Glioblastoma Multiforme (GBM) is the most common and lethal of human primary central nervous system tumours, with a median survival of 14-16 months despite surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, is not fully explained on the grounds of an oncogenic stimulus. Interaction with the tumour microenvironment, as well as inherent characteristics of the tumour cell of origin are likely a source of this heterogeneity. In this thesis we describe the use of a novel mouse model which integrates Cre-Lox mediated and Tet-regulated gene expression. This system in combination with germline and somatic strategies has enabled us to interrogate how the state in glial development and the region in the brain where transformation occurs influence the process of gliomagenesis. The findings of this thesis suggest that the state of glial development at which a mutation is introduced is an important determinant of gliomagenesis. In support of this, we showed that early progenitors in the radial glial lineage are more susceptible to transformation than those, which have committed to a gliogenic lineage and are presumably further along in the process of differentiation. Highlighting the interplay between genetic alterations and the molecular changes that accompany the process of differentiation. Despite findings that suggest that neurogenic regions of the adult brain are more susceptible to transformation, we show that this is not always the case and instead, transformation is dependent on an interaction between specific combinations of genetic mutations and susceptible cell types regardless of the region of origin. Results from this thesis highlight the need to view the tumourigenic process of gliomas in the context of normal brain development as the cell context of oncogene expression may determine the phenotype and biologic aggressiveness of the tumour. Thus, the results of genetic or epigenetic alterations leading to brain tumours may be quite different in different cells of the hierarchy, suggesting unique treatment targets and strategies depending on the cell of origin.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Ravi V. Bellamkonda; Committee Member: Ananth V. Annapragada; Committee Member: Andrew Lyon; Committee Member: Gang Bao; Committee Member: Niren Murthy. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (p. 172-190).

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciencias Biológicas. Programa de Pós-Graduação em Neurociências. / Made available in DSpace on 2013-07-16T01:33:41Z (GMT). No. of bitstreams: 1 223924.pdf: 1758222 bytes, checksum: ed30108c6dc3035c99570a4c4cf9dfb3 (MD5) / Efeito do Chumbo na modulação de proteínas da família das MAPKs, Akt, Hsp27 e Hsp70 em cultura de células de glioma C6 de rato, bem como análise comparativa da viabilidade de linhagem tumoral C6, U373 e cultura primária de astrócitos frente à exposição a este metal.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2017. / Made available in DSpace on 2017-09-05T04:12:11Z (GMT). No. of bitstreams: 1 348187.pdf: 10629585 bytes, checksum: 7a6d459b8d7a269bdaae02626adf406e (MD5) Previous issue date: 2017 / Gliomas são tumores altamente agressivos e malignos, de ocorrência no Sistema Nervoso Central. As baixas taxas de sobrevida são relacionadas à alta capacidade proliferativa, migratória e invasiva e consequente resistência a drogas. Dentre os fármacos mais utilizados está a Temozolomida (TMZ). A TMZ é um agente alquilante, eficaz na terapia de diversas neoplasias, entretanto mecanismos de resistência a esse composto acarretam em prognósticos ruins. Assim, o estudo de compostos citotóxicos é substancialmente importante para compreender os mecanismos da doença e o estudo de novas terapias. A Guanosina (GUO) e a Atorvastatina (ATOR) já demonstraram ter potencial modulatório sobre o sistema glutamatérgico. Sendo este um sistema importante no desenvolvimento dos gliomas, compostos que o modulem podem ter potencial citotóxico sobre as células de glioma. Dessa maneira, investigou-se: 1. O potencial citotóxico da ATOR em células de glioma A172 e o efeito desta em processos do desenvolvimento do glioma; 2. O potencial citotóxico da GUO em células de glioma A172, o efeito dela nos processos de desenvolvimento do glioma e mecanismos envolvidos nesse efeito. Os resultados revelam que a ATOR apresenta efeito