O carcinoma espinocelular de cabeça e pescoço (CECP) é um problema de saúde pública que apresenta alta taxa de mortalidade, frequentemente relacionado à presença de recorrências locais e metástases. A descoberta de um pequeno subconjunto de células tumorais com características semelhantes às células-tronco, conhecidas como células-tronco tumorais (CTTs), tem sido relatadas como as principais responsáveis pelo início, progressão e recidiva do CECP. O processo de metástase nestas neoplasias é bastante complexo e envolve o desprendimento de células epiteliais tumorais do local de aparecimento primário devido à subexpressão ou superexpressão de algumas proteínas específicas nestas células, caracterizando um processo conhecido como transição epitélio-mesenquimal (EMT). A compreensão dos mecanismos envolvidos no processo de EMT têm sido investigados para o desenvolvimento de terapias específicas. O ácido retinoico (AR) vem sendo empregado em diversas terapias devido a sua capacidade de controlar a proliferação e promover diferenciação celular, entretanto, anormalidades na expressão ou função de seus receptores são relatadas em muitos tipos de células do câncer. Este estudo tem por objetivo correlacionar a expressão de marcadores do processo de EMT, marcador de célula tronco tumoral (ALDH1) e receptores do ácido retinoico e de retinoide X (isoformas α e β) em amostras teciduais provenientes de portadores de CECP primários, além, de correlacionar os resultados obtidos com os parâmetros clínicos, características histopatológicas e prognóstico destes pacientes em um período de acompanhamento de 7 anos. / The head and neck squamous cell carcinoma (HNSCC) is a public health problem that presents high mortality rates in relation to the presence of local recurrences and metastases. A finding of a small subset of tumor cells with stem like-cells characteristics, known as cancer stem cells (CTTs), has been reported as being primarily responsible for the onset, progression and recurrence of CECP. The metastasis process in these neoplasms is quite complex and involves the tumor epithelial cells detachment from the primary site of appearance due to underexpression or overexpression of some specific proteins in these cells, characterizing the epithelial-mesenchymal transition (EMT) process. An understanding of the mechanisms involved in the EMT process has been investigated for the development of specific therapies. Retinoic acid (AR) has been used in several therapies because its ability to control the proliferation and promote cell differentiation, however, abnormalities in the expression and function of its receptors are reported in many types of cancer cells. The aim of this study was to correlate the expression EMT process markers, tumor stem cell marker (ALDH1), retinoic acid and retinoic acid X receptors (α and β isoforms) in tissue samples from primary CECP, in addition, to correlate the results with the clinical parameters, histopathological and prognostic characteristics of these patients in a 7 years follow-up.

A instabilidade genética é um importante evento associado ao carcinoma epidermóide de boca, sendo alterações na região cromossômica 11q constantemente relatadas. Neste estudo, genes localizados na região cromossômica 11q, especificamente os genes CTTN, PPFIA1, SHANK2, TAOS1 e MMP-7, foram investigados quanto a diferenças de expressão de transcritos entre carcinomas epidermóides de boca e suas margens correspondentes. A expressão desses genes foi relacionada com aspectos clínicos e histológicos, com critérios de agressividade estabelecidos, e com a sobrevida dos pacientes. Foram analisadas pela técnica de qRT-PCR 29 amostras congeladas de tumores e 25 margens. Todos os genes apresentaram maiores valores de expressão nos tumores em comparação com as margens, embora apenas o gene MMP-7 tenha exibido valores estatisticamente significantes. A expressão do gene MMP-7 mostrou fraca associação com tumores menos agressivos, e os outros genes apresentaram maiores valores de expressão em tumores mais agressivos, sem significância estatística. Não houve diferença estatística entre a freqüência das variáveis clínicas e histopatológicas com a expressão dos genes estudados, porém o PPFIA1 demonstrou maiores níveis de expressão em tumores de assoalho. Em relação à sobrevida, a expressão elevada de PPFIA1 pode implicar em um maior risco de óbito. Assim, é possível a participação do gene MMP-7 no desenvolvimento da neoplasia, e a relação do PPFIA1 com o risco de óbito, porém a expressão de transcritos dos genes CTTN, SHANK2, TAOS1 e MMP-7 não pode ser relacionada com agressividade tumoral e prognóstico. / Genetic instability is an important event associated with oral squamous cell carcinoma, and alterations in the chromosome region 11q are constantly reported. In this study, genes located on chromosome region 11q, specifically genes CTTN, PPFIA1, SHANK2, TAOS1 and MMP-7, were investigated for differences in the expression of transcripts in oral squamous cell carcinoma and their corresponding margins. The expression of these genes was correlated with clinical and histological aspects, aggressiveness criteria established, and with patient survival. Twenty-nine frozen samples of tumors and 25 samples of margin tissue were analyzed using qRT-PCR. All genes showed a higher expression in tumors, compared with the margins, although only the MMP-7 gene demonstrated statistically significant values. The expression of the MMP-7 gene showed weak association with less aggressive tumors, and the other genes showed higher expression in more aggressive tumors, without statistical significance. There was no statistical difference between the frequency of clinical and histopathological variables and the expression of genes studied, however the PPFIA1 gene demonstrated higher levels of expression in tumors of the floor of mouth. With regard to survival, the high expression of PPFIA1 may imply a greater risk of death. Thus, it is possible that the MMP-7 gene participates in the development of malignancy, and PPFIA1 expression may also be associated with risk of death, however, the expression of transcripts of the CTTN, SHANK2, TAOS1 and MMP-7 genes may not be related to tumor aggressiveness and prognosis.

