Caracterização citogenética e análise da expressão dos genes Mycn e Erbb2 da linha celular tumoral de Rattus norvegicus, CLS-ACI-1
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2014-12-11
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O cancro da mama é uma doença genética e celular de carácter multifatorial, na qual a desregulação génica desempenha um papel fulcral na sua iniciação e progressão. As linhas celulares de ratazana (RNO) têm, devido à similaridade mecanística da tumorigénese verificada entre Homem (HSA) e RNO, vindo a ser utilizadas como modelos em vários estudos de carácter fundamental e no desenvolvimento de novas terapias. A caracterização das linhas celulares usadas como modelos, é fundamental na seleção dos modelos celulares mais adequados para determinado estudo e para a interpretação correta de qualquer resultado.
Neste trabalho caracterizou-se citogeneticamente e ao nível da expressão dos genes Mycn e Erbb2 uma linha celular tumoral comercial de RNO, proveniente de um adenocarcinoma mamário, denominada CLS-ACI-1.
Os resultados obtidos mostram que a linha CLS-ACI-1 apresenta, pelo menos, três subclones e um complemento cromossómico quase triploide, caracterizado pela presença de alterações cromossómicas estruturais e numéricas. Alguns dos cromossomas alterados na linha CLS-ACI-1, encontram-se recorrentemente alterados noutras linhas tumorais de RNO e, em cromossomas homólogos, alterados no cancro da mama humano. Uma análise in silico utilizando várias bases de dados revelou a existência de 29 genes potencialmente afetados pelos "breakpoints" e que se encontram envolvidos em vários processos celulares (e.g. ciclo, proliferação, estabilidade genómica, vias WNT, RAS e MAPK).
Posteriormente analisou-se a expressão relativa e regulação epigenética dos oncogenes MYCN e ERBB2 (envolvidos na tumorigénese humana) por "Real-Time Reverse Transcription - quantitative" PCR na linha em estudo.
No que diz respeito ao gene Mycn, a análise revelou que este se encontra sobrexpresso na linha CLS-ACI-1 (181 vezes superior a uma linha normal). A sobrexpressão detetada pode ser explicada pela possível amplificação génica evidenciada pela caracterização citogenética e pela análise in silico dos "breakpoints" das alterações cromossómicas detetadas. No que diz respeito à sua regulação, este gene
não parece encontrar-se diretamente regulado por metilação das citosinas, à semelhança do que foi descrito em HSA e Mus musculus. No entanto, o aumento significativo observado após remoção da 5-azacitidina pode ser consequência de alterações ao nível da (des)metilação de genes envolvidos na regulação da expressão do Mycn.
Por sua vez o Erbb2 não apresentou na linha CLS-ACI-1 variação biologicamente significativa de expressão, o que não seria expectável pelos dados citogenéticos, que evidenciam o ganho de cópias do cromossoma onde este gene se localiza. Quando o genoma da linha CLS-ACI-1 foi globalmente desmetilado, verificou-se um aumento da expressão do gene, o que é indicativo de que o Erbb2 se encontrava transcripcionalmente suprimido, por metilação das citosinas; tal como em algumas linhas de HSA e MMU. Este aumento continuou após remoção da 5-azacitidina, o que é indicativo de mecanismos compensatórios na linha tumoral em análise.
Os resultados obtidos permitem concluir que a linha CLS-ACI-1 é um bom modelo para o estudo do cancro da mama, pois apresenta similaridade a nível citogenético com rearranjos cromossómicos recorrentes no cancro da mama humano. Esta linha é, também, um bom modelo para o estudo epigenético dos oncogenes Mycn e Erbb2, pois o seu perfil de regulação é similar ao descrito em HSA.