citotóxico às células de glioma, ainda, ATOR não apresenta efeito citotóxico a astrócitos em concentração similar. ATOR reduz a migração e a proliferação celular do glioma. ATOR induz aumento nos níveis de apoptose e a co-incubação com TMZ acarreta em uma potenciação deste efeito. A ATOR aumenta a presença de organelas vesiculares ácidas, indicando aumento da autofagia. Além disso, a inibição de receptores de glutamato foi capaz de prevenir parcialmente o efeito citotóxico da ATOR, demonstrando uma possível interação desta estatina com tais receptores na indução do efeito citotóxico. A GUO também demonstrou efeito citotóxico e redução da migração das células de glioma nos mesmos níveis que a TMZ. GUO reduziu a proliferação celular em células de glioma. GUO não alterou os níveis de apoptose, contudo a co-incubação com TMZ mostrou um efeito sinérgico. Esta sinergia foi também observada na diminuição do potencial de membrana mitocondrial e na indução de dano ao DNA. O aumento da liberação de glutamato observado por TMZ foi prevenido com a co-incubação com GUO. A inibição dos receptores de adenosina A1 ou A2A levou a um bloqueio parcial do efeito citotóxico da GUO. Assim sendo, esta tese contribuiu para demonstrar o efeito antitumoral da ATOR e da GUO, bem como elucidar alguns dos mecanismos de ação envolvidos neste efeito.<br> / Abstract : Gliomas are a group of tumors considered the most aggressive and malignant type, occurring in the Central Nervous System. Low patients overall survival rates are related to high proliferative, migratory and invasive behavior of these cells and drug resistance. Among the drugs most used to treat this disease is Temozolomide (TMZ). TMZ is an alkylant agent that demonstrated efficacy on several neoplasias treatment, however resistance mechanism to TMZ lead to bad prognoses. In this way, the study of compounds that show cytotoxic potencial is considerably relevant to understand the disease mechanisms and to enable the study of new therapies. Guanosine (GUO) and Atorvastatin (ATOR) have already been shown modulatory potential on glutamatergic system of neurotransmission. As this system is so important to glioma development, modulating drugs may show cytotoxic potential against glioma cells. Thus we investigated: 1. ATOR cytotoxic potential to human A172 glioma cells and its effects on development glioma processes. 2. GUO cytotoxic potential to human A172 glioma cells, it effects on development glioma processes and signaling pathways involved. ATOR showed cytotoxic effect to glioma cells in similar levels to TMZ. ATOR did not show cytotoxic effect to normal astrocytes. ATOR decreased migration and proliferation of glioma cells, similarly to TMZ levels. ATOR increases apoptosis and ATOR plus TMZ potentiates this effect. ATOR also increases acid vesicular organelles, indicating induction of autophagy. Furthermore, the ionotropic and metabotropic glutamate receptors inhibition partially prevents ATOR cytotoxic effect, suggesting a possible interaction between ATOR and glutamate receptors on ATOR cytotoxic effect. GUO also showed cytotoxic effects and reduces migration similar to TMZ levels to human A172 glioma cells. GUO decreases cell proliferation of glioma cells, and this effect is abolished when GUO and TMZ are co-incubated. GUO do not change apoptosis, nonetheless GUO+TMZ show synergistic effect in increasing apoptosis. The same effect was observed on decreasing mitochondrial membrane potential and DNA damage induction. GUO does not change glutamate release. TMZ increases glutamate release and this is prevented by GUO+TMZ co-incubation. A1 or A2A adenosine receptors inhibition partially blockade GUO cytotoxic effect. The A1R and A2AR blockade reduces cell viability and the A1R inhibition and A2AR activation do not change cell viability or GUO effect. Therefore, this study contributed to unravel the ATOR and GUO antitumoral effect as well as elucidate some of the mechanism involved in this effect.

Made available in DSpace on 2015-12-10T14:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-17. Added 1 bitstream(s) on 2015-12-10T14:29:14Z : No. of bitstreams: 1 000851655.pdf: 1706170 bytes, checksum: 990d3d441221c8d5284639c96fcf57c8 (MD5) / Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento... / Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation.

