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Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.
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Hepatoblastoma , Neoplasias Hepáticas , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hepatoblastoma/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Transición Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genéticaRESUMEN
Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2+ CD90+ CD45- CD235a- DAPI- cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.
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Células Neoplásicas Circulantes , Neuroblastoma , Humanos , Células Neoplásicas Circulantes/patología , Recurrencia Local de Neoplasia , Neuroblastoma/genética , Neuroblastoma/patología , Mutación , Secuenciación de Nucleótidos de Alto Rendimiento , Biomarcadores de Tumor/genéticaRESUMEN
Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial-mesenchymal transition (EMT), suggesting a role of ADAM32 in cancer stemness and EMT. Furthermore, knockdown of ADAM32 increased cisplatin-induced apoptosis, and this effect was attenuated by a caspase-8 inhibitor, suggesting that ADAM32 plays a role in extrinsic apoptosis signaling. We conclude that ADAM32 plays a crucial role in progression of HBL, so it might be a promising molecular target in anticancer therapy.
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We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.
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Neuroblastoma , Vitamina A , Línea Celular , Humanos , Neuroblastoma/patología , Ácido Retinoico 4-Hidroxilasa/genética , Tetraciclinas , TransfecciónRESUMEN
Lactoferrin (LF) is an iron binding glycoprotein of the transferrin family with a wide spectrum of biological effects, including anti-cancer activity. However, the detailed molecular mechanisms of anti-cancer activity of LF have not been fully determined. In this study, we tried to clarify cytotoxic functions of LF on various cell lines under hypoxic conditions and elucidate those molecular mechanisms. Cytotoxic activity of LF on cell lines was found to have a range of sensitivities. Hypoxia decreased sensitivity to LF in KD (lip fibroblast) but increased that in HSC2 (oral squamous cell carcinoma). Expression analyses further revealed that LF treatments increased hypoxic HIF-1α, -2α and p53 proteins in KD but attenuated them in HSC2 cells, and decreased HIF-1 target gene, DEC2, in KD but increased it in HSC2, suggesting a possible relationship between LF-modified DEC2 expression and HIF-α protein. MTT assay strikingly demonstrated that cells expressing mutant-type p53 (MT5) were more sensitive to LF than control HepG2 (hepatoma), suggesting an important role of the p53 signal. Knock-down of TP53 (p53 gene) interestingly reduced sensitivity to LF in HepG2, suggesting that p53 may be a target of LF cytotoxic activity. Further analyses with a ferroptosis promoter or inhibitor demonstrated that LF increased ACSL4 in hypoxic MT5, suggesting LF-induced ferroptosis in cells expressing mutant-type p53. In conclusion, hypoxia was found to regulate cytotoxic activities of LF differently among various cell lines, possibly through the p53 signaling pathway. LF further appeared to regulate ferroptosis through a modification of ACSL4 expression.
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The biological effects of low-dose-rate (LDR) radiation exposure in nuclear power plant accidents and medical uses of ionizing radiation (IR), although being a social concern, remain unclear. In this study, we evaluated the effects of LDR-IR on global gene expression in human cells and aimed to clarify the mechanisms. RNA-seq analyses demonstrated that relatively low dose rates of IR modify gene expression levels in TIG-3 cells under normoxic conditions, but those effects were attenuated under hypoxia-mimicking conditions. Gene set enrichment analysis demonstrated that LDR-IR significantly decreased gene expression related to cell division, cell cycle, mitosis, and the Aurora kinase B and FOXM1 pathways. Quantitative RT-PCR confirmed the down-regulation of AURKB and FOXM1 genes in TIG-3 cells with LDR-IR or hypoxia-mimicking treatments without any dose-rate effect. Knock-down experiments suggested that HIF-1α and HIF-2α, as well as DEC1, participated in down-regulation of AURKB and FOXM1 under DFOM treatments, but to a lesser extent under LDR-IR treatment. FACS and microscopic analyses demonstrated that LDR-IR induced G0/G1 arrest and increased micronucleus or chromosome condensation. Finally, MTT assays demonstrated that LDR-IR decreased sensitivity to paclitaxel or barasertib in TIG-3 cells but not in A549 cells. In conclusion, LDR-IR modifies global gene expression and cell cycle control, resulting in a reduction of sensitivity to anti-cancer chemotherapy in non-cancer cells and thus a reduction in untoward effects (GA).
