RESUMEN

In medical imaging, clinicians, researchers and technicians have begun to use 3D printing to create specialized phantoms to replace commercial ones due to their customizable and iterative nature. Presented here is the design of a 3D printed open source, reusable magnetic resonance imaging (MRI) phantom, capable of flood-filling, with removable samples for measurements of contrast agent solutions and reference standards, and for use in evaluating acquisition techniques and image reconstruction performance. The phantom was designed using SolidWorks, a computer-aided design software package. The phantom consists of custom and off-the-shelf parts and incorporates an air hole and Luer Lock system to aid in flood filling, a marker for orientation of samples in the filled mode and bolt and tube holes for assembly. The cost of construction for all materials is under $90. All design files are open-source and available for download. To demonstrate utility, B0 field mapping was performed using a series of gadolinium concentrations in both the unfilled and flood-filled mode. An excellent linear agreement (R2>0.998) was observed between measured relaxation rates (R1/R2) and gadolinium concentration. The phantom provides a reliable setup to test data acquisition and reconstruction methods and verify physical alignment in alternative nuclei MRI techniques (e.g. carbon-13 and fluorine-19 MRI). A cost-effective, open-source MRI phantom design for repeated quantitative measurement of contrast agents and reference standards in preclinical research is presented. Specifically, the work is an example of how the emerging technology of 3D printing improves flexibility and access for custom phantom design.

RESUMEN

We conducted a genome-wide meta-analysis of cognitive empathy using the 'Reading the Mind in the Eyes' Test (Eyes Test) in 88,056 research volunteers of European Ancestry (44,574 females and 43,482 males) from 23andMe Inc., and an additional 1497 research volunteers of European Ancestry (891 females and 606 males) from the Brisbane Longitudinal Twin Study. We confirmed a female advantage on the Eyes Test (Cohen's d=0.21, P<2.2 × 10-16), and identified a locus in 3p26.1 that is associated with scores on the Eyes Test in females (rs7641347, Pmeta=1.58 × 10-8). Common single nucleotide polymorphisms explained 5.8% (95% CI: 4.5%-7.2%; P=1.00 × 10-17) of the total trait variance in both sexes, and we identified a twin heritability of 28% (95% CI: 13%-42%). Finally, we identified significant genetic correlation between the Eyes Test and anorexia nervosa, openness (NEO-Five Factor Inventory), and different measures of educational attainment and cognitive aptitude.

Asunto(s)

Empatía/genética , Empatía/fisiología , Adulto , Anciano , Anorexia Nerviosa/genética , Cognición/fisiología , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Trastornos Mentales/genética , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Factores Sexuales , Gemelos , Población Blanca/genética

RESUMEN

A molecular approach was employed to investigate stock structure in Siamese mud carp Henicorhynchus siamensis populations collected from 14 sites across mainland south-east Asia, with the major focus being the lower Mekong River basin. Spatial analysis of a mitochondrial DNA fragment (ATPase 6 and 8) identified four stocks in the Mekong River basin that were all significantly differentiated from a population in the nearby Khlong River, Thailand. In the Mekong River basin, populations in northern Lao People's Democratic Republic and northern Thailand represent two independent stocks, and samples from Thai tributaries group with those from adjacent Mekong sites above the Khone Falls to form a third stock. All sites below the Khone Falls constituted a single vast stock that includes Cambodia and the Mekong Delta in Vietnam. While H. siamensis is considered currently to undertake extensive annual migrations across the Mekong River basin, the data presented here suggest that natural gene flow may occur over much more restricted geographical scales within the basin, and hence populations may need to be managed at finer spatial scales than at the whole-of-drainage-basin level.

