Follow-up study of a patient with early onset cerebral amyloid angiopathy following childhood cadaveric dural graft.

We retrospectively studied the T2 star (T2*)-weighted magnetic resonance imaging (MRI) of a 40-year-old patient diagnosed with symptomatic early-onset cerebral amyloid angiopathy (CAA), occurring 34 years following childhood neurosurgery using a cadaveric dural patch. Our findings revealed that CAA associated with cadaveric dural transplantation could progress rapidly, sometimes with bilateral bleeding. This microbleed evolution is suggestive of water-soluble amyloid-ß transmission via cerebrospinal fluid alongside perivascular drainage pathways with deposition in the cerebral artery walls due to clearance disturbances. Multiple intracerebral hemorrhages associated with CAA with a childhood cadaveric dural graft should be considered a life-threatening medical complication.

Prognostic significance of the immunohistochemical expression of O6-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas.

We studied the expression of O(6)-methylguanine-DNA methyltransferase (O(6)-MGMT), P-glycoprotein (Pgp), and multidrug resistance protein-1 (MRP-1) in 23 glioblastomas using RT-PCR, methylation-specific PCR, and immunohistochemistry, and analyzed their association with overall patient survival. Univariate analysis of collected data demonstrated that the expressions of O(6)-MGMT and MRP-1 detected by immunohistochemistry, in addition to the consistent factors, including preoperative Karnofsky performance scale (KPS), radical surgery, and tumor location and extension, were significant prognostic factors for the overall survival (OS) of patients with glioblastoma, who received nimustine (ACNU)-based chemotherapy in association with surgery and radiotherapy. Among them, following multivariate analysis, preoperative KPS, radical surgery, tumor location, and the expression of O(6)-MGMT remained as significant prognostic factors. These findings suggest that immunohistochemical analysis of O(6)-MGMT in patients with glioblastoma can be a useful method to predict the effects of chemotherapy and identify alternative chemotherapeutic regimens for O(6)-MGMT-positive patients.

Expression of vascular endothelial growth factor-A and mRNA stability factor HuR in human astrocytic tumors.

High-grade astrocytic tumors, such as glioblastoma, possess rich vascular components, which are necessary for their growth. VEGF-A is considered to be the major mediator of angiogenesis in malignant neoplasms including high-grade astrocytic tumors. The upregulation of VEGF-A expression in tumor cells is induced by two mechanisms: the transcriptional activation and the post-transcriptional stabilization of VEGF-A mRNA. While the former mechanism mediated by hypoxia inducible factor-1 alpha (HIF-1alpha) has been revealed, the latter mediated by mRNA stability factor HuR remains unclear in astrocytic tumors. In the present study, we investigated the expression of VEGF-A and mRNA stability factor HuR in supratentorial astrocytic tumors of 27 adults using RT-PCR, ELISA, and immunohistochemistry. Furthermore, we studied the involvement of HuR in the upregulation of VEGF-A expression using malignant astrocytoma cell lines. In higher-grade astrocytic tumors, the level of VEGF-A and microvascular density were elevated, cytoplasmic expression of HuR, which potentially means the protection of VEGF-A mRNA from degradation by ribonucleases, appeared, and they were correlated positively. In in vitro experiments, the inhibition of the cytoplasmic translocation of HuR protein by leptomycin B (LMB) reduced the upregulation of VEGF-A expression in malignant astrocytic tumor cells under hypoxic conditions. These findings suggest that the expression of VEGF-A and cytoplasmic translocation of HuR relates to the histological grade, and that HuR is involved in the upregulation of VEGF-A expression, in human astrocytic tumors.

Expression of vascular endothelial growth factor-A and mRNA stability factor HuR in human meningiomas.

We studied the expression of vascular endothelial growth factor-A (VEGF-A) and mRNA stability factor HuR in 40 supratentorial meningiomas using RT-PCR, ELISA and immunohistochemistry, and analyzed their associations with the clinicopathological characteristics, including microvascular density (MVD), peritumoral brain edema (PTBE), histological subtypes and grades, and the performance of preoperative arterial embolization. Furthermore, we investigated the involvement of HuR in the upregulation of VEGF-A expression using primary meningioma cell cultures. The level of VEGF-A is elevated in meningiomas with PTBE and in higher grade meningiomas. Preoperative arterial embolization did not significantly increase the level of VEGF-A, but it did increase the expression of HuR in tumor tissues. HuR expression was correlated positively with VEGF-A expression in meningioma tissues. In in vitro experiments, hypoxia induced the upregulation of VEGF-A expression and the cytoplasmic translocation of HuR protein in meningioma cells, and inhibition of the cytoplasmic translocation of HuR reduced the upregulation of VEGF-A expression in meningioma cells. These findings suggest that the expression of VEGF-A relates to the development of PTBE with meningiomas and the histological grade, and that HuR is involved in the upregulation of VEGF-A expression in human meningiomas.