O carcinoma de células escamosas de cavidade bucal tem alta prevalência e índice de mortalidade considerável entre as principais neoplasias malignas. Acredita-se que carcinógenos químicos promovem a carcinogênese através de danos oxidativos e mutações genéticas, entre outros mecanismos. O organismo evita o acúmulo de espécies reativas de oxigênio (EROs), ou seja, o estresse oxidativo, através da produção endógena de antioxidantes como o -tocoferol, o retinol e a glutationa. Diversos estudos têm demonstrado que a administração de glutamina pode aumentar a produção de glutationa, proteger células de danos oxidativos e alterações genéticas, e, assim, inibir ou retardar a carcinogênese. Esse aminoácido é considerado um agente quimiopreventivo por atuar em alvos moleculares e celulares específicos, no processo de inflamação ou transdução de sinais, além de induzir atividade de enzimas de detoxificação e suprimir proliferação celular. O protocolo de indução de carcinogênese bucal induzida por DMBA, dimetilbenzantraceno dilúido em acetona, aplicado três vezes por semana, foi bem estabelecido em hamsters e sabe-se que a formação de EROs durante sua metabolização pode se difundir a partir do local de geração, para fora da célula, e iniciar a cadeia de peroxidação lipídica. Sabe-se que existe metabolização hepática do DMBA quando aplicado em mucosa jugal. Nesse estudo avaliou-se o efeito da suplementação de glutamina via oral e intragástrica em carcinogênese DMBA-induzida em mucosa de bolsa jugal de hamsters. Dosaram-se níveis de malondialdeído (MDA), principal produto de peroxidação lipídica, e antioxidantes -tocoferol, retinol e glutationa. Obteve-se como resultado a formação de carcinoma de células escamosas na maioria dos animais, independentemente da administração de glutamina, na dosagem testada. Não foram observadas diferenças na quantidade de MDA, glutationa, proteína e retinol no fígado dos animais. Tampouco houve diferença entre os níveis plasmáticos de glutationa entre os grupos. Esses resultados demonstram que não houve estresse oxidativo significativo nos tecidos testados. Porém houve acúmulo de -tocoferol no fígado dos animais tratados com o carcinógeno por 15 semanas, independentemente da administração de glutamina. Conclui-se, portanto, que pode ter ocorrido agressão ao tecido hepático desses animais e que a glutamina não foi eficiente na prevenção de carcinogênese ou agressão hepática. Possivelmente o tecido tumoral apresentaria alteração do equilíbrio oxidante/antioxidante, mas não foi avaliado nesse estudo. / Oral cavity squamous cell carcinoma has a high prevalence and substantial mortality rate among the major malignancies. It is believed that chemical carcinogens promote carcinogenesis by oxidative damage and mutation, among other mechanisms. The body prevents the accumulation of reactive oxygen species (ROS), oxidative stress, through the production of endogenous antioxidants such as -tocopherol, retinol, and glutathione. Several studies have demonstrated that glutamine administration can increase the production of glutathione, protecting cells from oxidative damage and genetic changes, and thus inhibit or delay the carcinogenesis. This amino acid is considered a chemopreventive agent to act on specific molecular and cellular targets, in inflammation or signal transduction, and activity to induce detoxification enzymes and suppress cell proliferation. The dimethylbenzanthracene (DMBA) induced carcinogenesis protocol consists in three times a week application of DMBA diluted in acetone, in hamsters. It is known that the formation of ROS during its metabolism can diffuse from the site of generation, outside the cell, and initiate the lipid peroxidation chain. It is known that there is hepatic metabolism of DMBA when applied to the buccal mucosa. This study evaluated the effect of oral and intragastric glutamine supplementation in DMBA-induced carcinogenesis on the cheek pouch mucosa of hamsters. Levels were assayed for malondialdehyde (MDA), the main product of lipid peroxidation, and the antioxidants -tocopherol, retinol and glutathione. The results show the formation of squamous cell carcinoma in most animals, regardless of this glutamine amount administration. There were no differences in the amount of MDA, glutathione, protein and retinol in the liver of animals. There was no difference between plasma levels of glutathione between groups. These results demonstrate that there was no significant oxidative stress in the tissues tested. However, there was accumulation of -tocopherol in the liver of animals treated with the carcinogen for 15 weeks, regardless of the administration of glutamine. Therefore that may have occurred aggression liver tissue of animals and that glutamine was not effective in the prevention of carcinogenesis or liver injury. Possibly the tumor tissue present shift in the balance oxidant / antioxidant, but it was not evaluated in this study.

