Uma variedade de agentes antitumorais é capaz de induzir danos no material genético e estimular respostas como o bloqueio do ciclo celular, reparo do DNA ou apoptose. A resposta inicial das células é o bloqueio no ciclo em uma tentativa de reparar o dano, no entanto, se esse dano for muito extenso ou comprometer o metabolismo celular, uma cascata de sinalização aciona mecanismos alternativos que inibem a proliferação das células e acionam vias de morte. Os astrocitomas malignos são os tumores cerebrais mais comuns que afetam o sistema nervoso central, compreendendo mais de 60% dos tumores cerebrais primários. O tratamento padrão é a radioterapia seguida de quimioterapia, no entanto, o prognóstico para pacientes portadores desse tipo de câncer ainda continua desanimador. A cisplatina é um agente genotóxico largamente empregado no tratamento de gliomas, além de outros tipos de câncer. Essa droga liga-se ao DNA, formando aductos, os quais levam a um bloqueio na duplicação e na transcrição, podendo induzir apoptose nas células dependendo da extensão do dano. No presente trabalho foram avaliadas as respostas celulares ao tratamento com a cisplatina em linhagem de glioma (U343 MG-a) e em fibroblastos normais transformados por SV40 (MRC-5). Foram avaliadas as respostas em termos de sobrevivência celular, indução de apoptose e expressão gênica em larga escala pela técnica de micro-arranjos de cDNA, sendo esta última realizada somente para a linhagem U343. A cisplatina causou uma redução acentuada na sobrevivência das células MRC-5 (~1%) e U343 (< 1%) após cinco dias de tratamento (teste de sobrevivência celular) com concentrações que variavam de 12,5 a 300 ?M. O tratamento por 24 h com iguais concentrações de cisplatina reduziu a sobrevivência das linhagens em cerca de 20-80% (teste de citotoxicidade). Ambas as linhagens sofreram apoptose após o tratamento com diversas concentrações de cisplatina (12,5, 25 e 50 ?M). A linhagem U343 apresentou uma freqüência máxima de apoptose de 20,4% após o tratamento com 25 ?M de cisplatina por 72 h, enquanto a linhagem MRC-5 apresentou 11,0% de apoptose após 50 ?M de cisplatina por 48 h. Os dados de expressão gênica analisados pelo método de micro-aranjos de cDNA, obtidos 48 h após o tratamento das células U343 com 25 ?M de cisplatina, mostraram genes significativamente (p ?0,05) reprimidos relacionados principalmente com alterações no citoesqueleto (TBCD, RHOA, LIMK2 e MARK1), apoptose ou sobrevivência celular (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 e NFKBIE), invasão celular ou metástase (LIMK2, TIMP2 e CALU), reparo de DNA (SMC1L1) e metabolismo celular (DYRK3, MARK1, TBCD, LIMK2, VDP e P4HB), entre outros processos. Esses dados apontam para um sério comprometimento da maquinaria celular como um todo após os danos induzidos pela cisplatina. Embora o mecanismo de apoptose justifique cerca de 20% da extensão de morte celular, conforme foi comprovado nos ensaios de apoptose (induzida por 25 ?M de cisplatina), a maior parte das células são eliminadas em conseqüência da ação da droga em vários níveis do metabolismo e manutenção da integridade celular, visto o elevado grau de citotoxicidade da cisplatina, demonstrado nos testes de sobrevivência. / A variety of antitumoral agents is capable of inducing DNA damage and eliciting cell cycle arrest, DNA repair or apoptotic responses. The initial response is a cell cycle arrest in an attempt to repair the DNA damage, but under conditions of extensive DNA lesions and high drug cytotoxicity, a signaling cascade triggers alternative mechanisms that inhibit cell proliferation and activate cell death pathways. Astrocytomas are the most common neoplasm of the central nervous system, comprising more than 60% of primary brain tumors. The standard treatment for theses tumors are radiotherapy followed by chemotherapy, however, the prognostic for these patients is still very discouraging. Cisplatin is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers, including gliomas. This drug binds to DNA, producing diverse