Breast cancer is a genetic and cellular disease characterized by a multi-factorial nature, being gene deregulation one of its widespread marks. Due to the mechanistic similarity verified between the tumoral process in human (HSA) and rat (RNO), cell lines derived from RNO tumours can be successfully applied in breast cancer fundamental research and new therapy development. For a cell line to reach its full potential as a model, it should be properly characterized, as that characterization enable the choice of the most appropriate cell lines to a specific study and the correct result interpretation. In this work, the commercial RNO tumour cell line CLS-ACI-1, derived from a breast adenocarcinoma, was cytogenetically characterized and an expression analysis of the Mycn and Erbb2 genes was also performed. Our results show that, at least, three sub-clones are present in the CLS-ACI-1 cell line. A near triploid chromosomal content was detected, in which several structural and numerical alterations were present. Some of these chromosomes are recurrently implicated in chromosomal alterations in RNO tumour cell lines, and those homologous HSA chromosomes, have been implied in human breast cancer. An in silico analysis was performed in several databases and it revealed that 29 genes were potentially affected by CLS-ACI-1's breakpoints. Those genes have been associated to several cellular processes (e.g. cycle, proliferation, genomic stability and WNT, RAS and MAPK pathways). Afterwards, the relative expression and the epigenetic regulation of MYCN and ERBB2 oncogenes (implied in human tumourigenesis) was analyzed by Real-Time Reverse Transcription - quantitative PCR (RT-qPCR), in the tumour cell line under study. The RT-qPCR analysis revealed that the gene Mycn presents a 181 times-fold expression increase in the cell line CLS-ACI-1. This expression increment can be explained by the possible gene amplification (indicated by the cytogenetic characterization) and by the deregulation of several regulatory genes (revealed by the in silico analysis). Mycn does not seems to be directly epigenetically regulated by DNA methylation. This lack of epigenetic regulation, through cytosine methylation, have been described in HSA and Mus musculus (MMU). The significant expression increase, detected 10 days after 5-azacytidine removal, can be due to (de)methylation changes of several genes implied in Mycn regulation. In the CLS-ACI-1 cell line, Erbb2 doesn't show significant biological expression variation. These results are the opposite of what was expected, as the cytogenetic results show a copy gain of the chromosome where Erbb2 is located. After CLS-ACI-1's global genome demethylation, a gene expression increase was detected. This indicates that Erbb2 is transcriptionally suppressed by DNA methylation, as described in HSA and MMU. Erbb2 gene up-fold expression was also verified after removal of 5-azacytidine, indicating the existence of compensatory mechanisms in CLS-ACI-1 cell line. The similarity of the chromosomal rearrangements detected in this cell line with those described in human breast cancer and the similar epigenetic regulation, of the Mycn and Erbb2 genes, with the epigenetic marks described in HSA, render the CLS-ACI-1 tumor cell line as a well suited in vitro model for human breast cancer research.
Breast cancer is a genetic and cellular disease characterized by a multi-factorial nature, being gene deregulation one of its widespread marks. Due to the mechanistic similarity verified between the tumoral process in human (HSA) and rat (RNO), cell lines derived from RNO tumours can be successfully applied in breast cancer fundamental research and new therapy development. For a cell line to reach its full potential as a model, it should be properly characterized, as that characterization enable the choice of the most appropriate cell lines to a specific study and the correct result interpretation. In this work, the commercial RNO tumour cell line CLS-ACI-1, derived from a breast adenocarcinoma, was cytogenetically characterized and an expression analysis of the Mycn and Erbb2 genes was also performed. Our results show that, at least, three sub-clones are present in the CLS-ACI-1 cell line. A near triploid chromosomal content was detected, in which several structural and numerical alterations were present. Some of these chromosomes are recurrently implicated in chromosomal alterations in RNO tumour cell lines, and those homologous HSA chromosomes, have been implied in human breast cancer. An in silico analysis was performed in several databases and it revealed that 29 genes were potentially affected by CLS-ACI-1's breakpoints. Those genes have been associated to several cellular processes (e.g. cycle, proliferation, genomic stability and WNT, RAS and MAPK pathways). Afterwards, the relative expression and the epigenetic regulation of MYCN and ERBB2 oncogenes (implied in human tumourigenesis) was analyzed by Real-Time Reverse Transcription - quantitative PCR (RT-qPCR), in the tumour cell line under study. The RT-qPCR analysis revealed that the gene Mycn presents a 181 times-fold expression increase in the cell line CLS-ACI-1. This expression increment can be explained by the possible gene amplification (indicated by the cytogenetic characterization) and by the deregulation of several regulatory genes (revealed by the in silico analysis). Mycn does not seems to be directly epigenetically regulated by DNA methylation. This lack of epigenetic regulation, through cytosine methylation, have been described in HSA and Mus musculus (MMU). The significant expression increase, detected 10 days after 5-azacytidine removal, can be due to (de)methylation changes of several genes implied in Mycn regulation. In the CLS-ACI-1 cell line, Erbb2 doesn't show significant biological expression variation. These results are the opposite of what was expected, as the cytogenetic results show a copy gain of the chromosome where Erbb2 is located. After CLS-ACI-1's global genome demethylation, a gene expression increase was detected. This indicates that Erbb2 is transcriptionally suppressed by DNA methylation, as described in HSA and MMU. Erbb2 gene up-fold expression was also verified after removal of 5-azacytidine, indicating the existence of compensatory mechanisms in CLS-ACI-1 cell line. The similarity of the chromosomal rearrangements detected in this cell line with those described in human breast cancer and the similar epigenetic regulation, of the Mycn and Erbb2 genes, with the epigenetic marks described in HSA, render the CLS-ACI-1 tumor cell line as a well suited in vitro model for human breast cancer research.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Citogenética , Cancro da mama , Linha celular tumoral , "breakpoints" cromossómicos , Expressão génica , Epigenética , Oncogenes (Mycn e Erbb2)