RESUMO
Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25,000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.
Assuntos
Ilhas de CpG , Metilação de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular , Genes Neoplásicos , Genoma Humano , HumanosRESUMO
Amplification of the receptor tyrosine kinase ErbB2 is frequently observed in breast cancer. Amplification of erbB2 is also associated with multiple genomic gains and losses; however, the importance of these associated changes is largely unknown. We demonstrate that Brk, a cytoplasmic tyrosine kinase, is coamplified and coexpressed with ErbB2 in human breast cancers. ErbB2 interacts with Brk and increases its intrinsic kinase activity. Expression of Brk enhances the ErbB2-induced activation of Ras/MAPK signaling and cyclin E/cdk2 activity to induce cell proliferation of mammary 3-dimensional acini in culture. In a murine model of breast cancer, expression of Brk was found to shorten the latency of ErbB2-induced tumors by promoting cell proliferation, with no effect on protection from apoptosis. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation. Thus, we identified Brk as a drug target for ErbB2-positive cancers.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Amplificação de Genes/fisiologia , Neoplasias Mamárias Experimentais/patologia , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinases/fisiologia , Receptor ErbB-2/fisiologia , Quinases da Família src/fisiologia , Animais , Biópsia , Neoplasias da Mama/etiologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/análise , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/fisiologia , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética , Receptor ErbB-2/análise , Receptor ErbB-2/genética , Quinases da Família src/genéticaRESUMO
UNLABELLED: PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.edu