O carcinoma epidermóide da mucosa oral (CEMO) é uma neoplasia maligna comum; no Brasil, são estimados, para 2016, 15.490 novos casos. A invasão óssea ocorre em casos avançados.; esta é classificada em erosiva e infiltrativa. Aparentemente, o processo de transição epitélio-mesenquimal, com o envolvimento da E-caderina, é implicado. Foi investigada a expressão de E-caderina, por meio da imunoistoquímica em 15 casos avançados de CEMO e avaliada sua correlação com as características clínicas e histológicas da invasão óssea. A imunoexpressão da E-caderina foi estudada nos 15 casos de CEMO com evidência histológica de invasão óssea. A maioria dos pacientes eram homens (10 pacientes) e apresentavam invasão em mandíbula (9 casos). A expressão de E-caderina foi negativa em CEMOs com invasão erosiva e positiva nos casos que apresentavam infiltração óssea. A expressão de E-caderina na invasão óssea sugere que a participação do fenômeno de transição epitélio-mesenquimal é um fator diretamente envolvido com o tipo de invasão óssea. / Oral squamous cell carcinoma (OSCC) is a common malignancy; in Brazil it is estimated, in 2016,15.490 new cases. Bone invasion occurs in advanced cases; it is classified in erosive and infiltrative patterns. Apparently, the epithelial-mesenchymal phenomenon, with important participation of E-cadherin is implicated. We investigated the expression of E-cadherin in advanced OSSC and correlated its expression with the clinical characteristics and histologic patterns of bone invasion. Immunoexpression of E-cadherin was studied in 15 cases of OSCC with histological evidence of bone invasion. Most patients were men (10 patients) and presented mandible invasion (9 cases). The expression of E-cadherin was negative in OSCC in erosive bone invasion and positive in the infiltrative bone invasion. E-cadherin expression in bone invasion suggests that participation of epithelial-mesenchymal phenomenon is dependent on the patterns of tumour bone invasion.

Os carcinomas de cabeça e pescoço (CECPs) compreendem um grupo de tumores que atingem vários sítios do trato aerodigestivo superior, incluindo cavidade oral, orofaringe, hipofaringe e laringe. Esses carcinomas são clinicamente heterogeneous e resultam de modificações cumulativas em genes que regulam proliferação, migração celular e apoptose. São estimados aproximadamente 500.000 novos casos de CECP anualmente no mundo. No Brasil, cerca de 14.000 novos casos são esperados em 2010, somente para cavidade oral. As taxas de morbidade e mortalidade e as limitações das estratégias terapêuticas enfatizam a necessidade de um melhor entendimento dos padrões moleculares envolvidos na iniciação e na progressão desses tumores, e de abordagens preventivas e terapêuticas efetivas. Infelizmente, apesar da intensa pesquisa nessa área, poucos marcadores moleculares são conhecidos que exibam sensibilidade e especificidade para diagnóstico e prognóstico de CECP. Em um estudo prévio, nós avaliamos dados de três bibliotecas SAGE de carcinoma de laringe com a finalidade de identificar eventos associados ao desenvolvimento e à agressividade de CECP. Utilizando abordagens estatísticas e de Bioinformática, nós identificamos 60 genes com expressão elevada ou reduzida em tumores metastáticos versus não-metastáticos e em ambos os grupos versus tecidos normais. O objetivo do presente estudo foi avaliar a expressão de quatro genes desta lista, os das queratinas 6A (KRT6A) e 19 (KRT19), da mesotelina (MSLN) e da calicreína 8 (KLK8), em um conjunto de 63 carcinomas primários de cabeça e pescoço e suas margens cirúrgicas e em quarto linhagens celulares (Hep-2, FaDu, SCC9 e UM-SSC-38) por RT-PCR em tempo real. Como amostra de referência para as linhagens, foram utilizados queratinócitos orais humanos normais, cultivados sobre uma camada de sustentação de fibroblastos irradiados. Todos os genes exibiram níveis de transcritos reduzidos ou ausentes nas linhagens celulares, exceto MSLN, que mostrou um padrão irregular de expressão. Em tumores primários, os genes KRT19 e MSLN apresentaram expressão diminuída em laringe, o mesmo sendo observado para o gene KLK8 em tumores de língua metastático. Além disso, foi detectada expressão elevada de MSLN e KLK8 em tumores não metastáticos de soalho de boca e expressão reduzida de KRT19 em tumores de soalho de boca e língua metastáticos. Os resultados levantam questões sobre o papel desses genes em processos biológicos associados com a tumorigênese de cabeça e pescoço e sobre sua participação no fenótipo neoplásico. / Head and neck squamous cell carcinomas (HNSCCs) encompass a group of tumors that affect a variety of sites in the upper aero-digestive tract, including oral cavity, oropharynx, hypopharynx and larynx. These carcinomas are clinically heterogeneous and result from cumulative changes in genes that regulate cell proliferation, migration and death. It is estimated that approximately 500,000 new cases of HNSCC are diagnosed worldwide each year. In Brazil, about 14,000 new cases are expected in the year 2010, only in oral cavity. The morbidity and mortality rates and the limitations of therapeutic strategies emphasize the need for a better