TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

Aberrant DNA methylation of microRNA genes in human breast cancer – a critical appraisal

Aberrant DNA methylation of regulatory sequences is a well-documented mechanism of functional deletion of genes with anti-tumourigenic properties including microRNAs. This review discusses the publications describing aberrant methylation of microRNA genes in human breast cancer cells. Among the anti-tumourigenic properties of epigenetically inactivated microRNA genes, the inhibition of proliferation and of epithelial-to-mesenchymal transition (EMT) are the best studied. Several studies are conceptually very interesting and present a comprehensive functional characterization of anti-tumorigenic microRNAs. The link between microRNA expression and gene methylation is not addressed directly by all studies and a number of studies are limited in their strength by not including primary breast cancer specimens or by analysing very small sets of primary human specimens. The publications cover a wide range of DNA methylation detection techniques, often making direct comparison of results challenging. Despite the identification and thorough characterization of many interesting candidates and functionally important microRNA genes affected by DNA methylation, the translation of microRNA gene methylation as a new biomarker into the daily routine practice has not yet worked out.     

Impact of 900 MHz electromagnetic field exposure on main male reproductive hormone levels: a Rattus norvegicus model

This work analyzes the effects of radiofrequency-electromagnetic field (RF-EMF) exposure on the reproductive system of male rats, assessed by measuring circulating levels of FSH, LH, inhibin B, activin B, prolactin, and testosterone. Twenty adult male Sprague–Dawley rats (180?±?10 g) were exposed to 900 MHz RF-EMF in four equal separated groups. The duration of exposure was 1, 2, and 4 h/day over a period of 30 days and sham-exposed animals were kept under the same environmental conditions as the exposed group except with no RF-EMF exposure. Before the exposure, at 15 and 30 days of exposure, determination of the abovementioned hormone levels was performed using ELISA. At the end of the experiment, FSH and LH values of the long time exposure (LTE) group were significantly higher than the sham-exposed group (p?p?p?相似文献   

ROS release and Hsp70 expression after exposure to 1,800 MHz radiofrequency electromagnetic fields in primary human monocytes and lymphocytes

The aim of this study is to investigate if 1,800 MHz radiofrequency electromagnetic fields (RF-EMF) can induce reactive oxygen species (ROS) release and/or changes in heat shock protein 70 (Hsp70) expression in human blood cells, using different exposure and co-exposure conditions. Human umbilical cord blood-derived monocytes and lymphocytes were used to examine ROS release after exposure to continuous wave or different GSM signals (GSM-DTX and GSM-Talk) at 2 W/kg for 30 or 45 min of continuous or intermittent (5 min ON/5 min OFF) exposure. The cells were exposed to incubator conditions, to sham, to RF-EMF, or to chemicals in parallel. Cell stimulation with the phorbol ester phorbol-12-myristate-13-acetate (PMA; 1 μM) was used as positive control for ROS release. To investigate the effects on Hsp70 expression, the human monocytes were exposed to the GSM-DTX signal at 2 W/kg for 45 min, or to heat treatment (42°C) as positive control. ROS production and Hsp70 expression were determined by flow cytometric analysis. The data were compared to sham and/or to control values and the statistical analysis was performed by the Student’s t-test (P<0.05). The PMA treatment induced a significant increase in ROS production in human monocytes and lymphocytes when the data were compared to sham or to incubator controls. After continuous or intermittent GSM-DTX signal exposure (2 W/kg), a significantly different ROS production was detected in human monocytes if the data were compared to sham. However, this significant difference appeared due to the lowered value of ROS release during sham exposure. In human lymphocytes, no differences could be detected if data were compared either to sham or to incubator control. The Hsp70 expression level after 0, 1, and 2 h post-exposure to GSM-DTX signal at 2 W/kg for 1 h did not show any differences compared to the incubator or to sham control.     

Stable loss of global DNA methylation in the radiation-target tissue--a possible mechanism contributing to radiation carcinogenesis?

