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1.
Genes Dev ; 30(1): 34-51, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26701265

RESUMO

Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.


Assuntos
Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/fisiopatologia , Regulação Neoplásica da Expressão Gênica/genética , RNA Longo não Codificante/genética , Animais , Adesão Celular/genética , Movimento Celular/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Morfogênese/genética , Metástase Neoplásica/genética , Processamento de Proteína/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais/genética
2.
Cell ; 134(4): 657-67, 2008 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-18724938

RESUMO

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.


Assuntos
Senescência Celular , Cirrose Hepática/imunologia , Fígado/citologia , Animais , Tetracloreto de Carbono , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Fígado/fisiologia , Cirrose Hepática/metabolismo , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/metabolismo , Camundongos
3.
PLoS Genet ; 13(3): e1006635, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28301478

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.


Assuntos
Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Doenças Neurodegenerativas/genética , Retroelementos/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
EMBO J ; 32(14): 1990-2000, 2013 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23673358

RESUMO

While the Polycomb complex is known to regulate cell identity in ES cells, its role in controlling tissue-specific stem cells is not well understood. Here we show that removal of Ezh1 and Ezh2, key Polycomb subunits, from mouse skin results in a marked change in fate determination in epidermal progenitor cells, leading to an increase in the number of lineage-committed Merkel cells, a specialized subtype of skin cells involved in mechanotransduction. By dissecting the genetic mechanism, we showed that the Polycomb complex restricts differentiation of epidermal progenitor cells by repressing the transcription factor Sox2. Ablation of Sox2 results in a dramatic loss of Merkel cells, indicating that Sox2 is a critical regulator of Merkel cell specification. We show that Sox2 directly activates Atoh1, the obligate regulator of Merkel cell differentiation. Concordantly, ablation of Sox2 attenuated the Ezh1/2-null phenotype, confirming the importance of Polycomb-mediated repression of Sox2 in maintaining the epidermal progenitor cell state. Together, these findings define a novel regulatory network by which the Polycomb complex maintains the progenitor cell state and governs differentiation in vivo.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células de Merkel/citologia , Células de Merkel/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética , Gravidez , Fatores de Transcrição SOXB1/deficiência , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
RNA ; 21(11): 1966-79, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26392588

RESUMO

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion.


Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , RNA/metabolismo , Comunicação Celular/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Células K562
6.
Proc Natl Acad Sci U S A ; 106(9): 3615-20, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19218459

RESUMO

Cell-to-cell transport in plants occurs through cytoplasmic channels called "plasmodesmata" and is regulated by developmental and environmental factors. Callose deposition modulates plasmodesmal transport in vivo, but little is known about the mechanisms that regulate this process. Here we report a genetic approach to identify mutants affecting plasmodesmal transport. We isolated 5 mutants, named gfp arrested trafficking (gat), affected in GFP unloading from the phloem into the meristem. gat1 mutants were seedling lethal and carried lesions in an m-type thioredoxin that is expressed in non-green plastids of meristems and organ primordia. Callose and hydrogen peroxide accumulated in gat1 mutants, and WT plants subjected to oxidative conditions phenocopied the gat1 trafficking defects. Ectopic expression of GAT1 in mature leaves increased plasmodesmal permeability and led to a delay in senescence and flowering time. We propose a role for the GAT1 thioredoxin in the redox regulation of callose deposition and symplastic permeability that is essential for meristem maintenance in Arabidopsis.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Tiorredoxinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Meristema/genética , Fenótipo , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Tiorredoxinas/genética
7.
Life Sci Alliance ; 4(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33653689

RESUMO

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.