types of adducts, which can block replication, transcription, and lead to apoptosis induction. In the present work, we analyzed cellular responses to treatments with the anticancer agent cisplatin in MRC-5 (normal human fibroblasts SV40 transformed) and U343 MG-a (glioma cell line). The responses were evaluated in terms of cell survival, apoptosis induction and profiles of gene expression by the cDNA microarrays method (only for U343 cell line). Cisplatin treatment resulted in a pronounced reduction in MRC-5 cell survival (~ 1%) and U343 (< 1%) after five days of treatment (cell survival test) with several concentrations of cisplatin, ranging from 12.5 to 300 ?M. Following 24h of treatment under similar cisplatin concentrations the survival was reduced at about 20-80% (cytotoxicity test). Both cell lines underwent apoptosis after treatment with different concentrations of cisplatin (12.5; 25 and 50 ?M), but U343 cells presented a maximal frequency of 20.4% apoptosis (25 ?M cisplatin treatment for 72h), while MRC-5 cells presented 11.0% (50 ?M cisplatin treatment for 48h). Analysis of gene expression performed for U343 cells treated with 25 ?M cisplatin for 48h showed several genes that were found significantly (p ? 0,05) down-regulated, most of them related with cytoskeleton alterations (TBCD, RHOA, LIMK2 and MARK1), apoptosis or cell survival (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 and NFKBIE), cell invasion or metastasis (LIMK2, TIMP2 and CALU), DNA repair (SMC1L1), and cell metabolism (DYRK3, MARK1, TBCD, LIMK2, VDP and P4HB), among others. As a whole, these data demonstrate a serious commitment of the cell machinery after cisplatin-induced cellular damage. About 20% of the cell death corresponds to apoptosis, as was showed by the present assays. However, most of the cells are eliminated by the action of the drug in various levels of the metabolism and maintenance of cell integrity, due to the elevated degree of cisplatin citotoxicity, as demonstrated in cell survival tests.

Uma variedade de agentes antitumorais é capaz de induzir danos no material genético e estimular respostas como o bloqueio do ciclo celular, reparo do DNA ou apoptose. A resposta inicial das células é o bloqueio no ciclo em uma tentativa de reparar o dano, no entanto, se esse dano for muito extenso ou comprometer o metabolismo celular, uma cascata de sinalização aciona mecanismos alternativos que inibem a proliferação das células e acionam vias de morte. Os astrocitomas malignos são os tumores cerebrais mais comuns que afetam o sistema nervoso central, compreendendo mais de 60% dos tumores cerebrais primários. O tratamento padrão é a radioterapia seguida de quimioterapia, no entanto, o prognóstico para pacientes portadores desse tipo de câncer ainda continua desanimador. A cisplatina é um agente genotóxico largamente empregado no tratamento de gliomas, além de outros tipos de câncer. Essa droga liga-se ao DNA, formando aductos, os quais levam a um bloqueio na duplicação e na transcrição, podendo induzir apoptose nas células dependendo da extensão do dano. No presente trabalho foram avaliadas as respostas celulares ao tratamento com a cisplatina em linhagem de glioma (U343 MG-a) e em fibroblastos normais transformados por SV40 (MRC-5). Foram avaliadas as respostas em termos de sobrevivência celular, indução de apoptose e expressão gênica em larga escala pela técnica de micro-arranjos de cDNA, sendo esta última realizada somente para a linhagem U343. A cisplatina causou uma redução acentuada na sobrevivência das células MRC-5 (~1%) e U343 (< 1%) após cinco dias de tratamento (teste de sobrevivência celular) com concentrações que variavam de 12,5 a 300 ?M. O tratamento por 24 h com iguais concentrações de cisplatina reduziu a sobrevivência das linhagens em cerca de 20-80% (teste de citotoxicidade). Ambas as linhagens sofreram apoptose após o tratamento com diversas concentrações de cisplatina (12,5, 25 e 50 ?M). A linhagem U343 apresentou uma freqüência máxima de apoptose de 20,4% após o tratamento