Radiation-induced lymphomagenesis and leukemogenesis are complex processes involving both genetic and epigenetic changes. Although genetic alterations during radiation-induced lymphoma- and leukemogenesis are fairly well studied, the role of epigenetic changes has been largely overlooked. Rodent models are valuable tools for identifying molecular mechanisms of lymphoma and leukemogenesis. A widely used mouse model of radiation-induced thymic lymphoma is characterized by a lengthy "pre-lymphoma" period. Delineating molecular changes occurring during the pre-lymphoma period is crucial for understanding the mechanisms of radiation-induced leukemia/lymphoma development. In the present study, we investigated the role of radiation-induced DNA methylation changes in the radiation carcinogenesis target organ--thymus, and non-target organ--muscle. This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue. The presence of stable DNA hypomethylation in the radiation-target tissue, even though DNA damage resulting from initial genotoxic radiation insult was repaired, suggests of the importance of epigenetic mechanisms in the development of radiation-related pathologies. The possible role of radiation-induced DNA hypomethylation in radiation-induced genome instability and aberrant gene expression in molecular etiology of thymic lymphomas is discussed.     

Can prenatal exposure to a 900 MHz electromagnetic field affect the morphology of the spleen and thymus,and alter biomarkers of oxidative damage in 21-day-old male rats?

We investigated the effects of a 900 Megahertz (MHz) electromagnetic field (EMF), applied during the prenatal period, on the spleen and thymus of 21-day-old male rat pups. Pregnant Sprague-Dawley rats were divided into control and EMF groups. We applied 900 MHz EMF for 1 h/day to the EMF group of pregnant rats. Newborn male rat pups were removed from their mothers and sacrificed on postnatal day 21. Spleen and thymus tissues were excised and examined. Compared to the control group, thymus tissue malondialdehyde levels were significantly higher in the group exposed to EMF, while glutathione levels were significantly decreased. Increased malondialdehyde and glutathione levels were observed in splenic tissue of rats exposed to EMF, while a significant decrease occurred in superoxide dismutase values compared to controls. Transmission electron microscopy showed pathological changes in cell morphology in the thymic and splenic tissues of newborn rats exposed to EMF. Exposure to 900 MHz EMF during the prenatal period can cause pathological and biochemical changes that may compromise the development of the male rat thymus and spleen.     

Structural changes in condylar cartilage following prolonged exposure to the human parathyroid hormone fragment (hPTH) 1–34 in vitro

Summary This investigation presents the structural changes in condylar cartilage incubated in the presence of human parathyroid hormone (1–34) in an organ culture system for 6 to 12 days. Control cultures maintained their cartilaginous characteristics whereas human parathyroid hormone (1–34)-treated cultures revealed the following modifications: (1) The chondroprogenitor cell zone at the apical region of the explant underwent a substantial enlargement. The cells changed from a mesenchyme-like morphology into polygonal, glycogen-rich cells that were tightly attached to each other by a fibrillar intercellular matrix, but even by 12 days the apical region was comprised of healthy cells. (2) The mineralizing zone in the hypertrophic cartilage revealed a change in its cellular population. Hypertrophic chondrocytes were replaced by cells with amoeboid extensions and large numbers of secretory granules or vesicles. Based upon the above findings it appears that the condroprogenitor cells that are initially stimulated to proliferate, are being suppressed from subsequent differentiation into chondroblasts; and that hypertrophic chondrocytes apparently undergo a dedifferentiation process followed by development into an as yet unknown cell population.     

Design and dosimetric analysis of a 385 MHz TETRA head exposure system for use in human provocation studies

A new head exposure system for double‐blind provocation studies investigating possible effects of terrestrial trunked radio (TETRA)‐like exposure (385 MHz) on central nervous processes was developed and dosimetrically analyzed. The exposure system allows localized exposure in the temporal brain, similar to the case of operating a TETRA handset at the ear. The system and antenna concept enables exposure during wake and sleep states while an electroencephalogram (EEG) is recorded. The dosimetric assessment and uncertainty analysis yield high efficiency of 14 W/kg per Watt of accepted antenna input power due to an optimized antenna directly worn on the subject's head. Beside sham exposure, high and low exposure at 6 and 1.5 W/kg (in terms of maxSAR10g in the head) were implemented. Double‐blind control and monitoring of exposure is enabled by easy‐to‐use control software. Exposure uncertainty was rigorously evaluated using finite‐difference time‐domain (FDTD)‐based computations, taking into account anatomical differences of the head, the physiological range of the dielectric tissue properties including effects of sweating on the antenna, possible influences of the EEG electrodes and cables, variations in antenna input reflection coefficients, and effects on the specific absorption rate (SAR) distribution due to unavoidable small variations in the antenna position. This analysis yielded a reasonable uncertainty of <±45% (max to min ratio of 4.2 dB) in terms of maxSAR10g in the head and a variability of <±60% (max to min ratio of 6 dB) in terms of mass‐averaged SAR in different brain regions, as demonstrated by a brain region‐specific absorption analysis. Bioelectromagnetics 33:594–603, 2012. © 2012 Wiley Periodicals, Inc.     