Isocitrate Dehydrogenase-1 has been found to be mutated in over 70% of lower grade gliomas and has become an important diagnostic tool for tumor prognosis, however its role in glioma development, and its impact on response to therapy, is still not fully understood. Unmutated IDH1 functions to convert isocitrate to alpha-ketoglutarate (a-KG) in the tricarboxylic acid (TCA) cycle. Mutated IDH1R132H changes the enzymatic equilibrium and converts a-KG to 2-hydroxyglutarate (2-HG), an oncometabolite. IDH1R132H mutated tumors show an elevated production of 2-HG and epigenetic alterations in DNA and histone methylation. This mutation is predominantly seen in the proneural glioma subtype in which oligodendrocytes progenitors (OPCs) are considered the cell of origin due to phenotypic similarities. The effect of IDH1R132H mutation in cellular transformation has not been fully established. Epigenetic modifications connect genotype to phenotype by genetic expression alterations and epigenetic modifications are necessary for OPC differentiation. Tri-methylation of lysine residue K27 on histone H3 (H3K27me3) is a repressive mark associated with cell pluripotency. H3K27me3 is trimethylated by Enhancer of Zeste 2 (EZH2) and demethylated by the a-KG-dependent demethylases UTX/KMD6A and JMJD3/KDM6B. 2-HG is a competitive inhibitor of a-KG-dependent demethylases, providing a mechanistic link between IDH mutations and increases H3K27me3 by inhibiting demethylation. In this thesis, we evaluated the epigenetic changes in mouse models of IDH mutant and wildtype glioma and genetically-transformed OPCs and tested the effects of drugs that target specific epigenetic marks. We developed a mouse model of glioma to compare IDH1R132H cells to wildtype glioma cells and found that although there was no difference in survival, IDH1R132H gliomas have increased levels of 2-HG by MALDI-IMS and metabolomic analysis. Interestingly, based on RNA-sequencing analysis our IDH1R132H model has a more OPC-restricted expression profile compared the wildtype glioma model which have higher enrichment of genes from other cell lineages, including neurons, astrocytes, myelinating oligodendrocytes and microglia. We used the EZH2 inhibitor (Tazemetostat, EPZ-6438) and found that this treatment was not cytotoxic or cytostatic to our cells although H3K27me3 was reduced. Interestingly, Tazemetostat treatment increased the expression of non-OPC genes (genes normally expressed by other lineages as assessed using the Barres transcriptomic database). To better understand how IDH1R132H influences OPC transformation, we transformed OPCs in vitro. OPCs were isolated from floxed p53 postnatal day 5 mice from and retrovirally infected with viruses to delete p53 alone or to also express IDH1R132H. OPCs that express IDH1R132H had increased levels of 2-HG by metabolomics and showed alteration in H2K27 methylation and acetylation that resembled those seen in glioma cells. Standard methods of western blot analysis consist of analyzing whole cell lysate, cytoplasmic and nuclear fractionation, or histone acid extraction. To analyze both the cytoplasmic fraction as well as histone modification, I developed a cellular extraction method in which cells were fractionated and the nuclear fraction was acid extracted. This method allows for the analysis of both cytoplasmic proteins as well as histone modifications by western blot. Using this method, we found that treating glioma cells or OPCs with synthetic cell permeable octyl-2HG, or expressing IDH1R132H, caused cells to have increased H3K27me3, while treatment with Tazemetostat caused a decrease in H3K27me3. Based on the RNA-sequencing data we found that increased H3K27me3 (ID1R132H mutation) express more OPC-like phenotype while reduced H3K27me3 (Tazemetostat treatment) induced an upregulation of genes associated with other lineages making them less restricted to the OPC transcriptional phenotype. We found that in both the glioma cells and OPCs, Tazemetostat treatment decreased H3K27me3 and increased H3K27ac. Based on the increase of H3K27ac after Tazemetostat treatment, we hypothesized that a Histone deacetylase inhibitor (HDACi) would be synergistic. We found that although the HDACi Panobinostat was less cytotoxic to IDH1R132H mutated glioma cells and OPCs, co-treatment with Tazemetostat is synergistic in mutant and wildtype models. We also saw that in IDH1R132H ex vivo slices, the co-treatment reduced tumor marker composition. These findings point to a novel therapeutic strategy for IDH1-mutated proneural gliomas that targets the specific epigenetic alteration in these tumors.

