The Histone Deacetylase Inhibitor Sodium Butyrate Promotes Cell Death and Differentiation and Reduces Neurosphere Formation in Human Medulloblastoma Cells

Increasing evidence suggests that alterations in epigenetic mechanisms regulating chromatin state play a role in the pathogenesis of medulloblastoma (MB), the most common malignant brain tumor of childhood. Histone deacetylase (HDAC) inhibitors, which increase chromatin relaxation, have been shown to display anticancer activities. Here we show that the HDAC inhibitor sodium butyrate (NaB) markedly increases cell death and reduces colony formation in human MB cell lines. In addition, NaB increased the mRNA expression of Gria2, a neuronal differentiation marker, in D283 and DAOY cells and reduced the number of neurospheres in D283 cell cultures. Finally, NaB reduced the viability of D283 cells when combined with etoposide. These data show that NaB displays pronounced inhibitory effects on the survival of human MB cells and suggest that NaB might potentiate the effects of etoposide. In addition, our study suggests that HDAC inhibition might promote the neuronal differentiation of MB cells and provides the first evidence that an HDAC inhibitor might suppress the expansion or survival of MB cancer stem cells.     

Deranged hypothetical proteins Rik protein, Nit protein 2 and mitochondrial inner membrane protein, Mitofilin, in fetal Down syndrome brain.

Down syndrome (DS) is the most common genetic disorder with mental retardation and a host of deranged proteins has already been described. Protein hunting leads to rapid accumulation of aberrant proteins and proteomics methods not only allow unambiguous identification of proteins, they are also a powerful tools to identify new or predicted proteins. We applied two-dimensional gel electrophoresis with in-gel digestion of proteins and subsequent MALDI-TOF mass-spectrometrical identification and quantification of spots using specific software on cortical brain samples from 7 controls and 7 samples from fetal DS at the early second trimester. Nine hypothetical proteins were identified: three of them (4833418L03Rik protein Q9D614, mitochondrial inner membrane protein Q16891 and Nit protein 2 Q8WUF0) were significantly and about doublefold reduced in fetal DS brain. Hypothetical proteins CGI 99, FLJ10463, 70 kDa WD-repeat tumor rejection antigen homolog, KSRP, Hypothetical protein 49.6 kDa and Elongin A were comparable between groups. Domain analysis of deranged structures revealed a t_SNARE domain for the Rik protein, indicating involvement of this protein in the exocytotic-synaptic machinery impaired in DS, a CN hydrolase domain for Nit protein 2, possibly reflecting aberrant nitrilase-related metabolism and handling and an inner mitochondrial protein, extending knowledge on the mitochondrial deficit in in fetal DS early in life.     

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

Medulloblastoma cell invasion is inhibited by green tea (-)epigallocatechin-3-gallate

Epigallocatechin-3-gallate (EGCG), the major green tea polyphenol, can reach the brain following oral intake and could thus act as an anti-tumoral agent targeting several key steps of brain cancer cells invasive activity. Because integrin-mediated extracellular matrix recognition is crucial during the cell adhesion processes involved in carcinogenesis, we have investigated the effects of EGCG on different cellular integrins of the pediatric brain tumor-derived medulloblastoma cell line DAOY. Using flow cytometry, we report the levels of expression of several cell surface integrins in DAOY. These include high expression of alpha2, alpha3, and beta1 integrins, as well as alphav and beta3 integrins. Moreover, we provide evidence that EGCG can antagonize DAOY cell migration specifically on collagen by increasing cell adhesive ability through specific gene and protein upregulation of the beta1 integrin subunit. Our results suggest that this naturally occurring green tea polyphenol may thus be used as a nutraceutical therapeutic agent in targeting the invasive character of medulloblastomas.     

Assessing the intracellular concentration of platinum in medulloblastoma cell lines after Cisplatin incubation

Two different analytical approaches, external calibration and isotope dilution analysis both using flow-injection inductively coupled plasma mass spectrometry, have been developed and applied to determine the intracellular platinum concentration after Cisplatin incubation of two different medulloblastoma cell lines (UW228 and DAOY). As the internal or isotopically enriched standard was already used for cell lysis, maximum accuracy of the results was obtained, whereas a new home-built and inert injection system dramatically lowered carry-over effects and analyte loss. With limits of the detection well below 0.4 μg L⁻¹ and typical relative standard deviations of 2%, a strong correlation between the cell viability in MTT assays and the incorporated amount of Pt could be shown, which was subsequently normalized to the protein content of the samples. DAOY cells did significantly ingest more Pt and showed a higher mortality, which supports the fact that transporter expression needs to be taken into account in order to obtain meaningful results.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Synthesis, chaperoning, and metabolism of proteins are regulated by NT-3/TrkC signaling in the medulloblastoma cell line DAOY

The human medulloblastoma cell line DAOY was transfected with Tropomyosin receptor kinase (TrkC), a marker for good prognostic outcome. Following TrkC-activation by its ligand neurotrophin-3, protein extracts from DAOY cells were run on 2DE with subsequent MALDI-TOF-TOF analysis and quantification in order to detect downstream effectors. Protein levels of translational, splicing, processing, chaperone, protein handling, and metabolism machineries were shown to depend on neurotrophin-3-induced TrkC activation probably representing pharmacological targets.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

Inhibition of the MET Receptor Tyrosine Kinase as a Novel Therapeutic Strategy in Medulloblastoma

