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1.
Front Oncol ; 9: 245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024847

RESUMO

Acute leukemia is a heterogeneous set of diseases affecting children and adults. Current prognostic factors are not accurate predictors of the clinical outcome of adult patients and the stratification of risk groups remains insufficient. For that reason, this study proposes a multifactorial analysis which integrates clinical parameters, ex vivo tumor characterization and behavioral in vivo analysis in zebrafish. This model represents a new approach to understand leukemic primary cells behavior and features associated with aggressiveness and metastatic potential. Xenotransplantation of primary samples from patients newly diagnosed with acute leukemia in zebrafish embryos at 48 hpf was used to asses survival rate, dissemination pattern, and metastatic potential. Seven samples from young adults classified in adverse, favorable or intermediate risk group were characterized. Tumor heterogeneity defined by Leukemic stem cell (LSC) proportion, was performed by metabolic and cell membrane biomarkers characterization. Thus, our work combines all these parameters with a robust quantification strategy that provides important information about leukemia biology, their relationship with specific niches and the existent inter and intra-tumor heterogeneity in acute leukemia. In regard to prognostic factors, leukemic stem cell proportion and Patient-derived xenografts (PDX) migration into zebrafish were the variables with highest weights for the prediction analysis. Higher ALDH activity, less differentiated cells and a broader and random migration pattern are related with worse clinical outcome after induction chemotherapy. This model also recapitulates multiple aspects of human acute leukemia and therefore is a promising tool to be employed not only for preclinical studies but also supposes a new tool with a higher resolution compared to traditional methods for an accurate stratification of patients into worse or favorable clinical outcome.

2.
Elife ; 52016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27805568

RESUMO

Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Rim/metabolismo , Fatores de Transcrição/genética , Veias/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Mutação , Organogênese/genética , Fatores de Transcrição/metabolismo , Veias/crescimento & desenvolvimento , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
3.
Dev Biol ; 383(2): 214-26, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24075907

RESUMO

The endocardium forms the inner lining of the heart tube, where it enables blood flow and also interacts with the myocardium during the formation of valves and trabeculae. Although a number of studies have identified regulators in the morphogenesis of the myocardium, relatively little is known about the molecules that control endocardial morphogenesis. Prior work has implicated the bHLH transcription factor Tal1 in endocardial tube formation: in zebrafish embryos lacking Tal1, endocardial cells form a disorganized mass within the ventricle and do not populate the atrium. Through blastomere transplantation, we find that tal1 plays a cell-autonomous role in regulating endocardial extension, suggesting that Tal1 activity influences the behavior of individual endocardial cells. The defects in endocardial behavior in tal1-deficient embryos originate during the earliest steps of endocardial morphogenesis: tal1-deficient endocardial cells fail to generate a cohesive monolayer at the midline and instead pack tightly together into a multi-layered aggregate. Moreover, the tight junction protein ZO-1 is mislocalized in the tal1-deficient endocardium, indicating a defect in intercellular junction formation. In addition, we find that the tal1-deficient endocardium fails to maintain its identity; over time, a progressively increasing number of tal1-deficient endocardial cells initiate myocardial gene expression. However, the onset of defects in intercellular junction formation precedes the onset of ectopic myocardial gene expression in the tal1-deficient endocardium. We therefore propose a model in which Tal1 has distinct roles in regulating the formation of endocardial intercellular junctions and maintaining endocardial identity.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endocárdio/embriologia , Endocárdio/metabolismo , Junções Intercelulares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Endocárdio/patologia , Endocárdio/transplante , Regulação da Expressão Gênica no Desenvolvimento , Átrios do Coração/embriologia , Átrios do Coração/metabolismo , Morfogênese , Miocárdio/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Peixe-Zebra/embriologia
4.
Development ; 137(19): 3215-20, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20724450

RESUMO

Heart formation requires the fusion of bilateral cardiomyocyte populations as they move towards the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide in zebrafish the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that it plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fibronectinas/metabolismo , Coração/embriologia , Miocárdio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibronectinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Miocárdio/citologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
5.
Development ; 136(10): 1633-41, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19395641

RESUMO

Amongst animal species, there is enormous variation in the size and complexity of the heart, ranging from the simple one-chambered heart of Ciona intestinalis to the complex four-chambered heart of lunged animals. To address possible mechanisms for the evolutionary adaptation of heart size, we studied how growth of the simple two-chambered heart in zebrafish is regulated. Our data show that the embryonic zebrafish heart tube grows by a substantial increase in cardiomyocyte number. Augmented cardiomyocyte differentiation, as opposed to proliferation, is responsible for the observed growth. By using transgenic assays to monitor developmental timing, we visualized for the first time the dynamics of cardiomyocyte differentiation in a vertebrate embryo. Our data identify two previously unrecognized phases of cardiomyocyte differentiation separated in time, space and regulation. During the initial phase, a continuous wave of cardiomyocyte differentiation begins in the ventricle, ends in the atrium, and requires Islet1 for its completion. In the later phase, new cardiomyocytes are added to the arterial pole, and this process requires Fgf signaling. Thus, two separate processes of cardiomyocyte differentiation independently regulate growth of the zebrafish heart. Together, our data support a model in which modified regulation of these distinct phases of cardiomyocyte differentiation has been responsible for the changes in heart size and morphology among vertebrate species.


Assuntos
Diferenciação Celular/fisiologia , Coração/embriologia , Miócitos Cardíacos/citologia , Peixe-Zebra/embriologia , Animais , Proliferação de Células , Fatores de Crescimento de Fibroblastos/fisiologia , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra/fisiologia
6.
Anesth Analg ; 108(3): 997-1007, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19224816

RESUMO

BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC(50) for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity. METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca(2+)-responses were also evaluated. RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine < or = mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD(50), the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca(2+)-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca(2+)-evoked cytosolic Ca(2+)-level. CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons.


Assuntos
Anestésicos Locais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Carbacol/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Colorimetria , Ativação Enzimática/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Agonistas Muscarínicos/farmacologia , Neurônios/ultraestrutura , Bloqueadores dos Canais de Potássio/farmacologia , Tetraetilamônio/farmacologia , Sais de Tetrazólio , Tetrodotoxina/farmacologia , Tiazóis
7.
Anesthesiology ; 101(4): 895-901, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15448522

RESUMO

BACKGROUND: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca influx through the plasma membrane. METHODS: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]cyt was detected with fura-2 and experiments were carried out at 37 degrees C. RESULTS: Isoflurane reduced the peak and area of the carbachol-evoked [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.


Assuntos
Anestésicos Inalatórios/farmacologia , Cálcio/metabolismo , Carbacol/farmacologia , Isoflurano/farmacologia , Neurônios/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo N/fisiologia , Linhagem Celular Tumoral , Humanos , Elementos da Série dos Lantanídeos/farmacologia , Neurônios/metabolismo , Nitrendipino/farmacologia , ômega-Conotoxina GVIA/farmacologia
8.
Brain Res ; 1011(2): 177-86, 2004 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-15157804

RESUMO

In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis.


Assuntos
Cálcio/metabolismo , Tapsigargina/farmacologia , Atropina/farmacologia , Carbacol/farmacologia , Linhagem Celular Tumoral , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Fura-2/metabolismo , Humanos , Concentração Inibidora 50 , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Antagonistas Muscarínicos/farmacologia , Neuroblastoma , Potássio/farmacologia , Fatores de Tempo
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