EXPOSURE TO 50 Hz MAGNETIC FIELDS DOES NOT INDUCE THE PHOSPHORYLATION OF SEK1/MKK4 IN CULTURED CELLS

In our previous studies, we found that 50 Hz magnetic fields (MFs) could induce the phosphorylation of stress-activated protein kinase (SAPK) and enhance its enzymatic activity. In order to clarify the relationship between MF exposure and the SAPK pathway clearly, we studied the effects of 50 Hz MF exposure on phosphorylation (activation) of SEK1/MKK4 (the upstream kinase of SAPK). A Chinese hamster lung (CHL) cell line was exposed to 50 Hz MFs at two intensities (0.4 and 0.8 mT) for different durations, and Western blot analysis was used to measure the degree of phosphorylation (activation), and nonphosphorylation (non-activation) of SEK1/MKK4 with corresponding antibodies. The results showed that the exposures at both intensities could not induce the phosphorylation of SEK1/MKK4. However, treatment with high osmotic pressure NaCl could induce the phosphorylation of SEK1/MKK4 in cultured cells. It is suggested that 50 Hz MFs may activate the SAPK through a kinase other than SEK1/MKK4.     

EFFECTS OF 50 Hz MAGNETIC FIELD EXPOSURE ON PROTEIN TYROSINE PHOSPHORYLATION IN CULTURED CELLS

Protein phosphorylation is an extremely important and widely used mechanism of cellular regulation. Here, the effects of 50 Hz magnetic fields (MFs) on tyrosine phosphorylation were studied. A Chinese hamster lung (CHL) cell line was exposed to 50 Hz magnetic fields at two intensities (0.4 mT and 0.8 mT) for different exposure durations, and western blot analysis was used to measure the degree of tyrosine phosphorylation of cellular proteins. Results showed that both 0.4 mT and 0.8 mT 50 Hz magnetic fields could affect the protein tyrosine phosphorylation in cultured cells. Both intensities could affect the tyrosine phosphorylation of 38 and 97.4 kDa proteins. In addition, 0.4 mT could affect tyrosine phosphorylation of 61.7, 105, and 112 kDa proteins, and 0.8 mT affected the tyrosine phosphorylation of 79 and 150 kDa proteins. Moreover, all the tyrosine phosphorylation changes of these proteins were time-dependent. The findings from this study demonstrated that under these experimental conditions, there was evidence that protein tyrosine phosphorylation was a possible process for ELF-EMF producing bioeffects.     

Extremely low frequency electromagnetic fields (ELF-EMFs) induce in vitro angiogenesis process in human endothelial cells

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.     

Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1–1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects. Bioelectromagnetics 19:85–91, 1998. © 1998 Wiley-Liss, Inc.     

Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study

To investigate the effects of low frequency electromagnetic fields (EMF) on the proliferation of epidermal stem cells, human epidermal stem cells (hESC) were isolated, expanded ex vivo, and then exposed to a low frequency EMF. The test and control cells were placed under the same environment. The test cells were exposed for 30 min/day to a 5 mT low frequency EMF at 1, 10, and 50 Hz for 3, 5, or 7 days. The effects of low frequency EMF on cell proliferation, cell cycle, and cell‐surface antigen phenotype were investigated. Low frequency EMF significantly enhanced the proliferation of hESC in the culture medium in a frequency‐dependent manner, with the highest cell proliferation rate at 50 Hz (P < 0.05). Exposure to a low frequency EMF significantly increased the percentage of cells at the S phase of the cell cycle, coupled with a decrease in the percentage of cells in the G1 phase (P < 0.05) but the effect was not frequency dependent. The percentage of CD29⁺/CD71^? cells remained unchanged in the low frequency EMF‐exposed hESC. The results suggested that low frequency EMF influenced hESC proliferation in vitro, and this effect was related to the increased proportion of cells at the S phase. Bioelectromagnetics 34:74–80, 2013. © 2012 Wiley Periodicals, Inc.     

