Human astrocytic brain tumors express APO2L/TRAIL

APO2 ligand (APO2L) is a CD95 ligand (CD95L)-related cytokine of the tumor necrosis factor family that interacts with agonistic (DR4, DR5) and antagonistic (DcR1, DcR2) receptors. Cultured malignant glioma cells preferentially express agonistic receptors and are susceptible to APO2L-induced apoptosis. Here, we report that 8 of 8 human glioma cell lines expressed APO2L mRNA and protein in vitro. Immunohistochemistry using a monoclonal antibody to APO2L revealed that all 23 primary astrocytic brain tumors analyzed, including low-grade astrocytomas and glioblastomas, express APO2L in vivo. With the exception of reactive astrocytes, non-neoplastic glia and neurons in the cerebrum lacked immunoreactivity of APO2L. Thus, in addition to the CD95/CD95L system, a second death ligand/death receptor pair may regulate susceptibility to apoptosis in human glial neoplasms. Received: 19 August 1998 / Accepted: 14 September 1998     

Pathologic analysis of primary brain tumors

Diffuse astrocytomas of the cerebrum, cerebellum, brain stem, and spinal cord are classified into three groups according to the degree of tumor anaplasia. These groups are the astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme. Juvenile pilocytic astrocytomas have a better prognosis and are clinically and biologically distinct from the diffuse, fibrillary astrocytomas. The prognosis of astrocytomas depends not only on histologic characteristics, but also age of the patient, location of the tumor, and extent of surgical resection. The pattern of invasion into surrounding brain distinguishes gliomas from metastatic carcinomas and sarcomas. Topographic correlations have shown that malignant gliomas may invade the brain for distances of up to several centimeters from the enhancing rim seen on CT scan. However, the junction between glioblastoma and adjacent brain may also be fairly abrupt, with a peripheral margin of less than 1 mm. Recurrent glioblastomas are more widely invasive and often extend into areas that appear normal on CT scan. The optimal site for tumor biopsy corresponds to areas of contrast enhancement. Primitive neuroepithelial tumors are malignant neoplasms with a poor prognosis. They tend to recur locally and metastasize throughout the neuraxis via the CSF. It remains controversial whether these tumors should be classified as a single entity with the potential for differentiation along different cell lines, or whether the categories of neuroblastoma, spongioblastoma, ependymoblastoma, pineoblastoma, and medulloblastoma should be retained as specific entities. The medulloblastoma is the most common of these neoplasms, its clinicopathologic features are well characterized, and the current 5-year survivals of 50 to 60 per cent are better than for other "primitive" neoplasms. Glial fibrillary acidic protein is a specific marker for immature, reactive, and neoplastic astrocytes and ependymal cells. Although the absence of GFAP in a neoplasm does not exclude an astrocytic origin, the presence of GFAP indicates astrocytic or ependymal differentiation. This has important diagnostic applications. The expression of GFAP is used to distinguish astrocytic neoplasms from epithelial or mesenchymal tumors that may on occasion mimic a glioma. The detection of GFAP is also useful in the investigation of tumor histogenesis and differentiation both in vivo and in vitro. Although meningiomas exhibit a wide variety of histologic patterns, most tumors exhibit similar biologic and clinical behavior regardless of the histologic subtype.(ABSTRACT TRUNCATED AT 400 WORDS)     

p53 protein expression in central nervous system tumors: an immunohistochemical study with CM1 polyvalent and DO-7 monoclonal antibodies

Summary Formalin-fixed, paraffin-embedded surgical specimens from 137 primary central nervous system tumors, including 26 astrocytomas (21 fibrillary, 1 protoplasmic, 1 gemistocytic and 3 pilocytic), 26 anaplastic astrocytomas, 9 glioblastomas, 1 gliosarcoma, 8 oligodendrogliomas, 4 ependymomas, 1 anaplastic ependymoma, 2 subependymomas, 3 paragangliomas, and 57 meningiomas, were immunostained with the CM1 polyclonal (pAb) and the DO-7 monoclonal (mAb) antibodies against the p53 protein, using the streptavidin/peroxidase method. In addition, two series of 17 and 9 medulloblastomas were also immunostained with the above pAb and mAb, respectively. p53 protein expression was observed in 7 fibrillary astrocytomas, 17 anaplastic astrocytomas, 5 glioblastomas, 1 gliosarcoma, 1 oligodendroglioma, 1 anaplastic ependymoma, and 4 meningiomas with the CM1 pAb. An additional 10 cases (i.e., 3 anaplastic astrocytomas and 7 meningiomas) were found to be p53 protein positive with the DO-7 mAb. Of the medulloblastomas, 8 (of the 17) and 4 (of the 9) were found to express p53 protein with CM1 pAb and DO-7 mAb, respectively. Our results indicate that p53 protein is expressed in a number of central nervous system neoplasms, and suggest that in astrocytic tumors a possible association may exist between p53 protein expression and tumor progression through increasing histological grades of malignancy.     

Immunohistochemical study of human brain tumors with vimentin and astroprotein (GFAP)

Distributions of two different subclasses of intermediate filaments, vimentin and glial filaments, were studied immunohistochemically in human brain tumors using specific antiserum to each protein subunit, vimentin and astroprotein (GFAP), Surgical specimens (5 meningiomas, 4 ependymomas, 5 benign astrocytomas, 5 anaplastic astrocytomas and 7 glioblastomas) were fixed in 95% ethanol or ethanol-acidic acid (95:5) and embedded in paraffin Avidin biotin peroxidase-complex (ABC) method (Vectastain) was carried out on 6 microns-thick paraffin sections. All meningioma cells were negative for astroprotein (GFAP) and positive for vimentin. Ependymoma cells showed various patterns of immunoreaction for astroprotein (GFAP) but were invariably positive for vimentin. In benign astrocytomas, many cells (or cell body and processes) were positive for astroprotein (GFAP). Immunoreaction for vimentin was, however, less frequent and intense. In anaplastic astrocytomas, population of astroprotein (GFAP)-positive cells decreased and vimentin-positive cells increased. Astroprotein (GFAP)-positive cells were further decreased in glioblastomas and the population of vimentin-positive cells varied among tissues. The present study suggests that the anaplastic change of astrocytoma cells were associated with decreased expression of glial filaments and increased expression of vimentin filaments. It was also suggests that the expression of both intermediate filaments may be suppressed in highly-malignant glial tumor cells.     

Expression of tenascin in human gliomas: its relation to histological malignancy,tumor dedifferentiation and angiogenesis

Summary The immunohistochemical distribution of tenascin (TN), fibronectin (FN), and laminin (LN) was investigated in 56 human gliomas (8 astrocytomas, 15 anaplastic astrocytomas, and 33 glioblastomas) with regards to the histological degree of malignancy and the degree of tumor cell differentiation evaluated by the staining of glial fibrillary acidic protein (GFAP). In 8 anaplastic astrocytomas and 28 glioblastomas, TN was predominantly immunolocalized in the basement membrane zone of the proliferating tumor vessels; sections of all astrocytomas were negative for TN staining. FN was localized in the basement membrane zone of the vessels in all astrocytomas, 12 anaplastic astrocytomas, and 22 glioblastomas. In 7 anaplastic astrocytomas and 19 glioblastomas, both TN and FN were expressed to various degrees in the tumor vessels. However, most of the TN-positive vessels did not express FN, and most of the FN-positive vessels were negative for TN staining. Furthermore, in 6 anaplastic astrocytomas and 12 glioblastomas, either TN of FN, but not both, were expressed in any area on serial sections. Most of the tumor cells around TN-positive, FN-negative tumor vessels did not express GFAP. On the other hand, GFAP was present in most tumor cells around TN-negative, FN-positive vessels. LN was detected in all vascular and pial-glial basement membrane zone of the tissues examined. These findings indicate that the degree of histological malignancy and the degree of cell dedifferentiation of human gliomas correlate well with the expression of TN, but are inversely correlated with the expression of FN. We postulate that the expression of TN, but not of FN, plays a role in the promotion of angiogenesis in malignant gliomas.     

