RESUMEN

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Asunto(s)

Complejo Antígeno-Anticuerpo/química , Inmunoglobulina G/química , Simulación de Dinámica Molecular , Receptores de IgG/química , Regulación Alostérica , Complejo Antígeno-Anticuerpo/inmunología , Humanos , Inmunoglobulina G/inmunología , Receptores de IgG/inmunología

RESUMEN

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERß1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERß1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERß1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells. CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERß1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERß1 may not be the most important miR-92 target in breast cancer.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , MicroARNs/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Carcinogénesis/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Células Epiteliales/patología , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Captura por Microdisección con Láser , MicroARNs/genética , Persona de Mediana Edad

RESUMEN

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation.

Asunto(s)

Neoplasias de la Mama/patología , Mama/citología , Técnicas de Cultivo de Célula/métodos , Mama/enzimología , Neoplasias de la Mama/enzimología , Femenino , Humanos , Imagenología Tridimensional/métodos , Inmunohistoquímica , Radioinmunodetección/métodos , Receptor ErbB-2/biosíntesis , Receptor ErbB-3/biosíntesis

RESUMEN

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I-hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the -34- to -40-kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible cyclosporin A-sensitive enhancer at -37 kb that increased IL-3 promoter activity 40-fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells, and myeloid progenitor cells.

Asunto(s)

Elementos de Facilitación Genéticos/inmunología , Regulación del Desarrollo de la Expresión Génica/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-3/biosíntesis , Interleucina-3/genética , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Desoxirribonucleasa I/genética , Elementos de Facilitación Genéticos/genética , Sitios Genéticos/inmunología , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Distribución Tisular/genética , Distribución Tisular/inmunología

RESUMEN

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγ and MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E(2) levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.

Asunto(s)

Ciclooxigenasa 2/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasa C gamma/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Urotelio/metabolismo , Actinas/metabolismo , Western Blotting , Línea Celular Tumoral , Movimiento Celular/fisiología , Dinoprostona/metabolismo , Activación Enzimática/genética , Humanos , Análisis por Micromatrices , Mutagénesis Sitio-Dirigida , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The closely linked IL-3 and GM-CSF genes are located within a cluster of cytokine genes co-expressed in activated T cells. Their activation in response to TCR signaling pathways is controlled by specific, inducible upstream enhancers. To study the developmental regulation of this locus in T lineage cells, we created a transgenic mouse model encompassing the human IL-3 and GM-CSF genes plus the known enhancers. We demonstrated that the IL-3/GM-CSF locus undergoes progressive stages of activation, with stepwise increases in active modifications and the proportion of cytokine-expressing cells, throughout the course of T cell differentiation. Looking first at immature cells, we found that the IL-3/GM-CSF locus was epigenetically silent in CD4/CD8 double positive thymocytes, thereby minimizing the potential for inappropriate activation during the course of TCR selection. Furthermore, we demonstrated that the locus did not reach its maximal transcriptional potential until after T cells had undergone blast cell transformation to become fully activated proliferating T cells. Inducible locus activation in mature T cells was accompanied by noncoding transcription initiating within the enhancer elements. Significantly, we also found that memory CD4 positive T cells, but not naive T cells, maintain a remodeled chromatin structure resembling that seen in T blast cells.

Asunto(s)

Diferenciación Celular/inmunología , Epigénesis Genética/inmunología , Silenciador del Gen/inmunología , Sitios Genéticos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-3/genética , Activación de Linfocitos/genética , Subgrupos de Linfocitos T/inmunología , Animales , Diferenciación Celular/genética , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Citocinas/biosíntesis , Desoxirribonucleasa I/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Memoria Inmunológica/genética , Interleucina-3/biosíntesis , Ratones , Ratones Transgénicos , Familia de Multigenes/inmunología , ARN/biosíntesis , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismo , Activación Transcripcional/inmunología