The combined effects of multiple chemotherapeutic agents for malignant glioma cells.

The prognosis of malignant gliomas remains poor, despite the progress of surgery and radiotherapy. Chemotherapy has been shown to prolong an overall survival, but the benefits are still small. To overcome this situation, the optimal regimen of antineoplastic agents is required. In the present study, we investigated the effect of the association of five chemotherapeutic drugs, including ACNU, CBDCA, CDDP, VCR, and VP-16, on cell survival of U87, YKG1, A172, and U251 human glioma cell lines, using median-effect analysis. A synergistic effect was obtained by treatment involving the association of VP-16 with ACNU or CDDP among the combinations of two drugs, and the association of ACNU, CBDCA, and VP-16 in the combination of three drugs. This preclinical screening using median-effect analysis supports the design of clinical trials by indicating more effective combinations of antineoplastic agents for malignant gliomas.

Mitotic activity of multinucleated giant cells with glial fibrillary acidic protein immunoreactivity in glioblastomas: an immunohistochemical double labeling study.

To investigate the mitotic activity of multinucleated giant cells (MNGCs) with glial fibrillary acidic protein (GFAP) in glioblastomas, double immunohistochemical staining for GFAP and Ki67 was performed in formalin-fixed and paraffin-embedded specimens obtained from 12 primary glioblastomas with MNGCs including three giant cell glioblastomas. The Ki67 labeling index (LI:%) of GFAP+ tumor cells ranged from 0 to 5.6 (2.5+/-1.7, mean+/-standard deviation). The Ki67 LI of GFAP- tumor cells ranged from 18.6 to 35.9 (24.7+/-6.6). The Ki67 LI of GFAP+ cells was significantly lower than that of GFAP- cells (P<0.0001). The number of Ki67+ GFAP- MNGCs ranged from 0 to 23 (8.6+/-8.2). The number of Ki67- GFAP+ MNGCs ranged from 0 to 15 (6.2+/-5.1). There was no significant difference between them (P=0.42). The Ki67 LI of GFAP+ MNGCs was 10.4+/-12.6. The Ki67 LI of GFAP- MNGCs was 45.9+/-29.9. The Ki67 LI of GFAP+ MNGCs was significantly lower than that of GFAP- MNGCs (P<0.01). Our study suggested that MNGCs with mitotic capacity were not dominant and that GFAP+ MNGCs had little mitotic activity in glioblastomas. MNGCs identified in glioblastomas may develop via not only the proliferation of abnormal nuclei in a single tumor cell but also other processes.

Gene expression profiles of 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant C6 rat glioma cells.

Chemotherapy in itself is suspected to cause the development or selection of drug-resistant tumor cells, which have more aggressive phenotypes. The authors investigated the differential changes of gene expression in the 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant subline of the C6 rat glioma (C6AR2), which was established from C6 rat glioma cells by exposure to ACNU in vitro. The resistance to ACNU of C6AR2 was confirmed by MTS assay. The increased expression of O6-methylguanine-DNA methyltransferase in C6AR2 cells was shown using RT-PCR. C6AR2 cells displayed a higher proliferative activity relative to C6 cells. Analysis with cDNA array showed that 19 genes were transcriptionally up-regulated and 16 genes down-regulated in C6AR2 cells compared to C6 cells. They belonged to various functional classes of genes beside the drug-resistant system. Among them, the down-regulation of several genes in C6AR2 cells, including c-kit, pleiotrophin, platelet-derived growth factor receptor-alpha, peripheral myelin protein-22 and NG2 chondroitin sulfate proteoglycan, which are expressed originally in developmental glial lineages, were verified using semi-quantitative RT-PCR. In addition, the gene expression of astroglial intermediate filament proteins, including GFAP, vimentin and nestin, were decreased in C6AR2 cells relative to C6 cells in semi-quantitative RT-PCR and immunocytochemistry. These findings may represent an undifferentiated state of ACNU-resistant glioma cells and a more aggressive phenotype in recurrent tumors following chemotherapy.

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation.

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor structural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H-CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-alpha) expression in U251 glioma cells. H-CM activated nuclear factor-kappaB (NFkappaB) protein and increased the gene expression of antiapoptotic factors including Bcl-2, Bcl-X(L), survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H-CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4-[(4'-chloro-2'-fluoro) phenylamino]-6, 7-dimethoxyquinazoline)} or NFkappaB inhibitors (ALLN and BAY 11-7082). These VEGF inhibitors did not block the activation of NFkappaB induced by H-CM in endothelial cells. On the contrary, TNF-alpha antagonist WP9QY enhanced the survival activity of H-CM for endothelial cells and blocked NFkappaB activation induced by H-CM under serum-starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFkappaB in endothelial cells induced by TNF-alpha are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.