RESUMEN
Lung and colorectal cancers are the most common types of cancer characterized by a poor prognosis and a high mortality rate. Mutations in the genes encoding components of the main intra- and extracellular signaling pathways, in particular the NOTCH1 gene (Notch1, a member of the Notch family of receptors), play one of the key roles in progression of these malignancies. Notch signaling is involved in maintaining homeostasis of the intestinal epithelium and structural and functional lung elements. Therefore, it is not surprising that the constitutive activity and hyperactivity of Notch signaling due to somatic mutations in genes coding for the products directly involved into its activation, could lead to the progression of these cancer types. The aim of our study was to investigate how the NOTCH1 downregulation via RNA interference (RNAi) affects the phenotype, characteristics, and Notch-dependent signaling of human A549 lung and HCT116 colorectal carcinoma cells. Several small harpin RNAs (shRNAs) were selected using the bioinformatic analysis and tested for their ability to suppress the NOTCH1 expression. The most efficient one was used to produce the A549 and HCT116 cells with NOTCH1 knockdown. The obtained cell lines demonstrated decreased proliferation rates, reduced colony-forming capacity under adhesive conditions, and decreased migration activity in a Boyden chamber. The NOTCH1 knockdown also significantly decreased expression of some Notch signaling target genes potentially involved in the acquisition and maintenance of more invasive and malignant cell phenotype. In vivo experiments in immunodeficient athymic female Balb/c nu/nu mice confirmed the results obtained in vitro: the NOTCH1 inhibition decreased the growth rates of the subcutaneous xenografts formed by A549 and HCT116 tumor cells. Therefore, downregulation of the gene encoding the Notch1 receptor potentially reduces malignant characteristics of human lung and colorectal carcinoma cells.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Pulmonares , Receptor Notch1 , Animales , Femenino , Humanos , Ratones , Células A549 , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Células HCT116 , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de SeñalRESUMEN
Here we have shown that ß-cytoplasmic actin acts as a tumor suppressor, inhibiting cell growth and invasion in vitro and tumor growth in vivo. In contrast, γ-cytoplasmic actin increases the oncogenic potential via ERK1/2, p34-Arc, WAVE2, cofilin1, PP1 and other regulatory proteins. There is a positive feedback loop between γ-actin expression and ERK1/2 activation. We conclude that non-muscle actin isoforms should not be considered as merely housekeeping proteins and the ß/γ-actins ratio can be used as an oncogenic marker at least for lung and colon carcinomas. Agents that increase ß- and/or decrease γ-actin expression may be useful for anticancer therapy.
Asunto(s)
Actinas/metabolismo , Transformación Celular Neoplásica/metabolismo , Cofilina 1/metabolismo , Genes Supresores de Tumor/fisiología , Microscopía Confocal/métodos , Neoplasias/genética , Isoformas de Proteínas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Cofilina 1/genética , Humanos , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenes of the RAS family regulate many of the cell's activities, including proliferation, survival and differentiation. Activating mutations in these genes are common events for many types of cancer. One of the contradictory points concerning the biological significance of Ras activation is its dual effect (pro- or anti-proliferative) on cell reproduction. One of mechanisms by which Ras proteins influence cell growth is a regulation of intracellular level of reactive oxygen species (ROS), second messengers affecting variety of cellular processes including cell proliferation. Recently it was shown that repression of SESN1 and SESN3 genes, whose protein products control regeneration of peroxiredoxins, can play a critical role in Ras-induced ROS upregulation. In the present study we have found that Ras-induced repression of SESN3 expression and ROS upregulation is mediated via the modifications of transcriptional activity of HSF1. Interestingly, mutant Ras overexpression altered the activity of HSF1 in opposite directions in different cell contexts, in particular in human normal fibroblasts and HaCaT immortalized keratinocytes, but these opposite changes caused similar repression of SESN3 expression followed by elevation of ROS content and inhibition of cell proliferation in corresponding cell types. The inhibitory effect on cell proliferation was mediated by upregulation of p21(Cip1/WAF1). Thus, HSF1/SESN3/ROS/p21(Cip1/WAF1)-mediated deceleration of cell growth may contribute to cell defense systems protecting the organism from excessive proliferation of cells that overexpress activated Ras oncoproteins.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proliferación Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Fluorescencia , Genes Reporteros , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Espacio Intracelular/metabolismo , Cinética , Proteínas Mutantes/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Activación Transcripcional/genética , Transducción Genética , Regulación hacia Arriba/genéticaRESUMEN
Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.