Pituicytomas,a mis-diagnosed benign tumor of the neurohypophysis: report of three cases

Pituicytoma is a rare benign primary tumor of the neurohypophysis, occurring in the sellar and suprasellar spaces. We report here three new cases with immunohistochemical and electron microscopic study. Particular attention was paid to the expression of some cell adhesion molecules. These tumors were characterized by bundles of elongated cells strongly immunoreactive to anti-vimentin, S-100 protein, neural cell adhesion molecule and neuron-specific enolase antibodies. Glial fibrillary acidic protein (GFAP) was not recorded. It expressed the very late antigen alpha2 (VLAalpha2), but not VLAalpha5, and lacked epithelial markers expression (epithelial membrane antigen, E-cadherin), and specific neuronal markers (synaptophysin, chromogranin, neurofilament). Staining for pituitary hormones was negative. At the ultrastructural level, tumor/blood vessel basal lamina and cytoplasmic intermediate filaments were observed but desmosome or pericellular basal lamina were lacking. In one case few secretory granules were recorded. Differential diagnoses include granular cell tumors, pilocytic astrocytomas and spindle cell tumors such as solitary fibrous tumors, fibroblastic meningiomas and schwannomas. However, the unique pattern of antigenic expression and ultrastructural features of pituicytomas distinguish this rare tumor. As a subpopulation of pituicytes (which are distinctive glial cells of the neurophypophysis), some pituicytomas do not expressed GFAP. This suggests that pituicytomas presumably arise from pituicytes at various stages of their differentiation.     

Galectin‐3: A useful biomarker for differential diagnosis of brain tumors

Galectin‐3 (gal‐3) is a 31 kDa β‐galactoside‐binding lectin that is immunohistochemically expressed in macrophages, lymphocytes, and endothelial cells, and also in some neoplastic cells. Gal‐3's expression in and significance to brain tumors has not been fully addressed. Here, we investigated its immunohistochemical expression in 409 cases of surgically resected primary brain tumors, including various glioneuronal tumors, pituitary adenomas, meningiomas and Schwannomas, among others. In normal brain tissues, gal‐3 was robustly expressed in normal astrocytes, endothelial cells and macrophages. It showed consistent and diffuse positivity in 100% of the pilocytic astrocytomas, pleomorphic xanthoastrocytomas (PXA), Schwannomas, meningiomas, capillary hemangioblastomas, as well as in ependymomas, but it was completely negative in the diffuse astrocytomas, anaplastic astrocytomas, both low‐ and high‐grades of the oligodendrogliomas, central neurocytomas, and medulloblastomas. Definitely positive but heterogeneous expression was found in various tumors including subependymal giant cell astrocytomas (SEGA), classic glioblastoma multiforme, anaplastic oligoastrocytomas, CNS primitive neuroectodermal tumors (CNS PNETs), and hemangiopericytomas. Eighty percent of small cell glioblstomas were completely negative, but 20% showed heterogeneous positivity for gal‐3. Focal positivity for gal‐3 was also found in dysembryoplastic neuroepithelial tumors (DNTs) and gangliogliomas, in which the positive cells were the astrocytic component. On the basis of our immunohistochemical data in conjunction with previous reports, we therefore conclude that gal‐3 is differentially expressed in various brain tumors, and thereby, is a helpful biomarker in making differential diagnoses, especially in cases where a morphological diagnosis is controversial.     

Human parathyroid hormone-related protein and human parathyroid hormone receptor type 1 are expressed in human medulloblastomas and regulate cell proliferation and apoptosis in medulloblastoma-derived cell lines