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Antineoplásicos , Antineoplásicos/farmacología , Ciclo Celular/genética , Hipoxia de la Célula , Humanos , Hipoxia , Paclitaxel/farmacologíaRESUMEN
Single-cell sequencing of circulating tumor cells can precisely represent tumor heterogeneity and provide useful information for cancer treatment and research. After spiking TGW neuroblastoma cells into blood derived from healthy volunteer, the cells were isolated by fluorescence-activated cell sorting. DNA and mRNA were amplified by four different whole-genome amplifications (WGA) and three whole-transcriptome amplifications (WTA) methods, followed by single-cell DNA and RNA sequencing. Multiple displacement amplification (MDA)-based WGA methods showed higher amplification efficiency than other methods with a comparable depth of coverage as the bulk sample. The uniformity of coverage greatly differed among samples (12.5-89.2%), with some samples evaluated by the MDA-based WGA method using phi29 DNA polymerase and random primers showing a high (> 80%) uniformity of coverage. The MDA-based WTA method less effectively amplified mRNA and showed non-specific gene expression patterns. The PCR-based WTA using template switching with locked nucleic acid technology accurately amplified mRNA from a single cell. Taken together, our results present a more reliable and adaptable approach for CTC profiling at the single-cell level. Such molecular information on CTCs derived from clinical patients will promote cancer treatment and research.
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Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Células Neoplásicas Circulantes/química , Neuroblastoma/genética , ARN Neoplásico/genética , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Línea Celular Tumoral , Heterogeneidad Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Neoplásicas Circulantes/patología , Neuroblastoma/sangre , Neuroblastoma/patología , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Secuenciación del ExomaRESUMEN
Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.
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Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Tratamiento Farmacológico de COVID-19 , Carbazoles/farmacología , Omeprazol/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , COVID-19/virología , Carbazoles/uso terapéutico , Chlorocebus aethiops , Citocromo P-450 CYP1A1/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Omeprazol/uso terapéutico , RNA-Seq , Receptores de Hidrocarburo de Aril/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/patogenicidad , Transducción de Señal/efectos de los fármacos , Células Vero , Internalización del Virus/efectos de los fármacosRESUMEN
We previously demonstrated that expression of a Krüppel-like zinc finger transcription factor, GLIS1, dramatically increases under hypoxic conditions via a transcriptional mechanism induced by HIF-2α cooperating with AP-1 members. In this study, we focused on the functional roles of GLIS1 in breast cancer. To uncover its biological function, the effects of altered levels of GLIS1 in breast cancer cell lines on cellular growth, wound-healing and invasion capacities were assessed. Knockdown of GLIS1 using siRNA in BT-474 cells resulted in significant growth stimulation under normoxia, while attenuation was found in the cell invasion assay under hypoxic conditions. In MDA-MB-231 cells expressing exogenous 3xFLAG-tagged GLIS1, GLIS1 attenuated cell proliferation and enhanced cell mobility and invasion capacities under normoxia. In addition, breast cancer cells expressing GLIS1 acquired resistance to irradiation. Whole transcriptome analysis clearly demonstrated that downstream signals of GLIS1 are related to various cellular functions. Among the genes with increased expression, we focused on WNT5A. Knockdown of WNT5A indicated that enhancement of acquired cell motility in the MDA-MB-231 cells expressing GLIS1 was mediated, at least in part, by WNT5A. In an analysis of publicly available data, patients with estrogen receptor-negative breast cancer showing high levels of GLIS1 expression showed much worse prognosis than those with low levels. In summary, hypoxia-induced GLIS1 plays significant roles in breast cancer cells via regulation of gene expression related to cell migration and invasion capacities, resulting in poorer prognosis in patients with advanced breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/metabolismo , Proteína Wnt-5a/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/genética , Femenino , Humanos , Invasividad Neoplásica , Pronóstico , Tasa de Supervivencia , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteína Wnt-5a/genéticaRESUMEN
Although the biological systems in the human body are affected by the earth's gravity, information about the underlying molecular mechanisms is limited. For example, apoptotic signaling is enhanced in cancer cells subjected to microgravity. We reasoned that signaling regulated by p53 may be involved because of its role in apoptosis. Therefore, we aimed to clarify the molecular mechanisms of modified cis-diamminedichloroplatinum (CDDP)-sensitivity under simulated microgravity by focusing on p53-related cell death mechanisms. Immunoblotting analyses indicated that, under microgravity, CDDP-induced ATM/p53 signaling increased and caspase-3 was cleaved earlier. However, microgravity decreased the levels of expression of p53 targets BAX and CDKN1A. Interestingly, microgravity increased the PTEN, DRAM1, and PRKAA1 mRNA levels. However, microgravity decreased the levels of mTOR and increased the LC3-II/I ratio, suggesting the activation of autophagy. The CDDP-induced cleavage of caspase-3 was increased during the early phase in Group MG (+), and cleaved caspase-3 was detected even in Group MG (+) with constitutive expression of a mutant type of p53 (hereafter, "+" indicates CDDP treatment). These results interestingly indicate that microgravity altered CDDP sensitivity through activation of caspase-3 by p53-independent mechanism.