Assuntos
Cílios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Traqueia/metabolismo , Animais , Polaridade Celular , Cílios/fisiologia , Células Epiteliais , Feminino , Masculino , Camundongos , Camundongos Knockout , Muco , Óxido Nítrico Sintase Tipo I/fisiologia , Traqueia/citologia , Traqueia/fisiologia
8.
Curr Biol ; 17(14): 1253-8, 2007 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-17629483

RESUMO

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments [1-3]. However, how microenvironments are shaped is poorly understood. In testes of Drosophila melanogaster, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells [4]. We show that germ cells signal via the Egf class ligand Spitz (Spi) and provide evidence that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppressed the germ cell enclosure defects of the conditional spi allele. We propose that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. Our novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Drosophila melanogaster/citologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Células Germinativas/citologia , Masculino , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/fisiologia , Testículo/anormalidades , Testículo/citologia , Testículo/metabolismo
9.
J Cell Biol ; 171(3): 493-503, 2005 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16275753

RESUMO

Amodel that explains both the origin and sporadic nature of cancer argues that cancer cells are a chance result of events that cause genomic and epigenetic variability. The prevailing view is that these events are mutations that affect chromosome segregation or stability. However, genomic and epigenetic variability is also triggered by cell fusion, which is often caused by viruses. Yet, cells fused by viruses are considered harmless because they die. We provide evidence that a primate virus uses both viral and exosomal proteins involved in cell fusion to produce transformed proliferating human cells. Although normal cells indeed fail to proliferate after fusion, expression of an oncogene or a mutated tumor suppressor p53 in just one of the fusion partners is sufficient to produce heterogeneous progeny. We also show that this virus can produce viable oncogenically transformed cells by fusing cells that are otherwise destined to die. Therefore, we argue that viruses can contribute to carcinogenesis by fusing cells.


Assuntos
Transformação Celular Neoplásica , Vírus dos Macacos de Mason-Pfizer/fisiologia , Animais , Fusão Celular , Linhagem Celular , Sobrevivência Celular , Transformação Celular Viral , Genes p53 , Humanos , Células Híbridas , Vírus dos Macacos de Mason-Pfizer/genética , Mutação , Oncogenes
10.
Cancers (Basel) ; 12(1)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936750

RESUMO

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

11.
Mol Carcinog ; 48(9): 832-42, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19263435

RESUMO

PPP1R13L was initially identified as a protein that binds to the NF-kappaB subunit p65/RelA and inhibits its transcriptional activity. It also binds p53 and inhibits its action. One set of experimental findings based on overexpression of PPP1R13L indicates that PPP1R13L blocks apoptosis. Another set of experiments, based on endogenous production of PPP1R13L, suggests that the protein may sometimes be pro-apoptotic. We have used primary mouse embryonic fibroblasts (MEFs), dually transformed by HRAS and adenovirus E1A and differing in their p53 status, to explore the effects of PPP1R13L overexpression, thus examining the ability of PPP1R13L to act as an oncoprotein. We found that overexpression of PPP1R13L strongly accelerated tumor formation by RAS/E1A. PPP1R13L overexpressing cells were depleted for both p53 and active p65/RelA and we found that both p53-dependent and -independent apoptosis pathways were modulated by PPP1R13L. Finally, studies with the proteasome inhibitor MG132 revealed that overexpression of PPP1R13L causes faster p53 degradation, a likely explanation for the depletion of p53. Taken together, our results show that increased levels of PPP1R13L can increase tumorigenesis and furthermore suggest that PPP1R13L can influence metastasis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Experimentais/patologia , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Apoptose , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Camundongos Nus , Invasividade Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Proteínas ras/metabolismo
12.
Mol Biol Cell ; 15(6): 2664-73, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15034145

RESUMO

The organization of the pre-mRNA splicing machinery has been extensively studied in mammalian and yeast cells and far less is known in living plant cells and different cell types of an intact organism. Here, we report on the expression, organization, and dynamics of pre-mRNA splicing factors (SR33, SR1/atSRp34, and atSRp30) under control of their endogenous promoters in Arabidopsis. Distinct tissue-specific expression patterns were observed, and differences in the distribution of these proteins within nuclei of different cell types were identified. These factors localized in a cell type-dependent speckled pattern as well as being diffusely distributed throughout the nucleoplasm. Electron microscopic analysis has revealed that these speckles correspond to interchromatin granule clusters. Time-lapse microscopy revealed that speckles move within a constrained nuclear space, and their organization is altered during the cell cycle. Fluorescence recovery after photobleaching analysis revealed a rapid exchange rate of splicing factors in nuclear speckles. The dynamic organization of plant speckles is closely related to the transcriptional activity of the cells. The organization and dynamic behavior of speckles in Arabidopsis cell nuclei provides significant insight into understanding the functional compartmentalization of the nucleus and its relationship to chromatin organization within various cell types of a single organism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Splicing de RNA , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/ultraestrutura , Ciclo Celular , Núcleo Celular/metabolismo , Diclororribofuranosilbenzimidazol/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas Nucleares/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Especificidade de Órgãos , Fosfoproteínas/ultraestrutura , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Transporte Proteico , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-Arginina , Transcrição Gênica/efeitos dos fármacos
13.
Dev Cell ; 41(6): 652-664.e5, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28633019

