The concept of tumor stem cells (TSCs) provides a new paradigm for understanding tumor biology, although it remains unclear whether TSCs will prove to be a more robust model than traditional cancer cell lines. We demonstrate marked phenotypic and genotypic differences between primary human tumor-derived TSCs and their matched glioma cell lines. Unlike the matched, traditionally grown tumor cell lines, TSCs derived directly from primary glioblastomas harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, and in vivo biology of human glioblastomas. These findings suggest that TSCs may be a more reliable model than many commonly utilized cancer cell lines for understanding the biology of primary human tumors.
The Müller glia of fish provide a source for neuronal regeneration after injury, but they do not do so in mammals. We previously showed that lentiviral gene transfer of the transcription factor Achaete‐scute homolog 1 (Ascl1/Mash1) in murine Müller glia cultures resulted in partial reprogramming of the cells to retinal progenitors. The microRNAs (miRNAs) miR‐124‐9‐9* facilitate neuronal reprogramming of fibroblasts, but their role in glia reprogramming has not been reported. The aim of this study was to test whether (1) lentiviral gene transfer of miR‐124‐9‐9* can reprogram Müller glia into retinal neurons and (2) miR‐124‐9‐9* can improve Ascl1‐induced reprogramming. Primary Müller glia cultures were generated from postnatal day (P) 11/12 mice, transduced with lentiviral particles, i.e., miR‐124‐9‐9*‐RFP, nonsense‐RFP, Ascl1‐GFP, or GFP‐control. Gene expression and immunofluorescence analyses were performed within 3 weeks after infection. Overexpression of miR‐124‐9‐9* induced the expression of the proneural factor Ascl1 and additional markers of neurons, including TUJ1 and MAP2. When Ascl1 and miR‐124‐9‐9* were combined, 50 to 60% of Müller glia underwent neuronal reprogramming, whereas Ascl1 alone results in a 30 to 35% reprogramming rate. Analysis of the miR‐124‐9‐9* treated glial cells showed a reduction in the level of Ctdsp1 and Ptbp1, indicating a critical role for the REST pathway in the repression of neuronal genes in Müller glia. Our data further suggest that miR‐124‐9‐9* and the REST complex may play a role in regulating the reprogramming of Müller glia to progenitors that underlies retinal regeneration in zebrafish. GLIA 2016;64:743–762 We show that the combination of the microRNAs miR‐124‐9‐9* with the transcription factor Ascl1 significantly increase neuronal reprogramming of Müller glia and that the underlying mechanism appears to involve members of the REST silencing complex.
Background Pyridoxine (VB6), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. Objective This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. Methods Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2 ',7 '-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6. Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5 '-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. Results Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. Conclusion These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.
In this study, the effect of myrosinase-treated glucoerucin (GER+MYR), which releases the isothiocyanate (ITC) erucin, on heme oxygenase 1 (HO-1) gene expression and Nrf2 signaling was investigated in cultured cells and in mice. Treatment of HT-29 cells with GER+MYR resulted in a significant increase in the mRNA and protein levels of nuclear Nrf2 and HO-1. GER+MYR was more potent at enhancing the nuclear Nrf2 levels than were the following myrosinase-treated glucosinolates: sinigrin, glucoraphanin and gluconasturtiin, which are the precursors of allyl-ITC, -sulforaphane and 2-phenylethyl ITC, respectively. GER+MYR also significantly induced HO-1 gene expression in the mouse intestinal mucosae and liver but not in the brain. Mechanistic studies suggest that GER+MYR induces Nrf2 via ERK1/2-, p38- and JNK-dependent signal transduction pathways. The GER+MYR-mediated increase in HO-1 expression is primarily attributable to p38 signaling.
The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.
► Regulation of astrocytic e-5NT by TNF-α, LPS, IFN-γ, Glu and H O is investigated. ► LPS, IFN-γ, Glu and H O decrease, whereas TNF-α increases e-5NT activity. ► LPS, IFN-γ and Glu down-regulate e-5NT, while H O modulates its enzymatic efficiancy. ► TNF-α increases e-5NT protein abundance, without alteration in mRNA expression level. Ecto-5′-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H O ) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H O decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.
This study reports that the spontaneous 50‐fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13‐day chicken embryo, relies on a Ca 2+ ‐dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant‐negative form of the cAMP ‐responsive element‐binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca 2+ /calmodulin‐dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca 2+ rather than cAMP . This was confirmed by the inhibitory effect of general and T‐type selective Ca 2+ channel blockers. Oscillations in Ca 2+ fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization‐activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage‐gated Ca 2+ ‐channels, activation of Ca 2+ /calmodulin‐dependent kinase II and phosphorylation of cAMP ‐responsive element‐binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+‐dependent mechanism whereby hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) activate T‐type voltage‐dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin‐dependent kinase II (CaMKII) to phosphorylate the cAMP‐responsive element‐binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo . Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+‐dependent mechanism whereby hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) activate T‐type voltage‐dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin‐dependent kinase II (CaMKII) to phosphorylate the cAMP‐responsive element‐binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo .
The complex network of neuronal cells in the retina makes it a potential target of neuronal toxicity - a risk factor for visual loss. With growing use of nanoparticles (NPs) in commercial and medical applications, including ophthalmology, there is a need for reliable models for early prediction of NP toxicity in the eye and retina. Metal NPs, such as gold and silver, gain much of attention in the ophthalmology community due to their potential to cross the barriers of the eye. Here, NP uptake and signs of toxicity were investigated after exposure to 20 and 80 nm Ag- and AuNPs, using an in vitro tissue culture model of the mouse retina. The model offers long-term preservation of retinal cell types, numbers and morphology and is a controlled system for delivery of NPs, using serum-free defined culture medium. AgNO3-treatment was used as control for toxicity caused by silver ions. These end-points were studied; gross morphological organization, glial activity, microglial activity, level of apoptosis and oxidative stress, which are all well described as signs of insult to neural tissue. TEM analysis demonstrated cellular- and nuclear uptake of all NP types in all neuronal layers of the retina. Htx-eosin staining showed morphological disruption of the normal complex layered retinal structure, vacuole formation and pyknotic cells after exposure to all Ag- and AuNPs. Significantly higher numbers of apoptotic cells as well as an increased number of oxidative stressed cells demonstrated NP-related neuronal toxicity. NPs also caused increased glial staining and microglial cell activation, typical hallmarks of neural tissue insult. This study demonstrates that low concentrations of 20 and 80 nm sized Ag- and AuNPs have adverse effects on the retina, using an organotypic retina culture model. Our results motivate careful assessment of candidate NP, metallic or-non-metallic, to be used in neural systems for therapeutic approaches.
During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.
This study reports the metabolic engineering of carotenoid biosynthesis in cultured cells of sweet potato by down-regulation of β-carotene hydroxylase. Transgenic cells exhibited increases in carotenoid content, antioxidant activity and salt stress tolerance. ► Sweetpotato RNAi transgenic calli increased -carotene and total carotenoids. ► Transgenic cell lines also showed enhanced antioxidant capacity under salt stress. ► Interestingly, the transgenic calli accumulated more ABA content. ► It is suggested that enhanced levels of carotenoids contribute to stress tolerance in transgenic plants. Sweetpotato ( Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. β-Carotene hydroxylase ( ) is a key regulatory enzyme in the beta–beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin. To increase the β-carotene content of sweetpotato through the inhibition of further hydroxylation of β-carotene, the effects of silencing in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference- construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene β-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 μg/g dry weight, of which β-carotene measured 34.43 μg/g dry weight. In addition, -silenced calli showed elevated β-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA- calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3′-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of increased β-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity.