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1.
Ann Oncol ; 29(5): 1286-1291, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29509837

RESUMO

Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , DNA Tumoral Circulante/genética , Neoplasias Hepáticas/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biópsia/métodos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA/métodos , Estudos de Viabilidade , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fígado/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Projetos Piloto , Carga Tumoral/genética
2.
Gene Ther ; 19(6): 642-8, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22378345

RESUMO

Engineered recombinant viral vectors are a powerful tool for vehiculating genetic information into mammalian cells. Because of their ability to infect both dividing and non-dividing cells with high efficiency, lentiviral vectors have gained particular interest for basic research and preclinical studies in the cardiovascular field. We review here the major applications for lentiviral-vector technology in the cardiovascular field: we will discuss their use in trailing gene expression during the induction of differentiation, in protocols for the isolation of cardiac cells and in the tracking of cardiac cells after transplantation in vivo; we will also describe lentivirally-mediated gene delivery uses, such as the induction of a phenotype of interest in a target cell or the treatment of cardiovascular diseases. In addition, a section of the review will be dedicated to reprogramming approaches, focusing attention on the generation of pluripotent stem cells and on transdifferentiation, two emerging strategies for the production of cardiac myocytes from human cells and for the investigation of human diseases. Finally, in order to give a perspective on their future clinical use we will critically discuss advantages and disadvantages of lentivirus-based strategies for the treatment of cardiovascular diseases.


Assuntos
Doenças Cardiovasculares/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Miócitos Cardíacos/fisiologia , Diferenciação Celular , Transdiferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/transplante
3.
Int J Immunopathol Pharmacol ; 25(2): 467-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22697078

RESUMO

MicroRNAs are required for vascular smooth muscle growth, differentiation and function. MiR143-145 modulates cytoskeletal dynamics and acquisition of the contractile phenotype by smooth muscle cells. Lack of this miRNA cluster results in decreased blood pressure and reduced vasocontraction. As all these observations point to a key role for miR143-145 in the vasculature, we investigated whether miR143-145 deficiency is associated with impaired vascular tone. Vasocontraction was assessed in isolated aortic rings from miR143-145 KO and wild type animals incubated with increasing concentrations of phenylephrine (10(-9)M to 10(-5)M) or KCl 0.3M. In both cases, aortic vessel contraction was dramatically reduced in miR143-145 KO animals compared to controls. Next, aortic rings were pre-contracted with phenylephrine (EC60: 10(-7)M) and concentration responses for acetylcholine were obtained. A significantly reduced vasodilation was observed in miR143-145 KO animals compared to controls and similar results were obtained when an exogenous donor of nitric oxide (sodium nitroprusside) was used. Endothelial nitric oxide synthase or guanylate cyclase mRNA expression were not different between the animal groups thus suggesting to investigate the effect of other vasodilators. Isoprenaline mediated vasodilation was significantly reduced in miR143-145 KO animals compared to controls in the absence or in the presence of the guanylate cyclase inhibitor ODQ (10(-4)M), suggesting that also beta adrenergic vasodilation is impaired following miR143-145 deficiency. Finally, the effect of a stable mimetic prostacyclin, namely iloprost, was investigated and again a reduced vasodilation was observed in miR143-145 KO animals. MiR143-145 deficiency is associated not only with altered vasocontraction but also with impaired vasodilation, which probably reflects the impaired VSMC differentiation phenotype reported in miR143-145 KO animals.