Células tumorais têm sua proliferação e mobilidade modificada por diversos fatores de crescimento, citocinas e mediadores inflamatórios, dentre os quais a amilóide sérica A (SAA). Estudos prévios do nosso grupo mostraram o efeito direto da SAA em processos de proliferação, migração e invasão de células de glioblastoma multiforme (GBM), A172 e T98G. Neste estudo nós complementamos resultados prévios de migração e invasão; avaliamos SAA como possível indutora de moléculas importantes para a invasividade do tumor, como as MMP-2 e -9 e ROS; realizamos ensaio clonogênico com a intenção de investigar uma possível contribuição da rSAA no estágio inicial de desenvolvimento do tumor; avaliamos o impacto da hipóxia na expressão dos diferente genes da SAA; estimulamos as células com indutores hepáticos clássicos da SAA e analisamos a possibilidade destes induzirem os diferentes genes da SAA em células tumorais; avaliamos possíveis receptores e vias de sinalizações envolvidas nos processos de proliferação, migração e invasão. Construímos knockdowns (KDs) dos genes da SAA de fase aguda (SAA1 e 2) e constitutiva (SAA4) e avaliamos a função de cada um deles para a morfologia e para os processos de proliferação, migração e invasão de GBM. Por fim investigamos SAA como possível biomarcadora de gliomas em amostras clínicas. Nossos resultados sugerem que rSAA afetou a atividade das MMP-2 e -9 e a produção de ROS em ambos GBM, mas não se mostrou clonogênica. As citocinas IL-6, TNF-&#945; e IL-1&#946;, mas não a hipóxia, foram capazes de induzir os diferentes genes da SAA. A adição de rSAA às culturas celulares estimulou a transrição dos diferentes genes da SAA, sugerindo a ativação de mecanismos intracelulares retroalimentados. Efeitos pró-tumorais da rSAA parecem ser viabilizados via RAGE, enquanto efeitos anti-tumorais parecem ser induzidos via TLR-4. Pela primeira vez mostramos que SAA induz aumento de RAGE. KDs da SAA inibiram proliferação, migração e invasão, sugerindo que SAA seja um produto tumoral importante para a manutenção do fenótipo invasivo de GBM. A adição de SAA exógena reverteu grande parte dos efeitos nas células T98G KD, enquanto células A172 KD responderam parcialmente à rSAA. KDs da SAA sugerem a mesma como mantenedora da morfologia das células de GBM. De maneira inédita mostramos que o gene SAA4 até então descrito como um gene constitutivo de função desconhecida é importante para a proliferação, migração e invasão de GBM. Nós especulamos que os efeitos diferenciados induzidos por rSAA nos GBM estejam associados à natureza multiligante da SAA e às diferenças genéticas dos GBM. Pacientes com GBM apresentaram aumento significativo na transcrição e expressão de SAA1 no tecido tumoral, bem como aumento sérico de SAA. A correlação na expressão de SAA1 com moléculas importantes para progressão tumoral, como CXCR4, CXCR7, CD163 e HIF-1&#945; também a identificam como uma proteína associada à malignidade. / Tumor cells have their proliferation and migration modified by several growth factors, cytokines and inflammatory mediators, such as serum amyloid A (SAA). Previous studies from our group showed the direct effect of SAA on proliferation, migration and invasion of glioblastoma multiforme (GBM) cells, A172 and T98G. In this study we complemented previous migration and invasion data; evaluated SAA as possible inducer of MMP-2, -9 and ROS; performed clonogenic assay to investigate a possible contribution of rSAA in the early stage of tumor development; evaluated the impact of hypoxia on the expression of different genes of SAA; stimulated the cells with classics inducers of hepatic SAA and analyzed the possibility of these different genes to induce SAA in tumor cells; evaluated possible receptors and signaling pathways