in a parallel array. On the other hand, only a small number of the processes were seen in G10A10 tumor, which showed a few IFs arranged either randomly or sparsely in the processes. Both tumors commonly had the IFs accompanied by visible sidearms, but there was a difference in that the smooth and firm IFs were confined to part of F6B3 tumor. Thus, the comparison made between the two models presented differences in the content, arrangement and morphology of IFs, as well as in the manner of GFAP expression, suggesting correlation between these differences.     

An unusual meningioma variant with glial fibrillary acidic protein expression

We studied a recurrent meningioma located in the right frontal lobe. The tumor showed high cellularity and the cells had plump, hyalinous cytoplasm. Immunohistochemically, almost all the tumor cells were positive for epithelial membrane antigen and vimentin, and unexpectedly, glial fibrillary acidic protein (GFAP). Ultrastructural investigation revealed abundant 8- to 10-nm filaments in the cytoplasm. Conspicuous interdigitations with numerous desmosomes were present. Frequently, intracellular and intercellular lumina lined by microvilli were also found. We considered the present case to be an unusual variant of meningioma with GFAP expression. A few cases of meningioma with triple expression of GFAP, vimentin and cytokeratin have been reported previously. However, the present case showed obvious pathological differences from these, and had no immunoreactivity for cytokeratin. Received: 30 December 1996 / Revised, accepted: 20 May 1997     

Ultrastructural characteristics of glial fibrillary acidic protein expression in epoxy resin-embedded human brain tumors

Summary Thirteen surgically removed, epoxy resin (Durcupan ACM or Epon 812)-embedded human brain tumors were examined for glial fibrillary acidic protein (GFAP) content in semithin and ultrathin sections with the immunogold-silver staining method. Mild aldehyde fixation and the hydrophobic resin embedding did not interfere with the antigenicity, since silver intensification of the immunogold marker provided excellent visualization of the reaction on both light microscopic and ultrastructural levels. The GFAP reaction was usually localized on the glial intermediate filament bundles, usually correlating well with the amount of filaments. The unstained filamental regions of two ependymomas might correspond to the vimentin expression revealed by double labeling in semithin sections. Occasional GFAP immunopositivity without filamental appearance was observed in one of the oligodendrogliomas, as patchy electron-dense cytoplasmic corpuscules, in Rosenthal fibers and in some mainly necrobiotic tumor cells, reflecting a possible connection with glial filaments.     

Contrary effect of lactic acid on expression of neuron-specific enolase and glial fibrillary acidic protein in human glioma cells

Summary We examined the effect of lactic acid on cultured human glioma cell lines expressing glial fibrillary acidic protein (GFAP), vimentin and neuronspecific enolase (NSE). The growth of the cells was inhibited by the lactic acid in a dose-dependent manner. At 56 mM of lactic acid, the surviving cells of the KNS-42-c2 cell line developed slender processes and increasingly formed bizzar giant cells. In an immunofluorescence study of the lactic acid-resistant cells, the GFAP-positive cells prominently decreased in number, while the NSE-positive cells clearly increased. The vimentin was not affected throughout the experiment. After removing lactic acid from the medium, the GFAP-positive cells gradually increased in number. The method of dot immunoassay was useful for quantifying GFAP in cellular extracts. It indicated that the amount of GFAP decreased in the cells cultured with lactate-containing media and increased to the primary values after removing the lactic acid. These results may suggest that the morphological and immunochemical diversities of glioma cells are secondarily affected by cellular microenvironments such as lactic acid.Supported in part by Grant-in aid 624380309 from the Ministry of Education, Science and Culture, Japan     

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene

Summary We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)⁺ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5 and 3 ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5 non-coding, 1296 bp of coding and 1717 bp of 3 non-coding regions. Using cDNA probes made from both the coding and the 3 non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and-negative cells. It was shown that the 3 non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.     