Assuntos
MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição , Vasodilatação , Animais , Aorta Torácica/metabolismo , Diferenciação Celular , Relação Dose-Resposta a Droga , Regulação para Baixo , Genótipo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fenótipo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
4.
Cardiovasc Res ; 118(4): 1004-1019, 2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33757121

RESUMO

The human transcriptome comprises a complex network of coding and non-coding RNAs implicated in a myriad of biological functions. Non-coding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathological states, including in the cardiovascular system. This review defines the current knowledge gaps, unmet methodological needs, and describes the challenges in dissecting and understanding the role and regulation of the non-coding transcriptome in cardiovascular disease. These challenges include poor annotation of the non-coding genome, determination of the cellular distribution of transcripts, assessment of the role of RNA processing and identification of cell-type specific changes in cardiovascular physiology and disease. We highlight similarities and differences in the hurdles associated with the analysis of the non-coding and protein-coding transcriptomes. In addition, we discuss how the lack of consensus and absence of standardized methods affect reproducibility of data. These shortcomings should be defeated in order to make significant scientific progress and foster the development of clinically applicable non-coding RNA-based therapeutic strategies to lessen the burden of cardiovascular disease.


Assuntos
Doenças Cardiovasculares , RNA Longo não Codificante , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/terapia , Humanos , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Reprodutibilidade dos Testes , Transcriptoma
5.
Nat Med ; 1(6): 541-5, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7585120

RESUMO

Proliferation of smooth muscle cells of the arterial wall in response to local injury is an important aetiologic factor of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. Ras proteins are key transducers of mitogenic signals from membrane to nucleus in many cell types. We investigated the role of ras proteins in the vascular response to arterial injury by inactivating cellular ras of rats in which the common carotid artery was subjected to balloon injury. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury. Our results indicate a key role for ras in smooth muscle cell proliferation and show that the local delivery of transdominant negative mutants of ras in vivo might prevent some of the acute vascular injury caused by balloon injury.


Assuntos
Genes ras , Terapia Genética , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Lesões das Artérias Carótidas , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/patologia , Cateterismo/efeitos adversos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , DNA Recombinante/genética , DNA Recombinante/uso terapêutico , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão , Transfecção
6.
Nat Med ; 3(7): 775-9, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9212106

RESUMO

Injury of the arterial wall induces the formation of the neointima. This structure is generated by the growth of mitogenically activated smooth muscle cells of the arterial wall. The molecular mechanism underlying the formation of the neointima involves deregulated cell growth, primarily triggered by the injury of the arterial wall. The activated gene products transmitting the injury-induced mitogenic stimuli have been identified and inhibited by several means: transdominant negative expression vectors, antisense oligodeoxynucleotides, adenovirus-mediated gene transfer, antibodies and inactivating drugs. Results of our study show that local administration of 3',5'-cyclic AMP and phosphodiesterase-inhibitor drugs (aminophylline and amrinone) to rats markedly inhibits neointima formation after balloon injury in vivo and in smooth muscle cells in vitro. The growth inhibitory effect of aminophylline was completely reversed by the inhibition of cAMP-dependent protein kinase A (PKA). These findings indicate an alternative approach to the treatment of diseases associated with injury-induced cell growth of the arterial wall, as stimulation of cAMP signaling is pharmacologically feasible in the clinical setting.


Assuntos
Divisão Celular , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Músculo Liso Vascular/citologia , Transdução de Sinais , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aminofilina/farmacologia , Amrinona/farmacologia , Animais , Artérias Carótidas , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores do Crescimento/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos
7.
Int Endod J ; 43(10): 866-73, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618880

RESUMO

AIM: To assess the failure mechanism of rotary NiTi instruments by chemical, structural and morphological analyses to provide a rational explanation of the effects of surface and bulk treatments on their resistance to fatigue fracture. METHODOLOGY: Thermal treatment (350-500 °C) was performed on electropolished (EP) and non-electropolished (Non-EP) NiTi endodontic instruments. Bulk and surface chemical composition and crystallographic structures were determined by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) to evaluate the effects of thermal treatment and electropolishing on the NiTi alloy. Fatigue tests of all instruments were performed. Surface morphology before and after the tests, and fractured section were analysed using scanning electron microscopy to determine crack extensions. Results were analysed statistically using analysis of variance (anova) and post hoc Student-Newman-Keuls test. RESULTS: Before thermal treatment, significant differences (P < 0.05) in fatigue resistance between EP and Non-EP instruments (the number of revolutions to failure, N(f) , was 385 and 160, respectively) were attributed to differences in the surface morphology of the instruments. SEM analysis of the fracture surfaces indicated that flexural fatigue fractures occurred in two steps: first by a slow growth of initial cracks and then rapid rupture of the remaining material. Thermal treatment did not affect the surface morphology but resulted in significant changes in the instrument bulk with the appearance of an R-phase and an improved fatigue resistance; indeed after treatment at 500 °C, N(f) increased up to 829 and 474 for EP and Non-EP instruments, respectively. CONCLUSIONS: Both thermal treatment and electropolishing improved the resistance of NiTi rotary instruments against fatigue fracture.