understanding of the molecular pathways involved in the initiation and progression of these tumors and for effective preventive and therapeutic approaches. Unfortunately, despite intense research, few molecular markers are known to exhibit sensitivity and specificity for the diagnosis or prognosis of HNSCC. In a previous study, we evaluated data from three SAGE libraries of larynx carcinoma in order to identify events associated with the development and aggressiveness of HNSCCs. Using statistical and bioinformatic tools, we identified sixty top-up and 60 top-downregulated genes in metastatic versus non-metastatic tumors and in both these tumors versus normal tissues. The objective of the present study was to evaluate the expression of four genes from this list, keratin 6A (KRT6A), keratin 19 (KRT19), mesothelin (MSLN) and kallikrein 8 (KLK8), in a set of 63 primary carcinomas of head and neck and their surgical margins and in four cell lines (Hep-2, FaDu, SCC9 and UM-SSC-38) by real time RT-PCR. As a reference sample for cell lines, we used normal human oral keratinocytes grown on irradiated fibroblast feeder layer. All genes exhibited no or decreased levels of transcripts in the cell lines, except MSLN, which displayed an irregular pattern of expression. In primary tumors, KRT19 and MSLN genes were downregulated in larynx, and KLK8 in metastatic tongue tumors. In addition, MSLN and KLK8 were upregulated in non-metastatic floor of the mouth tumors and KRT19 was down regulated in metastatic floor of the mouth and tongue tumors. The results open questions about the role of these genes on biological processes related to head and neck tumorigenesis and on neoplastic phenotype.

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

Fung Ka Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 159-179). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABBREVIATIONS --- p.III / ABSTRACTS --- p.V / LIST OF FIGURES --- p.VIII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / CONTENTS / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1. --- Review: Cellular mechanisms of drug resistance in cancer cells --- p.2 / Chapter 1.1.1. --- Drug resistance by decrease of drug accumulation --- p.2 / Chapter 1.1.2. --- Drug resistance conferred by detoxification of drug in cells --- p.5 / Chapter 1.1.3. --- Drug resistance conferred by alteration of drug targets or by enhancement of target repair --- p.6 / Chapter 1.1.4. --- Drug resistance conferred by genes alteration in apoptotic pathways --- p.9 / Chapter 1.2. --- Objective and review --- p.11 / Chapter CHAPTER TWO: --- PROFILING AND IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES OF HUMAN SQUAMOUS CARCINOMA A431 CELLS BY mRNA DIFFERENTIAL DISPLAY AND cDNA MICROARRAY / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- Changes in gene expression and drug resistance --- p.16 / Chapter 2.1.2. --- Current states of technologies in gene expression analysis --- p.17 / Chapter 2.1.3. --- Monitoring the gene expression profile in the field of drug resistance by cDNA microarray --- p.18 / Chapter 2.1.4. --- Monitoring the gene expression profile in the field of drug resistance by differential display --- p.22 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Materials --- p.27 / Chapter 2.2.2. --- Methods / Chapter 2.2.2.1. --- Cell culture and cell lines --- p.29 / Chapter 2.2.2.2. --- cDNA microarray / Chapter 2.2.2.2.1. --- RNA preparation --- p.29 / Chapter 2.2.2.2.2. --- Differential hybridization of ResGen GenFilters Mammalian micro array --- p.30 / Chapter 2.2.2.3. --- mRNA differential display / Chapter 2.2.2.3.1. --- RNA preparation --- p.31 / Chapter 2.2.2.3.2. --- RT-PCR based mRNA differential display --- p.31 / Chapter 2.2.2.3.3. --- Reamplification of cDNA probes --- p.32 / Chapter 2.2.2.3.4. --- Subcloning of differentially expressed cDNAs / Chapter A. --- Preparation of ultra-competent E.coli cells for transformation --- p.33 / Chapter B. --- Preparation of cloning vector and DNA transformation --- p.33 / Chapter 2.2.2.3.5. --- Verification of cDNA expression by colony-PCR and cDNA probes / Chapter A. --- colony-PCR --- p.34 / Chapter B. --- Bacterial glycerol stock and plasmid preparation --- p.34 / Chapter C. --- cDNA probes preparation for Northern blot analysis --- p.35 / Chapter 2.2.2.3.6. --- Northern blot analysis --- p.35 / Chapter 2.2.2.3.7. --- Sequencing of cDNA cloned inserts --- p.36 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Differential gene expression profile of A431 cells --- p.43 / Chapter 2.3.2. --- Identification of differentially expressed genes by mRNA differential display --- p.53 / Chapter 2.4. --- Discussion --- p.60 / Chapter CHAPTER THREE: --- CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES.。