involved in proliferation, migration and invasion processes. SAA knockdowns (KDs) were made for acute phase (SAA1 and 2) and constitutive protein (SAA4) and evaluated their role in cell proliferation, migration, morfology and invasion. Finally it was investigated SAA as a possible biomarker of glioma grade in clinical samples. Our results suggest that rSAA affects MMP-2 and -9 activity and ROS production in both GBM, but did not affect clonogenicity. IL-6, TNF-&#945; and IL-1&#946;, but not hypoxia, were able to induce SAA expression. rSAA addition to cell cultures stimulated transcription of the three different SAA genes, suggesting the activation of intracellular feedback mechanisms. Pro-tumor effects of rSAA seem to occur via RAGE and anti-tumor effects appear to be induced via TLR-4. This was de first time that induction of RAGE triggered by rSAA was shown. Proliferation, migration and invasion were inhibited in SAA KDs, suggesting that SAA is an important tumoral product for the maintenance of the invasive phenotype of GBM. The addition of exogenous SAA largely reversed the effects on SAA KDs T98G cells, whereas SAA KDs A172 cells partially responded to the rSAA. The findings with SAA KDs suggest that SAA affect cell morphology. Another new contribution from our study was that SAA4, a constitutive gene with unknown function, was important for the proliferation, migration and invasion of GBM and it can be induced by rSAA, IL-6, TNF-&#945; and IL-1&#946;. We speculate that the different effects induced by rSAA in GBM are associated with the affinity of SAA to different receptors and the different genetic backgrounds of GBM. Patients with GBM showed a significant increase in the transcription and expression of SAA1 in tumor tissue as well as increased serum SAA. The correlation between the expression of SAA1 with important molecules for tumor progression, such as CXCR4, CXCR7, CD163 and HIF-1&#945; also identified SAA as a protein associated with malignancy.

São poucos os avanços obtidos em intervenções cirúrgicas ou terapias para melhora na qualidade de vida e/ou prognósticos dos pacientes acometidos por gliomas, que são os tumores mais comuns e fatais que acometem o sistema nervoso central. O modelo celular ST1 de glioma de rato responde ao tratamento com hormônio glicocorticóide (GC) com uma reversão fenotípica tumoral-normal in vitro e in vivo e o modelo celular P7 é resistente a este tratamento. Ambas as linhagens são utilizadas, no laboratório, como modelos de estudo de genes associados à origem de neoplasias e controle de proliferação celular, com potenciais aplicações na doença humana. Este trabalho possui dois objetivos gerais: a) clonagem e caracterização funcional do gene NRP/B de rato, previamente descrito no laboratório como sendo induzido durante o tratamento das células ST1 com GC; b) identificação de genes humanos no locus 22q13, frequentemente deletado e associado à progressão tumoral de gliomas. A identificação, clonagem e determinação da seqüência completa de NRP/B de rato, até então desconhecida, foi realizada neste trabalho e depositada no GenBank (número de acesso AY669396). Sua expressão parece ser cérebro-específica, sendo modulada positivamente durante o tratamento com GC nas células ST1. A superexpressão de NRP/B em células ST1 foi realizada para avaliar o papel do gene na reversão fenotípica tumoral-normal induzida por GC. Os resultados obtidos sugerem que NRP/B provoca diminuição da capacidade de crescimento independente de ancoragem em meio semi-sólido e do potencial tumorigênico das células ST1 em ensaios de tumorigênese in vivo. Experimentos preliminares sugerem o envolvimento da expressão de NRP/B no processo de progressão celular e em tumores humanos cerebrais e de mama. Para a identificação