Proliferation of gemistocytic cells and glial fibrillary acidic protein (GFAP)-positive oligodendroglial cells in gliomas: a MIB-1/GFAP double labeling study

Large gemistocytic cells are well-known elements of glial tumors. Recently, miniature gemistocytic cells and neoplastic glial fibrillary acidic protein (GFAP)-positive oligodendroglial cells, which are regularly seen in oligodendrogliomas, have been termed "transitional cells". The proliferative activity of the gemistocytic cell types and the GFAP-positive (gliofibrillary) oligodendrocytes was determined in eight astrocytomas, seven gemistocytic astrocytomas, eight glioblastomas, two monstrocellular glioblastomas, seven oligodendrogliomas and three mixed oligo-astrocytomas by immunohistochemical staining of the proliferation marker MIB-1 in combination with immunostaining for GFAP. Both large gemistocytic cells and the transitional cells showed cytoplasmic GFAP-positive staining. Neither in the classic gemistocytes nor in the minigemistocytes nuclear immunostaining for the MIB-1 antibody was observed. In contrast, MIB-1 staining was seen in the gliofibrillary oligodendrocytes. It is concluded that both large and miniature gemistocytic cell types contrast with gliofibrillary oligodendrocytes by their inability to proliferate. Received: 16 March 1995 / Revised: 24 May 1995 / Revised, accepted: 21 August 1995     

Suprasellar meningioma with expression of glial fibrillary acidic protein: a peculiar variant

A 24-year-old female presented with a 3-year history of a suprasellar and intraventricular solid midline process measuring about 3×4 cm. At surgery, this tumour was sharply delineated and of stone-like firmness and was removed completely. Histology suggested meningioma, featuring nests and cords of epithelium-like cells with prominent cytoplasm amidst abundant fibrous stroma with prominent lymphoplasmocellular infiltration. Immunocytochemically, the tumour cells expressed vimentin, S-100 protein, epithelial membrane antigen, cytokeratins, and most surprisingly, glial fibrillary acidic protein (GFAP). Ultrastructural investigation revealed abundant intermediate filaments and occasionally dense secretory granules in tumour cells with short, finger-like cytoplasmic processes joined by very rare small, but well-developed desmosomes. This tumour most likely represents a peculiar variant of meningioma with prominent production of GFAP, as previously described [Budka H (1986) Acta Neuropathol (Berl) 72: 43–54].This observation was presented by H. Budka at the Slide Seminar on Tumours of the XII^th International Congress of Neuropathology, Toronto, September 1994     

Laminin expression by glial fibrillary acidic protein positive cells in human gliomas

Extracellular matrix components are regarded as important substrates for invasive tumor cells. The present work focuses on the expression of laminin in the brain in response to invading brain tumors. Biopsies obtained from tissue macroscopically evaluated as the border zone between tumor and normal brain, in 5 patients undergoing surgery for glioblastoma multiforme, were examined by immunocytochemistry and scanning confocal microscopy for the expression of laminin and glial fibrillary acidic protein. Laminin was mainly found in all the specimens associated with the basal lamina of blood vessels, but a variable degree of punctate laminin deposits were also observed in the parenchyma not associated with blood vessels. In the specimens with substantial deposits, scanning confocal microscopy showed that some of the laminin co-localized with intracellular glial fibrillary acidic protein. Punctate deposits of laminin were also seen in an intracranial BT4C rat glioma model, where it was particularly abundant in the brain/tumor confrontation zone. Previous in vitro studies have shown that laminin, among several extracellular matrix components, represent a highly permissive substrate for glioma cell migration. The presented results indicate that laminin can be produced by glial fibrillary acidic protein positive cells during glioma cell invasion in humans. This glycoprotein may thus represent one important substrate among many, which contribute to the invasive phenotype of gliomas.     

DNA content and marker expression in human glioma explants

Summary Immunohistochemical studies of astrocytoma tissue have predominately shown fibronectin (FN) positivity restricted to vessels and glial fibrillary acidic protein (GFAP) positivity in the parenchyma. Cultured glioma cell lines, however, express both FN and GFAP. We measured the DNA content of explants of gliomas to determine if the ploidy of the FN-positive and GFAP-positive cells differed. Thirty-three explants from four high grade gliomas were cultured on slides. FN and GFAP markers were determined by double immunofluorescence. The slides were stained by the Feulgen method, the explants relocated and the DNA content measured by microdensitometry using the CAS-100 instrument. Human leukocytes applied to the slides were used as a diploid standard. Eleven GFAP-positive explants were hyperdiploid and one hypodiploid. Five FN-positive explants were diploid, three hypodiploid and ten hyperdiploid. One FN-positive explant was biclonal with aneuploid subpopulations. Two hyperdiploid explants, each of which had monoclonal histogram patterns, expressed both FN and GFAP. We conclude that most FN-positive cells, in addition to GFAP-positive cells, from cultured gliomas represent neoplastic cells. These may be present in the tumor in low numbers or may result from marker switching in culture.     