不同强度工频磁场对神经元延迟整流钾通道特性的影响

工频磁场是人类生活中接触最多的一类磁场,其引起的生物效应与人类健康的关系备受关注．本文选用1 mT、5 mT及10 mT工频磁场照射急性分离的小鼠皮层神经元(15 min),应用全细胞膜片钳技术离线记录通道电流,研究了工频磁场对神经元延迟整流钾通道特性的影响．结果显示,1 mT、5 mT及10 mT 3个强度的工频磁场对I_k均有抑制作用,但随着去极化电压的增加,发现1 mT和5 mT工频磁场的抑制率几乎不变,抑制率分别为(30 ± 4.2)%和(20 ± 2.2)%,而10 mT工频磁场的抑制率增加,最大抑制率为43.4%．另外,1 mT和5 mT工频磁场影响了延迟整流钾通道的激活特性,通道的半数激活电压变大,斜率因子不变．而10 mT工频磁场对通道的激活特性没有影响,半数激活电压和斜率因子均不改变．研究表明,工频磁场可能影响了细胞膜上离子通道蛋白质的结构和功能,并且不同强度工频磁场对通道的影响不同,存在强度窗口效应．     

Bidirectional frequency-dependent effect of extremely low-frequency electromagnetic field on E. coli K-12

In the present work, the frequency-dependent effects of extremely low-frequency electromagnetic field (ELF EMF) on Escherichia coli K-12 growth have been studied. The frequency-dependent effects of ELF EMF have shown that it can either stimulate or inhibit the growth of microbes. However, the mechanism by which the ELF EMF affects the bacterial cells is not clear yet. It was suggested that the aqua medium can serve as a target through which the biological effect of ELF EMF on microbes may be realized. To check this hypothesis, the frequency-dependent effects (2, 4, 6, 8, 10 Hz, B = 0.4 mT, 30 min) of ELF EMF on the bacterial growth were studied in both cases where the microbes were in the culture media during the exposure and where culture media was preliminarily exposed to the ELF EMF before the addition of bacteria. For investigating the cell proliferation, the radioactive [³H]-thymidine assay was carried out. It has been shown that EMF at 4 Hz exposure has pronounced stimulation while at 8 Hz it has inhibited cell proliferation.     

Effects of extremely low-frequency pulsed electromagnetic fields on morphological and biochemical properties of human breast carcinoma cells (T47D)

This study was carried out to investigate the effects of 100 and 217 Hz extremely low-frequency pulsed electromagnetic fields (ELF-PEMF) on cell proliferation, actin reorganization, and ROS generation in a human breast carcinoma cells (T47D). Cells were exposed for 24–72 h, at 100 and 217 Hz, 0.1 mT. The treatment induced a time dependent decrease in cell growth after 72 h and revealed an increase in fluorescence intensity in cytoplasm and actin aggregations around the nucleus as detected by fluorescence microscopy. The amount of actin in T47D cells increased after 48 h exposure to 100 Hz and 24 h to 217 Hz while no changes in nuclear morphology were detected. Exposing the cells to 217 Hz for 72 h caused a dramatically increase of intracellular ROS generation while with exposure to 100 Hz it remained nearly unchanged. These results suggest that exposure to ELF-PEMF (100, 217 Hz, 0.1 mT) are able inducing an increase of actin level, its migration toward nucleus but despite of these changes and dramatically increase in ROS generation the symptoms of apoptosis were not observed. Our results support the hypothesis that cell response to EMF may only be observed at certain window effects; such as frequency and intensity of EMF parameters.     

Suppression of natural killer cell activity on Candida stellatoidea by a 50 Hz magnetic field

Exposure to electromagnetic fields (EMF) is ubiquitous for almost all individuals living in industrialized countries. Epidemiological and laboratory studies suggest that exposure to Extremely Low Frequency (ELF) EMF increase cancer risk. The immune system functions as one of the body's main protective mechanisms, and Natural Killer (NK) cells are a subset of lymphocytes that can destroy several types of tumor cells. In this study, we investigated, NK cell activity after exposure to a 50 Hertz (Hz), 2 mT magnetic field generated by a Helmholtz Coil. Nineteen male, 10-12 week old guinea pigs were used, and NK cytotoxic activity of splenocytes was measured in vitro by natural anticandidial colorimetric index. The Mann-Whitney U test was applied for statistical analysis. NK cell cytotoxic activity was decreased in exposed compared to controls. Our data suggests that part of the immune system, the NK cell, can be suppressed by a 50 Hz magnetic field.     