de genes humanos no locus 22q13 associados ao desenvolvimento de gliomas, foram adotadas duas estratégias: utilização do Banco de Dados do Transcriptoma para identificação de mRNAs e ESTs alinhadas no locus associado ao câncer cerebral 22q13 e utilização dos dados de genes localizados no cromossomo 22 diferencialmente expressos em microarrays entre as linhagens ST1 e P7, ambas tratadas com GC. Como resultado da análise in silico, foram selecionados alguns genes para análise por Q-PCR em linhagens celulares e amostras clínicas de gliomas humanos, o que evidenciou a existência de algumas correlações positivas ao nível transcricional entre os genes. Três genes (KIAA1644, SULT4A1 e PARVG) apresentaram maior expressão em amostras clínicas de cérebro não-tumoral quando comparadas com amostras de gliomas, o que sugere um possível envolvimento destes na progressão de gliomas humanos. A caracterização dos alvos moleculares na ação dos genes analisados neste estudo é importante para melhor entendimento dos mecanismos moleculares que controlam a proliferação celular e, futuramente, para o desenvolvimento de novas estratégias terapêuticas para estes tumores cerebrais. / Gliomas are the most fatal central nervous system tumors and to date, satisfactory results from chemotherapies or surgical interventions are still not available. The ST1 rat glioma cell line is hyper-responsive to treatment with glucocorticoids (GC), undergoing a complete tumoral to normal phenotypic reversion, both in vitro and in vivo. The P7 glioma cell line is resistant to the action of GC. Both cell models are used in our lab to search for targets which are important to understand the origin of tumors and the control of cell proliferation. For the present study, we aimed at: a) cloning and functional characterization of the rat NRP/B gene, which had previously been identified as being induced during GC-treatment of the ST1 cells; b) identification of human genes located at the 22q13 locus, known to be involved in gliomagenesis. The previously unknown cDNA for rat NRP/B was identified, cloned, sequenced, and deposited in the GenBank (Acc. Nº AY669396). It displays brain-specific expression and positive modulation during GC treatment of ST1 cells. The role of NRP/B in the tumoral to normal phenotypic reversion induced by GC was analyzed by its overexpression in ST1 cells. The results suggest that induction of NRP/B partially impairs cell growth in semi-solid substrate and lowers its tumorigenic potential in vivo. Preliminary studies suggest that NRP/B expression is involved in cell cycle progression and in brain and human breast tumors. To identify additional human genes located in the glioma-related 22q13 locus, two strategies were used: a) analysis of the Ludwig - Fapesp Transcriptome Project database to identify mRNAs and ESTs that align to the referred locus and b) analysis of microarray data of differentially expressed genes in ST1 and P7 cells (both treated with GC) that also localize in chromosome 22. To validate the in silico analysis, Q-PCR was used to evaluate the expression of several genes in human brain tumor cell lines and clinical samples, being possible to find some positive correlations between genes at the transcriptional level. Three genes (KIAA1644, SULT4A1 e PARVG) displayed significantly higher expression levels in non-tumoral clinical samples, when compared to their tumor counterparts suggesting their involvement in human glioma progression. A better understanding of the molecular mechanisms by which these genes are involved may contribute with important information to understand the control of cell proliferation and may lead, in the future, to the development of new therapeutical strategies for brain cancer therapy and diagnosis.