Assuntos
Ligas Dentárias/química , Níquel/química , Preparo de Canal Radicular/instrumentação , Titânio/química , Carbono/análise , Cobre/análise , Cristalografia , Ligas Dentárias/análise , Polimento Dentário/métodos , Eletricidade , Microanálise por Sonda Eletrônica , Desenho de Equipamento , Falha de Equipamento , Temperatura Alta , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Níquel/análise , Oxigênio/análise , Silício/análise , Espectrometria por Raios X , Estresse Mecânico , Propriedades de Superfície , Titânio/análise , Difração de Raios X
8.
Dalton Trans ; 49(25): 8652-8660, 2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32555848

RESUMO

In this work, multiferroic bismuth ferrite (BFO) was functionalized with meso-tetraphenylporphine-4,4',4'',4'''-tetracarboxylic acid (TCPP). This new hybrid organic-inorganic material shows an enhanced photocatalytic activity for the degradation of organic dyes as it combines the properties of BFO which is an efficient visible light photocatalyst with peculiar porphyrin absorption in visible light. The anchoring of TCPP to the OH-terminations of the BFO surface through its carboxylic tethering groups was demonstrated using X-ray photoelectron spectroscopy and FT-IR spectroscopy. The photocatalytic activity of the material was also demonstrated through the photocatalytic degradation of Methylene Blue (MB) and Rhodamine-B (Rhd-B) in water under simulated solar light illumination. The TCPP molecules anchored to BFO slightly decrease (∼0.06 eV) the bandgap energy of the system and act as new catalytic centres, thus improving its photocatalytic activity. A photodegradation mechanism was also proposed. This new material is reusable and stable, as it maintains an unmodified photo-activity after several MB discoloration runs.

9.
Cell Death Differ ; 15(4): 762-72, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18202702

RESUMO

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.


Assuntos
Apoptose , Comunicação Autócrina , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Interleucina-4/metabolismo , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Anticorpos Monoclonais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Morte Celular , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Proteína bcl-X/metabolismo
10.
Cell Death Differ ; 26(12): 2808-2809, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31395960

RESUMO

Authors have only now noticed that in the Figure 3a, the immunohistochemical analysis of IL-4Rα on paraffin-embedded sections from breast is incorrect: IL-4 from breast was duplicated and used for the IL-4Rα staining. The correct Figure 3a has been included in the amendment to this paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

12.
Gene Ther ; 15(3): 161-70, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18033312

RESUMO

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.


Assuntos
Células-Tronco Embrionárias/citologia , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Miócitos Cardíacos/citologia , Actinas/genética , Animais , Diferenciação Celular , Linhagem Celular , Elementos Facilitadores Genéticos , Citometria de Fluxo , Engenharia Genética , Vetores Genéticos/farmacologia , Humanos , Lentivirus/genética , Camundongos , Regiões Promotoras Genéticas , Transdução Genética/métodos , Troponina I/genética
13.
Br J Pharmacol ; 153(4): 625-6, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18193077