RESUMO

The unfolded protein response (UPR), which protects cells against accumulation of misfolded proteins in the ER, is induced in several age-associated degenerative diseases. However, sustained UPR activation has negative effects on cellular functions and may worsen disease symptoms. It remains unknown whether and how UPR components can be utilized to counteract chronic ER proteinopathies. We found that promotion of ER-associated degradation (ERAD) through upregulation of ERAD-enhancing α-mannosidase-like proteins (EDEMs) protected against chronic ER proteinopathy without inducing toxicity in a Drosophila model. ERAD activity in the brain decreased with aging, and upregulation of EDEMs suppressed age-dependent behavioral decline and extended the lifespan without affecting the UPR gene expression network. Intriguingly, EDEM mannosidase activity was dispensable for these protective effects. Therefore, upregulation of EDEM function in the ERAD protects against ER proteinopathy in vivo and thus represents a potential therapeutic target for chronic diseases.


Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Expressão Gênica/fisiologia , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster/metabolismo , Dobramento de Proteína
14.
J Exp Med ; 214(3): 579-596, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28232471

RESUMO

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.


Assuntos
Carcinoma Ductal Pancreático/patologia , Fibroblastos/fisiologia , Miofibroblastos/fisiologia , Neoplasias Pancreáticas/patologia , Actinas/análise , Animais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Citocinas/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição STAT3/metabolismo
15.
Sci Transl Med ; 8(361): 361ra138, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27798263

RESUMO

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.


Assuntos
Armadilhas Extracelulares , Metástase Neoplásica , Neutrófilos/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Desoxirribonuclease I/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Neutrófilos/citologia
16.
J Spinal Cord Med ; 28(3): 241-5, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16048142

RESUMO

BACKGROUND/OBJECTIVES: Spasticity in patients with spinal cord injury (SCI) is difficult to manage. Exercise and stretching is advocated as a management tool, but these activities are difficult to perform for most patients as a result of multiple barriers. This report shows the effect of passive range-of-motion exercise in a walking-like pattern on frequency-dependent habituation of the H-reflex in the lower extremities of an individual with spastic tetraplegia due to SCI. METHODS: The participant, a man with a chronic ASIA B C7 SCI due to a gunshot wound, used a motorized bicycle exercise trainer (MBET) developed at the Jackson T. Stephens Spine & Neurosciences Institute at the University of Arkansas for Medical Sciences that could be operated from the individual's wheelchair. He used the MBET for 1 hour, 5 days a week, for 13 weeks. H-reflex habituation was tested at the beginning of the study and then periodically over the course of 17 weeks, including 4 weeks after exercise had ceased. RESULTS: Significant habituation of the H-reflex was evident beginning at the 10th week of training. The habituation in the H-reflex reached a normal level at 5- and 10-Hz frequencies at 12 weeks. Subjective assessment of spasticity indicated that it was significantly reduced. The H-reflex amplitude was maintained at normal levels during the remaining week of the course of exercise and for 2 additional weeks after exercise ceased. The H-reflex habituation, however, returned to near baseline when reassessed at week 17, 4 weeks after the exercise program had concluded. Subjective assessment indicated that spasticity also had returned to pretraining levels. CONCLUSIONS: Habituation of the H-reflex, and perhaps spasticity, can be managed by a routine passive range-of-motion exercise program using a MBET, but the exercise program may need to be continuous. The benefit of reduced medication for spasticity and possibly improved quality of life could be a motivating factor for an individual with SCI and spasticity to continue the program. Because of the low complexity of the program, ease of use, and small size, this system could be inexpensive and could be used by an individual in the home. Ongoing studies will determine the minimum amount of MBET training required for maintaining long-term H-reflex habituation.


