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| Id: |
lil-553314
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| Autor: |
Silva, Ana Paula Medeiros.
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| Título: |
Caraterização de genes diferencialmente expressos em linhagens celulares de mama que expressam diferentes níveis de C-erbB2 / Characterization of differentially expressed genes in strains cellular breast cancer that express different levels of c-erbB2.
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| Fonte: |
São Paulo; s.n; 2006. 163 p. ilus, tab.
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| Idioma: |
pt.
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| Tese: |
Apresentada a Fundação Antônio Prudente para obtenção do grau de Doutor.
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| Resumo: |
O oncogene c-erbB2 é um receptor de membrana com atividade de tirosinaquinase e pertence a família de receptores de fatores de crescimento epidermal. A super-expressão do oncogene c-erbB2 é observada em 25-30% dos tumores de mama e é também um fator de pior prognóstico. Para estudar os mecanismos moleculares de atuação desse oncogene, Harris et al (1999) desenvolveram um modelo de superexpressão de c-erbB2 em células epiteliais luminais imortalizadas da mama... A partir de ambas as linhagens celulares foi gerado um total de 24.521.236 tags, correspondendo a 24.065 tags distintas e válidas. Depois de estabelecidas as correlações entre tags e transcritos humanos conhecidos, 9% das tags não foram associadas a transcritos conhecidos (tags órfãs)... Para 41 das 83 amplificações de GLGI-MPSS, foi possível obter um fragmento predominante e de forte intensidade quando analisado em gel de agarose. Esses 41 fragmentos foram clonados e seqüenciados e as seqüências geradas foram analisadas através do programa BLAST, para verificar a presença de similaridade com transcritos humanos depositados em bancos de dados públicos... Já nos experimentos que avaliaram a expressão dessestranscritos em amostras tumorais, foi possível verificar que a média do nível de expressão desses transcritos em relação ao tecido normal de mama era maior no grupo de tumores c-erbB2 positivos e, para as tags 7 e 28, a diferença observada entre os dois grupos foi estatisticamente significativa. Esses resultados confirmam a expressão diferencial desses 4 transcritos em tumores c-erbB2 positivos e a caracterização funcional dos mesmos nos permitirá compreender melhor os mecanismos moleculares de atuação do c-erbB2, abrindo novas perspectivas para o acompanhamento e tratamento de pacientes com câncer de mama (AU)
The c-erbB2 oncogene is a membrane receptor with tyrosine quinase activity and belongs to the epidermal growth factor receptor family. C-erbB2 over-expression is observed in 25-30% of breast tumors and is an adverse prognostic factor. To study the molecular mechanisms of c-erbB2 over-expression, Harris et al. (1999) developed a model of c-erbB2 over-expression in conditionally immortalized mammary luminal epithelial cells. Two new cell lines, HB4a-C3.6 and HB4a-C5.2, expressing different levels of c-erbB2 were derived from the immortalized cell line HB4a. In order to identify transcripts differentially expressed between HB4a and HB4a-C5.2 cell lines, the MPSS (Massively Parallel Signature Sequencing) technique was used. A total of 24.521.236 MPSS tags, representing 24.065 unique reliable tags, was generated from both cell lines. After establishing reliable correlations between tags and known human transcripts, 9% of the tags could not be associated with a known transcripts (orphan tags). Among the orphan tags there were several that showed differential expression between HB4a and HB4a-C5.2 cell lines and probably represent novel transcripts regulated by c-erbB2. To further characterize some of these novel transcripts, the GLGI-MPSS (Generation of Longer cDNA fragments for Gene Identification) technique was developed in our laboratory and 83 orphan tags were converted into 3' cDNA fragments. The amplification of a dominant band was observed for 41 of 83 GLGI-MPSS amplifications, when analyzed in agarose gel. These 41 fragments were cloned and sequenced and the sequences were analyzed using BLAST to identify similarities with human transcripts submitted to public databases. The analysis of these fragments allowed the identification of 10 novel transcripts, putatively regulated by c-erB2, 3 polymorphic transcripts in which the presence of a SNP generated an MPSS alternative tag, 2 alternative polyadenylation isoforms of known transcripts and artefactual MPSS tags. GLGIMPSS also allowed us to identify 5 antisense transcripts from which 4 were further validated by strand specific RT-PCR. Differential expression of the 10 transcripts putatively regulated by c-erbB2, and of the 3 transcripts in which the presence of a SNP generated an MPSS alternative tag was evaluated by Real Time PCR in the HB4a and HB4a-C5.2 cell lines. Differential expression between the two cell lines was confirmed for 5 out of the 13 transcripts analyzed. Four out of the 5 validated transcripts were shown to be over-expressed in the HB4a-C5.2 cell line. Next, we examined, by Real Time PCR, the expression pattern of the 4 transcripts over-expressed in the HB4a-C5.2 cell line in breast tumor cell lines and breast tumor samples, over -expressing or not c-erbB2. Over-expression of the transcripts corresponding to tags 6 and 28 was observed in the majority of the breast tumor cell lines over-expressing c-erbB2. On the other hand, in the experiments carried out using tumor samples, we observed that the average expression level of these 4 transcripts, in relation to normal breast tissue, was higher in the c-erbB2 positive group and that the difference observed for tags 7 and 28 was statistically significant. These results confirm the differential expression of these 4 transcripts in c-erbB2 positive tumors and their functional characterization will allow us to better understand the molecular mechanisms behind c-erbB2 over-expression and will eventually open new perspectives in the management and treatment of breast cancer patients (AU)
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| Descritores: |
Cromossomos
Expressão Gênica
GENES ERBB-TEMEFOS
Linhagem Celular Tumoral
Neoplasias da Mama
Oncogenes
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-/imunologia
GENES ERBB-TEMEFOS/imunologia
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| Responsável: |
BR30.1 - Biblioteca |
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BR30.1 |
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