RESUMEN
Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Modelos Moleculares , Neoplasias/metabolismo , Pirazoles/farmacología , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Quinasas p21 Activadas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Pirazoles/química , Pirazoles/metabolismo , Pirroles/química , Pirroles/metabolismo , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Mammalian signal transduction occurs in the context of multiprotein complexes, yet currently available drug discovery strategies do not reflect this fact. We present a strategy for screening drugs and targets in living human cells by utilizing high content protein-fragment complementation assays. Synthetic fragments of a mutant fluorescent protein ("Venus" and/or enhanced yellow fluorescent protein) are used for protein-fragment complementation assay construction, allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. Here we describe the utility of this novel strategy for high-throughput screening of known targets, and for screening previously undrugable targets and profiling drug leads for improved selectivity and safety.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Complejos Multiproteicos/metabolismo , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Humanos , Unión Proteica , ARN Interferente Pequeño , Sensibilidad y Especificidad , Fracciones SubcelularesRESUMEN
Small GTPases of the Ras and Rho families are among the most studied signaling proteins and represent promising therapeutic targets for human neoplastic disease. Despite the high level of interest in these proteins, direct analysis of most aspects of Ras protein biology in living cells has not been possible, because much of the details of Ras signaling cannot be studied in vitro but requires simple cell-based assays. Here we describe a strategy for directly analyzing Ras signaling pathways in living cells using protein-fragment complementation assays (PCA) based on fragments of intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from Ras proteins, as well as complexes of Ras proteins with regulator and effector proteins. We describe high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity (high-content assays). Spatial and temporal changes in response to perturbations (chemical, siRNA, hormones) allow for delineation of Ras signaling networks and a general and high-throughput approach to identify drugs that act directly or indirectly on Ras pathways.
Asunto(s)
Prueba de Complementación Genética/métodos , Mapeo de Interacción de Proteínas/métodos , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Transfección/métodosRESUMEN
Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.
Asunto(s)
Técnicas Citológicas/métodos , Citometría de Flujo/métodos , Transporte Activo de Núcleo Celular , Animales , Arrestinas/metabolismo , Línea Celular Tumoral , Citofotometría/métodos , Epigénesis Genética , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Rayos Láser , Ligandos , Ratones , Receptores de Glucocorticoides/química , beta-ArrestinasRESUMEN
Cellular signal transduction occurs in the context of dynamic multiprotein complexes in highly ramified pathways. These complexes in turn interact with the cytoskeleton, protein scaffolds, membranes, lipid rafts, and specific subcellular organelles, contributing to the exquisitely tight regulation of their localization and activity. However, these realities of drug target biology are not addressed by currently available drug discovery platforms. In this article, we describe the use of protein-fragment complementation assays (PCAs) to assess drugs and drug targets in the context of their native environment. The PCA process allows for the detection of protein-protein complexes following the expression of full-length mammalian genes linked in-frame to polypeptide fragments of rationally dissected reporter genes. If cellular activity causes the association of two proteins linked to complementary reporter fragments, the interaction of the proteins of interest enables refolding of the fragments, which can then generate a quantifiable signal. Using a PCA based on a yellow fluorescent protein, we demonstrate that functional (p50/p65) complexes of the heterodimeric nuclear factor-kappaB transcription factor, as well as the transcription factor subunit p65 and its modulator IkappaBalpha, can be visualized and monitored in live cells. We observed similar responses of the PCA assays to the activities of the cognate endogenous proteins, including modulation by known agonists and antagonists. A proof-of-concept high throughput screen was carried out using the p50/p65 cell line, and potent inhibitors of this pathway were identified. These assays record the dynamic activity of signaling pathways in living cells and in real time, and validate the utility of PCA as a novel approach to drug discovery.
Asunto(s)
Prueba de Complementación Genética/métodos , Fragmentos de Péptidos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Tecnología Farmacéutica/métodos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/genética , Proteínas/análisis , Proteínas/genética , Proteínas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Altered gene expression contributes to the aetiology of inflammatory disease by modulation of the concentration of disease-related proteins. The expression of inflammatory genes is controlled through the concerted actions of specific transcription factors. Signal transduction networks positively or negatively regulate the activity of these transcription factors. Key components of these networks are protein kinases, which phosphorylate substrates on tyrosine, threonine or serine residues. During the disease process, pro-inflammatory signalling at the cell surface leads to a cascade of kinase activation, which ultimately culminates in modulation of the activity of transcription factors. Thus, pharmacological inhibition of protein kinases is a potential therapeutic strategy to treat inflammation. There are approximately 500 protein kinases in the human genome. Targeted small molecule inhibitors of these kinases should allow for tissue- and disease-specific therapies of unprecedented selectivity. Heralding this new era in molecular medicine is imatinib (Gleevec, Norvartis) a recently marketed tyrosine kinase inhibitor. This review focuses on kinase inhibitors that are currently in development for inflammatory diseases and the transcription factors that are involved.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inflamación/terapia , Fosfotransferasas/antagonistas & inhibidores , Transcripción Genética , Animales , Diseño de Fármacos , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Modelos Químicos , Fosforilación , Transducción de SeñalRESUMEN
Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERß loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERß and act as ERß antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.
Asunto(s)
Compuestos de Bencidrilo/química , Compuestos de Bencidrilo/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Fenoles/química , Fenoles/farmacología , Compuestos de Bencidrilo/efectos adversos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Células HeLa , Humanos , Células MCF-7 , Microscopía , Fenoles/efectos adversos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Despite rapid growth in our knowledge of potential disease targets following completion of the first drafts of the human genome over 10 years ago, the success rate of new therapeutic discovery has been frustratingly low. In addition to the widely reported costs and single-digit success rate of the entire drug discovery and development process, it has recently been estimated that even the preliminary process of transitioning new targets to preclinical development succeeds in less than 3% of attempts [Vogel (ed.) Drug Discovery and Evaluation: Pharmacological Assays. 3rd ed. Springer, Berlin (2007)]. At these early stages of development, poor understanding of therapeutic mechanisms and lack of compound selectivity are often to blame for failed compounds. It is worth noting than the emerging class of nucleic acid-based therapeutics, including miRNA and RNAi, are likely to be even more prone to unexpected system-wide and off-target activities. For all therapeutic approaches, it is clear that discovery strategies permitting the assessment of drug targets in their native context are required. At the same time, these strategies need to retain the high throughput of current reductionist approaches to enable broad assessment of chemical space for small molecule and genetic therapeutics. We describe here an integrated system based on high-content cellular analysis combined with system-wide pathway interrogation. The platform can be applied to novel therapeutic target and drug candidate identification, and for providing detailed mechanistic and selectivity information at an early stage of development.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas/análisis , Animales , Técnicas Citológicas/instrumentación , Técnicas Citológicas/métodos , Descubrimiento de Drogas/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Biología de Sistemas/instrumentación , Biología de Sistemas/métodosRESUMEN
Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Células HeLa , Humanos , Mutación , Fosforilación , Unión Proteica , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Interferente Pequeño/metabolismo , Treonina/químicaRESUMEN
Signal transduction pathways mediated by MAP kinases are among the most studied. Direct analysis of MAP kinase pathways has been difficult because some details of MAP kinase signaling cannot be studied in vitro. Here, we describe a strategy for directly analyzing MAP kinase signaling pathways in living cells using protein-fragment complementation assays (PCA) based on intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from MAPKs as well as complexes of MAPKs with regulator and effector proteins. We describe high-content assays, high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity. Spatial and temporal changes in response to perturbations (chemical, siRNA, and hormones) allow for delineation of MAPK signaling networks and a general and high-throughput approach to identify small molecules that act directly or indirectly on MAPK pathways.
Asunto(s)
Técnicas Químicas Combinatorias , Prueba de Complementación Genética/métodos , Técnicas Genéticas , Sistema de Señalización de MAP Quinasas , Fragmentos de Péptidos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , ARN Interferente Pequeño , TransfecciónRESUMEN
We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes. This strategy reproduced known structure-function relationships and also predicted 'hidden,' potent antiproliferative activities for four drugs with novel mechanisms of action, including disruption of mitochondrial membrane potential. A simple algorithm identified a 25-assay panel that was highly predictive of antiproliferative activity, and the predictive power of this approach was confirmed with cross-validation tests. This study suggests a strategy for therapeutic discovery that identifies novel, unpredicted mechanisms of drug action and thereby enhances the productivity of drug-discovery research.
