Gene expression in human breast epithelial cells exposed to 60 Hz magnetic fields
Lise I. Loberg,
James R. Gauger2,
James L. Buthod1,
William R. Engdahl and
David L. McCormick3
Experimental Toxicology and Carcinogenesis Division,
1 Microbiology and Immunology Division and
2 Electronics and Electromagnetics Section, IIT Research Institute, Chicago, IL 60616, USA
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Abstract
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Epidemiology suggests a possible relationship between exposure to power frequency magnetic fields (EMF) and breast cancer. One mechanism through which EMF could stimulate breast cancer induction is via altered expression of oncogenes and/or tumor suppressor genes that regulate normal and neoplastic growth. To evaluate the hypothesis that EMF action in the breast is mediated by alterations in gene expression, transcript levels of c-myc and a battery of other cancer-associated genes were quantitated in human breast epithelial cells exposed to pure, linearly polarized 60 Hz EMF with low harmonic distortion. HBL-100 cells and normal (non-transformed) human mammary epithelial cells were exposed to EMF flux densities of 0.1, 1.0 and 10.0 Gauss (G) for periods ranging from 20 min to 24 h; concurrent sham controls were exposed to ambient fields (<0.001 G) only. Gene expression was quantitated using ribonuclease protection assays. EMF exposure had no statistically significant effect on basal levels of c-myc transcripts in either human breast cell model, and had no effect on alterations in c-myc expression induced by 12-O-tetradecanoylphorbol-13-acetate. Transcript levels of c-erbB-2, p53, p21, GADD45, bax, bcl-x, mcl-1, and c-fos were also unaffected by EMF exposure. These results suggest that EMF is unlikely to influence breast cancer induction through a mechanism involving altered expression of these genes.
Abbreviations: EMF, 60 Hz magnetic field; G, Gauss; GADPH, glyceraldehyde-6-phosphate dehydrogenase gene; mG, milliGauss; HME cells, human mammary epithelial cells; RPA, ribonuclease protection assay; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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The possible association between exposure to power frequency (50 or 60 Hz) magnetic fields (EMF) and breast cancer risk has generated significant controversy. Several groups have reported increased breast cancer risk in women (1,2) and men (35) working in `electrical occupations' that involve presumed high levels of EMF exposure. However, other investigators studying similar occupational groups have failed to identify increases in breast cancer risk (6,7), and studies of the relationship between `environmental' EMF exposures and breast cancer have been uniformly negative. Plausible biochemical or molecular mechanisms through which EMF exposure may stimulate breast cancer induction remain to be identified.
Neoplastic development in the breast is commonly associated with altered expression of oncogenes and/or tumor suppressor genes (811). On this basis, alterations in the expression of cancer-related genes could serve as a mechanism for EMF action in the breast epithelium. Exposure to EMF has been reported to increase transcript levels of several immediate early response genes in transformed cells (1214). Of these genes, c-myc is of particular interest, in view of reports of EMF enhancement of its expression (1214), and its aberrant expression in a high percentage of human breast cancers (11,15,16).
Several genes in addition to c-myc demonstrate aberrant expression patterns in human breast cancers (10,11,17); influences on the expression of such genes may also provide a molecular basis for EMF action. For example, c-erbB-2 (HER2/neu) and p53 are overexpressed in some aggressive breast cancers (11,16,1820), and low levels of p21 in breast cancers correlate (independently of p53) with poor prognosis (20). In contrast, overexpression of bcl-2 is correlated with low histological grade and favorable prognosis (21,22).
EMF may also influence breast carcinogenesis through altered expression of other genes that regulate cell proliferation or apoptosis. Such genes include c-erbB-2, which codes for an epidermal growth factor-related receptor (23), p53, GADD45 and p21, whose products regulate cell cycle progression and are involved in the cellular stress response, bax, bcl-x and mcl-1, whose products regulate induction of apoptosis in response to extrinsic and intrinsic stimuli, and c-fos, an immediate early response gene induced by exposure to growth promoting signals. Through alterations in gene expression, EMF exposure could modulate breast cancer initiation, promotion or progression; altered expression of one or more of these genes might also serve as a biomarker for EMF exposure.
To evaluate the hypothesis that EMF alters gene expression in the breast epithelial cell, transcript levels of c-myc and the other cancer-related genes described above were quantitated in two human breast epithelial cell models exposed to EMF or sham fields. HBL-100 cells are estrogen receptor negative and have a 4-fold c-myc amplification (24). Although immortalized (25), HBL-100 cells have been used widely as a near-normal model for gene expression in the breast (24,26). Human mammary epithelial (HME) cells are non-transformed, histologically normal cells obtained from reduction mammoplasties. Because they are not transformed, HME cells provide a suitable model for the study of processes related to neoplastic transformation and carcinogenesis in the breast.
