[Abstract unavailable

Glial cells play an important role in sequestering neuronally released glutamate via Naϩ -dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system x c Ϫ , an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system x c Ϫ for their glutathione synthesis. Inhibition of system x c Ϫ causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system x c Ϫ . One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system x c Ϫ may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

Prenatal hypoxia-ischemia to the developing brain has been strongly implicated in the subsequent development of the hypertonic motor deficits of cerebral palsy (CP) in premature and full-term infants who present with neonatal encephalopathy. Despite the enormous impact of CP, there is no animal model that reproduces the hypertonia and motor disturbances of this disorder. We report a rabbit model of in utero placental insufficiency, in which hypertonia is accompanied by marked abnormalities in motor control. Preterm fetuses (67-70% gestation) were subjected to sustained global hypoxia. The dams survived and gave spontaneous birth. At postnatal day 1, the pups that survived were subjected to a battery of neurobehavioral tests developed specifically for these animals, and the tests were videotaped and scored in a masked manner. Newborn pups of hypoxic groups displayed significant impairment in multiple tests of spontaneous locomotion, reflex motor activity, and the coordination of suck and swallow. Increased tone of the limbs at rest and with active flexion and extension were observed in the survivors of the preterm insult.Histopathological studies identified a distinct pattern of acute injury to subcortical motor pathways that involved the basal ganglia and thalamus. Persistent injury to the caudate putamen and thalamus at P1 was significantly correlated with hypertonic motor deficits in the hypoxic group. Antenatal hypoxia-ischemia at preterm gestation results in hypertonia and abnormalities in motor control. These findings provide a unique behavioral model to define mechanisms and sequelae of perinatal brain injury from antenatal hypoxia-ischemia.

Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The av#3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the av,63 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain. (J.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates with integrin receptors and recruits other molecules to the site of this interaction thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. Crk-associated substrate (CAS) family members appear to play a pivotal role in FAK regulation of cell migration. Cellular Src bound to FAK phosphorylates CAS proteins leading to the recruitment of a Crk family adaptor molecule and activation of a small GTPase and c-Jun N-terminal kinase (JNK) promoting membrane protrusion and cell migration. The relocalization of CAS and signaling through specific CAS family members appears to determine the outcome of this pathway. FAK also plays an important role in regulating cell cycle progression through transcriptional control of the cyclin D1 promoter by the Ets B and Kruppel-like factor 8 (KLF8) transcription factors. FAK regulation of cell cycle progression in tumor cells requires Erk activity, cyclin D1 transcription, and the cyclin-dependent kinase (cdk) inhibitor p27Kip1. The ability of FAK to integrate integrin and growth factor signals resulting in synergistic promotion of cell migration and proliferation, and its potential regulation by nuclear factor kappa B (NFkappaB) and p53 and a ubiquitously expressed inhibitory protein, suggest that it is remarkable in its capacity to integrate multiple extracellular and intracellular stimuli.

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors that are well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here we provide evidence that CSCs selectively utilize the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. We detected CD36 expression in GBM cells in addition to previously described cell types including endothelial cells, macrophages and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was co-expressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal and tumor initiation capacity. We confirmed that oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival and metabolic advantages.

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serumstarved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BBstimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.

The ongoing characterization of the genetic and epigenetic alterations in the gliomas has already improved the classification of these heterogeneous tumors and enabled the development of rodent models for analysis of the molecular pathways underlying their proliferative and invasive behavior. Effective application of the targeted therapies that are now in development will depend on pathologists' ability to provide accurate information regarding the genetic alterations and the expression of key receptors and ligands in the tumors. Here we review the mechanisms that have been implicated in the pathogenesis of the gliomas and provide examples of the cooperative nature of the pathways involved, which may influence the initial therapeutic response and the potential for development of resistance.