Human parathyroid hormone-related protein (hPTHrP), identified in patients with paraneoplastic hypercalcemia and expressed by different cell types during development and adult life, plays important roles in many human neoplasms. Immunohistochemical and RT-PCR analyses of hPTHrP and human parathyroid hormone receptor type 1 (PTHR-1) in primary medulloblastoma confirmed their expression in both classic and desmoplastic variants at RNA and protein levels. To evaluate the functional role of hPTHrP, DAOY and D283 medulloblastoma and U87MG glioma cells, expressing high levels of hPTHrP and PTHR-1, were treated with anti-sense oligonucleotides for hPTHrP. Anti-sense treatment produced in all cell lines a decrease of cell proliferation and clonogenic activity and an increase of apoptosis, while addition of exogenous hPTHrP (1-37) prevented these effects. Anti-sense induced the increase of Caspase-3, Fas (CD95) mRNAs and Bax/Bcl-2 mRNA ratio after 12 h of cell treatment. Exogenous hPTHrP (1-37) increased intracellular Ca(2+) concentration in DAOY cells as revealed by FURA. Anti-sense treated cells showed a significant decrease of steady-state levels of intracellular Ca(2+), which was reverted by addition of exogenous hPTHrP (1-37). This study indicates that hPTHrP and PTHR-1 are expressed in medulloblastoma and could promote tumor growth, protecting cells from apoptosis.     

Expression of neurofibromatosis 2 protein in human brain tumors: an immunohistochemical study

The neurofibromatosis 2 (NF2) gene-encoded protein, named merlin, may function as a molecular linkage connecting cytoskeleton and plasma membrane. Merlin is thought to play a crucial role as a tumor suppressor not only in hereditary NF2-related tumors, but also in sporadic tumors such as schwannomas, meningiomas and gliomas. Using a merlin-expression vector system, we raised specific antiserum against merlin. We observed the intracellular distribution of merlin in cultured glioma cells, and further investigated merlin expression in 116 human brain tumors. Immunofluorescence microscopy revealed that merlin was localized beneath the cell membrane and concentrated at cell-to-cell adhesion sites, where actin filaments are densely associated with plasma membrane. By immunohistochemistry, none of the schwannomas from either NF2 patients or sporadic cases showed any immunoreactivity, while normal Schwann cells of cranial nerves were immunopositive. In meningiomas, merlin expression was frequently seen in the meningothelial subtype (8/10, 80%), but no expression could be detected in either the fibrous or the transitional variant. Most normal astrocytes were negative; however, reactive astrocytes often expressed merlin. Glioblastomas and anaplastic astrocytomas were found to be strongly positive, and focal positive staining was observed in fibrillary and pilocytic astrocytomas. Thus, the loss of merlin appears to be integral to schwannoma formation and the differential pathogenesis of meningioma subtypes. However, merlin alterations do not appear to play a critical role in either the tumorigenesis or malignant transformation of neoplastic astrocytes. Received: 8 July 1996 / Revised, accepted: 23 September 1996     

Differential expression of cell adhesion molecules (CAM), neural CAM and epithelial cadherin in ependymomas and choroid plexus tumors

A series of frozen specimens of 18 ependymomas and 7 choroid plexus tumors were examined for their expression of cell adhesion molecules, such as neural cell adhesion molecule (NCAM), its polysialylated isoforms (PSA NCAM), and epithelial (E-) cadherin, and of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP) and cytokeratin, using various monoclonal and polyclonal antibodies. Normal choroid plexus and ependyma were taken as controls. Anti-E-cadherin immunoreactivity was observed on the basolateral part of most adult choroid plexus and benign choroid plexus papilloma cells. However, a small number of atypical papillomas and carcinoma cells showed anti- E-cadherin immunoreactivity throughout their cell surface membrane. NCAM were not expressed by adult choroid plexus and benign papilloma cells. Only a few cells expressed NCAM and PSA NCAM in developing choroid plexus, atypical papillomas and carcinomas. Cytokeratin expression was always observed in choroid plexus and their tumors; GFAP expression was variable from case to case. In contrast, ependymal cells and their tumors never expressed E-cadherin but strongly expressed NCAM. PSA NCAM was found in ependymomas exhibiting anaplastic features. All ependymomas strongly expressed GFAP and a few demonstrated slight expression of cytokeratin. These data suggest that, besides GFAP and cytokeratin, NCAM and E-cadherin are of potential diagnostic value in distinguishing choroid plexus tumors from ependymomas. E-cadherin and NCAM may play a role in the functional organization of normal choroid plexus and ependyma, respectively. In particular, incomplete or irregular anti-E-cadherin expression in choroid plexus tumors and PSA NCAM immunoreativity in ependymomas and choroid plexus tumors correlates with the emergence of anaplastic histological features.     