Asunto(s)
Envejecimiento/fisiología , Mitocondrias/fisiología , Envejecimiento/efectos de los fármacos , Animales , Antioxidantes/farmacología , Cloroplastos/efectos de los fármacos , Cloroplastos/fisiología , Transporte de Electrón/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/fisiología , Oxidantes/farmacología , Oxidación-Reducción , Plastoquinona/análogos & derivados , Plastoquinona/farmacología , Ratas , Ubiquinona/fisiologíaRESUMEN
Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2'-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant N-acetyl-L-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of SESN1 and SESN3 from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4-7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of SESN1 gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53.
Asunto(s)
Proteínas de Choque Térmico/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas ras/genética , Animales , Línea Celular , Rotura Cromosómica , ADN/genética , ADN/metabolismo , Humanos , Mutagénesis , Oxidación-Reducción , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Transcripción Genética , Regulación hacia Arriba , Proteínas ras/metabolismoRESUMEN
Expression of activated Ras causes an increase in intracellular content of reactive oxygen species (ROS). To determine the role of ROS up-regulation in mediation of Ras-induced morphological transformation and increased cell motility, we studied the effects of hydrogen peroxide and antioxidant NAC on morphology of REF52 rat fibroblasts and their ability to migrate into the wound in vitro. Treatment with low dosages of hydrogen peroxide leading to 1.5- to 2-fold increase in intracellular ROS levels induced changes of cell shape, actin cytoskeleton organization, cell adhesions and migration resembling those in Ras-transformed cells. On the other hand, treatment with NAC attenuating ROS up-regulation in cells with conditional or constitutive expression of activated Ras led to partial reversion of morphological transformation and decreased cell motility. The effect of ROS on cell morphology and motility probably results from modulation of activity of Rac1, Rho, and cofilin proteins playing a key role in regulation of actin dynamics. The obtained data are consistent with the idea that ROS up-regulation mediates two key events in Ras-induced morphological transformation and cell motility: it is responsible for Rac1 activation and is necessary (though insufficient) for realization of Ras-induced cofilin dephosphorylation.
Asunto(s)
Movimiento Celular/fisiología , Forma de la Célula/fisiología , Genes ras/fisiología , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba , Factores Despolimerizantes de la Actina/metabolismo , Animales , Línea Celular , Ratas , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Exposure to asbestos fibers increases the risk of development of mesotheliomas and lung carcinomas, but not fibrosarcomas. We present data suggesting that resistance of fibroblasts to asbestos-induced carcinogenesis is likely to be connected with their lower ability to generate reactive oxygen species (ROS) in response to asbestos exposure and stricter control of proliferation of cells bearing asbestos/ROS-induced injuries. In fact, chrysotile (Mg6Si4O10(OH)8) asbestos exposure (5-10 microg/cm2) increased intracellular ROS and 8-oxo-guanine contents in rat pleural mesothelial cells, but not in lung fibroblasts. Simultaneously, moderate dosages of chrysotile and other agents increasing ROS levels (hydrogen peroxide, H2O2 and ethyl-methanesulfonate, EMS) inhibited cell cycle progression, in particular G1-to-S transition, in fibroblasts, but not in mesothelial cells. The arrested fibroblasts underwent cell death, while the majority of chrysotile-treated mesothelial cells survived. The differences in cell cycle response to asbestos/ROS-induced injuries correlated with distinct activity of p53-p21Cip1/Waf1 pathway in the two cell types. Chrysotile, H2O2 and EMS caused p53 upregulation in both cell types, but mesothelial cells, unlike fibroblasts, showed no accumulation of p21Cip1/Waf1. Of note, treatment with doxorubicin caused similar p53-dependent p21Cip1/Waf1 upregulation and cell cycle arrest in both cell types. This suggests differential response of fibroblasts and mesothelial cells specifically to asbestos/ROS exposure rather than to all DNA-damaging insults.
Asunto(s)
Asbestos Serpentinas/toxicidad , ADN/efectos de los fármacos , Fase G1 , Especies Reactivas de Oxígeno , Animales , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Células Cultivadas , ADN/metabolismo , Oxidación-Reducción , Pleura/citología , Pleura/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Earlier we have found that in p53-deficient cells the expression of activated Ras attenuates the DNA damage-induced arrest in G(1) and G(2). In the present work we studied Ras-mediated effects on the G(2) checkpoint in two human cell lines, MDAH041 immortalized fibroblasts and Saos-2 osteosarcoma cells. The transduction of the H-Ras mutants that retain certain functions (V12S35, V12G37, and V12C40 retain the ability to activate Raf or RalGDS or phosphatidylinositol 3-kinase, respectively) as well as the activated or dominant-negative mutants of RalA (V23 and N28, respectively) has revealed that the activation of Ras-RalGEFs-Ral pathway was responsible for the attenuation of the G(2) arrest induced by ethyl metanesulfonate or doxorubicin. Noteworthy, the activated RalA V23N49 mutant, which cannot interact with RLIP76/RalBP1 protein, one of the best studied Ral effectors, retained the ability to attenuate the DNA damage-induced G(2) arrest. Activation of the Ras-Ral signaling affected neither the level nor the intracellular localization of cyclin B1 and CDC2 but interfered with the CDC2 inhibitory phosphorylation at Tyr(15) and the decrease in the cyclin B/CDC2 kinase activity in damaged cells. The revealed function of the Ras-Ral pathway may contribute to the development of genetic instability in neoplastic cells.