Medulloblastoma is the most common pediatric posterior fossa malignancy, with a 5-year overall survival of only 60% and many survivors experiencing treatment-related morbidity secondary to current therapeutic regimens. With an improved understanding of the molecular basis for this disease, the opportunity to develop novel treatments with more tolerable toxicity profiles that target key molecular pathways, now exists. Recently, the hepatocyte growth factor (HGF)/MET signaling pathway has been implicated in medulloblastoma pathogenesis. Several therapeutic strategies targeting this pathway exist, including small molecule inhibitor therapy against the MET receptor tyrosine kinase. We examined the in vitro efficacy of targeting the MET receptor using the highly specific small molecule inhibitor PHA665752 as a novel treatment strategy in medulloblastoma. MET inhibition using PHA665752 was effective at reducing the proliferative capacity of the D283, ONS76, and MED8A medulloblastoma cell lines as assessed by MTS assay. Furthermore, PHA665752 treatment reduced D283 and ONS76 cell motility and impaired the growth of D283 cells in soft agar. Pretreatment of D283, ONS76, and MED8A cells with PHA665752 blocked exogenous recombinant human HGF-induced up-regulation of the downstream RAS/mitogen-activated protein kinase signaling pathway in D283, ONS76 and MED8A cell lines. Similarly, PHA665752 prevented HGF-induced phosphatidylinositol 3-kinase/AKT signaling in ONS76 and MED8A cells. These results highlight the efficacy of targeting the MET receptor tyrosine kinase therapeutically in medulloblastoma and provide support for further preclinical testing of small molecule inhibitors targeting the MET receptor in medulloblastoma.     

Secreted meningeal chemokines,but not VEGFA,modulate the migratory properties of medulloblastoma cells

Leptomeningeal metastasis is a cause of morbidity and mortality in medulloblastoma, but the understanding of molecular mechanisms driving this process is nascent. In this study, we examined the secretory chemokine profile of medulloblastoma cells (DAOY) and a meningothelial cell line (BMEN1). Conditioned media (CM) of meningothelial cells increased adhesion, spreading and migration of medulloblastoma. VEGFA was identified at elevated levels in the CM from BMEN1 cells (as compared to DAOY CM); however, recombinant VEGFA alone was insufficient to enhance medulloblastoma cell migration. In addition, bevacizumab, the VEGFA scavenging monoclonal antibody, did not block the migratory phenotype induced by the CM. These results reveal that paracrine factors secreted by meningothelial cells can influence migration and adherence of medulloblastoma tumor cells, but VEGFA may not be a specific target for therapeutic intervention in this context.     

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.     

Identification of proteins encoded by Epstein-Barr virus trans-activator genes.

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.     

Expressional pattern of known and predicted signaling proteins in seven human cell lines

Although a variety of signaling systems and signaling proteins have been described, cell specific expression of these structures has not yet been systematically studied. Human amnion, bronchial epithelial, fibroblast, glial, kidney, lymphocyte and mesothelial cells were subjected to two-dimensional-gel electrophoresis followed by analysis of protein spots by MALDI-TOF and subsequent identification by specific software. A series of well-documented signaling proteins showed cell specific expressional patterns. Five hypothetical proteins--hypothetical 37.5 kDa protein, similar to calsyntenin 1, hypothetical armadillo repeat/plakoglobulin ARM-repeat profile containing protein, 11 days embryo cDNA clone 2700084k13, hypothetical protein flj22171--so far predicted from their nucleic acid sequence only, were identified, complementing already reported signaling cascades. An analytical tool for the concomitant determination of a large series of signaling structures by an antibody independent protein-chemical method is provided.     

The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.     

The nucleotide and deduced amino acid sequence of the cationic 19 kDa outer membrane protein OmpH of Yersinia pseudotuberculosis.

The OmpH proteins of enteric bacteria are recently described, small (16 kDa), cationic outer membrane proteins. Because a Yersinia pseudotuberculosis cell envelope protein of this size has been found to cross-react serologically with the human histocompatibility antigen HLA-B27 (B*2701), the sequence of Y. pseudotuberculosis OmpH was determined by sequencing the gene region which encodes mature OmpH. A protein consisting of 143 amino acid residues was found. It was 96% homologous with the OmpH of Y. enterocolitica and 62% homologous with that of Escherichia coli. Two separate OmpH regions had sequence similarity with B*2701; they were identical in both Yersinia species.     

Detection of hypothetical proteins in human fetal perireticular nucleus

There is a legion of hypothetical proteins (HP) in prokaryotic and eukaryotic proteomes and the aim of this study was to describe HP in the perireticular nucleus (PN), a key structure in human brain development. Tissue from four PNs was homogenized and extracted proteins were run on two-dimensional gel electrophoresis followed by in-gel digestion and mass spectrometrical identification of proteins. Several databases were used for obtaining bioinformatic information and searching for functional and structural domains. Five spots represented HP: KIAA0423 protein (Q9Y4F4), hypothetical protein KIAA0153 (Q14166), hypothetical protein DKFZp564A2416 (Q9NTW4), hypothetical protein DKFZp564H1122 (Q9H0W9), and hypothetical protein DKFZp564D1378 (Q9H0R4). These structures were predicted to serve in cell cycle, DNA-condensation, neurogenesis, or apoptosis. The existence of formerly HP proteins in the PN of human fetal brain is shown, thus extending knowledge of the brain proteome and proposing the method used as a suitable analytical tool for searching HP.     

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.