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1.
J Am Chem Soc ; 145(5): 2951-2957, 2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36705574

RESUMO

Macrophages are the most plastic immune cells by changing their characters in response to environmental stimuli. Broadly, macrophages are categorized into two different subsets based on M1/M2 paradigm, which exhibit completely contrary phenotypes. Whereas M1 macrophages are aggressive to offend invaders such as bacteria and tumors, M2 are anti-inflammatory cells and seemingly help tumor immunity. Tumor-associated macrophages are typical examples of M2 cells as the key components of forming and maintaining the tumor microenvironment. Despite the intensive interest, monitoring M2 macrophages in real time is hampered by the lack of competent detection tools. Here, we report the first M2 selective probe CDg18 with a novel mechanism of gating-oriented live-cell distinction through M2-favored fatty acid transporters. To demonstrate the potential of CDg18, we visualize the progressive phenotypic change of M2 toward M1 using a resveratrol analogue HS-1793 as a reprogramming effector. Combined together with M1 probe CDr17, the diminishing M2 character and emerging M1 markers could be simultaneously monitored in real time through the multicolor changes during macrophage reprogramming.


Assuntos
Corantes Fluorescentes , Macrófagos , Fenótipo
2.
Anal Chem ; 95(8): 4147-4154, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36800528

RESUMO

The differentiation of the distinct phenotypes of macrophages is essential for monitoring the stage of inflammatory diseases for accurate diagnosis and treatment. Recent studies revealed that the level of hypochlorite (OCl-) varies from activated M1 macrophages (killing pathogens) to M2 (resolution of inflammation) during inflammation. Thus, we developed a simple and efficient fluorescent probe for discriminating M1 from M0 and M2. Herein, fluorescent-based imaging is applied as an alternative to immunohistochemistry, which is challenging due to the tedious process and high cost. We developed a hypochlorite-specific probe PMS-T to differentiate M1 and M2, employing a metabolism-oriented live-cell distinction. This probe enables the detection of inflammatory rheumatoid arthritis in an ex vivo mouse model. Thus, it can be a potential chemical tool for monitoring inflammatory diseases, including rheumatoid arthritis, that may overcome the existing barriers of immunohistochemistry.


Assuntos
Artrite Reumatoide , Corantes Fluorescentes , Animais , Camundongos , Ácido Hipocloroso , Elétrons , Artrite Reumatoide/diagnóstico por imagem , Inflamação/diagnóstico por imagem
3.
J Am Chem Soc ; 144(11): 5067-5073, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35258954

RESUMO

Aggregation of amyloidogenic proteins causing neurodegenerative diseases is an uncontrollable and contagious process that is often associated with lipid membranes in a highly complex physiological environment. Although several approaches using natural cells and membrane models have been reported, systematic investigations focusing on the association with the membranes are highly challenging, mostly because of the lack of proper molecular tools. Here, we report a new supramolecular approach using a synthetic cell system capable of controlling the initiation of protein aggregation and mimicking various conditions of lipid membranes, thereby enabling systematic investigations of membrane-dependent effects on protein aggregation by visualization. Extending this strategy through concurrent use of synthetic cells and natural cells, we demonstrate the potential of this approach for systematic and in-depth studies on interrogating inter- and intracellularly transmittable protein aggregation. Thus, this new approach offers opportunities for gaining insights into the pathological implications of contagious protein aggregation associated with membranes for neurotoxicity.


Assuntos
Células Artificiais , Proteínas Amiloidogênicas/metabolismo , Membrana Celular/metabolismo , Humanos , Lipídeos , Agregados Proteicos , Agregação Patológica de Proteínas
4.
Angew Chem Int Ed Engl ; 61(36): e202202095, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35789526

RESUMO

T and B lymphocytes are two major adaptive immune cells in the human defense system. To real-time monitor their diverse functions, a live-cell-selective probe for only one cell type is need to investigate the complex interaction of the immune cells. Herein, a small-molecule probe CDyB for live B cells is developed by an unbiased fluorescence library screening. The cell selectivity was confirmed by multiparametric single-cell analysis using CyTOF. Through a systematic SLC-CRISPRi library screening, the molecular target of CDyB was identified as SLC35C2 transporter based on a gating-oriented live-cell distinction (GOLD) mechanism. The gene expression analysis and knock-out experiments validated that the SLC35C2 transporter was the target for CDyB distinction. Interestingly, when CDyB was applied to study B cell development, the CDyB fluorescence and SLC35C2 expression were positively correlated with the B cell maturation process, and not involved in the T cell development.