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Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Ingravidez , Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Hep G2 , Humanos , Proteínas de la Membrana/metabolismo , Mutación , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
[This corrects the article DOI: 10.1038/s41526-018-0045-0.].
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The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite® may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was significantly attenuated under simulated microgravity (10-3G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was specifically observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were significantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated with 5-AzaC, and further incubated with differentiation medium under 1 G or 10-3 G conditions. The results showed differences in expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10-3 G conditions, completely disappeared in this experimental condition. Modified HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation.
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BACKGROUND: Diabetes complications include various symptoms such as diabetic neuropathy and cognitive disorders. Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway and is one of the causal factors of diabetes complications. In this study, the bioactivities of eight selected Kampo formulations that are currently in clinical use for diabetes complications were assessed using human AR (hAR) inhibitory activity as the primary parameter to explore the possibilities of novel clinical applications of these formulations in the treatment of diabetes complications. METHODS: The hAR inhibitory activities of four Kampo formulations that are clinically used for diabetic neuropathy, four Kampo formulations that are used for cognitive disorders, and a total of 21 component crude drugs were measured. Furthermore, the hAR inhibitory activity of Glycyrrhizae Radix preparata was measured to determine the effect of frying, which is one of the specific processing of Glycyrrhizae Radix. hAR inhibitory activity was determined by measuring the rate of decline in the absorbance of NAPH at 340 nm using 0.5 mM NADPH, 10 mM D,L-glyceraldehyde, and 3.6 mU/mL hAR in phosphate buffer solution (0.2 M, pH 6.2). RESULTS: All of the Kampo formulations exhibited significant hAR inhibitory activity; Chotosan exhibited particularly strong activity. Among the 21 crude drugs tested, adequate inhibitory activities were found for the following, in descending order of activity: Glycyrrhizae Radix > Paeoniae Radix > Chrysanthemi Flos > Cinnamomi Cortex > Phellodendri Cortex > Uncariae Uncis cum Ramulus > Bupleuri Radix. Glycyrrhizae Radix preparata exhibited an inhibitory activity that was nearly identical to that of Glycyrrhizae Radix. CONCLUSIONS: Despite their seemingly different treatment objectives, all of the Kampo formulations that are clinically used for diabetes complications demonstrated significant hAR inhibitory activity. This activity might underlie the characteristic multi-target effects of Kampo formulations. Although the overall effect of a Kampo formulation is certainly difficult to evaluate based on specific herbal medications or components, the approach as taken in this study might nonetheless contribute to further advancement in the development of new drugs via the review of proper usage and re-examination of the chemical compounds from a new perspective.
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Aldehído Reductasa/antagonistas & inhibidores , Complicaciones de la Diabetes/enzimología , Inhibidores Enzimáticos/farmacología , Medicina Kampo , Extractos Vegetales/farmacología , Plantas Medicinales/química , Aldehído Reductasa/metabolismo , Complicaciones de la Diabetes/tratamiento farmacológico , HumanosRESUMEN
The therapeutic effect of rehabilitation after cell therapy for brain injury remains unclear. Here, we report the neural stem/progenitor cells transplantation into a brain injury mouse model followed by treadmill exercise training. Among all experimental groups, mice that underwent transplantation and treadmill exercise demonstrated significant functional motor and electrophysiological improvement. Transplanted cells at the brain injury site were observed and differentiated into neurons and astrocytes. Transplanted cells significantly differentiated into neurons in the mice that underwent transplantation and treadmill exercise compared with those treated with only transplantation. Furthermore, the expression of brain-derived neurotrophic factor and growth-associated protein 43 mRNAs were significantly up-regulated in the mice that underwent transplantation and treadmill exercise than in those in other experimental groups during the early recovery stage. These results suggest that rehabilitation after neural stem/progenitor cell transplantation enhances neurogenesis and promotes the recovery of motor function in brain injury model mice.
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Lesiones Encefálicas/rehabilitación , Lesiones Encefálicas/terapia , Células-Madre Neurales/trasplante , Neurogénesis , Condicionamiento Físico Animal , Animales , Astrocitos/patología , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Diferenciación Celular , Supervivencia Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Potenciales Evocados Motores , Proteína GAP-43/metabolismo , Ratones , Ratones Endogámicos C57BL , Actividad Motora , Células-Madre Neurales/citología , Neuronas/patología , Regulación hacia ArribaRESUMEN
The mechanism by which electrical stimulation affects formation of neuromuscular junctions (NMJs) remains unknown. NG108-15, a neural cell line, is commonly used in in vitro co-culture models of myotubes to observe synapse formation; therefore, we employed this model to observe the effects of electrical stimulation on NMJ formation. Initially, L6 cells were differentiated and NG108-15 cells were then added to the same culture dish. After 2 and 3 days of co-culture, the cells were electrically stimulated at 50 V and 0.5 Hz for 0, 5, 30, and 60 min (C, ES5, ES30, and ES60 groups, respectively) and were analyzed after co-culture for 4 days. Immunofluorescence experiments showed significantly increased aggregation of acetylcholine receptors and inhibition of neural outgrowth in the ES30 and ES60 groups. Furthermore, ADAM19 and phospho-ErbB3 were found to be specifically localized in co-cultured NG108-15 cells. Immunoblotting demonstrated that synapsin 1, ADAM19 precursor and its activated form, phospho-ErbB3, and ERK1 protein levels had increased in an electrical stimulation period-dependent manner. Thus, we found that electrical stimulation accelerated NMJ formation, possibly through activation of ADAM19/neuregulin/ErbB signaling in NG108-15 cells.