Asunto(s)
Daño del ADN , Proteína p53 Supresora de Tumor/metabolismo , Factor de Intercambio de Guanina Nucleótido ral/metabolismo , Proteínas ras/metabolismo , Western Blotting , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Separación Celular , Ciclina B/metabolismo , Ciclina B1 , ADN/metabolismo , Doxorrubicina/farmacología , Metanosulfonato de Etilo/farmacología , Fibroblastos/metabolismo , Citometría de Flujo , Fase G1 , Fase G2 , Genes Dominantes , Humanos , Microscopía Fluorescente , Mitosis , Mutación , Osteosarcoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Retroviridae/metabolismo , Transducción de Señal , Timidina/química , Factores de Tiempo , Tirosina/químicaRESUMEN
The p53 tumor suppressor plays a central role in the negative control of growth and survival of abnormal cells. Previously we demonstrated that in addition to these functions, p53 expression affects cell morphology and lamellar activity of the cell edge (Alexandrova, A., Ivanov, A., Chumakov, P. M., Kopnin, P. B., and Vasiliev, J. M. (2000) Oncogene 19, 5826-5830). In the present work we studied the effects of p53 and its homologue p73alpha on cell migration. We found that loss of p53 function correlated with decreased cell migration that was analyzed by in vitro wound closure test and Boyden chamber assay. The decreased motility of p53-deficient cells was observed in different cell contexts: human foreskin fibroblasts (BJ), human colon and lung carcinoma cell lines (HCT116 and H1299, respectively), as well as mouse normal fibroblasts from lung and spleen, peritoneal macrophages, and keratinocytes. On the other hand, overexpression of the p53 family member p73alpha stimulated cell migration. Changes in cell migration correlated directly with transcription activation induced by p53 or p73alpha. Noteworthy, p53 modulated cell motility in the absence of stress. The effect of p53 and p73alpha on cell migration was mediated through the activity of the phosphatidylinositol 3-kinase/Rac1 pathway. This p53/p73 function was mainly associated with some modulation of intracellular signaling rather than with stimulation of production of secreted motogenic factors. The identified novel activity of the p53 family members might be involved in regulation of embryogenesis, wound healing, or inflammatory response.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Western Blotting , Movimiento Celular , Células Cultivadas , Colágeno/farmacología , Medios de Cultivo Condicionados/farmacología , Daño del ADN , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Genes Supresores de Tumor , Proteínas Fluorescentes Verdes , Humanos , Inflamación , Laminina/farmacología , Proteínas Luminiscentes/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Estructura Terciaria de Proteína , Proteoglicanos/farmacología , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
Mutated forms of p53 are often expressed in a variety of human tumors. In addition to loss of function of the p53 tumor suppressor, mutant p53s contribute to malignant process by acquisition of novel functions that enhance transformed properties of cells and resistance to anticancer therapy in vitro, and increase tumorigenecity, invasiveness and metastatic ability in vivo. Searching for genes that change expression in response to p53 gain of function mutants may give a clue to the mechanisms underlying their oncogenic effects. Recently by subtraction hybridization cloning we found that the dUTPase gene is transcriptionally upregulated in p53-null mouse fibroblasts expressing the exogenous human tumor-derived His175 p53 mutant. Here we show that conditional expression of His175 and Trp248 hot-spot p53 mutants in p53-negative mouse 10(1) fibroblasts and human SK-OV3 and H1299 tumor cells results in increase in dUTPase gene transcription, an important marker predicting the efficacy of cancer therapy with fluoropyrimidine drugs. Using tetracycline-regulated retroviral vectors for conditional expression of p53 mutants, we found that transcription of the dUTPase gene is increased within 24 h after tetracycline withdrawal, and the cells acquire higher resistance to 5-FU. Additional inactivation of the N-terminal transcription activation domain of mutant p53 (substitutions in amino-acid residues 22 and 23) results in abrogation of both induction of dUTPase transcripts and 5-FU resistance.