Assuntos
Linfócitos B , Corantes Fluorescentes , Proteínas de Neoplasias , Proteínas de Transporte de Nucleotídeos , Linfócitos B/citologia , Corantes Fluorescentes/química , Biblioteca Gênica , Humanos , Proteínas de Neoplasias/química , Proteínas de Transporte de Nucleotídeos/química
5.
J Am Chem Soc ; 143(15): 5836-5844, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33834782

RESUMO

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.


Assuntos
Linfócitos B/citologia , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Linfócitos T/citologia , Animais , Linfócitos B/química , Linfócitos B/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Membrana Celular/química , Citometria de Fluxo , Lipidômica , Camundongos , Linfócitos T/química , Linfócitos T/imunologia
6.
Angew Chem Int Ed Engl ; 60(44): 23743-23749, 2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34415094

RESUMO

Human neutrophils are the most abundant leukocytes and have been considered as the first line of defence in the innate immune system. Selective imaging of live neutrophils will facilitate the in situ study of neutrophils in infection or inflammation events as well as clinical diagnosis. However, small-molecule-based probes for the discrimination of live neutrophils among different granulocytes in human blood have yet to be reported. Herein, we report the first fluorescent probe NeutropG for the specific distinction and imaging of active neutrophils. The selective staining mechanism of NeutropG is elucidated as metabolism-oriented live-cell distinction (MOLD) through lipid droplet biogenesis with the help of ACSL and DGAT. Finally, NeutropG is applied to accurately quantify neutrophil levels in fresh blood samples by showing a high correlation with the current clinical method.


Assuntos
Células Sanguíneas/metabolismo , Corantes Fluorescentes/metabolismo , Neutrófilos/metabolismo , Células Sanguíneas/química , Corantes Fluorescentes/química , Humanos , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Estrutura Molecular , Neutrófilos/química
7.
J Am Chem Soc ; 142(7): 3430-3439, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32040300

RESUMO

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.


Assuntos
Corantes Fluorescentes/química , Células Secretoras de Insulina/citologia , Xantenos/química , Animais , Diabetes Mellitus Experimental/patologia , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Corantes Fluorescentes/toxicidade , Humanos , Células Secretoras de Insulina/transplante , Transplante das Ilhotas Pancreáticas , Fígado/citologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Tomografia por Emissão de Pósitrons , Ratos , Xantenos/síntese química , Xantenos/farmacocinética , Xantenos/toxicidade
8.
J Am Chem Soc ; 141(37): 14673-14686, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31436967

RESUMO

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.


Assuntos
Corantes Fluorescentes/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Camundongos , Células-Tronco Neoplásicas/enzimologia , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Angew Chem Int Ed Engl ; 58(25): 8426-8431, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31025486

RESUMO

The rapid and sensitive classification of bacteria is the first step of bacterial community research and the treatment of infection. Herein, a fluorescent probe BacGO is presented, which shows the best universal selectivity for Gram-positive bacteria among known probes with a minimum staining procedure for sample detection and enrichment of the live bacteria. BacGO could also be used to assess of the Gram status in the bacterial community from wastewater sludge. Furthermore, BacGO could sensitively and selectively detect a Gram-positive bacterial infection, not only in vitro but also using an in vivo keratitis mouse model. BacGO provides an unprecedented research tool for the study of dynamic bacterial communities and for clinical application.


Assuntos
Corantes Fluorescentes/química , Bactérias Gram-Positivas/isolamento & purificação , Ceratite/diagnóstico por imagem , Animais , Corantes Fluorescentes/síntese química , Camundongos , Estrutura Molecular
10.
Angew Chem Int Ed Engl ; 57(11): 2851-2854, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29377425

RESUMO

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.


Assuntos
Corantes Fluorescentes/química , Células-Tronco Neoplásicas/patologia , Bibliotecas de Moléculas Pequenas/química , Vimentina/análise , Animais , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Neoplasias Pulmonares/patologia , Camundongos
11.
Methods Mol Biol ; 2779: 305-321, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38526792

RESUMO

The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).


