Modelos celulares e genéticos em tumores mamários de gata
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2009
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Os tumores espontâneos nos animais de companhia, e especificamente, no gato (Felis catus), têm sido apontados como os modelos possivelmente mais adequados para o estudo da carcinogénese. Porém, as amostras de tecido tumoral que representam o cancro in vivo, representam apenas um momento estanque da sua evolução e do seu processo de desenvolvimento. Surgem então as linhas celulares que reproduzem in vitro as características que os tumores apresentam in vivo. As linhas celulares exibem propriedades únicas como, a facilidade de propagação e de manipulação genética e a reprodutibilidade de resultados que as tornam num dos melhores modelos celulares actuais para o estudo da carcinogénese.
Um dos objectivos principais deste trabalho foi a estipulação de uma linha celular de tumor mamário de gata. Para a sua estipulação recorreu-se a diversa metodologia inerente ao processamento de cultura de células.
Uma vez que a importância causal de algumas alterações cromossómicas na carcinogénese está perfeitamente estabelecida no Homem, e comparativamente, os estudos citogenéticos e moleculares descritos em tumores mamários de animais de companhia, especialmente de gata são escassos, a caracterização destas alterações nos tumores mamários de gata é de extrema importância para a compreensão da base cromossómica deste tipo de tumor nesta espécie, fornecendo também um importante conhecimento dos tumores mamários humanos. Com o objectivo de caracterizar as alterações cromossómicas presentes na 15ª passagem da linha em estipulação (a primeira passagem onde foram detectadas alterações cromossómicas), foram utilizadas as técnicas de bandeamento-G e “Chromosome Painting”. É importante referir que a metodologia de “Chromosome Painting” nunca antes tinha sido aplicada para a caracterização de linhas celulares tumorais de Felis catus. A combinação de técnicas de citogenética clássica e molecular permitiu a identificação dos vários cromossomas de Felis catus, bem como a detecção e identificação de rearranjos cromossómicos, bem como dos cromossomas envolvidos nesses rearranjos. Esta informação permitiu a identificação de vários clones existentes na 15ª passagem da linha celular, com base no facto de que nas células tumorais, as anomalias cromossómicas adquiridas são consistentes e partilhadas por várias células sugerindo uma origem clonal.
Foi também pela primeira vez utilizado o “Comparative Genomic Hibridization” - CGH na caracterização de cinco carcinomas mamários de gata. Esta metodologia permitiu medir quantitativamente as alterações não equilibradas que ocorreram nas células destes tumores. Apresentou-se como uma valiosa metodologia para identificação de regiões no genoma padrão, que, quando alteradas a nível do número de sequências podem contribuir para a iniciação e/ou progressão de um tumor, nomeadamente, as regiões que apresentam um elevado número de cópias de uma sequência de DNA podem alojar oncogenes, e regiões que exibam um decréscimo no número de cópias de uma sequência de DNA podem conter genes supressores de tumores. A aplicação desta técnica em combinação com análises in silico em diversas bases de dados, permitiu localizar regiões que apresentavam alterações no número de cópias de sequências de DNA nos genomas tumorais em estudo, e através da extrapolação das regiões sinténicas entre a espécie Homo sapiens e a espécie em estudo, foi possível estabelecer uma correlação entre as anomalias verificadas e as sequências possivelmente envolvidas nessas regiões, nomeadamente a existência de genes críticos de cancro.
A caracterização de anomalias cromossómicas presentes nos genomas de tumores mamários de gata será de extrema relevância não só para o conhecimento dos tumores mamários de gata, mas fundamentalmente porque proporcionará conhecimento fundamental sobre a etiologia e o comportamento molecular das células cancerosas em tumores mamários humanos, ao nível de programas de oncologia comparada.