HBL-100 cells (American Type Culture Collection, Rockville, MD) were maintained in McCoy's 5a medium (BioWhittaker, Walkersville, MD) supplemented with 4mM L-glutamine, 1% penicillin/streptomycin and charcoal-stripped fetal bovine serum (FBS; Gemini BioProducts, Calabasas, CA). The doubling time of these cells was ~24 h. Two sets of EMF exposures were performed on log phase HBL-100 cells; the first series was conducted at high cell density (18x106 cells per 75 cm2 flask in 35 ml growth medium containing 2% FBS), whereas the second experimental series was conducted at low cell density (5x106 cells per 75 cm2 flask at 10% FBS). The different plating conditions had no effect on gene expression in HBL-100 cells (data not shown), and results from both experimental series are reported together.
HME cells (Clonetics, San Diego, CA; donor 4144) were cultured serum-free in Clonetics' recommended medium and supplements (52 µg/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.01 µg/ml human epidermal growth factor, 5 µg/ml insulin, 50 µg/ml gentamicin and 50 ng/ml amphotericin-B). HME cells were seeded at a density of 0.6x104 cells/cm2; growth medium was replenished three times per week. Doubling time was 3648 h. At ~80% confluency, HME cells were passaged or exposed to EMF or sham fields; cells were discarded at passage 13.
Cell cultures were exposed to EMF using an in vitro magnetic field linear exposure system model 1 (Electric Research and Management, State College, PA) (27). This exposure system includes two identical exposure modules, each consisting of an insulated acrylic chamber surrounded by a pair of nested Merritt coils, and an insulated acrylic cell stock chamber. Environmental conditions in the exposure modules and cell stock chamber were controlled by a single incubator. Experimentally generated 60 Hz fields were linearly polarized sine waves, and were transient-free with low (<3%) total harmonic distortion. Experimentally generated EMF, ambient EMF, temperature, humidity and CO2 levels in the exposure and stock chambers were continuously monitored throughout all studies.
EMF exposures were conducted at flux densities of 0.1 (100 mG), 1.0 or 10.0 G for periods of 20 min, or 1, 4 or 24 h; some studies involved exposures at the low and high flux densities only. In each experiment, sham controls were exposed concurrently to ambient fields (<1 mG) for an identical period. The identities of the sham module and the exposure module were selected randomly by the system control computer for each experiment; all endpoint analyses were performed in a `blinded' fashion by individuals who were unaware of the exposure history of the cells being analyzed. The 100 mG flux density was selected as a 50- to 100-fold multiple of 60 Hz field densities that are routinely encountered in residential environments (28) and a 5- to 10-fold multiple of the EMF flux densities encountered in certain occupational environments (29). Exposure times were selected to bracket those used in previous studies in which alterations of gene expression in response to EMF exposure have been reported (13,14).
After exposure, total cellular RNA was isolated from EMF- and sham-exposed cultures, and gene expression was analyzed using the ribonuclease protection assay (RPA) method. Transcript levels of c-myc, c-erbB-2, and the housekeeping gene glyceraldehyde-6-phosphate dehydrogenase (GAPDH) were quantitated using human antisense templates (Ambion, Austin, TX) and the MAXIscript In Vitro Transcription Kit (Ambion); [
-32P]UTP was purchased from ICN (Cleveland, OH). Transcript levels of p53, p21, GADD45, bax, bcl-x, mcl-1, c-fos and the housekeeping genes GAPDH and L32 were analyzed using the RiboQuant hStress-1 multi-probe ribonuclease protection assay system (Pharmingen, San Diego, CA). RNAprobe hybrids were separated by polyacrylamide gel electrophoresis, visualized by autoradiography and quantitated by scintillation counting of bands excised from the gel or by densitometric analysis of autoradiographs. After completion of endpoint analyses for each experiment, the exposure code was broken and the ratio of expression of each gene to GAPDH was calculated for EMF- and sham-exposed cells. Within each experiment, differences in gene:GAPDH ratios in sham- and EMF-exposed cultures were analyzed using Student's t-test.
EMF exposure had no statistically significant effects on basal expression of c-myc in either cell model system. In HBL-100 cells, levels of c-myc transcripts in cells exposed to EMF ranged from 66 to 124% of sham control (Figure 1A
). The results of c-myc expression studies in HME cells exposed to 100 mG or 10.0 G EMF were similar: c-myc expression in HME cells exposed to EMF ranged from 81 to 113% of sham control (Figure 1B
).