Asunto(s)
Proteínas Bacterianas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Proteínas Luminiscentes/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Diseño de Fármacos , Genética , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/efectos de los fármacos , Estructura Molecular , Fenotipo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
The hepatic stellate cell (HSC) is the predominant cell type responsible for excess collagen deposition during liver fibrosis. Both transforming growth factor-beta (TGF-beta), the most potent fibrogenic cytokine for HSCs, which classically activates Smad signaling, and p38 MAPK signaling have been shown to influence collagen gene expression; however, the relative contribution and mechanisms that these two signaling pathways have in regulating collagen gene expression have not been investigated. The aim of this study was to investigate the relative roles and mechanisms of both Smad and p38 MAPK signaling in alpha1(I) collagen gene expression in HSCs. Inhibiting either p38 MAPK or Smad signaling reduced alpha1(I) collagen mRNA expression in untreated or TGF-beta-treated HSCs, and when both signaling pathways were simultaneously inhibited, alpha1(I) collagen gene expression was essentially blocked. Both signaling pathways were found to independently and additively increase alpha1(I) collagen gene expression by transcriptional mechanisms. TGF-beta treatment increased alpha1(I) collagen mRNA half-life, mediated by increased stability of alpha1(I) collagen mRNA through p38 MAPK signaling but not through Smad signaling. In conclusion, both p38 MAPK and Smad signaling independently and additively regulate alpha1(I) collagen gene expression by transcriptional activation, whereas p38 MAPK and not Smad signaling increased alpha1(I) collagen mRNA stability.
Asunto(s)
Colágeno Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Hígado/citología , Transducción de Señal , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Células Cultivadas , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Imidazoles/farmacología , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Masculino , Músculo Liso/citología , Fosforilación , Piridinas/farmacología , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ribonucleasas/metabolismo , Proteínas Smad , Factores de Tiempo , Transcripción Genética , Activación Transcripcional , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND/AIMS: Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-kappaB and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors. METHODS: Male Wistar rats (200+/-25 g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90 min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90 min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation. RESULTS: In the I/R survival model, vehicle-treated rats have a 7-day survival of 20-40%, while rats treated with the three different JNK inhibitors have survival rates of 60-100% (P<0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid. CONCLUSIONS: Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/enzimología , Daño por Reperfusión/metabolismo , Animales , Apoptosis , Caspasa 3 , Caspasas/metabolismo , Citocromos c/metabolismo , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Peroxidación de Lípido/efectos de los fármacos , Hígado/patología , Masculino , Mitocondrias/metabolismo , Necrosis , Ratas , Ratas Wistar , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/mortalidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family, and its function is critical for signal transduction in tumor and endothelial cells. JNK is a serine/threonine protein kinase that phosphorylates c-Jun, a component of the activator protein-1 transcription factor complex. We hypothesize that inhibiting JNK will lead to the inhibition of tumor growth; therefore, we evaluated the efficacy of the recently described JNK inhibitor SP600125 [anthra[1,9-cd] pyrazol-6 (2H)-one]. SP600125 is an anthrapyrazole that is a reversible, ATP-competitive inhibitor of JNK1/2. SP600125 exhibited broad-based antiproliferative activity in human endothelial and tumor cell lines. SP600125 affects proliferation by arresting cells in the G2/M phase of the cell cycle. SP600125 also acts to inhibit endothelial cell migration. In cell lines, a correlation of cell growth inhibition with reduced JNK activity was observed. The systemic administration of SP600125 resulted in the inhibition of DU145 human prostate carcinoma xenografts and murine Lewis lung carcinoma. SP600125 also enhanced the potency of cyclophosphamide in the inhibition of Lewis lung tumor growth. These data indicate the therapeutic antitumor potential of small molecule inhibitors that act to block the cellular activity of JNK.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antracenos/farmacología , Antracenos/uso terapéutico , Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Femenino , Citometría de Flujo , Fase G2/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Trasplante de NeoplasiasRESUMEN
Activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, supporting the importance of Raf-independent pathways in mediating Ras transformation. The p38 and JNK mitogen-activated protein kinase cascades are activated by Ras via Raf-independent effector function. Therefore, we determined whether p38 and JNK activation are involved in Ras transformation of RIE-1 epithelial cells. Rather surprisingly, we found that pharmacologic inhibition of p38, together with Raf activation of ERK, was sufficient to mimic the morphologic and growth transformation caused by oncogenic Ras. p38 inhibition together with ERK activation also caused the same alterations in cyclin D1 and p21(CIP1) expression caused by Ras and induced an autocrine growth factor loop important for transformation. Finally, in contrast to p38, we found that JNK activation promoted Ras transformation, and that Ras deregulation of p38 and JNK was not mediated by activation of the Rac small GTPase. We conclude that a key action of Raf-independent effector pathways important for Ras transformation may involve inhibition of p38 and activation of JNK.