RESUMEN
In this study, we report the existence of a communication system among human smooth muscle cells that uses mechanical forces to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca2+ waves, causing smooth muscle cells from healthy human donors to falsely perceive a much higher agonist dose than they actually received. This aberrant sensing of contractile agonist dose on stiffer matrices is completely absent in isolated smooth muscle cells, although the isolated cells can sense matrix rigidity. We show that the intercellular communication that enables this collective Ca2+ response in smooth muscle cells does not involve transport across gap junctions or extracellular diffusion of signaling molecules. Instead, our data support a collective model in which mechanical signaling among smooth muscle cells regulates their response to contractile agonists.
RESUMEN
Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The beta integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired beta tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine beta subunit genes, the nematode Caenorhabditis elegans expresses only one beta subunit (betapat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-betatail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the betapat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-betatail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that betapat-3 plays important roles in post-embryonic organogenesis and tissue function.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Gónadas/metabolismo , Cadenas beta de Integrinas , Integrinas/fisiología , Músculos/metabolismo , Actinas/química , Alelos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Movimiento Celular , Citoplasma/metabolismo , ADN Complementario/metabolismo , Epítopos , Genes Dominantes , Hemaglutininas/química , Integrinas/biosíntesis , Integrinas/genética , Datos de Secuencia Molecular , Mutación , Ovulación/genética , Fenotipo , Fenilalanina/química , Estructura Terciaria de Proteína , Transgenes , Tirosina/químicaRESUMEN
Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.
Asunto(s)
Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Neoplasias de la Mama/genética , Clonación Molecular , Quinasa 6 Dependiente de la Ciclina , Cartilla de ADN , Humanos , Mutagénesis , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinasas CDC2-CDC28 , Ciclo Celular/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Indoles/farmacología , Tamoxifeno/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
Glucocorticoids can induce a G1 arrest in the cell cycle progression of BDS1 rat hepatoma cells. In these cells, dexamethasone, a synthetic glucocorticoid, stimulated a rapid and selective increase in expression of the p21 cyclin-dependent kinase (CDK) inhibitor mRNA and protein and virtually abolished CDK2 phosphorylation of the retinoblastoma protein. Expression of the p27 CDK inhibitor, and other G1-acting cell cycle proteins, remained unaffected. Dexamethasone stimulated p21 promoter activity in a p53-independent manner that required functional glucocorticoid receptors. Transforming growth factor-beta, which also induced a G1 cell cycle arrest of the hepatoma cells, failed to elicit this response. Analysis of 5' deletions of the p21 promoter uncovered a glucocorticoid responsive region between nucleotides -1481 and -1184, which does not contain a canonical glucocorticoid response element but which can confer dexamethasone responsiveness to a heterologous promoter. Fine mapping of this region uncovered three distinct 50-60-base pair transcriptional elements that likely function as targets of glucocorticoid receptor signaling. Finally, ectopic expression of p21 had no effect on hepatoma cell growth in the absence of glucocorticoids but facilitated the ability of dexamethasone to inhibit cell proliferation. Thus, our results have established a direct transcriptional link between glucocorticoid receptor signaling and the regulated promoter activity of a CDK inhibitor gene that is involved in the cell cycle arrest of hepatoma cells.
Asunto(s)
Ciclinas/genética , Dexametasona/farmacología , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión/genética , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Neoplasias Hepáticas Experimentales/genética , Luciferasas/genética , Ratas , Eliminación de Secuencia , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000-0000(478563) defined a glucocorticoid responsive region within teh promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/ enhancer binding protein-alpha (C/EBP alpha). Wild type rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBP alpha and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBP alpha. Overexpression of C/EBP alpha in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBP alpha and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBP alpha gene expression.
Asunto(s)
Ciclinas/genética , Proteínas de Unión al ADN/fisiología , Neoplasias Hepáticas Experimentales/genética , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Animales , Proteínas Potenciadoras de Unión a CCAAT , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN/metabolismo , Dexametasona/farmacología , Fase G1 , Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/patología , Mutagénesis , RatasRESUMEN
Macrophages are phagocytic hematopoietic cells involved in several immune processes, but they are also present early in mammalian development and may participate in embryonic tissue remodeling. We have isolated and characterized a cell line, Py-YSA, from the mouse yolk sac. Py-YSA cells have several functional properties of macrophages, including uptake of acetylated low density lipoprotein and phagocytic capability. They express the murine macrophage markers F4/80 and Mac-1, and they express RNA for the c-fms receptor. Their expansion in culture requires fibroblast conditioned medium or exogenous monocyte-colony stimulating factor. Murine ES (embryonic stem) cell cultures that undergo in vitro differentiation recapitulate yolk sac development, and during this process cells arise that express both Mac-1 and F4/80 and morphologically resemble the Py-YSA cells. The kinetics and distribution pattern of the Mac-1+ cells during a time course of ES cell differentiation suggest that they originate in the blood islands, and that they subsequently leave the blood islands and disperse to tissue sites. Both F4/80 and Mac-1 are first expressed in primary cultures from day 9.5 yolk sacs. The Py-YSA cultured cells thus resemble embryonic tissue macrophages by several criteria, and they share a marker profile with a cell type found in yolk sacs and differentiating ES cells. Py-YSA cells will be a useful reagent for further understanding the role of embryonic tissue macrophages in development.
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Macrófagos/citología , Proteínas de la Membrana , Receptores de Lipoproteína , Células Madre/citología , Saco Vitelino/citología , Células 3T3 , Animales , Biomarcadores , Diferenciación Celular , Línea Celular , Células Cultivadas , Femenino , Macrófagos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Fagocitos , ARN , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Células Madre/metabolismo , Saco Vitelino/metabolismoRESUMEN
Role restructuring can be the key to maximizing efficiency, productivity, and operational effectiveness. The clinical nurse specialist role was restructured from a divisional project focus to a unit-based design to enhance the care of specific patient populations. The authors describe the process used to make this change, the outcomes achieved, and the lessons learned.
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Reestructuración Hospitalaria , Unidades Hospitalarias/organización & administración , Perfil Laboral , Enfermeras Clínicas/organización & administración , Eficiencia Organizacional , Humanos , Innovación Organizacional , Evaluación de Procesos y Resultados en Atención de Salud , Carga de TrabajoRESUMEN
PCA use is increasingly common in the Intensive Care Unit. The authors review current PCA research and make recommendations for the optimal use of PCA in the critically ill patient.
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Analgesia Controlada por el Paciente/enfermería , Traumatismo Múltiple/tratamiento farmacológico , Dolor Postoperatorio/tratamiento farmacológico , Analgesia Controlada por el Paciente/efectos adversos , Investigación en Enfermería Clínica , Cuidados Críticos , HumanosRESUMEN
Collaborative research teams are an attractive means of conducting nursing research in the clinical setting because of the many opportunities that collaboration can supply. These opportunities include a chance to: (1) network with other nurses who have similar interests, (2) share knowledge and expertise for designing clinical studies that directly affect daily practice, (3) develop instruments, (4) write grant proposals, (5) collect and analyze data, and (6) prepare manuscripts for publication. The effectiveness of research teams, however, is strongly influenced by group functioning. This article describes the functioning of a collaborative family interventions research team of nursing faculty members and CNSs at a large Midwestern university setting. The formation of the group and membership characteristics are described, along with strategies used to identify the research focus and individual and group goals. Aspects related to the influence of the group on members and the internal operations of the group are also addressed. Future strategies to be explored will focus on the size of the group and joint authorship issues. The authors also set forth a number of recommendations for development of collaborative research groups.