RESUMEN

Receptor-associated coactivator 3 (RAC3) is a nuclear receptor coactivator usually overexpressed in tumors that exerts oncogenic functions in the cytoplasm and the nucleus. Although as part of its oncogenic actions it was previously identified as an inhibitor of apoptosis and autophagy, its expression is required in order to preserve the pluripotency and embryonic stem cell self-renewal. In this work we investigated its role in cellular senescence. We found that RAC3 overexpression in the nontumoral HEK293 cells inhibits the premature senescence induced by hydrogen peroxide or rapamycin. The mechanism involves not only the inhibition of autophagy early induced by these stimuli in the pathway to senescence, but also the increase in levels and nuclear localization of both the cell cycle suppressors p53/p21 and the longevity promoters FOXO1A, FOXO3A and SIRT1. Furthermore, we found that RAC3 overexpression is required in order to maintain the telomerase activity. In tumoral HeLa cells its activity was inhibited by depletion of RAC3 inducing replicative senescence. Moreover, we demonstrated that in vivo, levels of RAC3 are downregulated in the liver from aged as compared with young rats, whereas the levels of p21 are increased, correlating with the expected senescent cell contents in aged tissues. A similar downregulation of RAC3 was observed in the premature and replicative senescence of human fetal WI-38 cells and premature senescence of hepatocyte HepG2 cell line. Taken together, all these results demonstrate that RAC3 is an inhibitor of senescence whose downregulation in aged individuals could be probably a tumor suppressor mechanism, avoiding the clonal expansion of risky old cells having damaged DNA.

Asunto(s)

Proteínas de Unión al GTP rac/fisiología , Envejecimiento , Animales , Proliferación Celular , Senescencia Celular , Regulación hacia Abajo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Ratas Wistar , Sirolimus/farmacología

RESUMEN

The neuronal cytoskeleton regulates numerous processes that occur in normal homeostasis. Under pathological conditions such as those of Alzheimer's disease (AD), major alterations in cytoskeleton organization have been observed and changes in both microtubules and actin filaments have been reported. Many neurodegenerative consequences of AD are linked to the production and accumulation of amyloid peptides (Aß) and their oligomers, produced from the internal cleavage of the amyloid-ß protein precursor. We previously reported that fibrillar Aß1-42 (fAß) treatment of hippocampal neurons induced an increase in Rac1 and Cdc42 activities linking fAß effects with changes in actin dynamics. Here we show fAß-induces increased activity of PAK1 and cyclin-dependent kinase 5, and that p21-activated kinase (PAK1) activation targets the LIMK1-cofilin signaling pathway. Increased cofilin dephosphorylation under conditions of enhanced LIM-Kinase 1 (LIMK1) activity suggests that fAß co-stimulates bifurcating pathways impacting cofilin phosphorylation. Overexpression of slingshot (SSH) prevents the augment of F-actin induced by fAß after 24 h, suggesting that fAß-induced changes in actin assembly involve both LIMK1 and SSH. These results suggest that fAb may alter the PAK1/LIMK1/cofilin axis and therefore actin organization in AD.

Asunto(s)

Factores Despolimerizantes de la Actina/metabolismo , Actinas/fisiología , Péptidos beta-Amiloides/fisiología , Amiloide/fisiología , Neuropéptidos/fisiología , Fragmentos de Péptidos/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/fisiología , Animales , Células Cultivadas , Ratones , Ratones Transgénicos , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteína de Unión al GTP rac1

RESUMEN

RAC3 has been firstly characterized as a nuclear receptor coactivator that is found in limited amounts in normal cells, but is over-expressed in tumors and is also an NF-kB coactivator. Although the mechanisms involved in its over-expression are not clear, it is well known that it enhances resistance to apoptosis. In this work, we investigated if there are any additional mechanisms by which RAC3 may contribute to tumor development and if TNF-a, an inflammatory cytokine that is found at high levels in cancer could increase RAC3 levels. We found that enhancement of RAC3 levels by transfection of HEK293 cells with a RAC3 expression vector induces a significant increase of cell proliferation not only in the presence, but also in the absence of serum growth factors. Moreover, the cells were transformed showing an anchorage independent growth, similar to that observed in tumoral cells. The treatment of HEK293 cells with TNF-a induced an increase in the protein levels of RAC3 and this was blocked by an NF-kB specific inhibitor, suggesting that this transcription factor is involved in the cytokine effect. We conclude that RAC3, in addition to is anti-apoptotic action, is a transforming factor that promotes the proliferation and growth independent of anchorage, and that its levels could be elevated by the action of inflammatory cytokines that are involved in the anti-tumoral response.