RESUMO

Human embryonic stem cells (hESCs) are a pluripotent cell type considered to have high potential for the treatment of cardiovascular disease by cell replacement therapies. Several groups have shown that hESCs can be differentiated in vitro into cardiac myocytes, which may be used to facilitate tissue regeneration by injection directly into damaged myocardium. However, several hurdles still need to be overcome before these cells can be used in clinical trials. In particular, because transplanted hESC-cardiac myocytes should integrate fully within the damaged heart, these cells must be functionally compatible with the host myocardium. To assess this aspect of hESC-cardiac myocytes, Brito-Martins et al. (2008) in this issue of the BJP, describe the responses of hESC-cardiac myocytes to beta-adrenoceptor stimulation, compared to those of myocytes from adult human hearts. Data obtained using specific beta-adrenoceptor agonists showed good compatibility of hESC-cardiac myocytes with adult human myocardium in terms of beta-adrenoceptor response.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Animais , Carbacol/farmacologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Humanos , Imidazóis/farmacologia , Isoproterenol/farmacologia , Agonistas Muscarínicos/farmacologia , Miócitos Cardíacos/metabolismo , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Transplante de Células-Tronco
14.
Nanoscale Res Lett ; 13(1): 45, 2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29417388

RESUMO

A versatile synthetic route based on magnetic Fe3O4 nanoparticle (MNP) prefunctionalization with a phosphonic acid monolayer has been used to covalently bind the gH625 peptide on the nanoparticle surface. gH625 is a membranotropic peptide capable of easily crossing the membranes of various cells including the typical human blood-brain barrier components. A similar synthetic route was used to prepare another class of MNPs having a functional coating based on PEG, rhodamine, and folic acid, a well-known target molecule, to compare the performance of the two cell-penetrating systems (i.e., gH625 and folic acid). Our results demonstrate that the uptake of gH625-decorated MNPs in immortalized human brain microvascular endothelial cells after 24 h is more evident compared to folic acid-functionalized MNPs as evidenced by confocal laser scanning microscopy. On the other hand, both functionalized systems proved capable of being internalized in a brain tumor cell line (i.e., glioblastoma A-172). These findings indicate that the functionalization of MNPs with gH625 improves their endothelial cell internalization, suggesting a viable strategy in designing functional nanostructures capable of first crossing the BBB and, then, of reaching specific tumor brain cells.

15.
J Clin Invest ; 95(5): 2346-58, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7738198

RESUMO

We have explored the expression of the transcription factors GATA-1, GATA-2, and NF-E2 in purified early hematopoietic progenitor cells (HPCs) induced to gradual unilineage erythroid or granulocytic differentiation by growth factor stimulus. GATA-2 mRNA and protein, already expressed in quiescent HPCs, is rapidly induced as early as 3 h after growth factor stimulus, but then declines in advanced erythroid and granulocytic differentiation and maturation. NF-E2 and GATA-1 mRNAs and proteins, though not detected in quiescent HPCs, are gradually induced at 24-48 h in both erythroid and granulocytic culture. Beginning at late differentiation/early maturation stage, both transcription factors are further accumulated in the erythroid pathway, whereas they are suppressed in the granulopoietic series. Similarly, the erythropoietin receptor (EpR) is induced and sustainedly expressed during erythroid differentiation, although beginning at later times (i.e., day 5), whereas it is barely expressed in the granulopoietic pathway. In the first series of functional studies, HPCs were treated with antisense oligomers targeted to transcription factor mRNA: inhibition of GATA-2 expression caused a decreased number of both erythroid and granulocyte-monocytic clones, whereas inhibition of NF-E2 or GATA-1 expression induced a selective impairment of erythroid colony formation. In a second series of functional studies, HPCs treated with retinoic acid were induced to shift from erythroid to granulocytic differentiation (Labbaye et al. 1994. Blood. 83:651-656); this was coupled with abrogation of GATA-1, NF-E2, and EpR expression and conversely enhanced GATA-2 levels. These results indicate the expression and key role of GATA-2 in the early stages of HPC proliferation/differentiation. Conversely, NF-E2 and GATA-1 expression and function are apparently restricted to erythroid differentiation and maturation: their expression precedes that of the EpR, and their function may be in part mediated via the EpR.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Expressão Gênica , Substâncias de Crescimento/farmacologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/biossíntese , Adulto , Sequência de Bases , Ensaio de Unidades Formadoras de Colônias , Primers do DNA , Proteínas de Ligação a DNA/fisiologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Fator de Transcrição GATA2 , Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cinética , Masculino , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Valores de Referência , Fatores de Tempo , Fatores de Transcrição/fisiologia , Dedos de Zinco
16.
J Clin Invest ; 98(2): 256-61, 1996 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8755632