Spontaneous tumors in pet animals, and specifically in the cat (Felis catus), have been pointed as, probably, the most suitable models for the study of carcinogenesis. However, the samples of in vivo tumoral tissue are no more than the representation of a moment of the tumor evolution and of its development process. Cell lines arise as the possibility to create in vitro characteristics of in vivo tumors. Cell lines exhibit unique features as, readiness of propagation and genetic manipulation, and reproducibility of results, what make them one of the best cell models in the study of carcinogenesis, presently. One of the main objectives of this work was the stipulation of a cat mammary tumor cell line. For this purpose different methodologies inherent to the cell culture practice were used. Since the causal implication of some chromosome alterations in carcinogenesis is completely established in human, and, comparatively, cytogenetic and molecular studies described in mammary tumors of pets, and specially in cat are few, the characterization of these chromosome alterations is of primordial importance in the knowledge of the chromosomal basis of this type of tumor in Felis catus, consequently allowing an important understanding of human mammary tumors. With the aim of characterizing the chromosome alterations of the 15th passage of the cell line in stipulation (the first passage with noticeable chromosome alterations), G-banding and Chromosome Painting were applied. It’s important to notice that this molecular cytogenetics tool was used here for the first time in the characterization of a cat mammary tumor cell line. The combination of methodologies of classic and molecular cytogenetics allowed the identification of the cat chromosomes (and thus the constitution of karyotypes), the detection of chromosome rearrangements, as well as the identification of the chromosomes involved in these rearrangements. This information allowed the identification of the different clones present at the 15th passage of the cell line, based on the fact that in the tumor cells, the acquired chromosome abnormalities are consistent and shared by several cells, suggesting a clonal origin. Also for the first time was used Comparative Genomic Hibridization - CGH in the genetic characterization of five cat mammary carcinomas. This methodology permitted to quantify the non-balanced alterations occurring in the cells of these tumors, presenting a valuable tool in the identification of regions in the standard genome that, when altered in the sequence’s copy number may contribute to the initiation and/or progression of a tumor, namely, the regions presenting an increased DNA sequence copy number might enclose oncogenes, and regions with a decrease in the number of DNA sequences, might contain tumor suppressor genes. The application of this technique in combination with in silico analysis of several databases, allowed to annotate chromosome regions with DNA sequences copy number variations in the tumor’s genomes under investigation. Through the extrapolation of syntenic regions between the index species Homo sapiens and the species here in study, it was possible to establish a correlation between the identified abnormalities and the putative sequences in these regions, namely, the existence of critical genes in cancer. The characterization of chromosome abnormalities in the genomes of cat mammary tumors will be of major importance not only for the understanding of these tumors, but also and importantly, because it will provide fundamental knowledge about the etiology and molecular behavior of cancer cells in human mammary tumors.
Spontaneous tumors in pet animals, and specifically in the cat (Felis catus), have been pointed as, probably, the most suitable models for the study of carcinogenesis. However, the samples of in vivo tumoral tissue are no more than the representation of a moment of the tumor evolution and of its development process. Cell lines arise as the possibility to create in vitro characteristics of in vivo tumors. Cell lines exhibit unique features as, readiness of propagation and genetic manipulation, and reproducibility of results, what make them one of the best cell models in the study of carcinogenesis, presently. One of the main objectives of this work was the stipulation of a cat mammary tumor cell line. For this purpose different methodologies inherent to the cell culture practice were used. Since the causal implication of some chromosome alterations in carcinogenesis is completely established in human, and, comparatively, cytogenetic and molecular studies described in mammary tumors of pets, and specially in cat are few, the characterization of these chromosome alterations is of primordial importance in the knowledge of the chromosomal basis of this type of tumor in Felis catus, consequently allowing an important understanding of human mammary tumors. With the aim of characterizing the chromosome alterations of the 15th passage of the cell line in stipulation (the first passage with noticeable chromosome alterations), G-banding and Chromosome Painting were applied. It’s important to notice that this molecular cytogenetics tool was used here for the first time in the characterization of a cat mammary tumor cell line. The combination of methodologies of classic and molecular cytogenetics allowed the identification of the cat chromosomes (and thus the constitution of karyotypes), the detection of chromosome rearrangements, as well as the identification of the chromosomes involved in these rearrangements. This information allowed the identification of the different clones present at the 15th passage of the cell line, based on the fact that in the tumor cells, the acquired chromosome abnormalities are consistent and shared by several cells, suggesting a clonal origin. Also for the first time was used Comparative Genomic Hibridization - CGH in the genetic characterization of five cat mammary carcinomas. This methodology permitted to quantify the non-balanced alterations occurring in the cells of these tumors, presenting a valuable tool in the identification of regions in the standard genome that, when altered in the sequence’s copy number may contribute to the initiation and/or progression of a tumor, namely, the regions presenting an increased DNA sequence copy number might enclose oncogenes, and regions with a decrease in the number of DNA sequences, might contain tumor suppressor genes. The application of this technique in combination with in silico analysis of several databases, allowed to annotate chromosome regions with DNA sequences copy number variations in the tumor’s genomes under investigation. Through the extrapolation of syntenic regions between the index species Homo sapiens and the species here in study, it was possible to establish a correlation between the identified abnormalities and the putative sequences in these regions, namely, the existence of critical genes in cancer. The characterization of chromosome abnormalities in the genomes of cat mammary tumors will be of major importance not only for the understanding of these tumors, but also and importantly, because it will provide fundamental knowledge about the etiology and molecular behavior of cancer cells in human mammary tumors.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Citogenética , Neoplasias mamárias , Carcinogénese , Modelos genéticos , Modelos celulares