Assuntos
Corantes Fluorescentes , Citometria de Fluxo/métodos
12.
J Mater Chem B ; 12(25): 6155-6163, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38842019

RESUMO

Advanced glycation end products (AGEs) play a pivotal role in the aging process, regarded as a hallmark of aging. Despite their significance, the absence of adequate monitoring tools has hindered the exploration of the relationship between AGEs and aging. Here, we present a novel AGE-selective probe, AGO, for the first time. AGO exhibited superior sensitivity in detecting AGEs compared to the conventional method of measuring autofluorescence from AGEs. Furthermore, we validated AGO's ability to detect AGEs based on kinetics, demonstrating a preference for ribose-derived AGEs. Lastly, AGO effectively visualized glycation products in a collagen-based mimicking model of glycation. We anticipate that this study will enhance the molecular tool sets available for comprehending the physiological processes of AGEs during aging.


Assuntos
Corantes Fluorescentes , Produtos Finais de Glicação Avançada , Produtos Finais de Glicação Avançada/análise , Produtos Finais de Glicação Avançada/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Colágeno/química , Colágeno/metabolismo , Estrutura Molecular , Imagem Óptica
13.
JACS Au ; 4(4): 1450-1457, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38665660

RESUMO

B lymphocytes play a pivotal role in the adaptive immune system by facilitating antibody production. Young B cell progenitors originate in the bone marrow and migrate to the spleen for antigen-dependent maturation, leading to the development of diverse B cell subtypes. Thus, tracking B cell trajectories through cell type distinction is essential for an appropriate checkpoint assessment. Despite its significance, monitoring specific B cell subclasses in live states has been hindered by a lack of suitable molecular tools. In this study, we introduce CDoB as the first mature B cell-selective probe, enabling real-time discrimination of three classified stages in B-cell development: progenitor, transitional, and mature B cells, through a single analysis using CyTOF. The selective mechanism of CDoB, elucidated as gating-oriented live-cell distinction (GOLD), targets SLC25A16, identified through systematic screening of SLC-CRISPRa and CRISPRi libraries. CDoB selectively brightens mature B cells in the mitochondrial area using SLC25A16 as the main gate, and the staining intensity correlates positively with the expression level of SLC25A16 along the B cell maturation continuum. In spleen tissues, CDoB demonstrates selective marking in mature B cell areas in live tissue status, representing the first performance achieved by a small-molecule fluorescent probe.

14.
Indian J Biochem Biophys ; 50(6): 485-91, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24772972

RESUMO

Triptolide, a diterpene derived from Tripterygium wilfordii Hook f., a Chinese medicinal herb, has been reported to inhibit cell proliferation and induce apoptosis in various human cancer cells, but its anticancer effects on human osteosarcoma cells have not yet been elucidated. In this study, we investigated whether triptolide induces apoptosis in human osteosarcoma cells and the underlying molecular mechanisms. We firstly demonstrated that triptolide inhibited cell growth and induced apoptosis in U2OS cells. Western blot analysis showed that the levels of procaspase-8, -9, Bcl-2, Bid and mitochondrial cytochrome c were downregulated in triptolide-treated U2OS cells, whereas the levels of Fas, FasL, Bax, cytosolic cytochrome c, cleaved caspase-3 and cleaved PARP were upregulated. These results suggest that triptolide induces apoptosis in U2OS cells by activating both death receptor and mitochondrial apoptotic pathways.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Diterpenos/farmacologia , Osteossarcoma/patologia , Fenantrenos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteólise/efeitos dos fármacos
15.
Indian J Biochem Biophys ; 49(2): 86-91, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22650004

RESUMO

In this study, the effect of cordycepin (3'-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 microM and 80 microM, respectively. Cordycepin treatment at 120 microM and 80 microM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Melanoma/patologia , Neuroblastoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos
16.
Mater Today Bio ; 16: 100405, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36060107