Assuntos
Ciclismo , Vértebras Cervicais , Reflexo H , Habituação Psicofisiológica , Veículos Automotores , Educação Física e Treinamento/métodos , Traumatismos da Medula Espinal/fisiopatologia , Adulto , Humanos , Masculino , Quadriplegia/etiologia , Quadriplegia/fisiopatologia , Traumatismos da Medula Espinal/complicações , Fatores de Tempo
17.
J Appl Physiol (1985) ; 92(3): 1169-75, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11842055

RESUMO

Mechanical ventilation is an essential but potentially harmful therapeutic intervention for patients with acute lung injury. The objective of this study was to investigate the effects of mechanical ventilation on large-aggregate surfactant (LA) structure and function. Isolated rat lungs were randomized to either a nonventilated control group, a relatively noninjuriously ventilated group [1 h, 10 ml/kg tidal volume, 3 cmH(2)O positive end-expiratory pressure (PEEP)], or an injuriously ventilated group (1 h, 20 ml/kg tidal volume, 0 cmH(2)O PEEP). Injurious ventilation resulted in significantly decreased lung compliance compared with the other two groups. LA structure, as determined by electron microscopy, revealed that LA from the injurious group had significantly lower amounts of organized lipid-protein structures compared with LA obtained from the other groups. Analysis of the biophysical properties by using a captive bubble surfactometer demonstrated that adsorption and surface tension reduction were significantly impaired with LA from the injuriously ventilated lungs. We conclude that the injurious mechanical ventilation impairs LA function and that this impairment is associated with significant morphological alterations.


Assuntos
Pulmão/fisiologia , Surfactantes Pulmonares/química , Surfactantes Pulmonares/fisiologia , Respiração Artificial , Adsorção , Animais , Fenômenos Biofísicos , Biofísica , Técnicas In Vitro , Pulmão/ultraestrutura , Complacência Pulmonar , Lesão Pulmonar , Masculino , Respiração com Pressão Positiva , Ratos , Ratos Sprague-Dawley , Respiração Artificial/efeitos adversos , Respiração Artificial/métodos , Tensão Superficial , Volume de Ventilação Pulmonar , Ferimentos e Lesões/etiologia , Ferimentos e Lesões/patologia , Ferimentos e Lesões/fisiopatologia
18.
J Neurosci Methods ; 118(2): 189-98, 2002 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-12204309

RESUMO

Primary dissociated human fetal forebrain cultures were grown in defined serum-free conditions. At 4 weeks in vitro the cultures contained abundant morphologically well differentiated neurons with complex dendritic arbors. Astrocytic proliferation was negligible without the use of antimitotic agents. Confocal scanning laser microscopy (CSLM) and electron microscopy confirmed the presence of a dense neuropil, numerous cell-cell contacts and synapses. Neurons expressed a variety of proteins including growth associated protein-43 (GAP43), microtubule associated protein-2ab (MAP), class-III beta tubulin (C3BT), neurofilaments (NF), synaptophysin (SYN), parvalbumin (PA) and calbindin (CB). The cultures have proven to be reliable and simple to initiate and maintain for many weeks without passaging. They are useful in investigations of dendritic growth and injury of primary human CNS neurons.


Assuntos
Dendritos/ultraestrutura , Neurônios/ultraestrutura , Sinapses/ultraestrutura , Calbindinas , Técnicas de Cultura de Células/métodos , Feto , Proteína GAP-43/biossíntese , Humanos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neurofilamentos/biossíntese , Neuroglia/ultraestrutura , Neurônios/metabolismo , Parvalbuminas/biossíntese , Prosencéfalo , Proteína G de Ligação ao Cálcio S100/biossíntese , Sinaptofisina/biossíntese , Tubulina (Proteína)/biossíntese
19.
Nat Commun ; 5: 3812, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24818823

RESUMO

One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Acetilação , Animais , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Histonas/metabolismo , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Protaminas/metabolismo , Testículo/metabolismo
20.
Mol Biol Cell ; 23(18): 3694-706, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22855529

RESUMO

The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II-mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Células Cultivadas , Células HeLa , Humanos , Immunoblotting , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/genética , Ligação Proteica , Interferência de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina , Transcrição Gênica
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