RESUMO

While hypertension is a major risk factor for stroke, it is not its sole determinant. Despite similar blood pressures, spontaneously hypertensive rats (SHR) do not share the predisposition to cerebrovascular disease typical of stroke-prone spontaneously hypertensive rats (SHRSP). We investigated vascular function in male SHR and SHRSP as well as in SHRSP/SHR-F2 hybrid animals. Animals were maintained on the appropriate dietary regimen necessary for the manifestation of stroke. Among the hybrid animals, a group of stroke-prone and a group of stroke-resistant rats were selected. Blood pressure was similar in all groups. Endothelium-independent vascular reactivity tested on isolated rings of thoracic aorta and basilar artery after death showed similar contractile and dilatory responses to serotonin and nitroglycerin, respectively, in all groups. In contrast, endothelium-dependent relaxation, in response to acetylcholine or substance P, was markedly reduced in SHRSP compared with SHR. Similarly, reduced vasodilatory responses were present in aortae of F2 rats that had suffered a stroke when compared with SHR or F2 rats resistant to stroke. The observed association and cosegregation of stroke with significant and specific impairment of endothelium-dependent vasorelaxation among SHRSP and stroke-prone F2 hybrids, respectively, suggest a potential causal role of altered endothelium-dependent vascular relaxation in the pathogenesis of stroke.


Assuntos
Aorta Torácica/fisiopatologia , Artéria Basilar/fisiopatologia , Transtornos Cerebrovasculares/fisiopatologia , Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Vasodilatação , Acetilcolina/farmacologia , Animais , Aorta Torácica/patologia , Aorta Torácica/fisiologia , Artéria Basilar/fisiologia , Pressão Sanguínea , Transtornos Cerebrovasculares/genética , Transtornos Cerebrovasculares/patologia , Cruzamentos Genéticos , Suscetibilidade a Doenças , Endotélio Vascular/fisiologia , Feminino , Frequência Cardíaca , Hipertensão/genética , Hipertensão/patologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos , Serotonina/farmacologia , Substância P/farmacologia , Vasodilatação/efeitos dos fármacos
17.
Mol Cell Biol ; 20(14): 5330-42, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10866689

RESUMO

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb(-) SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1-E12-Lmo2 and tal-1-E12-Lmo2-Ldb1 complexes but not that of a tal-1-E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1-E12-Lmo2-Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritrócitos/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Proteínas com Domínio LIM , Masculino , Metaloproteínas/genética , Metaloproteínas/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/genética , Proteína do Retinoblastoma/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Mol Cell Biol ; 17(5): 2954-69, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9111367