Asunto(s)

Carpas/fisiología , Explotaciones Pesqueras , Variación Genética , Ríos , Animales , Asia Sudoriental , Carpas/genética , Explotaciones Pesqueras/métodos , Genes Mitocondriales/genética , Genética de Población , Datos de Secuencia Molecular

Asunto(s)

Anemia Ferropénica/epidemiología , Pueblo Asiatico , Niño , Preescolar , Humanos , Grupos de Población , Instituciones Académicas , Salud Urbana

RESUMEN

p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Etopósido/farmacología , Rayos gamma , Humanos , Ratones , Modelos Biológicos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Factores de Tiempo , Transcripción Genética , Transfección , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba

RESUMEN

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

Asunto(s)

Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Fibronectinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes p53/genética , Terapia Genética/métodos , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/terapia , Factor de Crecimiento Transformador beta/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células Epiteliales/metabolismo , Humanos , Masculino , Modelos Genéticos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta1

RESUMEN

The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Transducción de Señal/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Células Cultivadas , Dermis/citología , Proteína 1 de la Respuesta de Crecimiento Precoz , Etopósido/farmacología , Fibroblastos/citología , Rayos gamma , Regulación Neoplásica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Glándulas Mamarias Animales/citología , Ratones , Ratones Noqueados , Neoplasias/fisiopatología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfohidrolasa PTEN , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Transducción de Señal/fisiología , Rayos Ultravioleta

RESUMEN

A methodology is described that allows the in vivo trapping of transcription factors to their target regulatory elements in multiple genes simultaneously. Cross-linking using formaldehyde is the first of several steps to isolate, purify, clone and characterize multiple gene promoter DNA fragments. The example that we use indicates that the TGF beta 1 gene is a direct target induced by Egr-1 in HT1080 cells that express constitutive Egr-1, thus explaining the growth retardation that follows Egr-1 expression. The genes identified using this procedure reflect the specific activities of Egr-1 at that moment in the cell and provide a method for confirmation of genes that are the direct targets of Egr-1 action.

Asunto(s)

Clonación Molecular/métodos , Proteínas de Unión al ADN/genética , Proteínas Inmediatas-Precoces , Factores de Transcripción/genética , Sitios de Unión , Neoplasias de la Mama/metabolismo , Reactivos de Enlaces Cruzados , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma , Formaldehído , Expresión Génica/efectos de los fármacos , Humanos , Técnicas de Inmunoadsorción , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Transcripción Genética , Activación Transcripcional , Transfección , Factor de Crecimiento Transformador beta/genética , Células Tumorales Cultivadas

RESUMEN

EGR-1, a transcription factor with important functions in the regulation of growth and differentiation, is highly expressed in brain. Previous studies have shown that EGR-1 suppresses the transformed phenotype. However, the expression and role of EGR-1 in human glioblastoma cells are not yet determined. In this study, we found that the basal expression of the EGR-1 protein is undetectable, but is inducible in four human glioblastoma cell lines. To determine EGR-1 functions, we re-expressed EGR-1 in human glioblastoma U251 cells and found that the secretion of transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin (FN) was greatly enhanced. Addition of anti-TGF-beta antibodies completely inhibited the secretion of PAI-1, but had little effect on secretion of FN, indicating that PAI-1 is under the control of EGR-1-induced TGF-beta1. An examination of the promoter of the FN gene revealed two EGR-1-binding sites between positions -75 and -52 and positions -4 and +14 that specifically bound EGR-1 in gel mobility shift experiments. Utilizing wild-type and mutant FN promoter/luciferase reporter genes, we demonstrated that EGR-1 positively regulated the activity of the FN gene. In addition, cell adhesion and migration were greatly increased in the EGR-1-expressing cells, and adhesion was reversed by addition of RGD-containing peptides. These results suggest that EGR-1 may regulate cell interaction with the extracellular matrix by coordinated induction of TGF-beta1, FN, and PAI-1 in human glioblastoma cells.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Fibronectinas/genética , Proteínas Inmediatas-Precoces , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Anticuerpos/farmacología , Sitios de Unión , Adhesión Celular , Movimiento Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Genes Reporteros , Glioblastoma , Humanos , Mutación , Oligopéptidos/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/inmunología , Células Tumorales Cultivadas

RESUMEN

Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility.