com 25 ?M de cisplatina por 72 h, enquanto a linhagem MRC-5 apresentou 11,0% de apoptose após 50 ?M de cisplatina por 48 h. Os dados de expressão gênica analisados pelo método de micro-aranjos de cDNA, obtidos 48 h após o tratamento das células U343 com 25 ?M de cisplatina, mostraram genes significativamente (p ?0,05) reprimidos relacionados principalmente com alterações no citoesqueleto (TBCD, RHOA, LIMK2 e MARK1), apoptose ou sobrevivência celular (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 e NFKBIE), invasão celular ou metástase (LIMK2, TIMP2 e CALU), reparo de DNA (SMC1L1) e metabolismo celular (DYRK3, MARK1, TBCD, LIMK2, VDP e P4HB), entre outros processos. Esses dados apontam para um sério comprometimento da maquinaria celular como um todo após os danos induzidos pela cisplatina. Embora o mecanismo de apoptose justifique cerca de 20% da extensão de morte celular, conforme foi comprovado nos ensaios de apoptose (induzida por 25 ?M de cisplatina), a maior parte das células são eliminadas em conseqüência da ação da droga em vários níveis do metabolismo e manutenção da integridade celular, visto o elevado grau de citotoxicidade da cisplatina, demonstrado nos testes de sobrevivência. / A variety of antitumoral agents is capable of inducing DNA damage and eliciting cell cycle arrest, DNA repair or apoptotic responses. The initial response is a cell cycle arrest in an attempt to repair the DNA damage, but under conditions of extensive DNA lesions and high drug cytotoxicity, a signaling cascade triggers alternative mechanisms that inhibit cell proliferation and activate cell death pathways. Astrocytomas are the most common neoplasm of the central nervous system, comprising more than 60% of primary brain tumors. The standard treatment for theses tumors are radiotherapy followed by chemotherapy, however, the prognostic for these patients is still very discouraging. Cisplatin is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers, including gliomas. This drug binds to DNA, producing diverse types of adducts, which can block replication, transcription, and lead to apoptosis induction. In the present work, we analyzed cellular responses to treatments with the anticancer agent cisplatin in MRC-5 (normal human fibroblasts SV40 transformed) and U343 MG-a (glioma cell line). The responses were evaluated in terms of cell survival, apoptosis induction and profiles of gene expression by the cDNA microarrays method (only for U343 cell line). Cisplatin treatment resulted in a pronounced reduction in MRC-5 cell survival (~ 1%) and U343 (< 1%) after five days of treatment (cell survival test) with several concentrations of cisplatin, ranging from 12.5 to 300 ?M. Following 24h of treatment under similar cisplatin concentrations the survival was reduced at about 20-80% (cytotoxicity test). Both cell lines underwent apoptosis after treatment with different concentrations of cisplatin (12.5; 25 and 50 ?M), but U343 cells presented a maximal frequency of 20.4% apoptosis (25 ?M cisplatin treatment for 72h), while MRC-5 cells presented 11.0% (50 ?M cisplatin treatment for 48h). Analysis of gene expression performed for U343 cells treated with 25 ?M cisplatin for 48h showed several genes that were found significantly (p ? 0,05) down-regulated, most of them related with cytoskeleton alterations (TBCD, RHOA, LIMK2 and MARK1), apoptosis or cell survival (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 and NFKBIE), cell invasion or metastasis (LIMK2, TIMP2 and CALU), DNA repair (SMC1L1), and cell metabolism (DYRK3, MARK1, TBCD, LIMK2, VDP and P4HB), among others. As a whole, these data demonstrate a serious commitment of the cell machinery after cisplatin-induced cellular damage. About 20% of the cell death corresponds to apoptosis, as was showed by the present assays. However, most of the cells are eliminated by the action of the drug in various levels of the metabolism and maintenance of cell integrity, due to the elevated degree of cisplatin citotoxicity, as demonstrated in cell survival tests.