PART 1: THE ROLE OF VACUOLAR PROTON PUMP IN REGULATION OF DRUG SENSITIVITY AND APOPTOSIS IN A431 CELLS / Chapter 3.1. --- Introduction / Chapter 3.1.1. --- An overview of vacuolar proton pump/vacuolar-H+-ATPase (V-ATPase): Structure and function --- p.66 / Chapter 3.1.2. --- V-ATPases and drug resistance mechanisms --- p.69 / Chapter 3.1.3. --- Objective --- p.70 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Materials --- p.73 / Chapter 3.2.2. --- Methods / Chapter 3.2.2.1. --- Cell culture and cell lines --- p.74 / Chapter 3.2.2.2. --- "RNA isolation, mRNA differential display, cDNA reamplification and colony-PCR verification" --- p.74 / Chapter 3.2.2.3. --- Cellular pH measurement by flow cytometry --- p.75 / Chapter 3.2.2.4. --- MTT Drug Sensitivity Assay --- p.76 / Chapter 3.2.2.5. --- DNA fragmentation assay --- p.77 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Identification of cDNA band encoding vacuolar proton pump subunit c by differential display --- p.78 / Chapter 3.3.2. --- Effect of bafilomycin A1 (BAF) and concanamycin A (CON) on DOX- and CP-induced cytotoxicity and apoptosis ' --- p.79 / Chapter 3.3.3. --- Elevation of cellular pH in cisplatin-resistant cells correlated with cisplatin resistance mechanism of A431 cells --- p.94 / Chapter 3.4. --- Discussion --- p.98 / Chapter CHAPTER FOUR: --- CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES。PART 2: IDENTIFICATION OF A NOVEL cDNA OVEREXPRESSED IN HUMAN SQUAMOUS CARCINOMA A431 PARENT CELLS / Chapter 4.1. --- Introduction --- p.102 / Chapter 4.2. --- Materials and Methods / Chapter 4.2.1. --- Materials --- p.105 / Chapter 4.2.2. --- Methods / Chapter 4.2.2.1. --- Cell culture and cell lines --- p.105 / Chapter 4.2.2.2. --- "RNA isolation, mRNA differential display, cDNA reamplification and colony-PCR verification" --- p.105 / Chapter 4.2.2.3. --- Northern blot analysis --- p.106 / Chapter 4.2.2.4. --- Human tissue distribution of clone PA-8P19 --- p.107 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Identification of novel cDNA clone PA-8P19 in A431 cells by differential display --- p.108 / Chapter 4.3.2. --- Gene expression profile of clone PA-8P19 in human tissues and other tumor cell lines --- p.113 / Chapter 4.3.3. --- Regulation of PA-8P19 expression by DNA damaging agents --- p.117 / Chapter 4.4. --- Discussion --- p.122 / Chapter CHAPTER FIVE: --- PROFILING OF DIFFERENTIAL EXPRESSED PROTEINS BY TWO-DIMENSIONAL GEL ELECTPOPHORESIS / Chapter 5.1. --- Introduction / Chapter 5.1.1. --- Protein analysis on proteomic scale --- p.125 / Chapter 5.1.2. --- Protein expression profiling for studying drug resistance by Two- dimensional gel electrophoresis --- p.127 / Chapter 5.2. --- Materials and Methods / Chapter 5.2.1. --- Materials --- p.130 / Chapter 5.2.2. --- Methods / Chapter 5.2.2.1. --- Cell culture and cell lines --- p.132 / Chapter 5.2.2.2. --- Two-dimensional gel electrophoresis / Chapter I. --- Sample preparation --- p.132 / Chapter II. --- First-dimensional gel: isoelectric focusing (IEF) --- p.133 / Chapter III. --- Second-dimensional gel: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.134 / Chapter IV. --- Protein visualization and image analysis --- p.134 / Chapter V. --- In-gel digestion --- p.135 / Chapter VI. --- Matrix-assisted laser desorption ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and database search --- p.136 / Chapter 5.2.2.3. --- Western blot analysis --- p.137 / Chapter 5.3. --- Results / Chapter 5.3.1. --- Identification of differentially expressed proteins in A431 cells by two-dimensional gel electrophoresis --- p.140 / Chapter 5.4. --- Discussion / Chapter 5.4.1. --- Two-dimensional gel electrophoresis as a powerful tool for identification of differential protein expression in A431 cells --- p.150 / Chapter CHAPTER SIX: --- GENERAL CONCLUSION AND PERSPECTIVES --- p.155 / REFERENCES --- p.159

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-06-22T15:03:16Z No. of bitstreams: 1 vitorrafafelregiani_dissert.pdf: 1149352 bytes, checksum: 542e5f6273639354b00db640f941c027 (MD5) / Made available in DSpace on 2016-06-22T15:03:16Z (GMT). No. of bitstreams: 1 vitorrafafelregiani_dissert.pdf: 1149352 bytes, checksum: 542e5f6273639354b00db640f941c027 (MD5) Previous issue date: 2015-08-25 / Introduction: DNA methylation plays an important role in regulating gene expression. During tumorigenesis, the hypermethylation of CpG-rich islands in promoter regions (Cytosine phosphodiester Guanine) is a mechanism that can inactivate tumor suppressor genes and contribute to the development of cancer. Objective: This study aimed to evaluate the methylation of promoter regions of tumor suppressor genes RASSF1A (Ras association domain family member 1) and MGMT (O-6-methylguanine-DNA methyltransferase) in squamous cell carcinoma of the head and neck. Methods: The methylation analysis of promoter regions of RASSF1A and MGMT genes was performed using the High Resolution Melting (HRM) in 42 and 37 tumor tissue samples, respectively. Samples of tissue adjacent to the tumor or peripheral blood were used as controls. Results: For the RASSF1A gene, 50% (21 of 42) of tumor tissue samples analyzed were methylated, whereas for MGMT of 37 tumor tissue samples analyzed, 17 (46%) showed methylation. Statistical analysis between pairs of tumor samples and controls (tissue adjacent to the site of tumor and peripheral blood) showed significant differences in the presence of methylation for the RASSF1A gene (P = 0.027, Chi-square test) and the MGMT gene (P = 0.002, Chi-square test). The evaluation of the presence of