RESUMEN
Direct proliferative effects of estrogen (E(2)) on estrogen receptor-positive tumors are well documented; however, the potential for E(2) to mediate effects selective for the host (i.e., angiogenesis, vascular permeability, or stromal effects), which influence tumor growth and/or metastasis, has received less attention. In this study, we examine the capacity for E(2) to promote tumor growth and/or metastasis independent of direct effects on tumor cells. In these studies, we distinguish host versus tumor compartment components of E(2) action in tumor growth and metastasis by analysis of E(2)-nonresponsive tumor cells implanted in ovariectomized (OVX) mice that contain s.c. implants of placebo (OVX) or E(2)-containing slow-release pellets (OVX + E(2)). We show that the D121 lung carcinoma cell line is E(2)-nonresponsive, and following s.c. implantation in OVX versus OVX + E(2) mice, E(2) action on the host compartment leads to an increase in spontaneous metastasis but not primary tumor growth or neovascularization. Similarly, experimental lung metastasis of E(2)-nonresponsive 4T1 mammary carcinoma cells also leads to increased tumor burden in the lungs of OVX + E(2) mice. These results suggest that the E(2) status of the host compartment influences late steps in tumor cell metastasis that can provide important insights into the role of E(2) in the tumor versus host compartments.
Asunto(s)
Neoplasias de la Mama/patología , Estradiol/toxicidad , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Neoplasias Hormono-Dependientes/secundario , Animales , Neoplasias de la Mama/irrigación sanguínea , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Receptor alfa de Estrógeno/biosíntesis , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Neoplasias Mamarias Experimentales/irrigación sanguínea , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Hormono-Dependientes/irrigación sanguínea , Neoplasias Hormono-Dependientes/inducido químicamente , Neoplasias Hormono-Dependientes/patología , Neovascularización Patológica/patologíaRESUMEN
Malignant brain tumors, such as glioblastoma, are characterized by extensive angiogenesis and permeability of the blood-brain barrier (BBB). The infiltration of glioma cells away from the primary tumor mass is a pathological characteristic of glial tumors. The infiltrating tumor cells represent a significant factor in tumor recurrence following surgical debulking, radiation, and chemotherapy treatments. Vascular endothelial growth factor (VEGF)-mediated vascular permeability (VP) has been associated with the progression of glioma tumor growth and infiltration into surrounding normal brain parenchyma. While VEGF induces a robust VP response in control mice (src+/+ or src+/-), the VP response is blocked in src-/- mice that demonstrate a 'leakage-resistant phenotype' in the brain. We used the Src-deficient mouse model to determine the role of Src in the maintenance of the BBB following orthotopic implantation and growth of glioma cells in the brain. Although solid tumor growth was the same in control and src-/- mice, the infiltrating component of glioma growth was reduced in src-/- mice. Characterization of the expression and localization of the extracellular matrix (ECM) protein fibrinogen was evaluated to determine the effect of a Src-mediated VP defect in the host compartment. These studies indicate that the reduced VP of host brain blood vessels of src-/- mice mediates a reduction in glioma cell invasion in a mouse brain tumor xenograft model.
Asunto(s)
Neoplasias Encefálicas/patología , Permeabilidad Capilar/fisiología , Glioma/patología , Invasividad Neoplásica/patología , Familia-src Quinasas/deficiencia , Animales , Barrera Hematoencefálica/fisiología , Neoplasias Encefálicas/enzimología , Línea Celular Tumoral , Circulación Cerebrovascular/fisiología , Fibrinógeno/metabolismo , Técnica del Anticuerpo Fluorescente , Glioma/enzimología , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Trasplante de Neoplasias , Neovascularización Patológica/enzimología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Signaling through the ErbB family of tyrosine kinase receptors in normal and cancer-derived cell lines contributes to cell growth and differentiation. In this work, we altered the levels of ErbB2 and ErbB3 receptors, individually and in combination, by using 6-finger and 12-finger synthetic zinc finger protein artificial transcription factors (ATFs) in an epidermoid squamous cell carcinoma line, A431. We successfully designed 12-finger ATFs capable of coregulating ErbB3 and ICAM-1 or ErbB2 and ErbB3. With ATFs, the effects of changes in ErbB2 and ErbB3 receptor levels were evaluated by using cell proliferation, cell migration, and cell signaling assays. Cell proliferation was increased when ErbB2 and ErbB3 were both overexpressed. Cell migration on collagen was decreased when ErbB2 was down-regulated, yet migration on laminin was significantly increased with ErbB3 overexpression. ErbB2 and ErbB3 overexpression also stimulated the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Our ATF approach has elucidated differences in ErbB receptor-mediated proliferation, migration, and intracellular signaling that cannot be explained merely by the presence or absence of particular ErbB receptors and emphasizes the dynamic nature of the ErbB signaling system. The transcription factor approach developed here provides a gene-economical route to the regulation of multiple genes and may be important for complex gene therapies.
Asunto(s)
Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Diseño de Fármacos , Regulación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Ingeniería de Proteínas , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Transducción de Señal , Factores de Transcripción/síntesis química , Factores de Transcripción/química , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.
Asunto(s)
Regulación de la Expresión Génica , Ingeniería de Proteínas/métodos , Factores de Transcripción/química , Dedos de Zinc , Secuencia de Aminoácidos , Animales , ADN/química , Desoxirribonucleasa I/metabolismo , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Biblioteca de Genes , Marcación de Gen , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligonucleótidos/química , Biblioteca de Péptidos , Regiones Promotoras Genéticas , Conformación Proteica , Estructura Terciaria de Proteína , Receptor ErbB-2/metabolismo , Retroviridae/genética , Programas Informáticos , Regulación hacia ArribaRESUMEN
Regulation of endogenous gene expression has been achieved using synthetic zinc finger proteins fused to activation or repression domains, zinc finger transcription factors (TFZFs). Two key aspects of selective gene regulation using TFZFs are the accessibility of a zinc finger protein to its target DNA sequence and the interaction of the fused activation or repression domain with endogenous proteins. Previous work has shown that predicting a biologically active binding site at which a TF(ZF) can control gene expression is not always straightforward. Here, we used a library of preassembled three-finger zinc finger proteins (ZFPs) displayed on filamentous phage, and selected for ZFPs that bound along a 1.4 kb promoter fragment of the human ErbB-2 gene. Following affinity selection by phage display, 13 ZFPs were isolated and sequenced. Transcription factors were prepared by fusion of the zinc finger proteins with a VP64 activation domain or a KRAB repression domain and the transcriptional control imposed by these TFZFs was evaluated using luciferase reporter assays. Endogenous gene regulation activity was studied following retroviral delivery into A431 cells. Additional ZFP characterization included DNaseI footprinting to evaluate the integrity of each predicted protein:DNA interaction. The most promising TFZFs able to both up-regulate and down-regulate ErbB-2 expression were extended to six-finger proteins. The increased affinity and refined specificity demonstrated by the six-finger proteins provided reliable transcriptional control. As a result of studies with the six-finger proteins, the specific region of the promoter most accessible to transcriptional control by VP64-ZFP and KRAB-ZFP fusion proteins was elucidated and confirmed by DNaseI footprinting, flow cytometric analysis and immunofluorescence. The ZFP phage display library strategy disclosed here, coupled with the growing availability of genome sequencing information, provides a route to identifying gene-regulating TFZFs without the prerequisite of well-defined promoter elements.
Asunto(s)
Regulación de la Expresión Génica , Dedos de Zinc , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genes erbB-2 , Regiones Promotoras GenéticasRESUMEN
In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.