<p>Gliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in <italic>IDH1</italic> and <italic>IDH2</italic> as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study. </p><p><italic>IDH1/2</italic> mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to &#945;-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant <italic>IDH1</italic> or mutant <italic>IDH2</italic>. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant <italic>IDH1/2</italic> .</p><p>Having observed mutations in <italic>IDH1/2</italic> in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in <italic>IDH1/2</italic>, lending less credence to the first hypothesis. To elucidate whether mutations in <italic>IDH1/2</italic> can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant <italic>IDH2</italic> in different populations of cells in the brain, either alone or in combination with <italic>TP53</italic> deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant <italic>IDH2</italic> developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both <italic>IDH2</italic> mutation and <italic>TP53</italic> deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant <italic>IDH2</italic> and the cell-of-origin for progressive gliomas.</p> / Dissertation

Diffuse gliomas, the most common primary brain cancer, are classified into astrocytomas, oligodendrogliomas and oligoastrocytomas according to morphological similarity to glial cell, and categorized into grade II to IV according to histological features. While standard-of-care of surgery followed by chemo-radiotherapy exists for grade IV glioblastomas, lower grade gliomas (grades II and III) remain as a heterogeneous entity with variable treatment and outcome. The current WHO classification of lower grade glioma is purely based on histology and subject to interobserver variation, which can be aggravated by sampling error or small tissue biopsy. Objective prognostic biomarker guiding patient risk stratification is also relatively inadequate in lower grade gliomas. Since molecular markers will be supplemented into the future classification system of gliomas in order to facilitate clinico-pathological prediction and therapeutic planning in patient management and clinical trials, identification of clinically relevant biomarker is of paramount importance in neuro-oncology. In this study, we evaluated the clinical significance of several important molecular markers in a very large cohort of 453 lower grade gliomas (including 244 diffuse astrocytomas, 73 anaplastic astrocytomas, 31 oligodendrogliomas, 17 anaplastic oligodendrogliomas, 76 oligoastrocytomas and 12 anaplastic oligoastrocytomas) from Prince of Wales Hospital (Hong Kong) and Huashan Hospital (Shanghai). Mutational analysis was conducted in IDH, CIC, FUBP1 and TERT promoter by direct sequencing and 1p/19q codeletion and EGFR amplification were evaluated by fluorescent in-situ hybridization. Protein expressions of IDH1-R132H, CIC, FUBP1, EGFR, INA, p53 and PDGFRA were examined by immunohistochemistry. IDH mutation, detected in 69% of lower grade gliomas, represented a crucial marker in classifying the tumors into two groups with distinct prognosis, the favorable IDH mutated group and unfavorable IDH wild type group. 