RESUMO
A genome-wide case-control association study done in our laboratory has identified a single nucleotide polymorphism located in RAD21 as being significantly associated with breast cancer susceptibility. RAD21 is believed to function in sister chromatid alignment as part of the cohesin complex and also in double-strand break (DSB) repair. Following our initial finding, expression studies revealed a 1.25- to 2.5-fold increased expression of this gene in several human breast cancer cell lines as compared with normal breast tissue. To determine whether suppression of RAD21 expression influences cellular proliferation, RNA interference technology was used in breast cancer cell lines MCF-7 and T-47D. Proliferation of cells treated with RAD21-specific small inhibitory RNA (siRNA) was significantly reduced as compared with mock-transfected cells and cells transfected with a control siRNA (Lamin A/C). This inhibition of proliferation correlated with a significant reduction in the expression of RAD21 mRNA and with an increased level of apoptosis. Moreover, MCF-7 cell sensitivity to two DNA-damaging chemotherapeutic agents, etoposide and bleomycin, was increased after inhibition of RAD21 expression with a dose reduction factor 50 (DRF50) of 1.42 and 3.71, respectively. At the highest concentrations of etoposide and bleomycin administered, cells transfected with a single siRNA duplex targeted against RAD21 showed 57% and 60% survival as compared with control cells, respectively. Based on these findings, we conclude that RAD21 is a novel target for developing cancer therapeutics that can potentially enhance the antitumor activity of chemotherapeutic agents acting via induction of DNA damage.
Assuntos
Bleomicina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Mama/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Dano ao DNA , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Predisposição Genética para Doença , Genoma , Humanos , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.
Assuntos
Fragmentação do DNA/genética , DNA/análise , Genes/genética , Variação Genética/genética , Glicoproteínas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Compostos Organofosforados/química , Análise de Sequência de DNA/métodos , Regiões 3' não Traduzidas/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transferência de Ésteres de Colesterol , Genótipo , Humanos , Proteínas I-kappa B/genética , Canais Iônicos , Proteínas de Membrana Transportadoras/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , Polimorfismo de Nucleotídeo Único/genética , Proteína Desacopladora 2RESUMO
The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.