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1.
Cell ; 172(5): 952-965.e18, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29474921

RESUMO

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.


Assuntos
Encefalopatias Metabólicas Congênitas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , RNA/química , RNA/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Encefalopatias Metabólicas Congênitas/patologia , Tronco Encefálico/patologia , Encefalite Viral/genética , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1 , Humanos , Interferons/metabolismo , Íntrons/genética , Masculino , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Linhagem , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Receptor 3 Toll-Like/metabolismo , Replicação Viral
2.
Genes Dev ; 29(8): 791-802, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25854920

RESUMO

Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.


Assuntos
Senescência Celular/fisiologia , Pâncreas/citologia , Actinas/metabolismo , Animais , Comunicação Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Camundongos , Pâncreas/fisiologia , Polimerização , Transporte Proteico , Linfócitos T/citologia , Linfócitos T/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
3.
Genes Dev ; 27(21): 2356-66, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24186980

RESUMO

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16-pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.


Assuntos
Senescência Celular/fisiologia , Produtos do Gene env/metabolismo , Vírus do Sarampo/fisiologia , Proteínas da Gravidez/metabolismo , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Senescência Celular/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Produtos do Gene env/genética , Humanos , Sarampo/fisiopatologia , Placenta/citologia , Gravidez , Proteínas da Gravidez/genética , Trofoblastos/metabolismo
4.
Blood ; 122(15): 2694-703, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23974202

RESUMO

The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.


Assuntos
Regulação Leucêmica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos , Elementos Facilitadores Genéticos/genética , Genômica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células Progenitoras Mieloides/fisiologia , Transplante de Neoplasias , Transcrição Gênica/fisiologia , Regulador Transcricional ERG
5.
Int J Cancer ; 128(3): 691-701, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20473860

RESUMO

Histone deacetylase (HDAC) inhibitors, such as valproic acid (VPA), constitute a novel class of anticancer agents that cause an increase in acetylated histones and thus restore the expression of dormant tumor-suppressor and other genes related to cell differentiation, cell-cycle arrest or apoptosis of tumor cells. The Ras inhibitor farnesylthiosalicylic acid (FTS, salirasib) attenuates cancer cell proliferation in vitro and in vivo and, under certain circumstances, induces cell death. FTS by itself does not induce differentiation or complete growth arrest. The abovementioned activity of VPA as a differentiation agent suggested that it might be worth investigating its possible therapeutic potential in synergistic combination with FTS. Here, we examined whether the combined application of VPA and FTS could synergistically inhibit the proliferation of cancer cells that express oncogenic K-Ras (A549 nonsmall-cell lung carcinoma cells), DLD1 (colon carcinoma cells) or chronically active wild-type K-Ras and constitutively active B-Raf (ARO, thyroid carcinoma cells). The results showed that combined treatment with VPA and FTS synergistically reduces proliferation in all of these cancer cell lines by downregulating Ras and blocking the expression of Survivin and Aurora A. These alterations, which were most pronounced following the combined treatment, led to a mitotic crisis, as reflected by mislocalization of the chromosomal passenger complex. Our findings thus demonstrate that combination therapy with VPA and FTS might offer a promising therapeutic approach to the treatment of epithelial tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genética , Antineoplásicos/uso terapêutico , Aurora Quinases , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/patologia , Divisão Celular , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Regulação para Baixo/efeitos dos fármacos , Farneseno Álcool/análogos & derivados , Farneseno Álcool/uso terapêutico , Citometria de Fluxo , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Pulmonares/patologia , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/uso terapêutico , Survivina , Ácido Valproico/uso terapêutico
6.
Nat Cancer ; 2(7): 758-772, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34939038

RESUMO

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.


Assuntos
Leucemia Linfocítica Crônica de Células B , Linhagem Celular , Células Clonais , Genômica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Transcriptoma
7.
Cancer Res ; 81(24): 6117-6130, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34686499

RESUMO

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.


Assuntos
DNA Metiltransferase 3A/metabolismo , DNA Metiltransferase 3A/fisiologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Notch/metabolismo , Animais , Antibacterianos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , DNA Metiltransferase 3A/genética , Daptomicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , RNA-Seq , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Blood Cancer Discov ; 2(1): 54-69, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33604581

RESUMO

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.


Assuntos
Epigenoma , Leucemia Linfocítica Crônica de Células B , Ilhas de CpG/genética , Metilação de DNA/genética , Progressão da Doença , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico
9.
Methods Mol Biol ; 1884: 259-267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465209

RESUMO

Cellular senescence, a state of permanent growth arrest, is an important mechanism preventing the propagation of damaged cells. It suppresses cancer development in premalignant lesions in response to activated oncogenes and in tumors following therapy. The presence of senescent cells in premalignant lesions and tumors is controlled by the immune system. The ability to identify and quantify senescent cells more efficiently in vivo is necessary in order to evaluate the effect of these cells on tumorigenesis and cancer therapy. Through combining senescent-associated beta-galactosidase staining with ImageStream X analysis, we have developed an effective method to identify and quantify senescent cancer cells in vivo.


Assuntos
Senescência Celular/imunologia , Citometria de Fluxo/métodos , Neoplasias/patologia , Coloração e Rotulagem/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos , Citometria de Fluxo/instrumentação , Galactose/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias/imunologia , Coloração e Rotulagem/instrumentação , Transfecção/instrumentação , Transfecção/métodos , beta-Galactosidase/metabolismo
10.
Cancer Cell ; 36(4): 369-384.e13, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31543463

RESUMO

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Evolução Clonal/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Commun ; 9(1): 5435, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575733

RESUMO

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1-/- mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.


Assuntos
Senescência Celular , Imunossenescência , Perforina/fisiologia , Animais , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Inflamação/etiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/farmacologia , Nitrofenóis/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Progéria/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico
12.
Aging Cell ; 16(4): 661-671, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28455874

RESUMO

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.


Assuntos
Envelhecimento/genética , Senescência Celular/genética , Neoplasias/genética , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Biomarcadores/análise , Senescência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Etoposídeo/farmacologia , Fibrose , Citometria de Fluxo , Expressão Gênica , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos , Imagem Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Cultura Primária de Células , Células Estromais/metabolismo , Células Estromais/patologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
13.
Nat Commun ; 7: 11190, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27048913

RESUMO

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Nitrofenóis/farmacologia , Proteínas/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/patologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Piperazinas/farmacologia , Cultura Primária de Células , Proteínas/genética , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
14.
PLoS One ; 6(11): e27490, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22102901

RESUMO

BACKGROUND: Galectin-3 (Gal-3) and active (GTP-bound) K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs) and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60%) of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50%) of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1) induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription) and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.


Assuntos
Galectina 3/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Western Blotting , Caseína Quinase I/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Citoplasma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
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