The state of DNA methylation of human ϵ-globin gene in erythroid and non-erythroid cells

The extent of DNA methylation within the embryonic human ϵ-globin gene domain was studied in erythroid and non-erythroid cell lines. The results obtained show that the human ϵ-globin gene is totally methylated at all sites tested in tissues where it is not expressed, i.e. blood leucocytes. In the erythroid cell lines, K562 and PUTKO, both forced to embryonic differentiation by induction with haemin, the level of methylation is reduced compared with that observed in blood leucocytes. In the nonerythroid cell lines HeLa and Raji, where the human ϵ-globin gene is not expressed, the overall level of methylation in all sites tested is lower compared with that in erythroid cell lines.     

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation

Molecular and Cellular Biochemistry - DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1...     

Microwave electromagnetic field regulates gene expression in T-lymphoblastoid leukemia CCRF-CEM cell line exposed to 900 MHz

Electric, magnetic, and electromagnetic fields are ubiquitous in our society, and concerns have been expressed regarding possible adverse effects of these exposures. Research on Extremely Low-Frequency (ELF) magnetic fields has been performed for more than two decades, and the methodology and quality of studies have improved over time. Studies have consistently shown increased risk for childhood leukemia associated with ELF magnetic fields. There are still inadequate data for other outcomes. More recently, focus has shifted toward Radio Frequencies (RF) exposures from mobile telephony. There are no persuasive data suggesting a health risk, but this research field is still immature with regard to the quantity and quality of available data. This technology is constantly changing and there is a need for continued research on this issue. To investigate whether exposure to high-frequency electromagnetic fields (EMF) could induce adverse health effects, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of 900?MHz MW-EMF generated by a transverse electromagnetic (TEM) cell at short and long exposure times. We evaluated the effect of high-frequency EMF on gene expression and we identified functional pathways influenced by 900?MHz MW-EMF exposure.     

Growth variations in OF1 mice following chronic exposure of parental and filial generations to a 15 μT, 50 Hz magnetic field

The growth of a first filial generation (F1) of OF1 mice was studied following chronic exposure of their mothers and themselves to a magnetic field of 15?μT (rms) and 50?Hz. The parental generation (F0) remained for 98 days in this field, after that time they were mated, went through pregnancy, birth, lactation, and the weaning of their offspring in this field. The latter remained exposed to this field until reaching adulthood (220 days). Control animals were treated in the same way but were exposed only to the Earth's magnetic field. The growth data for the offspring were analyzed using a generalization of Koop's equation. Using this model, four phases were identified: lactation growth acceleration, post-weaning growth acceleration, growth stabilization, and a stationary phase. Exposure to the artificial magnetic field was associated with a marked increase in maximum growth rate in the exposed animals during the post-weaning growth acceleration phase, and with a reduction in mass gain in the F1 mice (especially in males) during the third of these phases. In addition, the growth stabilization phase was more extended in exposed females and shorter in exposed males than in the control animals. Furthermore, statistically significant differences were seen between the mean body masses of exposed and control F1 males from 49-123 days. Exposure to the artificial magnetic field might have been associated with the stimulated growth rate seen over the noticeably shortened second and third growth phases (leaving these animals lighter by the stationary phase compared to controls) and a possible acceleration of aging. Both processes could be responsible for the stationary phase being reached at an earlier age, especially in males.     

Azathioprine, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro

Chromosomal analyses in lymphocytes of 28 patients with multiple sclerosis were carried out before, during and after Azathioprine (Aza) therapy. Only a higher incidence of gaps was found in treated patients than in a group of healthy persons but not in comparison with untreated patients. Similarly, no significant clastogenic effect was observed in vitro after short-term and long-term treatment of unstimulated and stimulated lymphocytes with concentrations of 1--100 microgram Aza per ml. Treatment of cultures with 0.0001--4.0 microgram/ml did not yield increased SCE frequencies. The absence of any significant clastogenic effect of therapeutic doses of Aza on human somatic cells is deduced from an evaluation of previously published data and from the present results.     

