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1.
Int J Mol Sci ; 18(8)2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28786938

RESUMO

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with "high-risk" human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.


Assuntos
Deficiência de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Microscopia/métodos , Transporte Biológico , Linhagem Celular Transformada , Imunofluorescência , Humanos , Queratinócitos , Microscopia de Fluorescência
2.
Int J Mol Sci ; 18(2)2017 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-28208769

RESUMO

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- 1 ß application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ -irradiation or alternatively neuregulin- 1 ß application mobilized ErbB receptors in a nucleograde fashion-a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.


Assuntos
Epitélio/metabolismo , Junções Comunicantes/metabolismo , Glândulas Mamárias Humanas/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/farmacologia , Conexina 43/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/patologia , Epitélio/efeitos da radiação , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Glândulas Mamárias Humanas/efeitos da radiação , Microscopia Confocal , Neuregulina-1/metabolismo , Ligação Proteica , Multimerização Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Trastuzumab/farmacologia
3.
Nanoscale ; 8(48): 20037-20047, 2016 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-27883139

RESUMO

For receptor tyrosine kinases supramolecular organization on the cell membrane is critical for their function. Super-resolution fluorescence microscopy techniques have offered new opportunities for the analysis of single receptor localization. Here, we analysed the cluster formation of the epidermal growth factor receptor variant III (EGFRvIII), a deletion variant which is expressed in glioblastoma. The constitutively activated variant EGFRvIII is expressed in cells with an egfr gene amplification and is thought to enhance the tumorigenic potential especially of glioblastoma cells. Due to the lack of an adequate model system, it is still unclear how endogenous EGFRvIII expression alters cellular signalling and if it is organized in clusters like the wild type receptor. We have recently described the establishment of two pairs of iso-genetic cell lines (BS153 and DKMG), displaying endogenous EGFRvIII expression or not. Using these cell lines we investigated single receptor localization of EGFRvIII by high precision localization microscopy. Cluster analysis revealed that EGFRvIII is present in clusters on the surface of the cells, with about 60% or even more receptor molecules being assembled in clusters of approximately 100 nm in diameter whereby the cluster definition was iteratively determined. The signal to signal distance may indicate dimer formation while signal quantification indicates 1 × 106-5 × 106 EGFRvIII molecules per cell. Altogether, these data give unique insights into the membrane surface localization of EGFRvIII in glioblastoma cells. These insights will help to unveil the function of this tumour associated receptor variant which might lead to a better understanding of glioblastoma and therefore could lead to improved therapy approaches.


Assuntos
Neoplasias Encefálicas/metabolismo , Receptores ErbB/análise , Glioblastoma/metabolismo , Microscopia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
4.
Biochim Biophys Acta ; 1861(9 Pt A): 1161-1169, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27365309

RESUMO

Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.


Assuntos
Mucosa Intestinal/metabolismo , Neoplasias Intestinais/metabolismo , Fosfatidilcolinas/metabolismo , Junções Íntimas/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epitélio/metabolismo , Humanos , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Mucina-3/antagonistas & inibidores , Mucina-3/genética , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , RNA Interferente Pequeno/genética , Técnicas de Cultura de Tecidos
5.
Biophys J ; 110(4): 947-53, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26910431

RESUMO

Gold nanoparticles (GNPs) enhance the damaging absorbance effects of high-energy photons in radiation therapy by increasing the emission of Auger-photoelectrons in the nm-µm range. It has been shown that the incorporation of GNPs has a significant effect on radiosensitivity of cells and their dose-dependent clonogenic survival. One major characteristic of GNPs is also their diameter-dependent cellular uptake and retention. In this article, we show by means of an established embodiment of localization microscopy, spectral position determination microscopy (SPDM), that imaging with nanometer resolution and systematic counting of GNPs becomes feasible, because optical absorption and plasmon resonance effects result in optical blinking of GNPs at a size-dependent wavelength. To quantify cellular uptake and retention or release, SPDM with GNPs that have diameters of 10 and 25 nm was performed after 2 h and after 18 h. The uptake of the GNPs in HeLa cells was either achieved via incubation or transfection via DNA labeling. On average, the uptake by incubation after 2 h was approximately double for 10 nm GNPs as compared to 25 nm GNPs. In contrast, the uptake of 25 nm GNPs by transfection was approximately four times higher after 2 h. The spectral characteristics of the fluorescence of the GNPs seem to be environment-dependent. In contrast to fluorescent dyes that show blinking characteristics due to reversible photobleaching, the blinking of GNPs seems to be stable for long periods of time, and this facilitates their use as an appropriate dye analog for SPDM imaging.


Assuntos
Ouro/química , Ouro/metabolismo , Nanopartículas Metálicas , Microscopia , Transporte Biológico , Células HeLa , Humanos , Tamanho da Partícula , Coloração e Rotulagem
6.
PLoS One ; 10(6): e0128555, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26042422

RESUMO

It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are investigated.


Assuntos
Cromatina/química , Cromatina/efeitos da radiação , Raios gama , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Estatística como Assunto , Análise por Conglomerados , Eucromatina , Genoma Humano , Células HeLa , Heterocromatina , Humanos , Probabilidade
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