RESUMEN

Medulloblastomas are highly malignant, poorly differentiated childhood tumours arising in the cerebellum. These tumors rarely lose TP53, which is the most commonly mutated gene in cancer. Recent work has shown that the basal level of p53 plays an important role in maternal reproduction by maintaining the expression of LIF in the uterus. Since LIF can maintain the undifferentiated state of stem cells we set out to ask if p53 regulates LIF in the human medulloblastoma cell lines DAOY and D283MED. We also used p53-/- and p53+/+ isogenic HCT116 colorectal carcinoma cell lines, already reported to exhibit p53-dependent expression of the LIF D transcript, to establish the extent of p53-dependency for LIF M and T alternative transcripts. Whilst all three known, full-length alternative transcripts are more abundant in p53+/+ cells, the alternative LIF M and T transcripts appear particularly sensitive to p53. In the p53 wild-type medulloblastoma cell line D283MED chromatin immunoprecipitation experiments showed p53 binding to the LIF gene. The mutant p53 expressed in line DAOY did not bind to this region or to the p21(WAF1) p53 binding site. RNA interference against either WIP1 or SIRT1 stabilized p53 and enhanced the transcription of LIF in D283MED cells. Interestingly, siRNA against WIP1 or SIRT1 also induced increased apoptosis in the medulloblastoma line D283MED and, over a longer time period, in DAOY cells. We speculate that suppression of p53 function by combined WIP1-mediated dephosphorylation and SIRT1 deacetylation enables medulloblastoma cell survival but p53-dependent and independent apoptotic pathways remain intact. Thus small molecule inhibitors of SIRT1 may be useful in treatment of medulloblastoma.

Asunto(s)

Apoptosis/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Factor Inhibidor de Leucemia/metabolismo , Meduloblastoma/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Citometría de Flujo/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica/genética , Proteína Fosfatasa 2C , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Transfección/métodos , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética

RESUMEN

The human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating-factor (GM-CSF, or CSF2) gene cluster arose by duplication of an ancestral gene. Although just 10 kb apart and responsive to the same signals, the IL-3 and GM-CSF genes are nevertheless regulated independently by separate, tissue-specific enhancers. To understand the differential regulation of the IL-3 and GM-CSF genes we have investigated a cluster of three ubiquitous DNase I-hypersensitive sites (DHSs) located between the two genes. We found that each site contains a conserved CTCF consensus sequence, binds CTCF, and recruits the cohesin subunit Rad21 in vivo. The positioning of these sites relative to the IL-3 and GM-CSF genes and their respective enhancers is conserved between human and mouse, suggesting a functional role in the organization of the locus. We found that these sites effectively block functional interactions between the GM-CSF enhancer and either the IL-3 or the GM-CSF promoter in reporter gene assays. These data argue that the regulation of the IL-3 and the GM-CSF promoters depends on the positions of their enhancers relative to the conserved CTCF/cohesin-binding sites. We suggest that one important role of these sites is to enable the independent regulation of the IL-3 and GM-CSF genes.

Asunto(s)

Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Elementos Aisladores/genética , Interleucina-3/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Huella de ADN , Metilación de ADN , Desoxirribonucleasas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Elementos de Facilitación Genéticos , Humanos , Células Jurkat , Ratones , Datos de Secuencia Molecular , Unión Proteica , Cohesinas

RESUMEN

We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer.

Asunto(s)

Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos/genética , Factores de Transcripción GATA/metabolismo , Factores de Transcripción NFATC/metabolismo , Nucleosomas/metabolismo , Elementos de Respuesta/genética , Acetilación , Animales , Emparejamiento Base/genética , Secuencia de Bases , Desoxirribonucleasas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Células HeLa , Humanos , Células Jurkat , Células K562 , Mastocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Células Mieloides/metabolismo , Nucleosomas/genética , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Linfocitos T/metabolismo

RESUMEN

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the approximately 130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1alpha (HNF-1alpha) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1alpha/HNF-4 signaling cascade and the serpin LCR.

Asunto(s)

Cromosomas Humanos Par 14 , Histonas/metabolismo , Región de Control de Posición , Serpinas/genética , Acetilación , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 4 del Hepatocito , Humanos , Células Híbridas , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Ratas , Eliminación de Secuencia , Factores de Transcripción/fisiología

RESUMEN

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19(ARF)-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently pro-oncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFbeta indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

Asunto(s)

Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3 , Animales , Transformación Celular Neoplásica/genética , Senescencia Celular/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Ratones , Proteínas Proto-Oncogénicas/genética , Fase S/genética , Fase S/fisiología , Factores de Transcripción/genética