Asunto(s)

Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rac/fisiología , Células HEK293 , Humanos , Factores de Transcripción/efectos de los fármacos , Transfección/métodos , Proteínas de Unión al GTP rac/análisis

RESUMEN

RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kappaB coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kappaB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

Asunto(s)

Apoptosis/fisiología , Transformación Celular Neoplásica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Factores de Transcripción/fisiología , Proteínas de Unión al GTP rac/fisiología , Humanos , Riñón/citología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Receptores Citoplasmáticos y Nucleares

RESUMEN

RAC3 pertenece a la familia de coactivadores de receptores nucleares p160, y se encuentra sobreexpresado en varios tumores. Demostramos previamente que RAC3 es coactivador del factor de transcripción anti-apoptótico NF-kB. En este trabajo investigamos su rol en la apoptosis inducida por H2O2 en una línea celular no tumoral derivada de riñón embrionario humano (HEK293), y por el ligando inductor de apoptosis relacionado a TNF (TRAIL) en una línea de leucemia mieloide crónica humana (K562), naturalmente resistente a la muerte por este estímulo. Observamos que las células tumorales K562 poseen niveles altos de RAC3 comparados con las células no tumorales HEK293. La sobreexpresión normal de coactivador o por transfección, inhibe la apoptosis mediante una disminución de la activación de caspasas, translocación del factor inductor de apoptosis (AIF) al núcleo, aumento de la actividad de NF-kB y las quinasas AKT y p38 y disminución de la quinasa ERK. Lo opuesto fue observado por disminución de RAC3 mediante la técnica de ARN interferente (RNAi) en K562, aumentando así la apoptosis inducida por TRAIL. Estas evidencias sugieren que una sobreexpresión de RAC3 contribuye al desarrollo de tumores, participando en las cascadas que controlan la muerte celular por mecanismos no estrictamente dependientes de hormonas esteroideas y/o de acetilación, constituyendo esto un posible blanco de ataque para el tratamiento de tumores.

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RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kB coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

Asunto(s)

Humanos , Apoptosis/fisiología , Transformación Celular Neoplásica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Factores de Transcripción/fisiología , Proteínas de Unión al GTP rac/fisiología , Riñón/citología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Receptores Citoplasmáticos y Nucleares

RESUMEN

Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 microM), an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 microM, resveratrol at 5 microM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 microM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 microM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominant-negative Rac retain filopodia response to 50 microM resveratrol. Lamellipodia response to 5 microM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 microM) signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

Asunto(s)

Actinas/metabolismo , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Citoesqueleto/efectos de los fármacos , Estradiol/farmacología , Estrógenos , Neoplasias Hormono-Dependientes/patología , Estilbenos/farmacología , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/fisiología , Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/ultraestructura , Movimiento Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Femenino , Genes Dominantes , Humanos , Invasividad Neoplásica , Neoplasias Hormono-Dependientes/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , Proteínas Recombinantes de Fusión/genética , Resveratrol , Transfección , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/genética

RESUMEN

The molecular, biochemical, and cellular characterization of EhGEF1 protein is described. Complete cDNA sequence of 1890 bp revealed an open reading frame that encodes a protein of 69 kDa. EhGEF1 is constituted of Dbl homology domain, pleckstrin homology domain, and several putative regulation sites. Studies of guanine nucleotide exchange activity of EhGEF1 on several GTPases from Entamoeba histolytica and Homo sapiens showed preferential activation on EhRacG, suggesting that EhGEF1 protein could be involved in mechanisms related to actin cytoskeleton activation, cytokinesis, capping, and uroid formation in trophozoite. Confocal microscopy studies of pExEhNeo/HSV-tagged-EhGEF1-transfected cells showed that trophozoites stimulated with ConA, EhGEF1, and EhRacG were localized at plasma membrane. Cellular studies showed that F-actin content of pExEhNeo/HSV-tagged-EhGEF1-transfected trophozoites as well as cellular migration and cell damage capacity were significantly altered. The observations suggest that EhRacG was the principal target of EhGEF1 and that EhGEF1 may provide a link between F-actin dynamics and EhRacG signaling.

Asunto(s)

Entamoeba histolytica/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Concanavalina A/farmacología , Secuencia Conservada , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Entamoeba histolytica/genética , Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Microscopía Confocal , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Transfección , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiología

RESUMEN

The biological and functional properties of beta2-chimaerin, a novel phorbol ester/diacylglycerol receptor unrelated to protein kinase C isozymes, are largely unknown. It has previously been established that beta2-chimaerin accelerates the hydrolysis rate of GTP from Rac1 in vitro, leading to the inactivation of this GTPase, which plays important roles in the control of actin cytoskeleton organization, proliferation, motility, and invasiveness. To explore the potential role of beta2-chimaerin in invasion and metastasis, we generated stable transfectants for its catalytic domain (the beta-GAP domain) in F3II murine mammary carcinoma cells. Reduced Rac-GTP levels were observed upon stimulation with epidermal growth factor in the beta-GAP clones compared with control cells. Moreover, a marked alteration in actin polymerization in response to epidermal growth factor was observed in the beta-GAP clones, suggesting impairment of Rac-dependent responses. The beta-GAP transfectants also evidenced slower growth rates and a striking reduction in their migratory properties. Adenoviral delivery of the beta-GAP domain into F3II cells also led to reduced proliferative and migratory responses. Importantly, significant differences were found between beta-GAP transfectants and control cells regarding their tumorigenic and metastatic properties after s.c. inoculation in syngeneic BALB/c mice. Tumors originating from beta-GAP transfectants showed a significantly lower growth rate and reduced invasive ability; in addition, a lower incidence of spontaneous lung metastases was observed. Our results indicate that beta2-chimaerin impairs key steps in the metastatic cascade and provide evidence for a rational modulation of the Rac signaling pathway in cancer treatment.

Asunto(s)

Proteínas Activadoras de GTPasa/fisiología , Neoplasias Mamarias Experimentales/patología , Proteínas de Neoplasias/fisiología , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Femenino , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Estructura Terciaria de Proteína , Transfección , Células Tumorales Cultivadas , Proteínas de Unión al GTP rac/biosíntesis , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología