RESUMEN
The insulin-like growth factors (IGFs) have a central role in mammary gland growth and differentiation as well as in breast cancer development. The BRCA1 gene encodes a pleiotropic protein that functions as a transcription factor. Germline BRCA1 mutations are associated with inherited predisposition to breast and ovarian cancer and confer a substantially increased risk for developing these neoplasms. Several lines of evidence led us to hypothesize that there is a functional interaction between the BRCA1 and IGF-I systems relevant to breast cancer biology. The present study tested the notion that BRCA1 gene expression is regulated by the IGF-I signaling pathway. Results of Western immunoblotting and RT-PCR analyses show that IGF-I stimulates BRCA1 protein and mRNA levels. Transient transfection experiments using BRCA1 promoter-luciferase reporter constructs reveal that IGF-I enhances BRCA1 promoter activity, suggesting that the effect of IGF-I is mediated at the transcriptional level. In addition, we provide evidence that the Sp1 zinc-finger protein is directly involved in BRCA1 gene transactivation. Combined, our data suggests that, at least part of the biological actions of IGF-I in mammary gland cells may be mediated through BRCA1. Dysregulated BRCA1 expression resulting from aberrant IGF signaling may have important consequences relevant to breast cancer pathogenesis.
Asunto(s)
Proteína BRCA1/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor de Transcripción Sp1/genética , Activación Transcripcional/efectos de los fármacos , Proteína BRCA1/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Silenciador del Gen , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismoRESUMEN
The insulin-like growth factor I receptor (IGF-I-R) has an important role in breast cancer etiology. The receptor is overexpressed by most breast cancers, where it functions as a potent antiapoptotic agent. BRCA1 is a tumor suppressor gene that is mutated in a large fraction of familial breast and ovarian cancers. Cotransfection of Saos-2, MCF7, and CHO cells with IGF-I-R promoter constructs driving luciferase reporter genes, and with a BRCA1 expression vector, suppressed promoter activity in a dose-dependent manner. Functional interactions between BRCA1 and Sp1 in the regulation of the IGF-I-R gene were studied in Schneider cells, a Drosophila cell line which lacks endogenous Sp1. In these cells BRCA1 suppressed 45% of the Sp1-induced trans-activation of the IGF-I-R promoter. These results suggest that BRCA1 is capable of suppressing the IGF-I-R promoter in a number of cell lines, thus resulting in low levels of receptor mRNA and protein. Mutant versions of BRCA1 lacking trans-activational activity can potentially derepress the IGF-I-R promoter. Activation of the overexpressed receptor by locally produced or circulating IGFs may be a crucial step in breast and ovarian cancer progression.
Asunto(s)
Proteína BRCA1/metabolismo , Regiones Promotoras Genéticas , Receptor IGF Tipo 1/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Drosophila/citología , Regulación de la Expresión Génica , Humanos , Ratones , Unión Proteica , TransfecciónRESUMEN
Expression of the red+ and gam+ genes of bacteriophage lambda in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these lambda functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2'-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red+ or gam+ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam+ or red+ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (sigma-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red+ products, beta protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD- ExoI- cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3' single-stranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.