RESUMO
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.
Assuntos
Proteínas de Choque Térmico/metabolismo , Indóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidores de Proteases/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismo , Administração Oral , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Técnicas In Vitro , Indóis/administração & dosagem , Injeções Intravenosas , Leupeptinas/farmacologia , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Chaperonas Moleculares , Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/efeitos dos fármacos , Inibidores de Proteases/administração & dosagem , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Ciclo Celular , Quinase do Ponto de Checagem 2 , DNA Polimerase II , Fase G1 , Proteínas Quinases/genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Transcrição Gênica , Transformação Genética , Raios UltravioletaRESUMO
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.
Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Fase S , Saccharomyces cerevisiae/enzimologia , Alelos , Sequência de Aminoácidos , DNA Polimerase II , DNA Polimerase Dirigida por DNA/genética , Teste de Complementação Genética , Interfase , Mitose , Dados de Sequência Molecular , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Supressão GenéticaRESUMO
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.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Células COS , Catálise , Linhagem Celular , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Fosforilação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
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.
Assuntos
Ciclo Celular , Dano ao DNA , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Ribonucleotídeo Redutases/metabolismo , Saccharomyces cerevisiae/enzimologia , Regulação Alostérica , Reparo do DNA , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Fúngico/genética , RNA Mensageiro/genética , Ribonucleotídeo Redutases/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
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.