RESUMO

Intracellular micro-temperature is closely related to cellular processes. Such local temperature inside cells can be measured by fluorescent thermometers, which are a series of fluorescent materials that convert the temperature information to detectable fluorescence signals. To investigate the intracellular temperature fluctuation in various organelles, it is essential to develop site-specific organelle thermometers. In this study, we develop a new series of fluorescent thermometers, Thermo Greens (TGs), to visualize the temperature change in almost all typical organelles. Through fluorescence lifetime-based cell imaging, it was proven that TGs allow the organelle-specific monitoring of temperature gradients created by external heating. The fluorescence lifetime-based thermometry shows that each organelle experiences a distinct temperature increment which depends on the distance away from the heat source. TGs are further demonstrated in the quantitative imaging of heat production at different organelles such as mitochondria and endoplasmic reticulum in brown adipocytes. To date, TGs are the first palette batch of small molecular fluorescent thermometers that can cover almost all typical organelles. These findings can inspire the development of new fluorescent thermometers and enhance the understanding of thermal biology in the future.

17.
Nat Commun ; 13(1): 5974, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216803

RESUMO

Macrophages play crucial roles in protecting our bodies from infection and cancers. As macrophages are multi-functional immune cells, they have diverse plastic subsets, such as M1 and M2, derived from naïve M0 cells. Subset-specific macrophage probes are essential for deciphering and monitoring the various activation of macrophages, but developing such probes has been challenging. Here we report a fluorescent probe, CDr17, which is selective for M1 macrophages over M2 or M0. The selective staining mechanism of CDr17 is explicated as Gating-Oriented Live-cell Distinction (GOLD) through overexpressed GLUT1 in M1 macrophages. Finally, we demonstrate the suitability of CDr17 to track M1 macrophages in vivo in a rheumatoid arthritis animal model.


Assuntos
Corantes Fluorescentes , Macrófagos , Animais , Transportador de Glucose Tipo 1/genética , Inflamação , Ativação de Macrófagos , Plásticos
18.
Biochem Biophys Res Commun ; 414(1): 159-64, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21945615

RESUMO

In this study we investigated for the first time the transcriptional regulation of pig Galß1,3GalNAc α2,3-sialyltransferase (pST3Gal I) in response to TGF-ß1 in porcine kidney PK-15 cells. The pST3Gal I gene was found to span about 90kb and to be composed of 8 exons including 2 exons in the 5'-untranslated region. RT-PCR analysis indicated that the induction of pST3Gal I by TGF-ß1 is regulated at the transcriptional level. Functional analysis of the 5'-flanking region of the pST3Gal I gene revealed the -1257 to -976 region functions as the TGF-ß1-inducible promoter and that the Smad-binding site at -1020 is crucial for TGF-ß1-induced expression of pST3Gal I in PK-15 cells. In addition, the transcriptional activity of pST3Gal I induced by TGF-ß1 in PK-15 cells was strongly inhibited by SIS3, which is a specific Smad-3 inhibitor. In summary, our results identified the core promoter region in the pST3Gal I promoter and demonstrated that Smad-3 binding to the Samd-3 binding site at -1020 is essential for transcriptional activation of pST3Gal I in TGF-ß1-induced PK-15 cells.


Assuntos
Regulação da Expressão Gênica , Rim/enzimologia , Sialiltransferases/genética , Proteínas Smad/metabolismo , Suínos/genética , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Crescimento Transformador alfa/farmacologia , beta-Galactosídeo alfa-2,3-Sialiltransferase
19.
RSC Chem Biol ; 2(6): 1590-1593, 2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34977573

RESUMO

CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine. However, the stem cell selectivity mechanism of CDy1 has not been fully understood. Here, we report ALDH2 and ABCB1 as the molecular targets of CDy1, elucidated by live-cell affinity-matrix and ABC transporter CRISPRa library screening. The two unique orthogonal mechanisms provide the potential of multi-demensional cellular distinction of specific cell types.

20.
Mol Cells ; 27(1): 113-8, 2009 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-19214441

RESUMO

In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5'-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-kappaB binding site at -731 to -722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and GO6976, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.


Assuntos
Neuroblastoma/enzimologia , Neuroblastoma/genética , Sialiltransferases/genética , Ativação Transcricional/efeitos dos fármacos , Ácido Valproico/farmacologia , Linhagem Celular Tumoral , Gangliosídeos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Sialiltransferases/metabolismo
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