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Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/?-catenin signaling, and reduces the binding of ?-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. © 2013 Springer Basel.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients.Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis.REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed.These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD.

The discovery of somatic isocitrate dehydrogenase (IDH) mutations in gliomas is an example of the powerful impact of the next-generation sequencing on the comprehension of both tumor biology and human diseases. IDHs catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with production of NADH/NADPH. Thus, they are key enzymes in the Krebs cycle. For this family of metabolic genes, no previous role in human cancer has been described. However, in a recent genomewide study, recurrent somatic mutations in the IDH1 gene have been identified in patients affected by Glioblastoma Multiforme (GBM) [1]. In successive studies, IDH mutations have also been found in low-grade gliomas, as well as in acute myeloid leukemia. The aim of this chapter is to review the findings on the epidemiology and significance of IDH mutations in human gliomas, from the discovery to the current knowledge about their molecular pathogenesis. Special attention will be paid to the powerful diagnostic and prognostic relevance of the IDH mutations in the clinical practice of neuro-oncology.

The epithelial membrane protein 3 (EMP3) is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate theEMP3promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations ofEMP3hypermethylation with total 1p/19q codeletion,MGMTpromoter hypermethylation,IDH1/IDH2andTP53mutations, andEGFRamplification were studied, as well as its prognostic significance. TheEMP3promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with bothIDH1/IDH2mutations and total 1p/19q codeletion and inversely withEGFRgene amplification. No association was found withMGMThypermethylation andTP53mutations. In the whole series, theEMP3hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of theEMP3promoter hypermethylation was found in patients with oligodendroglial tumors.

Cancer stem cells (CSCs) in gliomas are associated with resistance to radio- and chemotherapy, based on O(6)-methylguanine-DNA methyltransferase (MGMT) hypermethylation and the Multidrug resistance (MDR) system activation.Samples from 21 glioblastomas (GBMs) were put in culture with growth factors or serum in order to obtain neurospheres or adherent cells. Both were genetically and immunohistochemically characterized for ATP-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1), ATP-binding cassette, sub-family C (CFTR/MRP), member 1 (ABCC1) and MGMT expression together with primary tumors.ABCB1 expression was positive in endothelial cells of primary tumors. ABCC1 expression was variably positive in tumor cells and positive in neurospheres, and less expressed in adherent cells. MGMT was methylated and unmethylated in primary tumors and in neurospheres, respectively, and unmethylated in adherent cells.Methylation is an epigenetic event affecting progenitors before the separation of the two glia lineages and maximally the future initiating cells. ABCB1 expression is limited to endothelial cells, whereas ABCC1 expression could mark a minority of tumor cells approaching a stem-like status.

Coeliac disease (CD) is a chronic inflammatory disorder where dietary gluten is not tolerated. In the lesion there are gluten reactive T cells predominantly secreting gamma-interferon. Both HLA and non-HLA genes contribute to CD susceptibility. Interleukin-12 (IL-12) regulates gamma-interferon production. The IL12B gene is located in a region (5q31.1-33.1) where there is evidence for linkage with CD. Allele 1 of an IL12B 3'UTR single-nucleotide polymorphism leads to increased expression of IL-12, and was recently implicated in susceptibility for type 1 diabetes (T1D). We found no evidence for association of allele 1 to CD by the transmission/disequilibrium test or case-control approach. No increased frequency was observed in patients belonging to families where the disease was linked to markers on chromosome 5q. Unlike T1D, allele 1 does not appear to confer susceptibility to CD.

Gliomas represent the classical intra-axial tumors of the brain and glioblastoma multiforme (GBM) is the most frequent and malignant glioma. It is an extremely aggressive tumor with a high invasive potential. After treatments, it invariably resumes proliferation and its recurrences occur most often within 2 cm from the resection margins (Hochberg & Pruitt, 1980; Wallner et al., 1989; Oppitz et al., 1999). The dispersion of glioma cells in the surrounding normal brain puts them out of reach of surgery, radioand chemotherapy, because outside of the limits of surgical resection and of the irradiated volume, established in order to avoid damages to the normal brain. The BAT, therefore, is the place where tumor cells migrate and invade and where a series of biological, pathological and molecular events occur as far as the interaction between host and tumor is concerned. Migrating cells from the tumor, reacting astroglia and microglial cells, elements of the immunological response or belonging to the monocytic phagocyting system reaching the tumor from the blood flow, and cells from gliogenetic zones of the brain make the BAT a melting pot of interactions among cells and factors. It has special importance also from the neuroimaging point of view for the recognition of the tumor limits and of peritumoral edema, which may have, at the same time, a prognostic significance (Ramakrishna et al., 2010). With MRI, beside the peripheral increase of T2 signal, uptake of gadolinium, hypodensities corresponding to vasogenic edema, necrotic cyst formation, other features can be shown by special technical procedures. The correlation, therefore, between neuroimaging and histopathology and molecular biology in the BAT is of the greatest interest.