1p/19q codeletion, present in 20% of lower grade gliomas, identified tumors with oligodendroglial histology and favorable prognosis within the IDH mutated group. This IDH mutated-1p/19q codeleted genetic signature was frequently found in incidentally-discovered low grade gliomas, a clinical subgroup of tumors with excellent prognosis. CIC and FUBP1 mutations, detected in 47% and 16% of oligodendroglial tumors respectively, were oligodendroglial markers which conferred unfavorable prognosis to patients within IDH mutated-1p/19q codeleted oligodendroglial tumors. Among IDH mutated-1p/19q intact gliomas, p53 positive expressing tumors exhibited worse prognosis and PDGFRA positive expressing tumors showed better prognosis. TERT promoter mutation, identified in 28% of lower grade gliomas, exhibited as an oligodendroglial marker and favorable prognostic factor within the IDH mutated group and highlighted a subgroup of aggressive astrocytic tumors with short survival within the IDH wild type group. EGFR amplification, observed in 7% of lower grade gliomas, occurred exclusively in IDH wild type tumors, correlated with TERT promoter mutation and contributed a subset of tumors with fatal prognosis. Taken together, this study identifies objective and clinically relevant biomarkers which define lower grade gliomas into molecular subgroups with distinct clinico-pathological features. The biomarkers not only provide adjuncts to tumor classification beyond histology but potentially facilitate standardization of treatment strategy. With continuous efforts, I believe that this information will ultimately contribute to patient care in neuro-oncology in the era of personalized medicine. / 瀰漫性腦膠質瘤是最常見的原發性腦癌，可根據形態學分為星形細胞瘤、少枝膠質細胞瘤和少枝星形細胞瘤，並根據惡性特徵分為II至IV級。雖然膠質母細胞瘤(Ⅳ級)有標準的手術和術後化放療方案，低級別膠質瘤(Ⅱ和Ⅲ級)在治療方案和臨床結果上仍有很大的變異。目前世界衛生組織就膠質瘤的分類是純基於組織學，因此存在著觀察者間的變異，而抽樣誤差和活檢組織樣本太小亦加劇此問題。客觀和俱臨床價值的標誌物在低級別膠質瘤亦相對缺乏。為了改善腫瘤診斷和治療，未來膠質瘤分類將會加入分子標誌物，因此探討預後標誌物在神經腫瘤研究領域極為重要。我們從香港威爾斯親王醫院和上海華山醫院採集了453例低級別膠質瘤樣本，並對幾個重要的分子標誌物的臨床意義進行評估。我們以直接測序分析IDH，CIC，FUBP1和TERT啟動子突變，以熒光原位雜交技術檢測染色體1p/19q雜合性缺失和EGFR基因擴增，並以免疫組化檢測IDH1-R132H，CIC，FUBP1，EGFR，INA，p53和PDGFRA蛋白表達。IDH的突變率為69%，它把膠質瘤分成兩組: 預後好的IDH突變型組和預後差的IDH野生型組。1p/19q雜合性缺失的發生率為20%，主要是IDH突變型組內的少枝膠質細胞瘤，有較好的預後。IDH突變-1p/19q雜合性缺失這個基因特徵經常出現在偶發低級別膠質瘤，是一組預後良好的臨床亞組。CIC和FUBP1在少枝膠質腫瘤的突變率分別為47%和16%，在IDH突變-1p/19q雜合性缺失的少枝膠質腫瘤有不良的預後。在IDH突變-1p/19q完整的膠質瘤，p53陽性表達的腫瘤預後較差，PDGFRA陽性表達的腫瘤則預後較好。TERT啟動子的突變率為28%。在IDH突變型組，TERT啟動子突變表現為少枝膠質標誌物，出現在預後良好的腫瘤;在IDH野生型組，TERT啟動子突變則出現在一組生存期很短的星形細胞腫瘤亞組。EGFR基因擴增的發生率為7%，只出現在IDH野生型腫瘤，與TERT啟動子突變有相關性，並有致命性的預後。綜上所述，本研究確定了客觀的臨床標誌物，為膠質瘤分類提供組織學以外的參考，更為制定標準治療方案奠定基礎。在這個體化醫療時代，本人相信這些資訊最終將有助於神經腫瘤病人的治理。 / Chan, Ka Yin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 159-186). / Abstracts also in Chinese. / Title from PDF title page (viewed on 02, December, 2016). / Detailed summary in vernacular field only.

by Liu Heng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 115-138). / Acknowledgments --- p.I / List of Abbreviations --- p.II / Abstract --- p.V / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Cytokines in the Central Nervous System --- p.1 / Chapter 1.1.1 --- Basic Properties of Cytokines --- p.1 / Chapter 1.1.2 --- The General Characteristics of Glial Cells --- p.4 / Chapter 1.1.2.1 --- Astrocytes --- p.4 / Chapter 1.1.2.2 --- Oligodendrocytes --- p.6 / Chapter 1.1.2.3 --- Microglial --- p.7 / Chapter 1.1.3 --- The Effects of Cytokines on Neural Cells --- p.7 / Chapter 1.1.3.1 --- TNF-α and Neural Cells --- p.8 / Chapter 1.1.3.2 --- LIF and Neural Cells --- p.10 / Chapter 1.1.3.3 --- IL-1 and Neural Cells --- p.12 / Chapter 1.1.3.4 --- IL-6 and Neural Cells --- p.14 / Chapter 1.1.4 --- Immune Response in the Central Nervous System --- p.16 / Chapter 1.2 --- The C6 Glioma as a Model for the Study of Glial Cell Growth and Differentiation --- p.21 / Chapter 1.2.1 --- The Rat C6 Glioma Cells --- p.21 / Chapter 1.2.2 --- The Differentiation and Proliferation of C6 Glioma Cells --- p.23 / Chapter 1.3 --- Signal Transduction Pathways in Cytokine-stimulated Glial Cells --- p.28 / Chapter 1.3.1 --- Intracellular Signalling Pathways of Cytokines --- p.28 / Chapter 1.3.1.1 --- Protein Kinase C Pathway --- p.29 / Chapter 1.3.1.2 --- Tyrosine Kinase Pathway --- p.30 / Chapter 1.3.1.3 --- Cyclic Nucleotide Pathway --- p.32 / Chapter 1.3.1.4 --- Nitric Oxide Pathway --- p.33 / Chapter 1.3.2 --- Intracellular Signalling Pathways in Cytokine-stimulated C6 Glioma Cells --- p.34 / Chapter 1.4 --- The Aims of This Thesis Project --- p.37 / Chapter Chapter 2: --- Materials and Methods --- p.41 / Chapter 2.1 --- Rat C6 Glioma Cell Culture --- p.41 / Chapter 2.1.1 --- Preparation of Culture Media --- p.41 / Chapter 2.1.1.1 --- Complete Dulbecco's Modified Eagle Medium --- p.41 / Chapter 2.1.1.2 --- Complete Roswell Park Memorial Institute1640 Medium --- p.42 / Chapter 2.1.2 --- Maintenance of the C6 Cell Line --- p.42 / Chapter 2.1.3 --- Cell Preparation for Assays --- p.43 / Chapter 2.2 --- Determination of Cell Proliferation --- p.44 / Chapter 2.2.1 --- Determination of Cell Proliferation by [3H]-Thymidine Incorporation --- p.44 / Chapter 2.2.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.45 / Chapter 2.2.3 --- Data Analysis --- p.45 / Chapter 2.3 --- Effects of Cytokines and Lipopolysaccharide on C6 Cell Proliferation --- p.46 / Chapter 2.4 --- Effects of Protein Kinase C Activators and Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.47 / Chapter 2.5 --- Effects of cAMP or cGMP on Cytokine-induced C6 Cell Proliferation --- p.48 / Chapter 2.6 --- Effects of Tyrosine Kinase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.48 / Chapter 2.7 --- Effects of Calcium Ion on Cytokine-induced C6 Cell Proliferation --- p.49 / Chapter 2.8 --- Effects of Nitric Oxide on Cytokine-induced C6 Cell Proliferation --- p.49 / Chapter 2.8.1 --- Effects of Sodium Nitroprusside and Nitric Oxide Synthase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.49 / Chapter 2.8.2 --- Nitric Oxide Production Assay --- p.50 / Chapter 2.9 --- Effects of β-Adrenergic Receptor Agonist and Antagonist on Cytokine-induced C6 Cell Proliferation --- p.51 / Chapter 2.10 --- Morphological Studies on Cytokine-Treated C6 Glioma Cells --- p.51 / Chapter 2.10.1 --- Wright-Giesma Staining --- p.52 / Chapter 2.10.2 --- Glial Fibrillary Acidic Protein Staining --- p.52 / Chapter 2.10.3 --- Hematoxylin Staining --- p.53 / Chapter Chapter 3: --- Results --- p.55 / Chapter 3.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.55 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.56 / Chapter 3.1.2 --- The Time Course of Cytokine-induced C6 Cell Proliferation --- p.59 / Chapter 3.1.3 --- Effects of Lipopolysaccharide on C6 Cell Proliferation --- p.61 / Chapter 3.1.4 --- Effects of Cytokines on the Growth of C6 Cells --- p.64 / Chapter 3.2 --- Morphology and GFAP Expression in Cytokine-treated C6 Glioma Cells --- p.64 / Chapter 3.2.1 --- Effects of Cytokines on the Morphology of C6 Cells --- p.64 / Chapter 3.2.2 --- Effects of Cytokines on GFAP Expression in C6 Glioma Cells --- p.66 / Chapter 3.3 --- The Signalling Pathway of Cytokine-induced C6 Cell Proliferation --- p.69 / Chapter 3.3.1 --- The Involvement