methylation between tumor tissue and surrounding tissue showed no significant difference for RASSF1A genes (P = 1.0) and MGMT (P = 0.38). For the subset of tumor tissue and peripheral blood statistic was significant for RASSF1A (P = 0.005) and MGMT (P = 0.002) genes. Analysis of the presence of methylation in tumors relative to non-tumor tissues (tissues adjacent to tumor or peripheral blood) in different anatomical sites of primary occurrence showed that in oral cavity and pharynx no statistically significant difference for the RASSF1A gene (P = 0.233) relative to non-tumor samples; MGMT gene statistical results were significant (P = 0.033). In the larynx, the statistical results were significant for the RASSF1A gene (P = 0.04) compared to non-tumor samples, while for the MGMT gene, the result was not statistically significant (P = 0.998). The presence of the methylation of the MGMT gene in both genes simultaneously, RASSF1A and MGMT, were associated with the category N (P = 0.045 and P = 0.035, respectively). For the RASSF1A gene was not observed this association (P = 0.093). The analysis by multiple logistic regression of the influence of epidemiological factors (gender and age), risk factors (smoking and drinking) and clinical parameters of the tumor for the presence of methylation showed no significant association for these variables. Conclusions: The prevalence presence of methylation in the promoter region of RASSF1A and MGMT genes in tumor tissue of the pairs of samples (tumor and non-tumor tissue) suggests the involvement of these genes in the process of squamous cells carcinoma of the head and neck, in particular of the MGMT gene in tumors of the oral cavity and pharynx and RASSF1A gene in larynx tumors. No was observed association between methylation of RASSF1A and MGMT genes and age, gender, the tobacco and alcohol consumption and clinical tumor parameters. / Introdução: A metilação do DNA desempenha um papel importante na regulação da expressão gênica. Durante a tumorigênese, a hipermetilação em regiões promotoras ricas em ilhas CpG (Cytosine phosphodiester Guanine) é um mecanismo que pode inativar genes supressores de tumor e contribuir para o desenvolvimento do câncer. Objetivo: O presente trabalho teve como objetivo avaliar a metilação das regiões promotoras dos genes supressores de tumor RASSF1A (Ras association domain family member 1) e MGMT (O-6-methylguanine-DNA methyltransferase) em carcinoma espinocelular de cabeça e pescoço. Casuística e Métodos: A análise de metilação das regiões promotoras dos genes RASSF1A e MGMT foi realizada pela técnica High Resolution Melting (HRM) em 42 e 37 amostras de tecidos tumorais, respectivamente. Amostras de tecido adjacente ao tumor ou de sangue periférico foram utilizadas como controle. Resultados: Para o gene RASSF1A, 50% (21 de 42) das amostras de tecidos tumorais analisadas foram metiladas, enquanto para o MGMT, das 37 amostras de tecidos tumorais analisadas, 17 (46%) apresentaram metilação. A análise estatística entre os pares de amostras tumorais e controles (tecido adjacente ao tumor e sangue periférico) mostrou diferença significante quanto à presença de metilação para o gene RASSF1A (P=0,027, teste de Qui-quadrado) e para o gene MGMT (P=0,002, teste de Qui-quadrado). A avaliação da presença de metilação entre subgrupos de tecido tumoral e tecido adjacente não mostrou diferença significante para os genes RASSF1A (P=1,0) e MGMT (P=0,38). Para o subgrupo de tecido tumoral e sangue periférico o resultado estatístico foi significante para os genes RASSF1A (P=0,005) e MGMT (P=0,002). A análise da presença de metilação nos tumores em relação aos tecidos não tumorais (tecidos adjacentes ou sangue periférico) nos diferentes sítios anatômicos de ocorrência primária mostrou que, em cavidade oral e faringe não houve diferença estatística significante para o gene RASSF1A (P=0,233) em relação às amostras não tumorais; para gene MGMT o resultado estatístico foi significante (P=0,033). Em laringe, o resultado estatístico foi significante para o gene RASSF1A (P=0,04) em relação às amostras não tumorais, enquanto para o gene MGMT, o resultado estatístico não foi significante (P=0,998). A presença de metilação no gene MGMT bem como em ambos os genes concomitantemente, RASSF1A e MGMT, foi associada com a categoria N (P=0,045 e P=0,035, respectivamente). Para o gene RASSF1A não foi observada tal associação (P=0,093). A análise por meio da regressão logística múltipla da influência dos fatores epidemiológicos (gênero e idade), dos fatores de risco (tabagismo e etilismo) e parâmetros clínicos do tumor quanto à presença de metilação não mostrou associação significante para essas variáveis. Conclusões: A presença de metilação nas regiões promotoras dos genes RASSF1A e MGMT apenas no tecido tumoral da maioria dos pares de amostras (tecido tumoral e não tumoral) sugere o envolvimento desses genes no processo neoplásico de cabeça e pescoço, em especial do gene MGMT em tumores da cavidade oral e faringe e do gene RASSF1A na laringe. Não há associação entre metilação dos genes RASSF1A e MGMT e a idade, o gênero, os hábitos tabagista e etilista e os parâmetros clínicos do tumor.