siRNA沉默CD99基因表达对髓母细胞瘤DAOY细胞增殖和凋亡的影响

目的 探讨siRNA沉默CD99基因表达对髓母细胞瘤细胞系DAOY细胞增殖和凋亡的影响。方法 利用CD99 siRNA转染髓母细胞瘤DAOY细胞,细胞分为沉默1组（转染CD99 siRNA-1#）、沉默2组（转染CD99 siRNA-2#）、对照组（转染siRNA阴性对照）,采用实时荧光定量PCR和免疫印迹法检测沉默效果。利用CCK-8细胞增殖实验、平板克隆形成实验检测细胞增殖能力,采用流式细胞术检测细胞周期和细胞凋亡。结果 沉默1组和沉默2组CD99蛋白和mRNA表达水平均明显低于对照组（P<0.05）,而沉默2组又明显低于沉默1组（P<0.01）;因此,选用CD99 siRNA-2#进行后续实验。沉默CD99基因表达后3、4、5 d,沉默2组细胞增殖水平较对照组明显降低（P<0.05）;平板克隆形成实验结果显示,沉默2组细胞克隆集落数明显减少（P<0.05）。沉默2组G0/G1期细胞百分比较对照组明显增加（P<0.05）,S期细胞、G2/M期细胞百分比明显低于对照组（P<0.05）。沉默2组细胞早期凋亡率（8.75%±1.47%）显著高于对照组（3.42%±1.21%;P<0.05）。结论 CD99基因在髓母细胞瘤DAOY细胞的增殖和凋亡中发挥重要作用,推测CD99基因可能成为治疗髓母细胞瘤的一个新靶点。     

Expression of prostaglandin H synthase-2 in human brain tumors

Numerous studies have demonstrated that prostaglandin H synthase-2 (PHS-2) is involved in gastrointestinal carcinogenesis, and that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PHS, can reduce the risk of colon cancer. In brain tumors, elevated prostaglandin production and its correlation to anaplastic grade of gliomas have been demonstrated. To determine whether the increased prostaglandin production is due to enhanced expression of PHS-2 and whether the up-regulation of PHS-2 has any correlation to histopathological findings in brain tumors, we evaluated the profile of PHS expression in several human glioma cell lines and surgical specimens from patients with various types of brain tumors. In glioma cell lines, five out of six cell lines showed constitutive expression of PHS-2, whereas PHS-1 was weakly expressed in all of them. All surgical specimens, except an ependymoma, which expressed both isozymes equally, expressed PHS-2 mRNA predominantly. Immunohistochemistry of various types of brain tumors, including six glioblastomas, nine astrocytomas, six meningiomas, five medulloblastomas, four craniopharyngiomas, three ependymomas, three neurinomas, two oligodendrogliomas, two malignant lymphomas, two dysembryoplastic neuroepitherial tumors and one metastatic brain tumor showed PHS-2 staining in most cases. In gliomas, astrocytomas (grade 2 and 3) were strongly stained, but the staining intensity of glioblastomas was relatively weak. Meningiomas and a metastatic brain tumor were also strongly stained. Our data thus suggest that most brain tumors express PHS-2, which may also play a role in tumorigenesis in the brain.     

Inhibition of enhancer of zest homologue 2 is a potential therapeutic target for high‐MYC medulloblastoma

MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3‐lysine 27‐tri‐methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high‐MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY‐MYC, UW228‐MYC, CB‐MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY‐MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.     

Immunohistochemical study of fibronectin in human glioma and meningioma

The presence of fibronectin (FN) and glial fibrillary acidic protein (GFAP) in two astrocytomas, 17 glioblastomas, and five meningiomas was studied by indirect immunoperoxidase staining of formalin-fixed and paraffin-embedded surgical specimens. Angiogenesis in tumor was scored by the microscopic angiogenesis grading system (MAGS), and plasma FN levels were measured by single radial immunodiffusion. In astrocytomas and glioblastomas, GFAP-positive tumor cells had no FN expression and FN was confined to proliferating vessel walls and the leptomeninges, showing a mutually exclusive FN and GFAP expression. GFAP-positive tumor cells were occasionally surrounded by a network of FN-positive matrix produced by cells derived from the leptomeninges or blood vessels. In meningiomas, FN expression was found in vessel walls and meningioma cells including whorl formations and psammoma bodies. In general, deep immunoperoxidase staining for FN was shown in the endothelial cells and the psammoma bodies. Plasma FN levels were correlated significantly not to the degree of leptomeningeal proliferation but to the MAGS scores in gliomas.     