of Protein Kinase C in Cytokine-induced C6Cell Proliferation --- p.71 / Chapter 3.3.2 --- The Involvement of Tyrosine Kinase in the Cytokine- induced C6 Cell Proliferation --- p.81 / Chapter 3.3.3 --- The Involvement of Calcium Ions in Cytokine-induced C6 Cell Proliferation --- p.87 / Chapter 3.3.4 --- The Involvement of Cyclic Nucleotides in Cytokine- induced C6 Cell Proliferation --- p.92 / Chapter 3.3.5 --- The Involvement of Nitric Oxide in Cytokine-induced C6 Cell proliferation --- p.94 / Chapter 3.3.6 --- The Involvement of P-Adrenergic Receptor in Cytokine- induced C6 Cell Proliferation --- p.101 / Chapter Chapter 4: --- Discussion and Conclusions --- p.104 / References --- p.115

Orientador : Valeria Valente / Banca: Cleslei Fernando Zanelli / Banca: Ana Lúcia Fachin Saltoratto / Resumo: 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... / Abstract: 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. / Mestre

La memoria del trabajo describe en detalle la estrategia seguida para mejorar a nivel preclínico la detección temprana de respuesta a la terapia en modelos animales de cáncer cerebral, más concretamente de tipo glioma. Así, la evolución de un tumor de tipo glioblastoma (glioma astrocítico de grado IV) inducible mediante inyección estereotáctica intracerebral a ratones C57BL/6 de células de la línea GL261 de ratón, ha sido caracterizada de manera no invasiva (mediante imágenes por RM, IRM) en cuanto a su aumento de volumen durante el tiempo de vida de los animales. Además, las curvas de supervivencia y la supervivencia promedio de estos ratones en ausencia de terapia han demostrado ser reproducibles y han permitido establecer el grupo control con el cual comparar el efecto de diversos agentes terapéuticos en la evolución del tumor. De los agentes y protocolos terapéuticos investigados, el más efectivo resultó ser, como era de esperar por la experiencia clínica previa, un agente alquilante del DNA, la Temozolomida (TMZ). La TMZ, en un protocolo de tres ciclos de administración, aumentó significativamente la supervivencia de los animales tratados, produciendo además una ralentización del crecimiento de los tumores durante una semana aproximadamente. Una vez establecido el modelo de respuesta a terapia, se investigó si alguna estrategia de imagen molecular basada en RM podía detectar dicha respuesta transitoria de los tumores a la terapia. Las dos principales estrategias de perturbación del metaboloma del tejido cerebral investigadas fueron la retención diferencial de dimetilsulfóxido (DMSO) y la variación del patrón espectral global tanto en condiciones de euglicemia como en hiperglicemia transitoria. En ambos casos se adquirieron imágenes moleculares de los tumores cerebrales antes, durante la respuesta a la terapia y al manifestarse la recidiva tumoral mediante imagen espectroscópica de RM (IERM). Finalmente, la información de las imágenes moleculares basadas en la perturbación con DMSO, aplicada a las imágenes moleculares previas a la perturbación en condiciones de euglicemia, permitió desarrollar un clasificador matemático que genera imágenes nosológicas de zonas que muestran respuesta a la terapia de tumores GL261. Este clasificador fue validado con éxito en un conjunto de datos independiente (tumores no utilizados para desarrollar el clasificador). Además, la tesis ha permitido avanzar en paralelo con la caracterización mediante IRM/ERM/IERM de otros modelos preclínicos de glioma (ratones modificados genéticamente que desarrollan oligodedndrogliomas de manera espontánea, y glioblastomas humanos desarrollados en animales inmunodeprimidos, nude mice), para poder aplicar en un futuro estrategias de predicción y seguimiento de la respuesta a la terapia. / This work describes in detail the strategy followed for improving the early response to therapy detection in brain tumors at preclinical level. Animal models of brain tumors, specifically glioma models, have been used for this purpose. The evolution of a high grade glioma model (glioblastoma (GBM), IV grade), induced by estereotatic injection of GL261 cells into the striatum of C57BL/6 WT mice, has been characterized using a non-invasive technique, consisting on MR images (MRI). Tumor volume growth rate and animal survival curves have been proved to be reproducible and have been used for establishing