A localização da podoplanina e da ezrina nas células malignas sugere uma ligação dessas proteínas nos processos de migração e invasão tumoral, ativadas mediante a fosforilação de Rho-A. O objetivo desse estudo foi avaliar a distribuição e a correlação da podoplanina, da ezrina e da Rho-A em 91 carcinomas espinocelulares de lábio inferior, e verificar a associação dessas proteínas com as variáveis clínicas e patológicas, com a evolução e com o prognóstico dos pacientes. Os pacientes foram analisados quanto ao gênero, idade, tabagismo, etilismo, classificação pelo sistema TNM, tratamento, ocorrência de recidivas locorregionais, segundo tumor primário, além da presença de embolização vascular, infiltração perineural, muscular, glandular e comprometimento linfonodal histopatológico. Analisou-se também as expressões imuno-histoquímicas de podoplanina, ezrina e Rho-A no front de invasão tumoral e o índice de malignidade tumoral. A associação entre a podoplanina, a ezrina e a Rho-A com as variáveis clínico-patológicas e a correlação entre as proteínas foram analisadas pelos testes do qui-quadrado e de Spearman, respectivamente. A análise da sobrevivência global em 5 e 10 anos foi feita pelo estimador produto-limite Kaplan-Meier e a comparação da curva de sobrevivência pelo teste log-rank. Os resultados demonstraram uma forte expressão de podoplanina, de ezrina e de Rho-A no front de invasão dos carcinomas espinocelulares de lábio inferior. Houve uma associação significativa entre a expressão citoplasmática de podoplanina com o etilismo (p=0,024), com a recidiva locorregional (p=0,028) e com comprometimento linfonodal histopatológico (p=0,010), porém não foi detectada nenhuma associação significativa entre a ezrina e a Rho-A com as variáveis clínicas e microscópicas analisadas. Uma correlação positiva e estatisticamente significativa entre a expressão de podoplanina membranosa (p=0,000 e r =0,384) e citoplasmática (p=0,000 e r=0,344) com a expressão de ezrina, e da podoplanina membranosa com a expressão de Rho-A (p=0,006 e r=0,282) foi detectada. Nenhuma das três proteínas se mostrou fator de prognóstico significativo para os pacientes com câncer de lábio. Concluímos que a forte expressão membranosa de podoplanina nos carcinomas espinocelulares de lábio inferior pode ajudar a identificar pacientes com menor risco de recidiva locorregional. Além disso, a correlação entre as expressões da podoplanina, da ezrina e da Rho-A pelas células neoplásicas sugere uma participação conjunta destas proteínas nos processos de movimentação celular e invasão tumoral do câncer de lábio. / Immunolocalization of podoplanin and ezrin suggests a connection between these proteins in migration and tumoral invasion process, activated through Rho-A phosphorylation. The aim of this study was to evaluate the distribution and the correlation of podoplanin, ezrin and Rho-A in 91 squamous cells carcinomas of the lower lip and to verify its association with clinical and pathological features, evolution and prognostic of the patients. Patients were analyzed concerning gender, age, tobacco, alcohol, TNM classification, local and regional recurrences, second primary tumor, perineural, muscle and glandular infiltration, and histopahological lymph node metastasis. The association of podoplanin, ezrin and Rho-A expressions at tumoral invasion front and histological risk assessment of tumors were verified by chi-square test. The association between podoplanin, ezrin and Rho-A expressions with clinical and pathological variables, and the correlation of these variables were analyzed by chi-square and Spearman test, respectively. Overall survival in 5 and 10 years was calculated by Kaplan Meier method and overall curves were compared by log rank test. The results showed strong expression of podoplanin, ezrin and Rho-A at tumoral invasion front of squamous cell carcinomas of the lower lip. A significant association of strong cytoplasmic podoplanin expression and alcoholism (p=0,024), local recurrences (p=0,028) and lymph node metastasis (p=0,010) was found, although ezrin and Rho-A expressions were not associated with clinical and microscopic features analyzed. A statistically significant correlation between membranous (p=0,000 e r =0,384) and cytoplasmic (p=0,000 e r=0,344) podoplanin expressions and ezrin, and membranous podoplanin and Rho-A (p=0,006 e r=0,282) was observed. None of the proteins analyzed can be considered as prognostic factor for lip cancer. We can conclude that strong membranous podoplanin expression in squamous cell carcinoma of the lower lip can help to identify patients with lower risk of local and regional recurrences. Furthermore, the correlation of podopolanin, ezrin and Rho-A expressions suggest a cooperative participation in cell movement and tumoral invasion.

