DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.

Alterations of the TP53 Gene in Gastric and Esophageal Carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Methylation status of CDH1 gene in samples of gastric mucous from brazilian patients with chronic gastritis infected by Helicobacter pylori

CONTEXTO:O câncer gástrico é uma das principais neoplasias que causam o óbito no Brasil e no mundo. Helicobacter pylori é um carcinógeno do tipo I relacionado à gastrite crônica. Diferenças no grau de virulência de suas cepas levam a maior risco de desenvolvimento de doenças gástricas. A metilação de ilhas CpGs está envolvida com o processo de tumorigênese em diferentes tipos de câncer. CDH1 é um gene supressor tumoral que, quando inativado, pode aumentar as chances de metástase. A metilação deste gene em estágios precoces da carcinogênese gástrica ainda não é totalmente compreendida. OBJETIVO: Investigar o padrão de metilação do gene CDH1 em amostras de gastrites crônicas e correlacionar com a presença do H. pylori. MÉTODOS: Foram usadas 60 biopsias de mucosas gástricas. A detecção de H. pylori foi realizada por PCR para o gene da urease C e a genotipagem com PCR para os genes cagA e vacA (região s e m). O padrão de metilação do gene CDH1 foi analisado usando a técnica de PCR e específica para a metilação e sequenciamento direto dos produtos de PCR. RESULTADOS: A bactéria H. pylori foi detectada em 90% das amostras de gastrites crônicas; destas, 33% portavam o gene cagA e 100% vacA s1. O genótipo vacA s2/m1 não foi detectado nas amostras analisadas. Metilação de CDH1 foi detectada em 63,3% das amostras de gastrites e 95% delas eram portadoras de H. pylori. CONCLUSÃO: Os resultados deste estudo sugerem que a metilação em CDH1 e a infecção pelo H. pylori são eventos frequentes em amostras de pacientes brasileiros com gastrite crônica e reforça a correlação entre infecção por H. pylori e inativação do gene CDH1 em estágios precoces da tumorigênese gástrica.

p53 gene analysis in childhood B non - Hodgkin's lymphoma

CONTEXTO: Alterações do gene supressor de tumor p53, como mutações e deleções, são lesões genéticas encontradas com maior freqüência nas neoplasias humanas, incluindo câncer de mama, pulmão e cólon. Entre as malignidades hematológicas, o gene 53 é freqüentemente mutado no linfoma de Burkitt, sendo detectadas mutações em 30-40% das amostras tumorais e em 70% das linhagens celulares. OBJETIVO: Analisar as alterações do gene p53 em crianças com linfoma não-Hodgkin de origem B. TIPO DE ESTUDO: Estudo descritivo. LOCAL: Centro de Oncologia Terciário. PARTICIPANTES: O estudo analisou 12 pacientes com linfoma não-Hodgkin B classificados como linfoma de Burkitt. A análise de possíveis mutações do gene p53 foi realizada pela técnica de PCR-SSCP dos exons 5, 6 ,7 e 8/9 do gene. RESULTADOS: Um padrão anormal de migração foi observado em quatro pacientes (33.3%), em um paciente no exon 6 e em três no exon 7. Os casos positivos incluíam dois pacientes que evoluíram para o óbito por progressão da doença. CONCLUSÃO: Esses resultados preliminares sugerem que as alterações do gene p53 são freqüentes em crianças com linfoma de Burkitt e podem contribuir para patogênese ou progressão da doença.

Expressão do gene FOXE1 em culturas de osteossarcoma canino

Pós-graduação em Medicina Veterinária - FMVZ

Metilação e expressão do gene BRCA1 em meningiomas

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

A correlação da expressão do gene p53 no carcinoma invasor de colo uterino com fatores clínicos e prognóstico

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

O gene PHF21B como candidato a predisposição familia em familial em cânceres de cabeça e pescoço

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

Alterações epigenéticas e do número de cópias do gene SFN em carcinomas mamários

Pós-graduação em Ciências Biológicas (Genética) - IBB

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl-2 and p63 proteins in canine transmissible venereal tumour

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.