The postnatal development of glial fibrillary acidic protein and neurofilament triplet proteins in rat brain stem

Cytoskeletal preparations containing both the glial fibrillary acidic protein and the neurofilament triplet proteins were prepared from brain stems of rats at different ages and the individual peptides separated in polyacrylamide gels. Stained peptide bands were quantitated as the area under peaks generated by densitometric scanning. Peak areas were converted to grams of protein based on total gel dye binding and total protein applied to the gels. Between 5 and 30 days, the concentration of the peptide (g of peptide/mg of tissue protein) of apparent molecular weight 51,000 (corresponding to the glial fibrillary acidic protein), increased 3 fold. The corresponding increase in total concentration of the three peptides corresponding to the neurofilament proteins was 4.5 fold. However, the increase in concentration of the individual neurofilament peptides was each different. Very little of the apparent molecular weight 210,000 neurofilament peptide was present at 5 days and its concentration increased 11 fold by 30 days compared to about 3.5 fold for the other two neurofilament peptides. These results are in general agreement with studies using immunological techniques and the methods have the advantages of using readily available techniques and allowing the simultaneous comparison of both neuronal and glial specific filaments during development.     

Peculiar pattern of glial fibrillary acidic protein production in extracranial metastatic tumor cells of malignant astrocytoma

Summary An 83-year-old woman suffered from malignant astrocytoma originating in the temporal lobe. Autopsy revealed its extracranial metastasis to the liver, lung and bone marrow. The tumor tissue at the primary site was composed of plump, process-forming cells and small cells with scanty cytoplasm, and showed dural invasion. In the metastatic areas, most of the tumor cells were small cells, although proliferation of the plump cells in contact with perivascular connective tissue was marked, particularly in the liver. These plump cells were positively stained with antiserum to glial fibrillary acidic protein (GFAP), showing that the collagenous tissue was able to induce increased production of GFAP by the glial tumor cells.     

Glial fibrillary acidic protein expression in a new human glioma cell line in culture before and after xenogenic transplantation into nude mice

A human glioma cell line, SA146, was initiated on precoated extracellular matrix from a stereotactic biopsy of a glioblastoma. We report modulation in the expression of glial fibrillary acidic protein (GFAP) by SA146 passed in vitro before or after xenogenic transplantation into nude mice. Immunofluorescence data show a decrease in the percentage of GFAP-expressing cells with increasing in vitro passages but a full reexpression (100% of GFAP-positive cells among vimentin-positive cells) was observed in cultures just derived from the xenotransplanted tumor. These changes are correlated with the mRNA content (Northern blot probed with a cDNA for GFAP) and with the protein level (cytoskeletal fraction analyzed by two-dimensional gel electrophoresis and Western blots probed with a monoclonal antibody). At the optimal level of GFAP expression, a large range of microheterogeneity in GFAP isoforms is reached for which post-translational events are clearly involved since mRNA translation in cell free system would provide at best three isomers. We suggest that SA146 would be an appropriate model to study the regulation of GFAP expression in the context of human glial tumor biology. Received: 11 June 1996 / Revised: 10 February 1997 / 1 April 1997 / Accepted: 1 April 1997     

Analysis of cerebrospinal fluid glial fibrillary acidic protein after seizures in children