Das diversas neoplasias denominadas câncer de boca, mais de 95% tem o diagnóstico de carcinoma epidermóide (CE). O carcinoma epidermóide apresenta etiologia e patogênese complexas e não totalmente compreendidas determinando altos índices de morbidade e mortalidade. Por isso crescentes esforços têm sido empregados para a melhor compreensão dos eventos celulares, da susceptibilidade genética e dos fatores de risco que atuando em conjunto dão origem e atuam na progressão do carcinoma epidermóide de boca. Buscando novas facetas da carcinogênese oral e baseando-se no papel pró-carcinogênico que os hormônios esteróides desempenham nas neoplasias dos órgãos reprodutivos, alguns estudos passaram a avaliar o papel desses hormônios na patogênese e progressão do CE. O propósito deste trabalho foi avaliar a presença dos receptores de androgênio, estrogênio (alfa e beta) e da enzima aromatase em carcinomas epidermóides de boca (CE), através das técnicas de imuno-histoquímica, western-blotting e imunofluorescência, e comparar, com base no gênero (masculino e feminino), os resultados encontrados. Da amostra de CE utilizada, 30 eram pertencentes a pacientes do gênero feminino e 30 do masculino. Para as reações de Western blotting e imunofluorescência foram utilizadas duas linhagens celulares: OSCC9 e OSCC25, derivadas de CE de língua. Nas reações imunohistoquímicas, apesar de um número maior de casos negativos do que positivos, observou-se que o receptor de estrogênio beta foi o mais expresso, seguido pelo receptor de androgênio e aromatase. O receptor de estrogênio alfa apresentou menor imunoexpressão. Houve diferença estatisticamente significante entre os gêneros apenas na expressão dos receptores de androgênio. Os resultados obtidos pelo western blotting e imunofluorescência evidenciaram que as duas linhagens celulares utilizadas apresentaram receptores de estrogênio beta no núcleo, bem como, a presença da enzima aromatase com localização citoplasmática. Os receptores de androgênio foram observados no núcleo somente na linhagem OSCC9 enquanto que os receptores de estrogênio alfa não foram evidenciados em nenhuma das linhagens. Esses resultados sugerem que em alguns casos de CE possa existir contribuição hormonal na progressão da doença, evidenciando a necessidade da utilização desses marcadores nesse tipo de neoplasia. / Oral squamous cell carcinoma (OSCC) is the main type of oral cancer presenting complex and not completely understood pathogenesis. OSCC implies quite significant mortality and morbidity rates and in spite of the vast amount of research and the advances in the field of oncology and surgery, the overall survival remains largely unchanged. Therefore, the identification of new pathways involved in the mechanisms of carcinogenesis can bring knowledge to the control and advent of new treatments. The role of androgens and estrogens in several endocrine-related malignancies is well known. The successful use of these hormones antagonists in the treatment of these neoplasms has prompted researches to investigate the presence of hormones receptors in other tissues and tumor types. The aim of this study was to asses through immunohistochemistry, western blot and immunoflorescence assays the expression, genderrelated, of androgen, estrogen alpha and beta receptors and aromatase enzyme in cases of OSSC. A total of 60 cases of OSCC (30 from males and 30 from females) were retrieved and submitted to immunohistochemical assay. Two OSCC cell lines were used for western blot and immunoflurescence techniques: OSCC-9 and OSCC-25, originated from the tongue. Immunohistochemical staining demonstrated that estrogen beta and androgen receptors and aromatase enzyme, respectively, were more frequently expressed in OSCC. Estrogen alpha receptor had the lower immuno expression. Only androgen receptors presented differences statistically significant between genders. Western blotting and immunofluorescence analysis demonstrated that estrogen beta receptor was abundantly present in nuclear region of SCC9 and SCC25 cell lines, as well as aromatase protein, although its localization was cytoplasmatic. Androgen receptor was observed in the nucleus of SCC9 cell line and absent in the SCC25 cell line. On the other hand, estrogen alpha receptor was not detected either in western blot assay or in immunofluorescence. Even though these proteins presented a variable expression in OSCC, it is interesting to conduct more studies about them, since we could have more possibilities to predict and control oral squamous cell carcinoma.