极低频磁场对人肝癌细胞生长、代谢及细胞周期的影响

目的研究一定参数的极低频磁场对人肝癌细胞(SK-HEP-1)在诸多方面的影响。方法在整个SK-HEP-1细胞的培养周期中用50Hz,20mT的极低频磁场对其进行作用,并检测作用后细胞的增殖活性、生长动力学、代谢以及细胞周期的变化。结果50Hz,20mT的极低频磁场对SK-HEP-1细胞的生长与代谢有抑制作用,并能阻碍其有丝分裂的进行。结论50Hz,20mT的极低频磁场为治疗人类恶性肿瘤提供了一种可能的手段。     

Effects and Mechanisms of Exogenous Electromagnetic Field on Bone Cells: A Review

Osteoporosis, fractures, and other bone diseases or injuries represent serious health problems in modern society. A variety of treatments including drugs, surgeries, physical therapies, etc. have been used to prevent or delay the progression of these diseases/injuries with limited effects. Electromagnetic field (EMF) has been used to non-invasively treat bone diseases, such as fracture and osteoporosis, for many years. However, because a variety of cellular and molecular events can be affected by EMF with various parameters, the precise bioeffects and underlying mechanisms of specific EMF on bone cells are still obscure. Here, we summarize the common therapeutic parameters (frequency and intensity) of major types of EMF used to treat bone cells taken from 32 papers we selected from the PubMed database published in English from 1991 to 2018. Briefly, pulse EMF promotes the proliferation of osteoblasts when its frequency is 7.5–15 Hz or 50–75 Hz and the intensity is 0.40–1.55 mT or 3.8–4 mT. Sinusoidal EMF, with 0.9–4.8 mT and 45–60 Hz, and static magnetic field with 0.1–0.4 mT or 400 mT, can promote osteoblast differentiation and maturation. Finally, we summarize the latest advances on the molecular signaling pathways influenced by EMF in osteoblasts and osteoclasts. A variety of molecules such as adenosine receptors, calcium channels, BMP2, Notch, Wnt1, etc., can be influenced by EMF in osteoblasts. For osteoclasts, EMF affects RANK, NF-κB, MAPK, etc. We speculate that EMF with different frequencies and intensities exert distinct bioeffects on specific bone cells. More high-quality work is required to explore the detailed effects and underlying mechanisms of EMF on bone cells/skeleton to optimize the application of EMF on bone diseases/injuries. Bioelectromagnetics. 2020;41:263–278 © 2020 Bioelectromagnetics Society.     

Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.     

Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1

A putative Akt kinase phosphorylation site ((64)ydRIRplSYp(73)) was found in Rac1/CDC42 and Rho family proteins (RhoA, RhoB, RhoC, and RhoG). Phosphorylation of Rac1 by Akt kinase was assayed with recombinant Rac1 protein and the fluorescein-labeled Rac1 peptide. It was shown that the Rac1 peptide and the recombinant protein were phosphorylated by the activated recombinant Akt kinase and the lysate of SK-MEL28 cells, a human melanoma cell line. The phosphorylation of Rac1 inhibited its GTP-binding activity without any significant change in GTPase activity. Both the GTP-binding and GTPase activities of Rac1 S71A protein (with the serine residue to be phosphorylated replaced with alanine) were abolished regardless of the treatment of Akt kinase. Akt kinase activity and Rac1 peptide phosphorylation were down-regulated by the treatment of SK-MEL28 cells with wortmannin or LY294002 (a phosphoinositide 3-kinase inhibitor), but JNK/SAPK kinase activity was up-regulated. Thus, the results suggest that Akt kinase of the phosphoinositide 3-kinase signal transduction pathway phosphorylates serine 71 of Rac1 as one of its authentic substrates and modulates the Rac1 signal transduction pathway through phosphorylation.     

Induction of cAMP-dependent protein kinase A activity in human skin fibroblasts and rat osteoblasts by extremely low-frequency electromagnetic fields

Sinusoidal extremely low-frequency electromagnetic fields (ELF-EMF; 7–8 mT, 20 Hz) have already been shown to inhibit proliferation and to accelerate terminal differentiation of human skin fibroblasts in vitro. In order to elucidate the underlying processes of signal transduction, we analysed the activity of cAMP-dependent protein kinase (PKA). EMF exposure for 60 min resulted in an increased PKA activity in human skin fibroblasts (2-fold) and rat embryonic osteoblasts (1.7-fold). Long-term exposure for up to 7 days with a constant 1 h-on/1 h-off EMF exposure rhythm indicated a transient stimulation of PKA activity during the first two exposure rhythms followed by a decrease to the baseline levels of sham-exposed controls. Based on these results, we postulate that a modulation of proliferation and differentiation processes in cells of mesenchymal origin is triggered by an immediate and transient EMF-induced increase in PKA activity. Received: 12 March 1999 / Accepted in revised form: 17 May 1999     

Signal transduction of the melatonin receptor MT1 is disrupted in breast cancer cells by electromagnetic fields

The growth of estrogen‐receptor positive breast cancer cells is inhibited by the pineal gland hormone, melatonin. Concern has been raised that power‐line frequency and microwave electromagnetic fields (EMFs) could reduce the efficiency of melatonin on breast cancer cells. In this study we investigated the impact of EMFs on the signal transduction of the high‐affinity receptor MT1 in parental MCF‐7 cells and MCF‐7 cells transfected with the MT1 gene. The binding of the cAMP‐responsive element binding (CREB) protein to a promoter sequence of BRCA‐1 after stimulation with melatonin was analyzed by a gel‐shift assay and the expression of four estrogen‐responsive genes was measured in sham‐exposed breast cancer cells and cells exposed to a sinusoidal 50 Hz EMF of 1.2 µT for 48 h. In sham‐exposed cells, binding of CREB to the promoter of BRCA‐1 was increased by estradiol and subsequently diminished by treatment with melatonin. In cells exposed to 1.2 µT, 50 Hz EMF, binding of CREB was almost completely omitted. Expression of BRCA‐1, p53, p21^WAF, and c‐myc was increased by estradiol stimulation and subsequently decreased by melatonin treatment in both cell lines, except for p53 expression in the transfected cell line, thereby proving the antiestrogenic effect of melatonin at molecular level. In contrast, in breast cancer cells transfected with MT1 exposed to 1.2 µT of the 50 Hz EMF, the expression of p53 and c‐myc increased significantly after melatonin treatment but for p21^WAF the increase was not significant. These results convincingly prove the negative effect of EMF on the antiestrogenic effect of melatonin in breast cancer cells. Bioelectromagnetics 31:237–245, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of extremely low frequency (50 Hz) magnetic field on morphological and biochemical properties of human keratinocytes

We investigated the effects on human keratinocytes (HaCaT) of exposure to a sinusoidal magnetic field of 2 mT (50 Hz). These cells are a good model for studying interaction of nonionising radiation, because they are not shielded from fields in vivo and also because they are resistant to both mechanical and thermal stimuli. We performed scanning microscopy which showed modification in shape and morphology in exposed cells. This modification is related to differential actin distribution as revealed by phalloidin fluorescence analysis. Moreover, the exposed cells show increased clonogenic capacity, as well as increased cellular growth as showed by clonogenicity assays and growth curves. Indirect immunofluorescence analysis using a fluorescent antibody against involucrin and beta4 integrin, which are respectively differentiation and adhesion markers, revealed an increase of involucrin expression and segregation of beta4 integrin in the cell membrane in cells exposed to 50 Hz; a higher percentage of the exposed cells shows a modified pattern of adhesion and differentiation markers. We also present evidence that exposure of HaCaT cells can interfere with protein kinase activity. Our observations confirm the hypothesis that electromagnetic fields at 50 Hz may modify cell membrane morphology and interfere with initiation of the signal cascade pathway and cellular adhesion.     

不同强度工频磁场对皮层神经元瞬时外向钾离子通道的影响

人们对电磁辐射越来越关注,但是工频磁场产生的生物效应并不确定.选用1、5、10 mT的工频磁场照射急性分离的小鼠皮层神经元(15 min),应用全细胞膜片钳技术离线记录瞬时外向钾通道电流,研究工频磁场对离子通道的影响.结果显示:工频磁场抑制通道的电流密度,并且1 mT、5 mT及10 mT工频磁场的抑制率分别为(63.0±2.2)%、(55.0±1.7)%和(38.0±1.8)%.工频磁场影响离子通道的激活和失活特性,半数激活电压和半数失活电压变小.不同强度工频磁场对离子通道产生的影响程度不同,其中1 mT工频磁场对通道电流的抑制率最大,5 mT工频磁场对通道的半数激活电压和半数失活电压影响最大,10 mT工频磁场增大了通道的失活斜率因子.研究结果表明,工频磁场影响了细胞膜上离子通道蛋白质构象的变化,进一步影响了离子通道的正常功能.     

Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.