Hana Lin

Increased levels of hsp70 transcripts induced when cells are exposed to low frequency electromagnetic fields

Abstract Previous experiments have shown that the steady state levels of some RNA transcripts are increased when cells are exposed to extremely low frequency electric or magnetic fields. Experiments have exposed a variety of cell types, including dipteran salivary gland cells, yeast and human HL-60 cells. The range of responsive transcripts includes oncogenes such as c-myc, as well as transcripts associated with growth and development. One hypothesized mechanism of how cells respond to electromagnetic (EM) fields assumes that the response represents or mimics a generalized physiological stress response. RNA from exposed HL-60 cells, previously shown to have increased transcript levels for c-myc, was analyzed for hsp70 transcripts levels. The hsp70 transcripts were found to be elevated in all cases, even though the cells were exposed to various fields at normal growth temperatures. The conditions of maximum induction for hsp70 were coordinate with those of c-myc. In yeast cells, the SSA1 gene (homologous to hsp70) was found to be elevated in cells exposed to EM fields at 0.8−80 μT. In the case of yeast, conditions for maximum induction of SSA1 were coordinate with those for URA3, the gene for uracil metabolism. Thus the model of cell interaction with electric and/or magnetic fields appears to be related to the stress response model for heat shock.

Effects of mobile phone radiation on reproduction and development in Drosophila melanogaster

In this report we examined the effects of a discontinuous radio frequency (RF) signal produced by a GSM multiband mobile phone (900/1,900 MHz; SAR ∼ 1.4 W/kg) on Drosophila melanogaster, during the 10‐day developmental period from egg laying through pupation. As found earlier with low frequency exposures, the non‐thermal radiation from the GSM mobile phone increased numbers of offspring, elevated hsp70 levels, increased serum response element (SRE) DNA‐binding and induced the phosphorylation of the nuclear transcription factor, ELK‐1. The rapid induction of hsp70 within minutes, by a non‐thermal stress, together with identified components of signal transduction pathways, provide sensitive and reliable biomarkers that could serve as the basis for realistic mobile phone safety guidelines. J. Cell. Biochem. 89: 48–55, 2003.

Regulating genes with electromagnetic response elements

A 900 base pair segment of the c‐myc promoter, containing eight nCTCTn sequences, is required for the induction of c‐myc expression by electromagnetic (EM) fields. Similarly, a 70 bp region of the HSP70 promoter, containing three nCTCTn sequences, is required for the induction of HSP70 expression by EM fields. Removal of the 900 base pair segment of the c‐myc promoter eliminates the ability of EM fields to induce c‐myc expression. Similarly, removal of the 70 bp region of the HSP70 promoter, with its three nCTCTn sequences, eliminates the response to EM fields. The nCTCTn sequences apparently act as electromagnetic field response elements (EMRE). To test if introducing EMREs imparts the ability to respond to applied EM fields, the 900 bp segment of the c‐myc promoter (containing eight EMREs) was placed upstream of CAT or luciferase reporter constructs that were otherwise unresponsive to EM fields. EMREs‐reporter constructs were transfected into HeLa cells and exposed to 8 μT 60 Hz fields. Protein extracts from EM field‐exposed transfectants had significant increases in activity of both CAT and luciferase, compared with identical transfectants that were sham‐exposed. Transfectants with CAT or luciferase constructs lacking EMREs remained unresponsive to EM fields, i.e., there was no increase in either CAT or luciferase activity. These data support the idea that EMREs can be used as switches to regulate exogenously introduced genes in gene therapy. J. Cell. Biochem. 81:143–148, 2001.

Electromagnetic field exposure induces rapid, transitory heat shock factor activation in human cells

Stimulation of human promyelocytic HL60 cells by a 60Hz magnetic field at normal growth temperatures results in heat shock factor 1 activation and heat shock element binding, a sequence of events that mediates the stress‐induced transcription of the stress gene HSP70 and increased synthesis of the stress response protein hsp70kD. Thus, the events mediating the electromagnetic field–stimulated stress response appear to be similar to those reported for other physiological stresses (e.g., hyperthermia, heavy metals, oxidative stress) and could well be the general mechanism of interaction of electromagnetic fields with cells. J. Cell. Biochem. 66:482–488, 1997.

A magnetic field‐responsive domain in the human HSP70 promoter

HSP70 gene expression is induced by a wide range of environmental stimuli, including 60‐Hz electromagnetic fields. In an earlier report we showed that the induction of HSP70 gene expression by magnetic fields is effected at the level of transcription and is mediated through c‐myc protein binding at two nCTCTn sequences at −230 and −160. in the human HSP70 promoter. We report on the identification of a third c‐myc binding site (between −158 and −162) that is an important regulator of magnetic field‐induced HSP70 expression. We also show that the heat shock element (HSE), lying between −180 and −203, is required for induction of HSP70 gene expression by magnetic fields. The HSE centered at −100 alone is insufficient. J. Cell. Biochem. 77:170–176, 1999.

Myc-mediated transactivation of HSP70 expression following exposure to magnetic fields.

We investigated c‐myc protein‐binding sites on the HSP70 promoter as modulators of the induction of HSP70 gene expression in response to magnetic field stimulation (8μT at 60Hz) and whether the presence of c‐myc protein potentiates transactivation of HSP70 expression. A 320 base pair region in the HSP70 promoter (+1 to −320) was analyzed. This region contains two c‐myc‐protein binding sites with consensus sequences located at −230 and −160 nucleotide positions (relative to the transcription initiation site) and overlapping with the region reported for the regulation of HSP70 gene expression by c‐myc protein. This promoter region is upstream of other regulatory sequences, including the heat shock element (HSE), AP‐2, and serum response element (SRE). Transfectants containing both c‐myc protein‐binding sites, HSP‐MYC A and HSP‐MYC B, and exposed to magnetic fields showed a 3.0‐fold increase in expression of CAT activity as compared with sham‐exposed control transfectants. Transfectants containing one c‐myc binding site, HSP‐MYC A, and exposed to magnetic fields showed a 2.3‐fold increase in CAT expression. Transfectants in which both HSP‐MYC A and HSP‐MYC B binding sites were deleted showed no magnetic field sensitivity; values were virtually identical with sham‐exposed controls. If the c‐myc expression vector was not co‐transfected with the constructs containing myc‐binding sites, there was no difference in the expression of CAT activity between magnetically stimulated and sham‐exposed controls, although both responded to heat shock. These data suggest that endogenous elevated levels of myc protein contribute to the induction of HSP70 in response to magnetic field stimulation. J. Cell. Biochem. 69:181–188, 1998.

Magnetic field activation of protein–DNA binding

The mechanisms involved in sensing, signaling, and coordinating changes resulting from magnetic field‐induced stress show substantial similarities to those of heat shock, e.g., magnetic field‐induced heat shock 70 gene (HSP70) expression involves heat shock factor (HSF) activation and heat shock element binding. However, an additional requirement for transactivation of HSP70 expression by magnetic fields is the binding of Myc protein, indicating that additional elements and/or pathways are involved in the induction of HSP70 expression by magnetic fields. To investigate the possible participation of additional genetic elements in magnetic field‐induced HSP70 expression, we examined both magnetic field exposure and heat shock on protein–DNA binding of the transcription factors HSF, AP‐1, AP‐2, and SP‐1 in four human cell lines. The binding sites for these transcription factors are present in the HSP70 promoter. AP‐1 binding activity, normally not increased by heat shock, was increased by magnetic fields; heat shock induced an increase only in HSF binding. Although intersecting and converging signaling pathways could account for the multiplicity of elements involved in magnetic field‐induced HSP70 transcription, direct interaction of magnetic fields with DNA is also a possible mechanism. Because magnetic fields penetrate the cell, they could well react with conducting electrons present in the stacked bases of the DNA. J. Cell. Biochem. 70:297–303, 1998.

Application of magnetic field–induced heat shock protein 70 for presurgical cytoprotection

To develop an alternative to hyperthermia for the induction of hsp70 for presurgical cytoprotection, we investigated the optimal exposure conditions for magnetic field induction of hsp70. Normal human breast cells (HTB124) were exposed to 60‐Hz magnetic fields and hsp70 levels were measured following three different exposure conditions: continuous exposure up to 3 h, a single 20‐min exposure, and a single 20‐min exposure followed by repeated 20‐min exposures at different field strengths. In cells exposed continuously for 3 h, hsp70 levels peaked (46%) within 20 min and returned to control levels by 2 h. Following a single 20‐min exposure, the return of hsp70 levels to control values extended to more than 3 h. When cells underwent a 20‐min exposure followed by repeated 20‐min exposures (restimulation) with different field strengths, additional increases in hsp70 levels were induced: 31% at 1 h, 41% at 2 h, and 30% at 3 h. J. Cell. Biochem. 71:577–583, 1998.

Changes in transcription in HL-60 cells following exposure to alternating currents from electric fields

Abstract The effect of varying field strength and exposure time on histone H2B and c- myc transcript levels in HL-60 cells exposed to 60 Hz electric fields (sine waves) is reported here. An increase in the basal levels of these normally expressed transcripts was observed, which was dependent on both field strength and time of exposure. β2-microglobulin, a transcript known to be uninducible, was unaffected by cellular exposure to the field strengths used in these experiments.

Cytoprotection by electromagnetic field‐induced hsp70: A model for clinical application

A unique approach to clinical application of cytoprotection is offered by electromagnetic (EM) field induction of stress proteins. EM fields are noninvasive and easily applied, as compared with the current hyperthermia protocols. Fertilized dipteran eggs and cultured rodent cardiomyocytes (H9c2 cells) were used as models to test EM fields for their ability to induce increased hsp70 levels for effective cytoprotection. Eggs preconditioned with an 8μT 60Hz EM field for 30 min had 114% increase in hsp70 levels, and an average 82% increase in survival, following a lethal temperature of 36.5°C. Thermal preconditioning at 32°C was not nearly as effective in dipteran eggs, inducing only a 44% increase in survival. Preconditioning of cultured murine cardiomyocytes (H9c2 cells) with an 8μT 60 Hz field induced a 77% average increase in hsp70 levels. J. Cell. Biochem. 79:453–459, 2000.