PURPOSE: To evaluate pediatric seizure patients for astrocytic injury by measuring cerebrospinal fluid (CSF) glial fibrillary acidic protein (GFAP), determine risk factors for GFAP elevation after seizures, and compare seizure-induced astrocyte injury with neuronal injury by concurrent measurement of CSF neuron-specific enolase (NSE). METHODS: CSF obtained from pediatric patients (n = 52) within 24 h of seizure was assayed for GFAP and NSE. Retrospective chart review was performed for seizure type, duration, and etiology. RESULTS: Overall, children with seizures had elevated CSF GFAP compared with controls (p = 0.0075), but no elevation of NSE (p = 0.1437). No effect of seizure type or etiology was found, but a significant positive effect of seizure duration (p = 0.0010) and status epilepticus (p = 0.0296) was seen on CSF GFAP. Individually, seven children (13%) had elevated GFAP (>440 pg/ml); in five children, the increased GFAP was not accompanied by elevations in NSE (<12 ng/ml). Five children with elevated GFAP had symptomatic etiologies for their seizures, but the etiology of one child with elevated GFAP was cryptogenic, and one had febrile seizures. CONCLUSIONS: Elevation of CSF GFAP after seizures suggests that astrocytic injury may occur in a subgroup of children, primarily in the context of prolonged seizures and symptomatic etiologies. Increased GFAP levels may occur in patients with normal NSE, suggesting that GFAP may be a more sensitive marker of brain injury in some cases.     

The role of glial fibrillary acidic protein in the diagnosis of central nervous system tumors

Summary Eighty glial and non-glial brain tumors have been studied to date using an immunologically specific and highly sensitive method of staining GFA protein which is applicable to formalin fixed and paraffin embedded tissue. Eight of these cases have been described and illustrated in some detail. GFA protein was present in all astrocytes and astrocytomas studied and in a proportion of ependymal cells and ependymomas. Some tumor cells have been demonstrated by this method to be glial despite the complete lack of blue fibrillar staining with PTAH and the absence of all morphological similarity to glial cells. In such cases the demonstration of GFA protein by this method has been valuable in establishing a diagnosis. In addition to its diagnostic value in specific cases, the method promises to shed light on unsolved problems of tumor cytogenesis.Supported in part by VA Research Grant, MRIS 2390.     

Purification of glial fibrillary acidic protein (GFAP) from normal bovine brain

Glial fibrillary acidic protein (GFAP) was purified from normal bovine brain by a modification of the procedure used to isolate vimentin in order to avoid contamination by other cytoskeletal components; vimentin, neurofilament triplet proteins, tubulin and actin. GFAP is thought to be separated from vimentin in the DE cellulose column chromatography step. The three other major proteins were also separable through ion exchange and gel filtration column chromatographies. A purified 49 kDa polypeptide was estimated to be GFAP from peptide mapping and subsequent immunoblotting analysis. We obtained 4.4 mg GFAP/1 g bovine brain white matter in less than 3 days. The polyclonal antibody raised against purified GFAP was able to detect 49 kDa GFAP by immunoblotting analysis. This isolation method is simpler and more rapid than previous methods.     

Biochemical and immunocytochemical changes in glial fibrillary acidic protein after sab wounds

Changes of glial fibrillary acidic protein (GFAP) in the forebrain of rats with stab wounds were determined by quantitative immunoblots and by immunohistochemistry. Bilateral stab wounds were made stereotaxically in the cortex and hippocampus. In control rats, the scalp was retracted and depressions were etched on the intact skull. At various times up to 21 days postoperation, one cerebral hemisphere was homogenized, proteins were separated by polyacrylamide gel electrophoresis and immunoblots were quantitated by densitometry. The contralateral hemisphere was immunostained for GFAP. Three hours postoperation GFAP⁺ cells were detected around the wound but there was no increase of total GFAP. At 6 h postoperation total GFAP in the fore brain decreased to 80% of the sham-operated control value and the number of GFAP⁺ cells was lower, compared to the controls, in layer 1 of the cortex, corpus callosum, cingulum, external capsule, internal capsule, hippocampus, optic tracts and around blood vessels. This early relative decrease in GFAP levels was actually due to an increase in GFAP in the sham-operated controls, which mounted a stronger gliotic response during the first 24 h. In neither group of animals did the GFAP levels drops below those of intact unoperated animals. At 24 h total GFAP began to increase. The number and intensity of reactive glia in the vicinity of the wound increased steadily, appearing to reach a maximum at about 7 days, then declining significantly by 21 days. The glial reaction was most pronounced in the hippocampus. Total GFAP reached 180% of the control value by 7 days and then declined to 117% by 21 days. The resolution of reactive gliosis and the changes in GFAP content were much faster than we observed in experimental autoimmune encephalomyelitis. The decreased ratio of GFAP levels in operated animals over sham-operated controls at 6 and 12 h was unexpected and may be due to more severe anoxic and ischemic damage and stress-related suppression of protein synthesis occurring in operated animals.