a control group for comparing the effect of different therapeutic agents on tumor evolution. Among all the agents investigated, the most effective was Temozolomide (TMZ), an oral alkylating agent commonly used in clinical trials for GBM treatment. A three cycle protocol using TMZ improves the survival rate of treated animals in comparison with the control group. This protocol also affects tumor growth rate, slowing down tumor evolution for a seven days period. After developing the appropriate response to therapy model, different molecular imaging strategies based on MR were investigated to determine if any of them could detect the transitory tumor response to therapy. The main strategies studied were the differential retention of dimethyl sulfoxide (DMSO) and global changes detected in the spectral pattern of tumors in euglicemia and after a hyperglicemic challenge. In both cases, molecular images from the tumors were acquired before, during the response to therapy and at tumor recurrence, using MR spectroscopic imaging (MRSI). Finally, the information obtained from molecular images based on DMSO perturbation (perturbation enhanced MRSI) was applied in euglicemic conditions for developing a mathematical classification system which generates nosologic images for GL261 tumors that show response to TMZ treatment. This classification system was successfully validated with an independent test set of data (tumors that were not used previously for developing the system). In addition, this work has also advanced in the characterization by MRI/MRS/MRSI of other preclinical glioma models (genetically engineered mice which spontaneously develop oligodendrogliomas, and immunosuppressed mice which develop human GBM) for a possible application in the future in tumor response to therapy prediction and follow up strategies.

Cell cycle-related kinase (CCRK) is a 42 KDa serine/threonine protein kinase homologous to Cdk1, 2 and 7. Previous work has shown that CCRK regulates cell cycle transition by phosphorylating Cdk2 and Rb. More importantly, it was found that CCRK was a candidate oncogene in both glioblastoma multiform (GBM) and human colorectal cancer. However, the mechanistic role of CCRK in tumorigenicity is still not completely understood. In the first part of this thesis, I found that casein kinas II beta (CKIIβ) was one of proteins that interact with CCRK using the high-throughput yeast-two-hybrid analysis. Then I confirmed their interaction by co-immunoprecipitation. CCRK phosphorylated CKIIβ at Ser-209 in a cell cycle-dependent manner. The phosphorylation of CKIIβ by CCRK enhanced the activity of CKII holoenzyme, protected CKIIβ against proteasome degradation, and facilitated CKIIβ translocation into the nucleus in U-87 MG and U-373 MG GBM cells. Importantly, CCRK de-sensitized GBM cells to the cytotoxic effect of three chemotherapy drugs, whereas knockdown of CCRK by siRNA reduced chemoresistance. Functionally, CKIIβ is responsible for CCRK-mediated inhibition of apoptosis, as suppression of CKIIβ by siRNA or CKIIβ inhibitor could re-sensitize cells to the cytotoxic effect of cisplatin in both wild type and CCRK-overexpressing U-87 MG cells. In vivo studies also showed that stable over-expression of CCRK increased tumor growth and decreased the anti-tumor efficacy of cisplatin in a nude mice GBM xenograft model. These results provide the first evidence that phosphorylation of CKIIβ is a new mechanism by which CCRK confers tumor growth and drug resistance to GBM cells. In the second part of this thesis I described a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600 and sebacoyl chloride. 1H-NMR, FT-IR and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. Cytotoxicity of the mPPS-FA/DNA nanoparticles was also evaluated on B16-F0, U87MG, CHO-1 and Ho-8910 cells. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910 and A549 cells was investigated in vitro as compared to the lipid-based transfection agent LipofectamineTM2000 and Linear PEI 22KDa (L-PEI 22KDa). I found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1 and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be significantly competed and blocked by the free folic acid molecules. All together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

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.