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Proteínas ADAM/metabolismo , Estimulación Eléctrica/métodos , Neurregulinas/metabolismo , Unión Neuromuscular/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Proteínas Oncogénicas v-erbB/metabolismo , Animales , Línea Celular , Ratones , Unión Neuromuscular/ultraestructura , Transducción de Señal/fisiologíaRESUMEN
GLI-similar 1 (GLIS1) is important for the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs). However, the molecular mechanisms of regulation of GLIS1 expression remain unclear. We have therefore examined GLIS1 expression in various cancer cell lines and demonstrated that GLIS1 expression was dramatically increased under hypoxic conditions. Importantly, GLIS1 expression was significantly attenuated in VHL-overexpressing renal cell carcinoma cells compared to the VHL-deficient parent control. Moreover, promoter analysis demonstrated that GLIS1 transcription was regulated by hypoxia through a hypoxia-inducible factors (HIFs)-dependent mechanism. Co-transfection experiments revealed that HIF-2α had greater potency on the GLIS1 promoter activation than HIF-1α. Subsequent studies using wild-type and mutant HIF-2α demonstrated that DNA binding activity was not necessary but TADs were critical for GLIS1 induction. Finally, co-transfection experiments indicated that HIF-2α cooperated with AP-1 family members in upregulating GLIS1 transcription. These results suggest that the hypoxic signaling pathway may play a pivotal role in regulating the reprogramming factor GLIS1, via non-canonical mechanisms involving partner transcription factor rather than by direct HIF transactivation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factor de Transcripción AP-1/metabolismoRESUMEN
Bone marrow stromal cells (BMSCs) have received significant attention for its use in neural regeneration. However, neural replacement by transplanted BMSCs was not very effective. Recently, the gene transfection method has improved the capability of cell transplantation; however, this method results in canceration and immune rejection. We induced the differentiation of mouse BMSCs into neural cells using electrical stimulation and transplanted the cells into traumatic brain injury (TBI) model mice. We found that the electrically stimulated cells have good potential to differentiate into neural cells and contribute to recovery from TBI without differentiating into astrocytes. In addition, we found that electrical stimulation enhanced neurogenin2 (Ngn2) expression. Ngn2 is involved in neural differentiation and inhibits astrocytic differentiation during cell growth. Furthermore, we found that this enhancement of Ngn2 expression occurred through ß-catenin signaling pathway. This study may contribute to the use of BMSCs for neural replacement in central nervous system diseases.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Lesiones Encefálicas/terapia , Proteínas del Tejido Nervioso/metabolismo , beta Catenina/fisiología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Estimulación Eléctrica , Masculino , Ratones , Ratones Endogámicos C57BL , Movimiento , Neuronas/citología , Neuronas/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/trasplanteRESUMEN
The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/genética , Factores de Tiempo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Treatment of soft tissue defects with exposed bones and joints, resulting from trauma, infection, and surgical complications, represents a major challenge. The introduction of negative pressure wound therapy has changed many wound management practices. Negative pressure wound therapy has recently been used in the orthopedic field for management of traumatic or open wounds with exposed bone, nerve, tendon, and orthopedic implants. This article describes a case of a patient with a large soft tissue defect and exposed knee joint, in which negative pressure wound therapy markedly improved wound healing. A 50-year-old man presented with an ulceration of his left knee with exposed joint, caused by severe wound infections after open reduction and internal fixation of a patellar fracture. After 20 days of negative pressure wound therapy, a granulated wound bed covered the exposed bones and joint.To our knowledge, this is the first report of negative pressure wound therapy used in a patient with a large soft tissue defect with exposed knee joint. Despite the chronic wound secondary to infection, healing was achieved through the use of the negative pressure wound therapy, thus promoting granulation tissue formation and closing the joint. We suggest negative pressure wound therapy as an alternative option for patients with lower limb wounds containing exposed bones and joints when free flap transfer is contraindicated. Our result added to the growing evidence that negative pressure wound therapy is a useful adjunctive treatment for open wounds around the knee joint.