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1.
FASEB J ; 33(6): 6919-6932, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30817226

RESUMO

T cells expressing invariant γδ antigen receptors (γδ T cells) bridge innate and adaptive immunity and facilitate barrier responses to pathogens. Macrophage migration inhibitory factor (MIF) is an upstream mediator of host defense that up-regulates the expression of pattern recognition receptors and sustains inflammatory responses by inhibiting activation-induced apoptosis in monocytes and macrophages. Surprisingly, Mif-/- γδ T cells, when compared with wild type, were observed to produce >10-fold higher levels of the proinflammatory cytokine IL-17 after stimulation with gram-positive exotoxins. High-IL-17 expression was associated with the characteristic features of IL-17-producing γδ T (γδ17) cells, including expression of IL-23R, IL-1R1, and the transcription factors RORγt and Sox13. In the gram-positive model of shock mediated by toxic shock syndrome toxin (TSST-1), Mif-/- mice succumbed to death more quickly with increased pulmonary neutrophil accumulation and higher production of cytokines, including IL-1ß and IL-23. Mif-/- γδ T cells also produced high levels of IL-17 in response to Mycobacterium lipomannan, and depletion of γδ T cells improved survival from acutely lethal Mycobacterium infection or TSST-1 administration. These data indicate that MIF deficiency is associated with a compensatory amplification of γδ17 cell responses, with implications for innate immunity and IL-17-mediated pathology in situations such as gram-positive toxic shock or Mycobacterium infection.-Kim, H. K., Garcia, A. B., Siu, E., Tilstam, P., Das, R., Roberts, S., Leng, L., Bucala, R. Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.


Assuntos
Imunidade Inata/imunologia , Inflamação/imunologia , Interleucina-17/metabolismo , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Enterotoxinas/administração & dosagem , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/imunologia , Receptores de Interleucina/metabolismo , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Choque Séptico/patologia , Superantígenos/administração & dosagem , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
2.
Mol Carcinog ; 58(3): 411-425, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30488488

RESUMO

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-ß (LTß), receptor LTßR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTßR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTß, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTßR, NIK, and RELB proteins. Recombinant LTß, and siRNA depletion of endogenous LTßR and NIK, modulated expression of LTßR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTß induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTß-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTß is expressed. Our findings show that LTß/LTßR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.


Assuntos
Carcinoma de Células Escamosas/secundário , Neoplasias de Cabeça e Pescoço/patologia , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-alfa/metabolismo , Linfotoxina-beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição RelB/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Receptor beta de Linfotoxina/genética , Linfotoxina-alfa/genética , Linfotoxina-beta/genética , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fator de Transcrição RelB/genética , Células Tumorais Cultivadas , Quinase Induzida por NF-kappaB
3.
Nucleic Acids Res ; 38(21): 7570-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20631007

RESUMO

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5' and 3' cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5' and 3' exons are tethered to RNAP II for splicing.


Assuntos
Splicing de RNA , Transcrição Gênica , Íntrons , Modelos Genéticos , RNA Polimerase II/metabolismo , Ribonuclease H/metabolismo , Spliceossomos/metabolismo
4.
Hum Vaccin ; 5(4): 237-41, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18948733

RESUMO

OBJECTIVES: To inform strategies for vaccinating elderly veterans, the objectives were to determine the proportion of veterans > or =65 years old vaccinated against influenza during the 2004-05 vaccine-shortage, the place they received vaccine, the reasons why they were/were not vaccinated and their attitudes to the shortage. RESULTS: Among 682 respondents, 87% were vaccinated during 2004-05. More participants received vaccine at the Veterans' Administration hospital than previously (67% vs. 57%, p < 0.0001). Only 28% perceived themselves as being at high risk from influenza even though all participants met > or =1 high-risk criteria. Among unvaccinated participants, 21% were unvaccinated so that someone else could have their dose ("altruism"). For 61%, the shortage led to feeling heightened urgency for vaccination; those participants were more apt to be vaccinated (96% v. 77%, p < 0.001). Of those not vaccinated during the previous season, only 20% reported feeling urgency associated with the shortage, versus 65% of those vaccinated previously (p < 0.0001). METHODS: Survey-questionnaire mailed to a random sample of veterans > or =65 years-old. The questionnaire was derived from qualitative interviews during December 2004. CONCLUSION: The climate of shortage led to a heightened sense of urgency for vaccination that was most prominent among veterans who were vaccinated in the past. It also may have led to vaccination for a small proportion not previously vaccinated against influenza. In contrast, other elderly veterans may have been motivated to avoid vaccination for altruistic reasons or because of confusion about their risk-status. Outreach and communication about vaccination should target both those who may feel urgency to be vaccinated as well as those who may inappropriately avoid vaccination.


Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Vacinas contra Influenza/provisão & distribuição , Influenza Humana/prevenção & controle , Vacinação/estatística & dados numéricos , Veteranos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Humanos , Influenza Humana/imunologia , Pessoa de Meia-Idade , Distribuição Aleatória , Inquéritos e Questionários
5.
JCI Insight ; 4(7)2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944253

RESUMO

Recruitment of myeloid-derived suppressor cells (MDSCs) into tumors induces local immunosuppression in carcinomas. Here, we assessed whether SX-682, an orally bioavailable small-molecule inhibitor of CXCR1 and CXCR2, could block tumor MDSC recruitment and enhance T cell activation and antitumor immunity following multiple forms of immunotherapy. CXCR2+ neutrophilic MDSCs (PMN-MDSCs) were the most abundant myeloid cell subset within oral and lung syngeneic carcinomas. PMN-MDSCs demonstrated greater suppression of tumor-infiltrating lymphocyte killing of targets compared with macrophages. SX-682 significantly inhibited trafficking of PMN-MDSCs without altering CXCR2 ligand expression. Trafficking of CXCR1+ macrophages was unaltered, possibly due to coexpression of CSF1R. Reduced PMN-MDSC tumor infiltration correlated with enhanced accumulation of endogenous or adoptively transferred T cells. Accordingly, tumor growth inhibition or the rate of established tumor rejection following programed death-axis (PD-axis) immune checkpoint blockade or adoptive cell transfer of engineered T cells was enhanced in combination with SX-682. Despite CXCR1/2 expression on tumor cells, SX-682 appeared to have little direct antitumor effect on these carcinoma models. These data suggest that tumor-infiltrating CXCR2+ PMN-MDSCs may prevent optimal responses following both PD-axis immune checkpoint blockade and adoptive T cell transfer therapy. Abrogation of PMN-MDSC trafficking with SX-682 enhances T cell-based immunotherapeutic efficacy and may be of benefit to patients with MDSC-infiltrated cancers.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Carcinoma/terapia , Movimento Celular/efeitos dos fármacos , Neoplasias Pulmonares/terapia , Neoplasias Bucais/terapia , Células Supressoras Mieloides/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular Tumoral/transplante , Movimento Celular/imunologia , Terapia Combinada/métodos , Modelos Animais de Doenças , Humanos , Tolerância Imunológica/efeitos dos fármacos , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
6.
Clin Cancer Res ; 25(9): 2860-2873, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30723145

RESUMO

PURPOSE: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. RESULTS: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. CONCLUSIONS: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/patologia , MicroRNAs/administração & dosagem , MicroRNAs/genética , Nanopartículas/administração & dosagem , Carcinoma de Células Escamosas de Cabeça e Pescoço/secundário , Animais , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Variações do Número de Cópias de DNA , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanomedicina , Nanopartículas/química , Prognóstico , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Sci Transl Med ; 9(391)2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28539475

RESUMO

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-3/metabolismo , Animais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias da Mama/complicações , Neoplasias da Mama/tratamento farmacológico , Feminino , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética
8.
J Colloid Interface Sci ; 292(1): 1-10, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16126217

RESUMO

The potential of thermally activated titanium rich bauxite (TRB) for adsorptive removal of excess fluoride from drinking water was examined. Adsorption with respect to variation of pH, adsorbent dose, initial fluoride concentration, presence of interfering ions and heat treatment were investigated by batch equilibrium experiments. Thermal activation at moderate temperatures (300-450 degrees C) greatly increased the adsorption capacity of TRB. The rate of adsorption was rapid and maximum level was attained within 90 min. The uptake of fluoride increased with increasing pH, reached to a maximum at pH 5.5-6.5 and thereafter decreased. The adsorption kinetics was found to follow first order rate expression and the experimental equilibrium adsorption data fitted reasonably well to both Langmuir and Freundlich isotherm models. The presence of common interfering ions in drinking water did not greatly affect the uptake of fluoride from aqueous solution indicating F specific sorption behaviour of TRB. Nearly complete desorption of adsorbed fluoride from loaded bauxite was achieved by treating with aqueous solutions of pH > or =11.1 ([NaOH] > or =0.015 mol/dm(3)).


