Le glioblastome est une tumeur de pronostic défavorable, qui dans plus de 75 % des cas récidive au sein même de l’isodose de prescription. Une des origines de cette radiorésistance réside en la présence d’un contingent cellulaire souche cancéreux. On estime que ces cellules pourraient tirer leur résistance d’une plus grande capacité à réparer les dommages à l’ADN induit par les thérapeutiques usuelles. L’objectif de cette étude était de lever la radiorésistance des cellules souches cancéreuses de glioblastome en évaluant l’association d’un faisceau d’hadrons, plus efficace et plus précis que la radiothérapie photonique, et différents inhibiteurs de poly (ADP-ribose) polymérase (PARP, protéine impliquée dans la réparation de l’ADN) et au témozolomide (alkylant). Cette combinaison est comparée au protocole de Stupp et al., usuel. Deux lignées souches de glioblastomes ont constitué le matériel cellulaire de cette étude. Trois inhibiteurs de PARP ont été étudiés : l’olaparib, l’AG14361, et le talazoparib. Les cellules, préalablement caractérisées, par analyse du cycle et d’un panel de 69 gènes impliqués dans la réparation de l’ADN, ont été irradiées, d’une part, classiquement par des photons, et d’autre part, avec des faisceaux d’ions carbone (INFN-LNS Catane, Ganil Caen). La viabilité, la capacité à former des sphères, reflet du potentiel souche de la cellule, et le cycle cellulaire ont été analysés pour chaque combinaison thérapeutique. Le traitement multimodal par association de témozolomide, d’inhibiteur de PARP et d’hadronthérapie permet de freiner la prolifération cellulaire. La proportion de cellules capables de former des sphères est effondrée lorsque les cellules ont reçu la combinaison thérapeutique par rapport au protocole de Stupp et al. Enfin, la distribution des cellules dans le cycle a été perturbée par l’association thérapeutique. L’association de faisceau de particules, d’inhibiteur de PARP et de témozolomide est une combinaison thérapeutique prometteuse. Les investigations doivent être poursuivies sur des modèles in vivo.

Objective: Intranasal route is an emerging option for brain cancer treatment to infuse directly telomerase inhibitors and/or viruses into the brain. Paradoxically, the standard chemotherapeutic Temozolomide (TMZ) widely used to treat glioma tumors is orally given. Here, we tested for the first time the intranasal administration of TMZ in nude mice xenograft models carrying human glioblastoma tumors generated from the human glioma stem-like cells TG16, TG1N and TG20. Methods: The resistance to TMZ of the different glioma stem-like cells was determined by WST-1 cell proliferation and cell viability assay. Tumour cells were stereotaxically injected intrastriatally and one month after graft, mice were anesthetized using Isofluorane and TMZ was infused into the nostrils three times a week during two weeks with a nano-injector using Hamilton syringe coupled to a cannula. Buried food pellet test was carried out to check the sense of smell. Animals were weighted and surveilled once a week and separated into two cohorts, one for histopathological analysis and the other for Kaplan-Meier survival analysis. Results: Intranasal administration of TMZ did not induce major adverse effects on the sense of smell of the animals. TMZ administred intranasally delayed tumour growth and significantly extended the lifespan of mice engrafted with TG16 and TG1N cells, which are sensitive in vitro to TMZ. By contrast, TMZ at the dose tested had no effects on the tumors generated by TG20 cells that are resistant to TMZ in vitro. Conclusion: Our results demonstrate that the intranasal route should be further considered as an option for TMZ delivery into the brain to treat intrastriatal brain tumours. Moreover, it consists of an easy, fast, and cost-less method to gain direct access to the brain.

Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.

Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFα), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFα-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFα proliferative effects to human astrocytes. After 3 days of permanent application, TGFα induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFα required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFα as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.

TheDrosophila(fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins fromDrosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 −/− mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.

Astrocyte gap junction communication (GJC) is thought to contribute to death signal propagation following central nervous system injury, noteworthy in some ischemia/anoxia models. The inhibition of p38/stress‐activated protein kinase 2 (p38/SAPK2) by a pyrimidyl imidazole derivative has been reported to reduce the extent of the lesion area after cerebral ischemia. Therefore, interleukin‐1β (IL‐1β), which contributes to stroke‐induced brain injury and activates p38/SAPK2, and hyperosmolarity induced by sorbitol, a potent stimulus of p38/SAPK2 in non‐neuronal cells, were used to investigate a possible involvement of p38/SAPK2 in GJC modulation in mouse cultured astrocytes. Both stimuli inhibited dye coupling within minutes. The IL‐1β effect was transient, while that of sorbitol lasted up to 90 min. Both stimuli induced a rapid p38/SAPK2 activation, the kinetic of which matched that of induction of dye coupling inhibition. Immunocytochemical studies showed that IL‐1β and sorbitol induced a p38/SAPK2 translocation from the nucleus to the cytoplasm. The pharmacological agent SB203580 specifically blocked p38/SAPK2 activation, cytoplasmic translocation and reversed the IL‐1β and sorbitol‐induced inhibition of GJC. Further characterization of the p38/SAPK2 mode of action on GJC, performed with sorbitol, revealed an increased phosphorylation of protein kinase C (PKC) substrates abolished by both PKC inhibitors and SB203580. Expression and serine phosphorylation of connexin 43, the main component of astrocyte gap junctions, were unchanged, suggesting the existence of additional intracellular signaling mechanisms modulating the channel gating. Altogether, these results demonstrate that p38/SAPK2 is a central mediator of IL‐1β and sorbitol inhibitory actions on GJC and establish PKC among the distal effectors of p38/SAPK2. © 2004 Wiley‐Liss, Inc.

PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes [Araujo et al., J Biol Chem 1993;268(8):5911–20], and subsequently shown to be widely expressed in different tissues and highly conserved among mammals [Estelles et al., J Biol Chem 1996;271(25):14800–6; Danziger et al., J Neurochem 1995;64(3):1016–25]. It is composed of a N-terminal death effector domain and a C-terminal tail of irregular structure. PEA-15 is regulated by multiple calcium-dependent phosphorylation pathways that account for its different forms: a non-phosphorylated form in equilibrium with a mono and a biphosphorylated variety. This already suggested that PEA-15 may play a major role in signal integration. Accordingly, it has been demonstrated to modulate signaling pathways that control apoptosis and cell proliferation. In particular, PEA-15 diverts astrocytes from TNFalpha-triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been modelized and recently determined using NMR spectroscopy, and may help to understand the various functions played by the protein through its molecular interactions.

PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes and subsequently shown to be widely expressed in different tissues and highly conserved among mammals. It is composed of an N-terminal death effector domain (DED) and a C-terminal tail of irregular structure. PEA-15 is regulated by multiple calcium-dependent phosphorylation pathways. PEA-15 is ideally positioned to play a major role in signal integration. Accordingly, it has been demonstrated that PEA-15 diverts astrocytes from TNFalpha-triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. Expression of PEA-15 directs TNFalpha outcomes toward survival, whereas its absence allows the development of the cytokine-induced cell death.