Asunto(s)

Actinas/metabolismo , Shigella flexneri/fisiología , Vinculina/fisiología , Animales , Western Blotting , Movimiento Celular , Sistema Libre de Células , Ratones , Microscopía Fluorescente , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Shigella flexneri/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Vinculina/farmacología

RESUMEN

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Asunto(s)

Factor de Crecimiento Epidérmico , Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Sitios de Unión , Línea Celular Transformada , Membrana Celular/metabolismo , Desarrollo Embrionario y Fetal , Proteínas Ligadas a GPI , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Proteínas de Neoplasias/genética , Fosfatidilinositol Diacilglicerol-Liasa , Conejos , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismo

RESUMEN

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

Asunto(s)

Apoptosis , Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de la radiación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Pruebas de Carcinogenicidad , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Ciclo Celular/efectos de la radiación , División Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Fragmentación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , MAP Quinasa Quinasa 4 , Proteína Quinasa 3 Activada por Mitógenos , Mutación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta

RESUMEN

Amphiregulin (Ar) and Cripto (Cr) are autocrine growth factors for mammary cells and both have been observed to exhibit high expression in human mammary tumors, in contrast with adjacent tissues. To investigate whether Ar and Cr play roles in the progression of mammary cell proliferation to unregulated growth and tumor formation, the levels of expression were examined in transgenic mice (TGM) that over-express several different oncogenes: MMTV-Polyoma virus middle T antigen (MMTV-PyMT), MMTV-c-ErbB2 (c-neu, HER2) and MT-hTGF alpha. These transgenic mice all produce mammary tumors but with different rates of progression. The levels of Ar were induced up to 10-fold in association with hyperplasia in 2 of the TGM. Cr overexpression was consistently observed in hyperplastic mammary glands in all the animal models, decreasing in overt tumors in 2 of the TGM models. In MMTV-PyMT mammary glands, the levels of Cr expression rose 7- to 10-fold in hyperplastic tissue and 25-fold the levels in tumors compared to age-matched transgene negative mice. Ar and especially Cr thus should have potential value as markers of preneoplastic change in mammary tissue.

Asunto(s)

Factor de Crecimiento Epidérmico , Glicoproteínas/genética , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Virus del Tumor Mamario del Ratón/genética , Glicoproteínas de Membrana , Proteínas de Neoplasias/genética , Anfirregulina , Animales , Antígenos Virales de Tumores/genética , Antineoplásicos/análisis , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , División Celular , Familia de Proteínas EGF , Femenino , Proteínas Ligadas a GPI , Genes erbB-2 , Glicoproteínas/análisis , Sustancias de Crecimiento/análisis , Humanos , Hiperplasia , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Proteínas de Neoplasias/análisis , Oncogenes , Poliomavirus/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Embarazo , Receptor ErbB-2/genética

RESUMEN

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

Asunto(s)

Transformación Celular Neoplásica , Proteínas de Unión al ADN/farmacología , Fibronectinas/biosíntesis , Fibrosarcoma/metabolismo , Proteínas Inmediatas-Precoces/farmacología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Factores de Transcripción/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Dedos de Zinc , Adhesión Celular/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligopéptidos/farmacología , Regiones Promotoras Genéticas , Transfección , Células Tumorales Cultivadas

RESUMEN

Cripto-1(Cr1) protein encoded by the tdgf1 gene, is a secreted growth factor that is expressed early in embryonic development and is re-expressed in some tumors of the breast and colon. During embryonic development, Cr1 is expressed in inner cell mass cells and the primitive streak, and later is restricted to the developing heart. To investigate the role of Cr1 during mouse development, mice were generated that contain a null mutation of both Cr1 genes, derived from homologous recombination in embryonic stem cells. No homozygous Cr1-/- mice were born, indicating that Cr1 is necessary for embryonic development. Embryos initiated gastrulation and some embryos produced mesoderm up to day E7.5. Increasingly aberrant morphogenesis gave rise to disordered neuroepithelium that failed to produce a recognizable neural tube, or head-fold. Although some biochemical markers of differentiating ectoderm, mesoderm and endoderm were expressed, all the cardiac-specific markers were absent from day E8.7 embryos: (&agr;)MHC, betaMHC, MLC2A, MLC2V and ANF, whereas they were expressed in wild-type embryos. The yolk sac and placental tissues continued development in the absence of the embryo until day E9.5 but lacked large yolk sac blood vessels. Chimeric mice were constructed by microinjection of double targeted Cr1(-/- )embryonic stem cells into normal C57BL/6 blastocysts. The Cr1 produced by the normal C57BL/6 cells fully rescued the phenotype of Cr1(-/-) cells, indicating that Cr1 protein acted in a paracrine manner. Cells derived from the embryo proliferated and migrated poorly and had different adhesion properties compared to wild type. Therefore, lethality in the absence of Cr1, likely resulted largely from defective precardiac mesoderm that was unable to differentiate into functional cardiomyocytes.