A glial fibrillary acidic protein-expressing and tumorigenic cell line derived from an avian sarcoma virus-induced rat astrocytoma

A permanent cell line, S635c15, was derived from an anaplastic astrocytoma induced by the Schmidt-Ruppin strain of avian sarcoma virus (ASV) in a female F-344 rat. Persistent expression of the astrocytic differentiation protein, glial fibrillary acidic protein (GFAP), was detected both in cultured cells after 100 passages in vitro and in transplanted tumors. Subcutaneous and intracerebral transplantation of S635c15 cells in syngeneic rats resulted in a 100% tumor incidence and a reproducible mortality distribution. S635c15 cells formed discrete masses after subcutaneous injection but grew intracranially as infiltrative lesions. Tumor blood flow and blood-to-tissue transport studies yield comparable values to other rat glioma models; S635c15 intracranial tumors proved to be a homogeneous model with little variation within and between tumors with respect to morphology, GFAP expression, blood flow, and permeability. This cell line provides a GFAP-expressing brain tumor model that extends the use of autochthonous ASV-induced astrocytomas by allowing in vitro and in vivo studies. It may be useful for further studies in neurobiology and brain tumor biology, diagnosis, and therapy.     

Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression

Podoplanin (aggrus) is a mucin-like transmembrane sialoglycoprotein that is expressed on lymphatic endothelial cells. Podoplanin is putatively involved in cancer cell migration, invasion, metastasis, and malignant progression and may be involved in platelet aggregation. Previously, we showed upregulated expression of podoplanin in central nervous system (CNS) germinomas, but not in non-germinomatous germ cell tumors, except for parts of immature teratomas in limited numbers. However, little information exists about its role in CNS astrocytic tumors. In this study, 188 astrocytic tumors (30 diffuse astrocytomas, 43 anaplastic astrocytomas, and 115 glioblastomas) were investigated using immunohistochemistry with an anti-podoplanin antibody, YM-1. In 11 of 43 anaplastic astrocytomas (25.6%) and in 54 of 115 glioblastomas (47.0%), podoplanin was expressed on the surface of anaplastic astrocytoma cells and glioblastoma cells, especially around necrotic areas and proliferating endothelial cells. However, the surrounding brain parenchyma was not stained by YM-1. On the other hand, podoplanin expression was not observed in diffuse astrocytoma (0/30: 0%). Furthermore, we investigated the expression of podoplanin using quantitative real-time PCR and Western blot analysis in 54 frozen astrocytic tumors (6 diffuse astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas). Podoplanin mRNA and protein expression were markedly higher in glioblastomas than in anaplastic astrocytomas. These data suggest that podoplanin expression might be associated with malignancy of astrocytic tumors.     

Increased endothelial expression of transglutaminase in glioblastomas

D. A. Hilton, S. Love and R. Barber (1997) Neuropathology and Applied Neurobiology , 23, 507–511
Increased endothelial expression of transglutaminase in glioblastomas
Transglutaminases are a family of calcium-dependent enzymes that catalyse the formation of covalent cross-links between proteins. They have several diverse functions and are thought to be involved in cell differentiation, apoptosis and blood coagulation. We have investigated the expression of tissue transglutaminase in five fibrillary astrocytomas, five anaplastic astrocytomas and seven glioblastomas by immunohistochemistry. Strongly labelled tumour cells were seen in most of the fibrillary and anaplastic astrocytomas and all of the glioblastomas. Labelling was particularly prominent in the pseudopalisading tumour cells that surrounded foci of necrosis and apoptosis in glioblastomas. Most of the immunostained cells did not themselves show morphological features of apoptosis. In addition, apoptotic cells were demonstrated using in situ end-labelling and by in situ hybridization with digoxigenin-labelled poly(A) oligonucleotide probes. Apoptotic cells demonstrated by both of these methods were most numerous in anaplastic astrocytomas and glioblastomas. However, their distribution did not correlate with that of the tumour cells showing transglutaminase labelling. Strong transglutaminase labelling was also observed in the endothelial cells of vessels showing microvascular proliferation in all of the glioblastomas studied. In contrast, endothelial transglutaminase labelling was weak or absent in lower grade astrocytic tumours. Enhanced expression of transglutaminase by endothelial cells in glioblastomas may contribute to the high prevalence of vascular thrombosis and necrosis in these tumours.     