Assuntos
Óxido de Alumínio/química , Fluoretos/química , Titânio/química , Purificação da Água/métodos , Adsorção , Ânions/química , Concentração de Íons de Hidrogênio , Cinética , Propriedades de Superfície , Temperatura
9.
Cancer Cell ; 27(4): 533-46, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25873175

RESUMO

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Cetuximab , Classe I de Fosfatidilinositol 3-Quinases , Resistencia a Medicamentos Antineoplásicos , Carcinoma de Células Escamosas do Esôfago , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase Axl
10.
Indian J Ophthalmol ; 51(2): 182-3, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12831152

RESUMO

Morning glory disc anomaly (MGDA) is usually known to be unilateral. We report an unusual case of a bilateral form of the disorder with an aim to distinguish it from other bilateral optic nerve head anomalies.


Assuntos
Anormalidades do Olho/diagnóstico , Criança , Diagnóstico Diferencial , Feminino , Fundo de Olho , Humanos , Disco Óptico/anormalidades , Tomografia Computadorizada por Raios X , Ultrassonografia
11.
Cancer Res ; 73(19): 6024-35, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23928993

RESUMO

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/patologia , Neuregulina-1/metabolismo , Conformação Proteica/efeitos dos fármacos , Receptor ErbB-3/antagonistas & inibidores , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS One ; 7(11): e48548, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23155392

RESUMO

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Imidazóis/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Everolimo , Humanos , Sirolimo/farmacologia
13.
Cell Rep ; 2(4): 799-806, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23022481

RESUMO

Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts in patients with SMA. Here, we report that FUS associates with the SMN complex, mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions among SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, providing strong support for the view that these motor neuron diseases are related.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Atrofia Muscular Espinal/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína DEAD-box 20/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Atrofia Muscular Espinal/patologia , Mutação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína FUS de Ligação a RNA/antagonistas & inibidores , Proteína FUS de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/genética
14.
Mol Cell ; 26(6): 867-81, 2007 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-17588520

RESUMO

Transcription and splicing are functionally coupled, resulting in highly efficient splicing of RNA polymerase II (RNAP II) transcripts. The mechanism involved in this coupling is not known. To identify potential coupling factors, we carried out a comprehensive proteomic analysis of immunopurified human RNAP II, identifying >100 specifically associated proteins. Among these are the SR protein family of splicing factors and all of the components of U1 snRNP, but no other snRNPs or splicing factors. We show that SR proteins function in coupling transcription to splicing and provide evidence that the mechanism involves cotranscriptional recruitment of SR proteins to RNAP II transcripts. We propose that the exclusive association of U1 snRNP/SR proteins with RNAP II positions these splicing factors, which are known to function early in spliceosome assembly, close to the nascent pre-mRNA. Thus, these factors readily out-compete inhibitory hnRNP proteins, resulting in efficient spliceosome assembly on nascent RNAP II transcripts.


Assuntos
RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcrição Gênica/fisiologia , Animais , Bovinos , Linhagem Celular , Sistema Livre de Células , Humanos , Modelos Biológicos , Proteômica , RNA Polimerase II/química , Precursores de RNA/química , Proteínas de Ligação a RNA/química , Ribonucleoproteína Nuclear Pequena U1/química , Spliceossomos/química , Spliceossomos/metabolismo
15.
Genes Dev ; 20(9): 1100-9, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16651655

RESUMO

The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent pre-messenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.


Assuntos
RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/fisiologia , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Células HeLa , Humanos , RNA Polimerase II/genética , Precursores de RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
Genes Dev ; 19(13): 1512-7, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15998806

RESUMO

In yeast, the TREX complex contains the THO transcription elongation complex, which functions in direct cotranscriptional recruitment of the mRNA export proteins Sub2 and Yra1 to nascent transcripts. Here we report the identification of the human THO complex and show that it associates with spliced mRNA, but not with unspliced pre-mRNA in vitro. Transcription is not required for this recruitment. We also show that the human THO complex colocalizes with splicing factors in nuclear speckle domains in vivo. Considering that splicing occurs cotranscriptionally in humans, our data indicate that recruitment of the human TREX complex to spliced mRNA is not directly coupled to transcription, but is instead coupled to transcription indirectly through splicing.


Assuntos
Exodesoxirribonucleases/genética , Fosfoproteínas/genética , Splicing de RNA , RNA Mensageiro/genética , Humanos , Imunoprecipitação
18.
Curr Protoc Mol Biol ; Chapter 27: Unit 27.1, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18265328

RESUMO

In many methods currently used to analyze RNA-protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA-protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA-protein complexes. Moreover, the use of low-melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.


Assuntos
Eletroforese em Gel de Ágar/métodos , RNA/isolamento & purificação , Ribonucleoproteínas/isolamento & purificação , Sistema Livre de Células , Células HeLa , Humanos , Indicadores e Reagentes , Marcação por Isótopo/métodos , Radioisótopos de Fósforo/análise , RNA/análise , RNA Mensageiro/isolamento & purificação , Spliceossomos/química , Transcrição Gênica
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