Asunto(s)

Factor de Crecimiento Epidérmico , Gástrula/fisiología , Sustancias de Crecimiento/fisiología , Corazón/embriología , Glicoproteínas de Membrana , Miocardio/citología , Proteínas de Neoplasias/fisiología , Alelos , Animales , Biomarcadores , Diferenciación Celular , Quimera/genética , Técnicas de Cultivo , Fibroblastos/citología , Genotipo , Sustancias de Crecimiento/genética , Homocigoto , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Proteínas de Neoplasias/genética , Neovascularización Fisiológica/genética , Fenotipo

RESUMEN

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Inmediatas-Precoces , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Sondas de ADN , Regulación hacia Abajo , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma/genética , Humanos , Fenotipo , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología

RESUMEN

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

Asunto(s)

Proteínas del Citoesqueleto/fisiología , Uniones Intercelulares/fisiología , Vinculina/fisiología , Sitios de Unión , Comunicación Celular , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales , Humanos , Proteínas de la Membrana/fisiología , Fosfoproteínas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , Vinculina/deficiencia , Vinculina/metabolismo , Proteína de la Zonula Occludens-1 , alfa Catenina

RESUMEN

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Asunto(s)

Adhesión Celular/fisiología , Transactivadores , Vinculina/fisiología , Actinas/fisiología , Animales , Cadherinas/fisiología , Carcinoma Embrionario/patología , Agregación Celular , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/fisiología , Técnica del Anticuerpo Fluorescente , Ratones , Organoides , Reología , Resistencia a la Tracción , Células Tumorales Cultivadas , Vinculina/deficiencia , alfa Catenina , beta Catenina

RESUMEN

The molecular events of cardiac lineage specification and differentiation are largely unknown. Here we describe the involvement of a growth factor with an EGF-like domain, Cripto-1 (Cr-1), in cardiac differentiation. During embryonic development, Cr-1 is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-1, we have generated Cr-1-negative embryonic stem (ES) cell lines by homologous recombination. The resulting double "knockout" ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing Cr-1 gene back into the Cr(-/-) cells. Furthermore, the lack of functional Cr-1 is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during differentiation. Differentiation into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-1 is essential for contractile cardiomyocyte formation in vitro.

Asunto(s)

Factor de Crecimiento Epidérmico , Proteínas Fetales , Regulación del Desarrollo de la Expresión Génica/fisiología , Corazón/embriología , Glicoproteínas de Membrana , Miocardio/citología , Proteínas de Neoplasias/fisiología , Células Madre/citología , Proteínas de Dominio T Box , Animales , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/genética , Marcación de Gen , Mesodermo/química , Ratones , Morfogénesis , Fibras Musculares Esqueléticas , Músculo Esquelético/citología , Contracción Miocárdica , Miocardio/química , Miogenina/genética , Cadenas Pesadas de Miosina/análisis , Cadenas Ligeras de Miosina/análisis , Cadenas Ligeras de Miosina/genética , Proteínas de Neoplasias/genética , Proteínas de Neurofilamentos/genética , Neuronas , ARN Mensajero/análisis , Células Madre/química , Factores de Transcripción/genética , Tretinoina/farmacología

RESUMEN

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

Asunto(s)

Movimiento Celular/fisiología , Vinculina/fisiología , Línea Celular , Humanos , Microscopía Confocal , Mutación , Transfección