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Fig. 1. Effects of EMF on basal c-myc expression in human breast epithelial cells. All data were normalized to sham-exposed controls. (A) EMF effects on c-myc expression in HBL-100 cells. 100 mG, n = 89 replicate cultures in two independent exposures; 1 G, n = 4 replicate cultures in a single exposure; 10 G, n = 1013 replicate cultures in three independent exposures. (B) EMF effects on c-myc expression in HME cells. n = 3 replicate cultures in a single exposure.
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To identify possible co-promoting effects of EMF in the breast epithelium, studies were conducted in which c-myc expression was quantitated in HBL-100 and HME cells exposed to EMF in combination with the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA; Alexis Corporation, San Diego, CA). Exposure of HBL-100 cells to TPA alone (0.01 µg/ml) induced a 3- to 6-fold increase in c-myc expression at 1 h, with a return to near baseline conditions after 24 h of exposure. Although the magnitude of TPA effects varied among exposure sets, concurrent exposure to EMF had no effect on c-myc expression in TPA-treated HBL-100 cells under any exposure condition (Figure 2A
).

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Fig. 2. Effects of EMF on c-myc gene expression in TPA-treated HBL-100 and HME cells. Within each exposure, a single cell culture was treated with TPA and exposed to EMF at the indicated field strengths and durations. All data were normalized to untreated sham-exposed controls. (A) HBL-100 cells were treated with 0.01 µg/ml TPA. Where error bars are present, results were compiled from two independent exposures. (B) HME cells were treated with 0.001 µg/ml TPA.
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In contrast to the induction of c-myc expression by TPA in the immortalized HBL-100 cells, exposure of non-transformed HME cells to TPA (0.001 µg/ml) decreased c-myc expression (Figure 2B
); higher concentrations of TPA were cytotoxic to HME cells. c-myc expression in HME cells exposed to TPA for 1 h was 62 ± 9% of that seen in solvent controls; this decrease in c-myc expression was not influenced by concomitant EMF exposure (77 ± 17% of solvent control). At 24 h, c-myc expression in TPA-treated HME cells had recovered to basal levels (106 ± 7% of untreated sham control); recovery of basal c-myc levels was not affected by simultaneous exposure to EMF flux densities of 100 mG or 10.0 G (112 ± 0.3% of untreated sham controls).
Relative transcript levels of c-erbB-2, p53, GADD45, p21, bcl-2, bcl-x, bax, mcl-1, c-fos and the housekeeping genes GAPDH and L32 in sham- and EMF-exposed HBL-100 and HME cells are presented in Tables I and II
; a representative autoradiograph is presented in Figure 3
. No patterns of statistically significant EMF effects on any gene were seen in either cell system; bcl-2 was undetectable in all samples analyzed. At the 10.0 G flux density, a 25% increase in bax expression was seen in HBL-100 cells; expression of all other genes in HBL-100 cells, and all genes (including bax) in HME cells, was comparable with levels measured in sham controls. At 100 mG, a 32% increase in c-fos was seen in HME cells; no other effects were seen in HME cells, and the expression of all assayed genes in HBL-100 cells was comparable with sham control. The increase in c-fos expression in HME cells exposed to 100 mG EMF is notable, since c-fos is a breast cancer-associated oncogene (10) whose induction by EMF has been reported in other in vitro systems (1214). However, the small magnitude of this increase, when considered with the lack of induction of c-fos in cells exposed to EMF at the higher flux density (10.0 G), appear to limit the biological significance of this finding.

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Fig. 3. Sample RPA autoradiograph from HBL-100 cells exposed to sham fields or 100 mG for 24 h. Five individual cell cultures were exposed under each condition. The multi-probe RPA gel was exposed to X-ray film for 8 h.
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The results of the present studies demonstrate that exposure to EMF has no statistically significant effects on the expression of c-myc and a battery of other cancer-related genes in two in vitro human breast epithelial cell model systems. These results supplement a growing body of evidence (3035) which suggests that alterations in oncogene or tumor suppressor gene expression are unlikely to be involved in a mechanism of EMF-induced cancer.
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Acknowledgments
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This research was supported by grant RO1 ES-07093 from the National Institute of Environmental Health Sciences, NIH.
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Notes
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3 To whom correspondence should be addressed Email: dmccormick{at}iitri.org 
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Received October 21, 1998;
revised April 23, 1999;
accepted April 30, 1999.