Immunohistochemical study of S-phase cells in human gliomas

Intermediate filament proteins are cytoskeletal components in most vertebrate eukaryotic cells and some of these proteins are recognized markers of cell differentiation. To investigate the expression of intermediate filament proteins of the S-phase cells in human glial tumors, we have examined fourteen patients with benign and malignant gliomas by immunohistochemical study using in vivo labeling with bromodeoxyuridine (BrdU). Five glioblastoma multiforme, five anaplastic astrocytoma, three fibrillary astrocytoma and one gemistocytic astrocytoma were studied. All patients were given intravenous infusion of BrdU (10 mg/kg) one hour before craniotomy for labeling the S-phase cells of the tumors. Surgical specimens were immersed in 70% ethanol, and embedded in paraffin. Four micron sections were immunostained with anti-BrdU monoclonal antibody (Mab) and anti-vimentin Mab by avidin-biotin complex (ABC) method, and anti-glial fibrillary acidic protein (GFAP) serum by peroxidase-antiperoxidase (PAP) method. All sections (except for case 4) were double-labeled with anti-BrdU Mab and anti-GFAP serum, or with anti-BrdU Mab and anti-vimentin Mab. The population of BrdU-labeled cells (i.e. S-phase cells), and double-labeled cells were analyzed. The proportions of BrdU-labeled cells ranged from 6.1% to 17.0% (average 11.1%) in glioblastoma multiforme, from 3.5% to 15.6% (average 8.8%) in anaplastic astrocytoma, and from 2.0% to 2.8% (average 2.5%) in fibrillary astrocytomas. One gemistocytic astrocytoma showed S-phase fraction of 1.7%. Two recurrent cases of anaplastic astrocytoma showed higher S-phase fractions than other non-recurrent cases of anaplastic astrocytoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Apoptosis in cerebral astrocytic tumours and its relationship to expression of the bcl-2 and p53 proteins

Apoptosis is an important determinant of tumour growth which can be regulated by the bcl-2 and p53 genes. This study examines the relationship between apoptosis, growth fraction (Ki-67 immunolabelling index), and accumulation of the bcl-2 and p53 proteins in a spectrum of cerebral astrocytic tumours (n=81). including fibrillary astrocytomas (n=16), anaplastic astrocytomas (n=19), and glioblastomas (n=46). Median apoptosis indices (AIs) increased across this spectrum of tumours, and a significant (P<0.0001) correlation was demonstrated between A1 and Ki-67 labelling index (LI). Immunolabelling with the bcl-2 antibody was found in 44% of fibrillary astrocytomas, 42% of anaplastic astrocytomas, and 28% of glioblastomas. It was also found in the vascular endothelial proliferation typically seen in glioblastomas, and in the giant, multinucleated cells of some glioblastomas. No clear relationship between AI and bcl- 2 accumulation was evident. Immunolabelling with the p53 antibody was found in 56% of fibrillary astrocytomas, 79% of anaplastic astrocytomas, and 50% of glioblastomas. No clear relationship between AI and patterns of p53 immunolabelling was evident. Equal proportions of p53-positive tumours were bcl- 2 positive and bcl- 2 negative, but a small proportion of p53-negative tumours was bcl- 2 positive. The correlation between A1 and Ki-67 LI is in line with findings in other malignant tumours. We suggest that the regulation of apoptosis in astrocytic tumours is too complex for a clear association between A1 and bcl- 2 and p53 protein expression to be demonstrated.