RESUMO

The TAL-1 gene specifies a basic helix-loop-helix domain (bHLH) transcription factor, which heterodimerizes with E2A gene family proteins. tal-1 protein is abnormally expressed in the majority of T-cell acute lymphoblastic leukemias (T-ALLs). tal-1 is expressed and plays a significant role in normal erythropoietic differentiation and maturation, while its expression in early myeloid differentiation is abruptly shut off at the level of late progenitors/early differentiated precursors (G. L. Condorelli, L. Vitelli, M. Valtieri, I. Marta, E. Montesoro, V. Lulli, R. Baer, and C. Peschle, Blood 86:164-175, 1995). We show that in late myeloid progenitors (the phenotypically normal murine 32D cell line) and early leukemic precursors (the human HL-60 promyelocytic leukemia cell line) ectopic tal-1 expression induces (i) a proliferative effect under suboptimal culture conditions (i.e., low growth factor and serum concentrations respectively), via an antiapoptotic effect in 32D cells or increased DNA synthesis in HL-60 cells, and (ii) a total or marked inhibitory effect on differentiation, respectively, on granulocyte colony-stimulating factor-induced granulopoiesis in 32D cells or retinoic acid- and vitamin D3-induced granulo- and monocytopoiesis in HL-60 cells. Furthermore, experiments with 32D temperature-sensitive p53 cells indicate that aberrant tal-1 expression at the permissive temperature does not exert a proliferative effect but causes p53-mediated apoptosis, i.e., the tal-1 proliferative effect depends on the integrity of the cell cycle checkpoints of the host cell, as observed for c-myc and other oncogenes. tal-1 mutant experiments indicate that ectopic tal-1 effects are mediated by both the DNA-binding and the heterodimerization domains, while the N-terminally truncated tal-1 variant (M3) expressed in T-ALL malignant cells mimics the effects of the wild-type protein. Altogether, our results (i) indicate proliferative and antidifferentiative effects of ectopic tal-1 expression, (ii) shed light on the underlying mechanisms (i.e., requirement for the integrity of the tal-1 bHLH domain and cell cycle checkpoints in the host cell, particularly p53), and (iii) provide new experimental models to further investigate these mechanisms.


Assuntos
Apoptose , Proteínas de Ligação a DNA/biossíntese , Sequências Hélice-Alça-Hélice , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Colecalciferol/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células HL-60 , Humanos , Interleucina-3/farmacologia , Leucemia Mieloide/metabolismo , Fenótipo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Tretinoína/farmacologia
19.
Mol Cell Biol ; 20(17): 6323-33, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10938109

RESUMO

In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Calpha (PKCalpha) and beta activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCalpha and -beta activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCbeta with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCbeta block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCbeta. Finally, blocking PKCalpha expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCalpha and -beta. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCbeta plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.


Assuntos
Insulina/metabolismo , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase C/metabolismo , Western Blotting , Divisão Celular , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteínas Substratos do Receptor de Insulina , Lovastatina/farmacologia , Músculos/metabolismo , Oligonucleotídeos Antissenso , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosforilação , Testes de Precipitina , Isoformas de Proteínas , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor de Insulina/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas ras/metabolismo
20.
Mol Cell Biol ; 21(7): 2485-95, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11259597

RESUMO

The high-mobility group I (HMGI) nonhistone chromosomal proteins HMGI(Y) and HMGI-C have been implicated in defining chromatin structure and in regulating the transcription of several genes. These proteins have been implicated in adipocyte homeostasis: a severe deficiency of fat tissue is found in mice with targeted disruption of the HMGI-C locus, and lipomagenesis in humans is frequently associated with somatic mutations of HMGI genes. The aim of this study was to examine the role of HMGI(Y) proteins in adipocytic cell growth and differentiation. First, we found that differentiation of the preadipocytic 3T3-L1 cell line caused early induction of HMGI(Y) gene expression. Suppression of HMGI(Y) expression by antisense technology dramatically increased the growth rate and impaired adipocytic differentiation in these cells. The process of adipogenic differentiation involves the interplay of several transcription factors, among which is the CCAAT/enhancer-binding protein (C/EBP) family of proteins. These factors are required for the transcriptional activation of adipocyte-specific genes. We also tested the hypothesis that HMGI(Y) might participate in transcriptional control of adipocyte-specific promoters. We found that HMGI(Y) proteins bind C/EBPbeta in vivo and in vitro. Furthermore, we show that HMGI(Y) strongly potentiates the capacity of C/EBPbeta to transactivate the leptin promoter, an adipose-specific promoter. Taken together, these results indicate that the HMGI(Y) proteins play a critical role in adipocytic cell growth and differentiation.


Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proteína HMGA1a , Camundongos
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