Azathioprine,a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro

Chromosomal analyses in lymphocytes of 28 patients with multiple sclerosis were carried out before, during and after Azathioprine (Aza) therapy. Only a higher incidence of gaps was found in treated patients than in a group of healthy persons but not in comparison with untreated patients. Similarly, no significant clastogenic effect was observed in vitro after short-term and long-term treatment of unstimulated and stimulated lymphocytes with concentrations of 1–100 μg Aza per ml. Treatment of cultures with 0.0001–4.0 μg/ml did not yield increased SCE frequencies. The absence of any significant clastogenic effect of therapeutic doses of Aza on human somatic cells is deduced from an evaluation of previously published data and from the present results.     

Effects of 837 and 1950 MHz radiofrequency radiation exposure alone or combined on oxidative stress in MCF10A cells

The aim of this study was to determine whether the exposure to either single or multiple radio‐frequency (RF) radiation frequencies could induce oxidative stress in cell cultures. Exposures of human MCF10A mammary epithelial cells to either a single frequency (837 MHz alone or 1950 MHz alone) or multiple frequencies (837 and 1950 MHz) were conducted at specific absorption rate (SAR) values of 4 W/kg for 2 h. During the exposure period, the temperature in the exposure chamber was maintained isothermally. Intracellular levels of reactive oxygen species (ROS), the antioxidant enzyme activity of superoxide dismutase (SOD), and the ratio of reduced/oxidized glutathione (GSH/GSSG) showed no statistically significant alterations as the result of either single or multiple RF radiation exposures. In contrast, ionizing radiation‐exposed cells, used as a positive control, showed evident changes in all measured biological endpoints. These results indicate that single or multiple RF radiation exposure did not elicit oxidative stress in MCF10A cells under our exposure conditions. Bioelectromagnetics 33:604–611, 2012. © 2012 Wiley Periodicals, Inc.     

Human keratinocytes in culture exhibit no response when exposed to short duration, low amplitude, high frequency (900 MHz) electromagnetic fields in a reverberation chamber

We exposed normal human epidermal keratinocytes to short duration, high frequency, and low amplitude electromagnetic fields, similar to that used by mobile phone technologies. We paid particular attention to the control of the characteristics of the electromagnetic environment generated within a mode stirred reverberation chamber (statistical homogeneity and isotropy of the field and SAR distribution). Two non‐thermal exposure conditions were tested on the epidermal cells: 10‐min exposure with a field amplitude of 8 V/m, and 30 min with 41 V/m. Corresponding specific absorption rates ranged from 2.6 to 73 mW/kg (continuous wave, 900 MHz carrier frequency). We collected RNA from cells subjected to these conditions and used it for a large‐scale microarray screening of over 47000 human genes. Under these conditions, exposure of keratinocytes to the electromagnetic field had little effect; only 20 genes displayed significant modulation. The expression ratios were very small (close to 1.5‐fold change), and none of them were shared by the two tested conditions. Furthermore, those assayed using polymerase chain reaction did not display significant expression modulation (overall mean of the exposed samples: 1.20 ± 0.18). In conclusion, the data presented here show that cultured keratinocytes are not significantly affected by EMF exposure. Bioelectromagnetics 32:302–311, 2011. © 2010 Wiley‐Liss, Inc.     

Host group formation decreases exposure to vector-borne disease: a field experiment in a ‘hotspot’ of West Nile virus transmission

Animals can decrease their individual risk of predation by forming groups. The encounter-dilution hypothesis extends the potential benefits of gregariousness to biting insects and vector-borne disease by predicting that the per capita number of insect bites should decrease within larger host groups. Although vector-borne diseases are common and can exert strong selective pressures on hosts, there have been few tests of the encounter-dilution effect in natural systems. We conducted an experimental test of the encounter-dilution hypothesis using the American robin (Turdus migratorius), a common host species for the West Nile virus (WNV), a mosquito-borne pathogen. By using sentinel hosts (house sparrows, Passer domesticus) caged in naturally occurring communal roosts in the suburbs of Chicago, we assessed sentinel host risk of WNV exposure inside and outside of roosts. We also estimated per capita host exposure to infected vectors inside roosts and outside of roosts. Sentinel birds caged inside roosts seroconverted to WNV more slowly than those outside of roosts, suggesting that social groups decrease per capita exposure to infected mosquitoes. These results therefore support the encounter-dilution hypothesis in a vector-borne disease system. Our results suggest that disease-related selective pressures on sociality may depend on the mode of disease transmission.     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

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