Lucy Paradiso

Glutamate is associated with a higher risk of seizures in patients with gliomas

Objective: To investigate the relationship of glutamate and glutamate transporter expression in human gliomas and surrounding peritumoral brain to the presence of tumor-associated seizures (TAS). Methods: We studied a retrospective (group 1: 190 patients) and then a prospective (group 2: 98 patients) cohort of patients who underwent a craniotomy for a supratentorial glioma. Tumor and peritumor tissue specimens were assayed for glutamate concentration and expression of glial glutamate transporters. Differences between the seizure (TAS) and seizure-free (non-TAS) groups were compared. Results: A total of 42% of patients had TAS, with 95% of seizures first occurring preoperatively. Clinical factors independently associated with risk of TAS were younger age, temporal lobe location, and tumors with oligodendroglial components. Molecular features in tumor specimens associated with TAS were higher glutamate concentrations, reduced EAAT2 expression, and increased system Xc− expression. In group 2, these results were also replicated in the peritumor tissue. Logistic regression analysis identified raised glutamate concentrations in tumor and peritumor tissue, increased expression of peritumor system Xc−, younger age, temporal lobe location, and tumors with oligodendroglial components as independently predictive of preoperative seizures. Conclusion: Relative increased glutamate concentration in gliomas, and altered glutamate transporter expression, are associated with the presence of TAS and may play a mechanistic role in their pathogenesis.

Overexpression of hyaluronan synthase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity.

OBJECTIVE The interactions of CD44 with hyaluronan are thought to be crucial for tumor cell attachment to the extracellular matrix, migration, and invasion. For migration to occur, however, the interactions between hyaluronan and cell surface receptors need to be transient. Hyaluronidases may facilitate the degradation of hyaluronan bound to the cell surface and thus reduce the interactions of the cells with the matrix, whereas the overproduction of hyaluronan in the absence of hyaluronidase activity may prevent cells from proliferating or invading normal surrounding tissue. METHODS We analyzed the effects in vitro and in vivo of hyaluronan synthase-2 (HAS2) overexpression on a murine glioma cell line that is deficient in hyaluronidase activity. In addition, we evaluated the expression levels of HAS and hyaluronidase genes in human glioma cell lines and in glioma specimens. RESULTS Increased hyaluronan synthesis had no effect on the in vitro proliferation of the cells but diminished their in vivo growth rate. Several human glioma cell lines were found to overexpress hyaluronan synthases, but they did so in conjunction with hyaluronidase Hyal2 and MGEA5 expression. Similarly, all glioblastomas multiforme expressed hyaluronidases MGEA5 and Hyal2. CONCLUSION The data suggest that an increased synthesis of hyaluronan by astrocytoma cells is only promoting tumor cell growth in vivo if the cells express hyaluronidases as well.

Formation of STAT5-containing DNA Binding Complexes in Response to Colony-stimulating Factor-1 and Platelet-derived Growth Factor

Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

ADAM22, Expressed in Normal Brain but not in High-Grade Gliomas, Inhibits Cellular Proliferation via the Disintegrin Domain

OBJECTIVE:To study the expression and function of the brain-specific proteinase deficient disintegrins, ADAM11 and ADAM22 (a disintegrin and metalloproteinase). METHODS:Specimens of low- and high-grade gliomas and normal brain were analyzed for ADAM11 and ADAM22 expression using Western blotting. The effects of overexpression of ADAM11 and ADAM22 in glioma cells on growth were analyzed using bromodeoxyuridine incorporation linked to immunocytochemistry. Similarly analyzed were the effects on cell proliferation of bacterially expressed glutathione S-transferase fusion proteins with the disintegrin domain of ADAM11 and ADAM22. RESULTS:ADAM22 is expressed in normal brain and some low-grade gliomas, but not in high-grade gliomas, whereas ADAM11 is expressed in all low- and high-grade gliomas. In vitro, ADAM22 inhibits cellular proliferation of glioma derived astrocytes. The growth inhibition appears to be mediated by interactions between the disintegrin domain of ADAM22 and specific integrins expressed on the cell surface. This growth inhibition can be avoided by over-expression of integrin linked kinase. CONCLUSION:ADAM22, a brain-specific cell surface protein, mediates growth inhibition using an integrin dependent pathway. It is expressed in normal brain but not in high-grade gliomas. A related protein, ADAM11, has only a minor effect on cell growth, and its expression is unchanged in low- and high-grade gliomas.

Regulation of glycogen synthase kinase-3 beta (GSK-3β) by the Akt pathway in gliomas

Gliomas are aggressive brain tumours that, despite advances in multimodal therapies, continue to portend a dismal prognosis. Glioblastoma multiforme (GBM) represents the most aggressive glioma and patients have a median survival of 14 months, even with the best available treatments. The phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3 beta (GSK-3β) and Wnt/β-catenin pathways are dysregulated in a number of cancers, and these two pathways share a common node protein, GSK-3β. This protein is responsible for the regulation/degradation of β-catenin, which reduces β-catenins translocation to the nucleus and influences the subsequent transcription of oncogenes. The non-specific small-molecule GSK-3β inhibitor, lithium chloride (LiCl), and the specific Akt inhibitor, AktX, were used to treat U87MG and U87MG.Δ2-7 human glioma cell lines. LiCl treatment significantly affected cell morphology of U87MG and U87MG.Δ2-7 cells, while also increasing levels of phospho-GSK-3β in a dose-dependent manner. Increased cell proliferation was observed at low-to-mid LiCl concentrations as determined by MTT cell growth assays. Treatment of U87MG and U87MG.Δ2-7 cells with AktX resulted in reduced levels of phospho-GSK-3β through its inhibition of Akt, in addition to decreased levels of phosphorylated (active) Akt in a dose-dependent fashion. We have shown in this study that GSK-3β regulation by phosphorylation is important for cell morphology and growth, and that LiCl enhances growth of U87MG and U87MG.Δ2-7 cells by inhibiting GSK-3β through its phosphorylation, whereas AktX reduces growth via activation of GSK-3β by inhibiting Akts kinase activity.

Efficient ADAM22 surface expression is mediated by phosphorylation-dependent interaction with 14-3-3 protein family members

ADAM22 is one of three catalytically inactive ADAM family members highly expressed in the brain. ADAM22 has numerous splice variants, all with considerable cytoplasmic tails of up to 148 amino acids. ADAM22 can act to inhibit cell proliferation, however, it has been suggested that it also acts as an adhesion protein. We identified three 14-3-3 protein members by a yeast two-hybrid screen and show by co-immunoprecipitation that the cytoplasmic domain of ADAM22 can interact with all six 14-3-3 proteins expressed in the brain. In addition, we show that 14-3-3 proteins interact preferentially with the serine phosphorylated precursor form of ADAM22. ADAM22 has two 14-3-3 protein binding consensus motifs; the first binding site, spanning residues 831-834, was shown to be the most crucial for 14-3-3 binding to occur. The interaction between ADAM22 and 14-3-3 proteins is dependent on phosphorylation of ADAM22, but not of 14-3-3 proteins. ADAM22 point mutants lacking functional 14-3-3 protein binding motifs could no longer accumulate efficiently at the cell surface. Deletion of both 14-3-3 binding sites and newly identified ER retention motifs restored localization of ADAM22 at the cell surface. These results reveal a role for 14-3-3 proteins in targeting ADAM22 to the membrane by masking ER retention signals.

Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Aberrant Activation of JAK/STAT Pathway Components in Response to G-CSF, Interferon-a/p and Interferon-y in NFS-60 Cells

AbstractThere is evidence that the cellular responses to cytokines, such as granulocyte colony stimulating factor (G-CSF) and interferons, depend on prior activation of components of the JAK/STAT signalling pathway. We report here that the myeloid cell line NFS-60 shows aberrant JAK/STAT signalling yet elicits expected biological responses to G-CSF and interferons-α/β and γ. Instead of increased phosphorylation of JAKl and JAK2 in response to G-CSF and interf eron-y, and JAK1 and JAk2 in response to interferon-α/β we observed only an increase of phosphorylation of Tyk2 in response to all of these cytokines in NFS-60 cells. The subset of STAT proteins being activated in response to these cytokines was unusual as well. G-CSF activated STAT3 and STAT5A, whereas interferons activated, in addition to STAT1 and STAT5 other, as yet unidentified, DNA binding proteins. However, NFS-60 cells show normal biological responses to these cytokines, such as proliferation in response to G-CSF, and reduction of proliferati...

Genetic absence epilepsy rats from Strasbourg have increased corticothalamic expression of stargazin

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.

Differential Ability of SOCS Proteins to Regulate IL-6 and CSF-1 Induced Macrophage Differentiation

M1/WT4 cells, derived from the murine myeloid leukemic M1 cells by over-expression of the receptor for CSF-1, were transfected with expression vectors encoding SOCS-1, SOCS-2, SOCS-3 or Cis-1. The differentiation response to CSF-1 and IL-6 was analyzed in the resulting cell lines. Myeloid differentiation in response to CSF-1 was not affected by any of the SOCS proteins, whereas the IL-6-mediated differentiation was inhibited by SOCS-1 and SOCS-3 and slightly delayed by SOCS-2 expression. In M1/WT4 cells IL-6 causes strong tyrosine phosphorylation of STAT3, whereas the response to CSF-1 is weaker. The expression of the SOCS proteins had no effect on CSF-1 mediated STAT3 tyrosine phosphorylation; however, SOCS-1 and SOCS-3 reduced the tyrosine phosphorylation of STAT3 in response to IL-6 but did not abolish it. It appears, therefore, that SOCS-1, -2 and -3 and Cis-1 do not inhibit tyrosine kinase activity involved in CSF-1 mediated cell differentiation, whereas SOCS-1 and -3 are inhibiting kinase activity required for IL-6-mediated differentiation.