MGMT (O6-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II–III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance.

The transcription factor SOX2 controls gene expression during development and has roles in both neurogenesis and gliogenesis.The current study utilized immunohistochemistry, immunofluorescence, molecular genetics (gene copy number analysis) and Western blotting to examine SOX2 expression in surgical samples from 133 brain gliomas of different grades of malignancy, and in cell lines from 16 glioblastomas.Our results reveal a positive correlation between SOX2 expression and malignancy grade in gliomas and identify the hypercellular and hyperproliferative areas of glioblastomas as the areas with the highest SOX2 expression. SOX2 gene amplification was found in neurospheres and 14.4% and 11.1% of glioblastomas and anaplastic oligodendrogliomas, respectively. In contrast, SOX2 expression was not observed in neuronal tumors and varied in medulloblastomas depending upon their neuronal differentiation status.It is concluded that SOX2 is a marker for undifferentiated and proliferating cells and that its expression is up-regulated in the most anaplastic areas of glioblastomas and oligodendrogliomas. The hypothesis is that antigenic and genetic analogy exist between these areas and neurospheres and that glioblastoma stem cells may actually be dedifferentiated tumor cells that re-acquire a stem cell-like status.

Formation of neurospheres (NS) in cultures of glioblastomas (GBMs), with self-renewal, clonogenic capacities, and tumorigenicity following transplantation into immunodeficient mice, may denounce the existence of brain tumor stem cells (BTSCs) in vivo. In sixteen cell lines from resected primary glioblastomas, NS showed the same genetic alterations as primary tumors and the expression of stemness antigens. Adherent cells (AC), after adding 10% of fetal bovine serum (FBS) to the culture, were genetically different from NS and prevailingly expressed differentiation antigens. NS developed from a highly malignant tumor phenotype with proliferation, circumscribed necrosis, and high vessel density. Beside originating from transformed neural stem cells (NSCs), BTSCs may be contained within or correspond to dedifferentiated cells after mutation accumulation, which reacquire the expression of stemness antigens.

This review begins with the description of the nervous cytogenesis, proliferation of primitive cells, migration and differentiation with antigen expression and regulation through factors. Emphasis is given to neural stem cells, progenitors and to radial glia as belonging to early stages of gliogenesis. Experimental production of brain tumors in the rat by transplacental administration of ethylnitrosourea and systemic administration of methylnitrosourea effective through neural stem cells and progenitors is briefly described as a preamble to the recent conception of gliomas as originating from stem cells or brain tumor stem cells (BTSCs). The nature and origin of BTSCs, their molecular regulation and their recognition in vivo are discussed. In the growth of tumors, the role of nestin, migration of neural stem cells toward the tumor from the subventricular zone or from the tumor to the brain and the migration from bone marrow of mesenchymal stem cells for angiogenesis of the tumors are considered. Special mention is made of the relationship between glio-vascular niches in the subventricular zone and the neo-niches around the tumors with their importance in the tumor growth as points of joint between cytogenesis and angiogenesis. Finally, besides the importance of hypoxia, cell traffic in the brain adjacent to tumor is emphasized in relation to the growth of gliomas.

The mammalian target of rapamycin (mTOR) controls cell growth through protein synthesis regulation. It can be activated by protein kinase B (AKT) or through ribosomal S6 kinase (S6K1). In malignant glioma, mTOR inhibitors have antiproliferative and proapoptotic effects and mTOR has been suggested as a target of therapies, thus it is worthwhile to verify its relations with the phosphatidylinositol-3-kinase (PI3)/AKT cascade, proliferation and apoptosis in human gliomas.In a series of 64 gliomas, including high- and low-grade tumors, AKT, mTOR, S6, caspase-3, poly(ADP-ribose) polymerase 1 (PARP1) and cleaved PARP1, Ki-67/MIB.1 and beclin 1 were studied by molecular biology techniques, quantitative immunohistochemistry and Western blotting.mTOR (phospho-mTOR), S6 (phospho-S6), AKT (phospho-AKT) levels and Ki-67/MIB.1 labelling index (LI) increased with increasing grade of malignancy. Epithelial growth factor receptor (EGFR) amplification correlated with EGFRwt and EGFRvIII immunohistochemistry, and with AKT expression; the latter correlated with mTOR expression, whereas S6 expression correlated with Ki-67/MIB.1 LI. Within the category of glioblastoma, S6 but not mTOR correlated with proliferation. mTOR did not show correlation with apoptosis, whereas it was inversely correlated with beclin 1, in line with the observation that autophagy is not activated in many malignancies.The relationship of S6 with the proliferation markers emphasizes the importance of the position of S6K1 downstream AKT in the PI3/AKT pathway.