São poucos os avanços obtidos em intervenções cirúrgicas ou terapias para melhora na qualidade de vida e/ou prognósticos dos pacientes acometidos por gliomas, que são os tumores mais comuns e fatais que acometem o sistema nervoso central. O modelo celular ST1 de glioma de rato responde ao tratamento com hormônio glicocorticóide (GC) com uma reversão fenotípica tumoral-normal in vitro e in vivo e o modelo celular P7 é resistente a este tratamento. Ambas as linhagens são utilizadas, no laboratório, como modelos de estudo de genes associados à origem de neoplasias e controle de proliferação celular, com potenciais aplicações na doença humana. Este trabalho possui dois objetivos gerais: a) clonagem e caracterização funcional do gene NRP/B de rato, previamente descrito no laboratório como sendo induzido durante o tratamento das células ST1 com GC; b) identificação de genes humanos no locus 22q13, frequentemente deletado e associado à progressão tumoral de gliomas. A identificação, clonagem e determinação da seqüência completa de NRP/B de rato, até então desconhecida, foi realizada neste trabalho e depositada no GenBank (número de acesso AY669396). Sua expressão parece ser cérebro-específica, sendo modulada positivamente durante o tratamento com GC nas células ST1. A superexpressão de NRP/B em células ST1 foi realizada para avaliar o papel do gene na reversão fenotípica tumoral-normal induzida por GC. Os resultados obtidos sugerem que NRP/B provoca diminuição da capacidade de crescimento independente de ancoragem em meio semi-sólido e do potencial tumorigênico das células ST1 em ensaios de tumorigênese in vivo. Experimentos preliminares sugerem o envolvimento da expressão de NRP/B no processo de progressão celular e em tumores humanos cerebrais e de mama. Para a identificação de genes humanos no locus 22q13 associados ao desenvolvimento de gliomas, foram adotadas duas estratégias: utilização do Banco de Dados do Transcriptoma para identificação de mRNAs e ESTs alinhadas no locus associado ao câncer cerebral 22q13 e utilização dos dados de genes localizados no cromossomo 22 diferencialmente expressos em microarrays entre as linhagens ST1 e P7, ambas tratadas com GC. Como resultado da análise in silico, foram selecionados alguns genes para análise por Q-PCR em linhagens celulares e amostras clínicas de gliomas humanos, o que evidenciou a existência de algumas correlações positivas ao nível transcricional entre os genes. Três genes (KIAA1644, SULT4A1 e PARVG) apresentaram maior expressão em amostras clínicas de cérebro não-tumoral quando comparadas com amostras de gliomas, o que sugere um possível envolvimento destes na progressão de gliomas humanos. A caracterização dos alvos moleculares na ação dos genes analisados neste estudo é importante para melhor entendimento dos mecanismos moleculares que controlam a proliferação celular e, futuramente, para o desenvolvimento de novas estratégias terapêuticas para estes tumores cerebrais. / Gliomas are the most fatal central nervous system tumors and to date, satisfactory results from chemotherapies or surgical interventions are still not available. The ST1 rat glioma cell line is hyper-responsive to treatment with glucocorticoids (GC), undergoing a complete tumoral to normal phenotypic reversion, both in vitro and in vivo. The P7 glioma cell line is resistant to the action of GC. Both cell models are used in our lab to search for targets which are important to understand the origin of tumors and the control of cell proliferation. For the present study, we aimed at: a) cloning and functional characterization of the rat NRP/B gene, which had previously been identified as being induced during GC-treatment of the ST1 cells; b) identification of human genes located at the 22q13 locus, known to be involved in gliomagenesis. The previously unknown cDNA for rat NRP/B was identified, cloned, sequenced, and deposited in the GenBank (Acc. Nº AY669396). It displays brain-specific expression and positive modulation during GC treatment of ST1 cells. The role of NRP/B in the tumoral to normal phenotypic reversion induced by GC was analyzed by its overexpression in ST1 cells. The results suggest that induction of NRP/B partially impairs cell growth in semi-solid substrate and lowers its tumorigenic potential in vivo. Preliminary studies suggest that NRP/B expression is involved in cell cycle progression and in brain and human breast tumors. To identify additional human genes located in the glioma-related 22q13 locus, two strategies were used: a) analysis of the Ludwig - Fapesp Transcriptome Project database to identify mRNAs and ESTs that align to the referred locus and b) analysis of microarray data of differentially expressed genes in ST1 and P7 cells (both treated with GC) that also localize in chromosome 22. To validate the in silico analysis, Q-PCR was used to evaluate the expression of several genes in human brain tumor cell lines and clinical samples, being possible to find some positive correlations between genes at the transcriptional level. Three genes (KIAA1644, SULT4A1 e PARVG) displayed significantly higher expression levels in non-tumoral clinical samples, when compared to their tumor counterparts suggesting their involvement in human glioma progression. A better understanding of the molecular mechanisms by which these genes are involved may contribute with important information to understand the control of cell proliferation and may lead, in the future, to the development of new therapeutical strategies for brain cancer therapy and diagnosis.