RESUMO
The CCAAT enhancer binding protein alpha (C/EBPalpha) is an important myeloid tumor suppressor that is frequently mutated in human acute myeloid leukemia (AML). We have previously shown that mice homozygous for the E2F repression-deficient Cebpa(BRM2) allele develop nonfatal AML with long latency and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6-driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa(+/+), Cebpa(+/BRM2), and Cebpa(BRM2/BRM2) mice, with respect to disease type, latency of tumor development, and identity of the retroviral insertion sites (RISs). Both Cebpa(+/BRM2) and Cebpa(BRM2/BRM2) mice preferentially develop myeloid leukemias, but with differing latencies, thereby demonstrating the importance of gene dosage. Determination of RISs led to the identification of several novel candidate oncogenes, some of which may collaborate specifically with the E2F repression-deficient allele of Cebpa. Finally, we used an in silico pathway analysis approach to extract additional information from single RISs, leading to the identification of signaling pathways which were preferentially deregulated in a disease- and/or genotype-specific manner.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Predisposição Genética para Doença , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Mutagênese Insercional , Mutação/genética , Retroviridae/genética , Alelos , Animais , Células Clonais , Biologia Computacional , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Genes Neoplásicos , Instabilidade Genômica , Imunoglobulinas/genética , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Lesões Pré-Cancerosas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Retroviridae/fisiologia , Latência ViralRESUMO
BACKGROUND: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. MATERIAL AND METHODS: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by beta-globin PCR. RESULTS: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non-amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. CONCLUSION: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples.