Survivin is expressed in proliferating tissues and in tumors. It is a member of the inhibitory apoptosis protein (IAP) family known to regulate mitosis and to inhibit apoptosis. It has therefore been regarded as a target for therapies. In malignant gliomas it increases with malignancy, even though in glioblastomas it does not seem to correlate with outcome.Survivin was immunohistochemically studied in 39 selected viable glioblastoma areas belonging to 20 cases which were assayed for apoptosis, using a TUNEL assay, caspase-3, poly(ADP-ribose)polymerase 1 (PARP-1), Bid (BH3-interacting domain death agonist) and with the proliferation index Ki-67/MIB-1 and mitotic index (MI).A positive linear correlation was found between the survivin labelling index (LI) and the Ki-67/MIB-1 LI and MI. No inverse correlation was found with apoptosis.This double behavior can be attributed to mechanisms mediating survivin activity, either as a mitosis regulator and apoptosis inhibitor, and should be taken into account in therapeutic strategies using survivin.

The direct and indirect regulation, as well as the mechanisms of apoptosis in the nervous tissue and their implications in the treatment and prognosis of brain tumors are reviewed.

We have systematically screened the genome for evidence of linkage disequilibrium (LD) with multiple sclerosis (MS) by typing 6000 microsatellite markers in case-control and family based (AFBAC) cohorts from the Italian population. DNA pooling was used to reduce the genotyping effort involved. Four DNA pools were considered: cases (224 Italian MS patients), controls (231 healthy Italians), index (185 index cases from trio families) and parents (the 370 parents of the patient included in the Index pool), respectively. After refining analysis of the most promising 14 markers to emerge from this screening process, only marker D2S367 retained evidence for association. © 2003 Elsevier B.V. All rights reserved.

Marta Mellai,1 Angela Piazzi,1 Cristina Casalone,2 Silvia Grifoni,2 Antonio Melcarne,3 Laura Annovazzi,1 Paola Cassoni,4 Tetyana Denysenko,1 Maria Consuelo Valentini,5 Angelina Cistaro,6,7 Davide Schiffer1 1Neuro-Bio-Oncology Center, Policlinico di Monza Foundation/Consorzio di Neuroscienze, University of Pavia, Vercelli, Italy; 2Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy; 3Department of Neurosurgery, CTO Hospital/Città della Salute e della Scienza, Turin, Italy; 4Department of Medical Sciences, University of Turin, Turin, Italy; 5Department of Neuroradiology, CTO Hospital/Città della Salute e della Scienza, Turin, Italy; 6Positron Emission Tomography Center IRMET S.p.A, Euromedic Inc., Turin, Italy; 7Institute of Cognitive Sciences and Technologies, National Research Council, Rome, Italy Abstract: The diagnosis of astroblastoma is based on a typical histological aspect with perivascular distribution of cells sending cytoplasmic extensions to the vessels and vascular hyalinization. These criteria are useful for standardizing the identification of the tumor, but, in spite of this, there are discrepancies in the literature concerning the age distribution and the benign or malignant nature of the tumor. Three cases are discussed in this study: Case 1 was a typical high-grade astroblastoma; Case 2 was an oligodendroglioma at the first intervention and an oligoastrocytoma at the second intervention with typical perivascular arrangements in the astrocytic component; Case 3 was a gemistocytic glioma with malignant features and typical perivascular arrangements. Genetic analysis showed genetic alterations that are typical of gliomas of all malignancy grades. Using the neurosphere assay, neurospheres and adherent cells were found to have developed in Case 1, while adherent cells only developed in Case 2, in line with the stemness potential of the tumors. The cases are discussed in relation to their diagnostic assessment as astroblastoma, and it is hypothesized that the typical perivascular distribution of cells may not indicate a separate and unique tumor entity, but may be a peculiarity that can be acquired by astrocytic gliomas when an unknown cause from the tumor microenvironment influences the relationship between vessels and tumor cells. Keywords: gliomas, cell lines, histology, genetics

By testing DNA pools rather than single samples the number of tests for a case–control association study can be decreased to only two for each marker: one on the patient and one on the control pool. A fundamental requirement is that each pool represents the frequency of the markers in the corresponding population beyond the influence of experimental errors. Consequently the latter must be carefully determined. To this aim, we prepared pools of different size (49–402 individuals) with accurately quantified DNAs, estimated the allelic frequencies in the pools of two SNPs by primer extension genotyping followed by DHPLC analysis and compared them with the real frequencies determined in the single samples. Our data show that (1) the method is highly reproducible: the standard deviation of repeated determinations was ±0.014; (2) the experimental error (i.e., the discrepancy between the estimated and real frequencies) was ±0.013 (95% C.I.: 0.0098–0.0165). The magnitude of this error was not correlated to the pool size or to the type of SNP. The effect of the observed experimental error on the power of the association test was evaluated. We conclude that this method constitutes an efficient tool for high-throughput association screenings provided that the experimental error is low. We therefore recommend that before a pool is used for extensive association studies, its quality, i.e., the experimental error, is verified by determining the difference between estimated and real frequencies for at least one marker.