RESUMEN
Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Indoles/farmacología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Mieloma Múltiple/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteína bcl-X/metabolismo , Administración Oral , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/administración & dosificación , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/efectos de los fármacos , Humanos , Técnicas In Vitro , Indoles/administración & dosificación , Inyecciones Intravenosas , Leupeptinas/farmacología , Ratones , Ratones Desnudos , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Chaperonas Moleculares , Mieloma Múltiple/enzimología , Proteínas de Neoplasias/efectos de los fármacos , Inhibidores de Proteasas/administración & dosificación , Pirazinas/administración & dosificación , Pirazinas/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/efectos de los fármacosRESUMEN
Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.
Asunto(s)
Proteínas de Drosophila , Flagelina/farmacología , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Animales , Células COS , Línea Celular , Colon , Regulación de la Expresión Génica/inmunología , Células HeLa , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Superficie Celular/fisiología , Receptor Toll-Like 5 , Receptores Toll-Like , TransfecciónRESUMEN
RIP2 is a serine-threonine kinase associated with the tumor necrosis factor (TNF) receptor complex and is implicated in the activation of NF-kappaB and cell death in mammalian cells. However, the function of its kinase domain is still enigmatic as it is not required in engaging these responses. Here we show that RIP2 activates the extracellular signal-regulated kinase (ERK) pathway and that the kinase activity of RIP2 appears to be important in this process. RIP2 activates AP-1 and serum response element regulated expression by inducing the activation of the Elk1 transcription factor. RIP2 directly phosphorylates and activates ERK2 in vivo and in vitro. RIP2 in turn is activated through its interaction with Ras-activated Raf1. Kinase-defective point and deletion variants of RIP2 also significantly blocked the activation of ERK2 by TNFalpha but not epidermal growth factor. These results describe a novel pathway of ERK activation and the first catalytic function ascribed to any of the RIP-like kinases associated with the TNF receptor superfamily.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Células COS , Catálisis , Línea Celular , Activación Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair. Examination of the inducibility of RNR3 in response to UV damage has revealed that the various checkpoint genes can be arranged in a pathway consistent with their requirement to arrest cells at different stages of the cell cycle. While RAD9, RAD24, and MEC3 are required to activate the DNA damage checkpoint when cells are in G1 or G2, POL2 is required to sense UV damage and replication blocks when cells are in S phase. The phosphorylation of the essential central transducer, Rad53p, is dependent on POL2 and RAD9 in response to UV damage, indicating that RAD53 functions downstream of both these genes. Mutants defective for both pathways are severely deficient in Rad53p phosphorylation and RNR3 induction and are significantly more sensitive to DNA damage and replication blocks than single mutants alone. These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal. Each of these pathways subsequently activates the central transducers Mec1p/Esr1p/Sad3p and Rad53p/Mec2p/Sad1p, which are required for both cell-cycle arrest and transcriptional responses.
Asunto(s)
Proteínas de Ciclo Celular , Daño del ADN , ADN Polimerasa Dirigida por ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Ciclo Celular , Quinasa de Punto de Control 2 , ADN Polimerasa II , Fase G1 , Proteínas Quinasas/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Transcripción Genética , Transformación Genética , Rayos UltravioletaRESUMEN
Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon). Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases. We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Fase S , Saccharomyces cerevisiae/enzimología , Alelos , Secuencia de Aminoácidos , ADN Polimerasa II , ADN Polimerasa Dirigida por ADN/genética , Prueba de Complementación Genética , Interfase , Mitosis , Datos de Secuencia Molecular , Mutación , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Supresión GenéticaRESUMEN
Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage.