Cell Biology International 1999, Vol. 23, No. 11, 739–748Article No. cbir.1999.0436, available online at http://www.idealibrary.com on

EFFECTS ON PROTEIN KINASE C AND GENE EXPRESSION IN A HUMAN MASTCELL LINE, HMC-1, FOLLOWING MICROWAVE EXPOSURE

CHARLES HARVEY* and PETER W. FRENCH†

Centre for Immunology, St Vincent’s Hospital, Sydney, NSW 2010, Australia

Received 10 May 1999; accepted 9 August 1999

We used a resonant cavity which delivered a continuous wave exposure at 864.3 MHz at anaverage specific absorption rate (SAR) of 7 W/kg to determine non-thermal biological effects ofmicrowave exposure. A human mast cell line, HMC-1, was used as the biological target. Cellswere given three exposures each of 20-min duration daily for 7 days. The temperature of the cellculture medium during the exposure fell to 26.5�C. Effects were seen on localization of proteinkinase C, and expression of three genes of 588 screened. The affected genes included theproto-oncogene c-kit, the transcription factor Nucleoside diphosphate kinase B and theapoptosis-associated gene DAD-1. Stress response genes were variably upregulated. Nosignificant effect on morphology or on F-actin distribution was detected. We conclude thatlow-power microwave exposure may act on HMC-1 cells by altering gene expression via amechanism involving activation of protein kinase C, and at temperatures well below thoseknown to induce a heat shock response. � 1999 Academic Press

K: electromagnetic fields; microwaves; gene expression; signal transduction; protein kinase C;c-kit.

*Current address: Department of Gastroenterology, Prince of WalesHospital, Sydney, NSW 2031, Australia.†To whom correspondence should be addressed.

INTRODUCTION

The biological significance of exposure to low-levelmicrowave and radiofrequency fields, in theabsence of heating of the target cells or tissue, iscontroversial. This issue has been brought sharplyinto focus by the enormous recent expansion ofmobile telecommunications technology. Exposureto electromagnetic fields in the microwave region isknown to impose stresses on living cells when thespecific absorption rate (SAR) is beyond thethreshold level required to induce heating of cellsand tissues. However, there is no clear agreementas to the biological (and, by implication, health)effects of chronic microwave exposures at powerlevels too low to register a temperature rise usingnormal thermometry.

In vitro and in vivo studies in the area of radio-frequency and microwave bioeffects have examineda wide range of biological endpoints over the past

1065–6995/99/110739+10 $30.00/0

30 years. There has been broad variability in fre-quency, specific absorption rates (SARs), generat-ing equipment used and biological model chosen asthe target. Whilst some studies have shown aneffect at apparent athermal levels on the outcomeexamined, some have not. This has resulted in along list of phenomenological studies. However, todate there has been no generally agreed mechanismby which non-ionizing radiation in the radio-frequency range could affect such biological out-comes in the absence of heating. Nevertheless, therehave been some significant recent studies whichpoint to an effect on DNA directly, as well as ongene expression. Low-level pulsed and continuousmicrowave exposures at 2.54 GHz have beenshown to induce breaks in both double-strandedand single-stranded DNA in brain cells from ratsexposed for 2 h, at SARs of 0.6 and 1.2 W/kg (Laiand Singh, 1995, 1996). The researchers proposedthat these effects could result from a direct effect ofradiofrequency electromagnetic energy on DNAmolecules and/or impairment of DNA-damagerepair mechanisms in brain cells (Lai and Singh,

� 1999 Academic Press

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1995, 1996). There is some doubt about thisproposed mechanism, however, as an attempt toreproduce the results failed (Malayapa et al., 1997).Ivaschuk et al. (1997) demonstrated an effect ofdownregulation or c-jun expression after 20-minexposure to a radiofrequency field, but no effect onc-fos expression. Repacholi et al. (1997) demon-strated a 2.4-fold increase in lymphoma incidencein transgenic mice exposed to a pulsed microwavefield at 900 MHz for 1 h per day at low SARs(0.0078–4.2 W/kg). The mice were transgenic forthe E�-Pim1 gene construct, which predisposesthem to develop lymphoma, although another mu-tagenic event is required. The exposed mice notonly developed significantly more lymphoma thanthe control group, they developed it significantlyearlier. The mechanism for this reported effect isunknown. However, a study using the same in vivomodel showed no similar effect for 50-Hz exposures(Harris et al., 1998), providing strong evidence fora specific mutagenic effect of microwave exposure.Effects on gene expression were apparently notexamined.

In vitro studies have reported a range of effectson cell growth, cell cycle, and DNA synthesis. Inhuman lymphocytes, 450-MHz fields at around1.0 mW/cm2 affected cAMP-independent proteinkinase activity (Byus et al., 1984). Athermal expo-sure of glioma cells or lymphocytes to 27- or2450-MHz fields caused dose-rate dependent effectson proliferation. DNA synthesis was increased inglioma cells and suppressed in lymphocytes (Clearyet al., 1992). Cell cycle alterations in CHO cellswere induced by a 27 MHz exposure (Cao et al.,1995). The major alteration was an increase in thenumber of cells in G0/G1, and a decrease of those inM phase. The maximum effect was seen 3 dayspost-irradiation. Cytogenetic damage has beenreported in human lymphocytes from bloodsamples exposed to a 954-MHz field (Maes et al.,1995, 1996). Very low power pulsed exposures ofhuman amnion cells at 960 MHz have recently beenreported to induce a decrease in cell growthrate with increased exposure time (Kwee andRasmark, 1998).

In previous studies (Donnellan et al., 1997), wehave reported in vitro cellular effects of an electro-magnetic field exposure in the microwave range(864.3 MHz) at an SAR at 7 W/kg (Anderson et al.,1998) on a rat mast cell line, RBL 2H3. The cellswere allowed to cool to 26�C during the exposure.Exposure of cells at temperatures well below 37�Censured that the cells were not heat stressed. Theeffects seen in the exposed cultures includedchanges to the rate of DNA synthesis, F-actin

distribution, cell morphology and secretion of�-hexosaminidase, a surrogate marker of histaminerelease. Some of these alterations were similar tothose induced by the protein kinase C (PKC)activator, PMA (Ludowyke et al., 1994). After7 days of exposure the morphological rearrange-ments persisted through subculture in the absenceof exposure to the electromagnetic field. To date, amechanism to account for these apparent ‘ather-mal’ effects of radiofrequency/microwave exposurehas not been demonstrated. Based on our results,we hypothesized that radiofrequency exposure mayaffect molecules associated with the signal trans-duction pathway (in particular PKC), leading to analteration of gene expression.

The aim of this study was to determine the effectof such exposure on protein kinase C and geneexpression in human mast cells using the samein vitro exposure conditions described previouslyfor RBL 2H3 cells, whilst taking care to minimizevariability arising from the resonant design of thechamber. To examine gene expression, we used acommercial cDNA array screening system bywhich we could examine effects on 588 humangenes which are under tight transcriptional control.

MATERIALS AND METHODS

Cell culture

The human mast cell line HMC-1 (a kind gift ofDr R. Ludowyke) was maintained in culture inIscove’s modified Dulbecco’s minimum essentialmedium supplemented with 10% fetal bovineserum, 4 m glutamine, 100 units/ml penicillin, and100 �g/ml streptomycin (all from PA Biologicals,Sydney, Australia) and 1 m HEPES buffer (CSL,Melbourne, Australia). Cells were cultured in 75-cm2 flasks (Falcon, Becton Dickinson, NJ, U.S.A.)and incubated at 37�C in a Stericult-200 incubator(Forma Scientific, Marietta, OH, U.S.A.) in ahumidified atmosphere of 5% CO2 in air.

For the exposures, a single flask at a time wasplaced into the exposure chamber and exposed toan electromagnetic field of 864.3 MHz at a nominaloutput power of 2 W as previously described(Donnellan et al., 1997). Three exposures each of20 min were performed at 4-h intervals daily for7 days. Sham exposed flasks were treated in anidentical fashion to the exposed flasks. They wereremoved from the incubator and placed in the sameposition in the exposure chamber as the exposedflask. However, the power to the antenna was notactivated. After 20 min they were returned to the

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incubator. This was done immediately prior to thereal exposure.

The cells were allowed to cool during the expo-sure. After 20 min there was less than 1�C differ-ence between the temperature of the culturemedium in the exposed (26.5�C) and sham (25.8�C)flasks (P=0.13, Mann–Whitney U Test), and thisdifference occurred only during the last 5 min of theexposure (Fig. 1).

24

36

240

Time (min)

Tem

pera

ture

(°C

)

10

26

34

32

30

28

2 4 6 8 12 14 16 18 20 22

Fig. 1. Comparison of the temperature of the cell culture medium (20 ml volume) in exposed and sham 75-cm2 culture flasksduring the 20-min exposure period, measured using non-perturbing alcohol thermometers. —�— exposed, —�— sham. Eachpoint represents the mean of three independent measurements.

Electromagnetic field exposure

The exposure chamber is described in Donnellanet al. (1997). It consisted of a cubic aluminium boxof 1.1 m internal side length and contained a hori-zontal loop antenna mounted on a horizontalmasonite shelf located at the mid point of thechamber. The chamber walls were reflective (aresonant chamber) and thus a complex modalpattern of fields is set up (Donnellan et al., 1997).Further exposimetry characterization of the expo-sure chamber was carried out by Telstra ResearchLaboratories, Melbourne (Anderson et al., 1998).The exact frequency was calculated using a fre-

quency counter and the forward and reflectedpower, and the SARs in localized regions weredetermined using two methods: calculation basedon changes in return loss; and direct measurementusing millimetre-sized fluoroptic temperature sen-sors. It was determined that the frequency of theexposure was 864.3 MHz. At this frequency thereturn loss without the flask was 9.3 dB, i.e. 88.2%of the input power was dissipated in the emptychamber. With the flask present the return loss was17.8 dB, i.e. 98.3% dissipated power. This meantthat approximately 10% of the input power wasabsorbed in the culture flask. Averaged over the20 g of medium in the flask, this gives an averageSAR of 7.3 W/kg for a 2-W nominal transmitterpower (Anderson et al., 1998). Three locationswithin the flask were directly assayed usingLuxtron model 790 fluoropotic probe sensors inculture medium gelled with Natrosol 250 HR,which minimizes thermal diffusion due to fluidconvective currents. After 20 min at 2 W inputpower, temperature rises of between 0.8–1.8�C werederived from the three probes, giving SARs at thethree points of 4.9, 11.4 and 12.1 W/kg. TheTelstra analysis confirmed modal variability in the

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electromagnetic field due to the resonant nature ofthe chamber, and this was taken into account in theexperimental design with respect to the operationof the chamber.

Confocal microscopy

Immunofluorescence and confocal microscopywere used to determine effects of 864.3-MHz expo-sure on F-actin distribution and cell morphology.Cells were cultured in eight-well glass chamberslides (Falcon, Becton Dickinson, NJ) and exposedover a 7-day period as described above. Cultureslides were plated at a cell concentration calculatedto give 5�104 cells per well on each day, in orderto avoid variation in degree of confluence betweendays of treatment. Cells were examined on consecu-tive days starting at day 5. On each day one slidefrom each group was fixed in 4% (v/v) paraformal-dehyde 0.1% (v/v) Triton X-100 for 25 min at roomtemperature. The fixed cells were washed twicewith phosphate-buffered saline, pH 7.4 (PBS) andF-actin visualized by adding a solution ofphalloidin–fluorescin isothiocyanate (phalloidin-FITC) (Sigma-Aldrich, Sydney, Australia) diluted1:200 (v/v) in PBS for 1 h at room temperature. Thecells were washed with PBS and mountedfor viewing in fluorescent mounting mediumcontaining 90% (v/v) glycerol, 10% (v/v) PBS atpH 8.0 containing 1 mg/ml p-phenylenediamine(Sigma-Aldrich). Samples were examined by con-focal laser scanning microscopy (CLSM) using aSarastro 2000 Confocal Microscope (MolecularDynamics, Sunnyvale, CA), as described previously(Kawasugi et al., 1995).

Cell lysis

Control and EMP exposed HMC-1 cells were lysedin 200 �l fresh, ice cold lysis buffer (20 m Tris–HCl, 5 m EGTA, 2 m EDTA, 1 m DTT, 10%glycerol (v/v), 5 m, 10 � pefabloc, 10 � leupep-tin, 10 � aprotonin, 10 � pepstatin) per 5�106

cells. Crude whole cell lysates were fractionatedinto membrane and non membrane proteins fol-lowing successive rounds of sonication and ultra-centrifugation (Huang et al., 1989). Proteinconcentrations were determined using a BCA pro-tein microtitre assay system (Pierce, Rockford, IL,U.S.A.). Membrane and non-membrane sampleswere diluted 1:1 in 2� electrophoresis samplebuffer (0.1 Tris–HCl pH 6.8, 2% (v/v) SDS, 0.2%(v/v) glycerol, 1% (w/v) bromophenol blue) and

boiled for 5 min and stored at �20�C untilrequired.

SDS-polyacrylamide gel electrophoresis andWestern blotting

Samples (20 �g) containing the same protein con-centration were electrophoresed in a 12% SDSpolyacrylamide gel and the proteins transferred topolyvinylidene difluoride membranes (DuPont,Boston, MA) by electrophoresis. Membranes wereblocked for 2 h at room temperature in a 5%solution of skim milk powder in PBS then washedtwice in PBS-Tween. Membranes were probed witha rabbit anti-Pan PKC antibody used at 1:100 (v/v)in PBS-1% bovine serum albumin (BSA)(Amersham, Buckinghamshire, U.K.) and incu-bated for 16 h at 4�C. Membranes were washed3� in PBS-Tween and incubated with an anti-Rabbit-Ig peroxidase conjugated antibody(Silenus, Melbourne, Australia) diluted at 1:200(v/v) in PBS-1% BSA for 1 h at room temperature.The membranes were washed as described aboveand the proteins visualized using the Rennaisance�

chemiluminescence kit according to the manu-facturers’ instructions (DuPont, Boston, MA,U.S.A.) followed by exposure to autoradiographyfilm.

Nucleic acid array analysis of gene expression

The effects of electromagnetic energy exposure ongene expression were determined using the HumanAtlas� cDNA Array (Clontech, CA). The Atlas�

Array consists of two identical membranes spottedwith cDNAs representing 558 well-characterizedgenes known to be expressed under tight transcrip-tional control. A number of groups representingkey regulatory molecules are arranged on the arrayinto six quadrants. These groups include: onco-genes and tumour suppressors, cell cycle regulators,stress response, signal transduction, apoptosis re-lated, DNA repair, transcription factors, receptors,cell adhesion, cell–cell communication, and tran-scriptional regulators. Identical membranes allowthe simultaneous analysis of gene expression in twomRNA populations.

Cells were exposed as described above in 75-cm2

culture flasks seeded at 5�105 cells per flask andnot subcultured over the 7-day period, although themedium was replaced once. A control flask wasseeded at the same concentration and mockexposed over the same period. Following the lastexposure on day 7 the cells were harvested and

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lysed, mRNA extracted, and a complex probeprepared and hybridized to the Atlas� Arraymembranes as described below.

Isolation of poly(A)+RNA

PolyA+ RNA was purified from HMC-1 cells in atwo step process. Total RNA was isolated usinga guanidinium-isothiocyanate phenol/chloroformmethod (1 ml/107 cells) (TRIZOL, Gibco–BRL,U.S.A.) according to the manufacturer’s protocoland mRNA further purified using the QIAGENoligotex� mini kit (CA, U.S.A.). Total RNA wasincubated with dT30 oligonucleotides covalentlylinked to latex particles in 1�binding buffer andheated to 65�C to disrupt RNA secondary struc-tures. The oligo dT30 was hybridized to the poly Atail of the mRNA at room temperature and thesamples washed twice before eluting the mRNAfrom the oligotex� resin in preheated (70�C) elu-tion buffer. The quantity and purity of isolatedmRNA was determined by spectrophotometry.

cDNA synthesis

Complex cDNA probes were then prepared bysimultaneous reverse transcription and labellingin the presence of [�-32P]dATP (3000 Ci/mmol,Amersham, Buckinghamshire, U.K.), using 1 �gpolyA+ RNA and 50 U MMLV reverse tran-scriptase. cDNAs were purified from unincorpo-rated 32P-labelled nucleotides and small (<0.1 kb)cDNA fragments by column chromatography. Aparallel labelling reaction with the supplied ControlPolyA+ RNA was performed. Fractions containinglabelled cDNAs were eluted from the column indeionized water and checked for 32P incorporationusing the Cerenkov method of scintillation count-ing on a Camberra Packard Tri Carb 1500 gammacounter. The fractions with the two highestCerenkov counts (usually 2–5�106 cpm in total)were pooled and used to hybridize with the Atlas�

Array membranes.

Hybridization

Atlas� Array membranes were prehybridized in10 ml ExpressHyb� solution prewarmed to 68�C,containing 100 �g/ml heat-denatured herring spermDNA, for 2 h. The labelled cDNA probe wasprepared by adding 10% of the total volume with10�denaturing solution (1 NaOH) and incubat-ing at 68�C for 20 min. An equal volume of2�neutralizing solution (1 NaH2PO4 (pH 7.0)and 5 �g Cot-1 DNA (1 �g/�l) was added and the

probe incubated for an additional 10 min at 68�C.Prehybridization solution was decanted andreplaced with 5 ml fresh prehybridization buffercontaining the labelled probe to give a final probeconcentration of approximately 1�106 cpm/ml.The probe was hybridized for 16 h with continuousagitation at 68�C. Membranes were washed fourtimes in prewarmed 2�SSC, 1% SDS and a furthertwo times in 0.1�SSC, 0.5% SDS. All washes wereperformed at 68�C for 20 min. Moist membraneswere then wrapped in plastic and exposed to X-rayfilm for various lengths of time between 12 and 72 hat �70�C.

Analysis of hybridization signals

To analyse differences in gene expression betweencontrol and exposed mRNA samples, autoradio-graphs were scanned using a Hewlett PackardScan Jet IICX scanner. Hybridization signals werenormalized according to the manufacturer’s in-structions using the expression of the nine house-keeping genes included on the blot. Thehybridization intensity of specific genes was thenquantified using NIH Image analysis software andthe results expressed as percentage increase ordecrease in expression relative to the same gene onthe control membrane.

RESULTS

Confocal microscopy

Figure 2 shows representative confocal micro-graphs of the actin distribution in control andexposed at the 7-day time point. Contrary toprevious results seen with rat mast cells (RBL 2H3cells) (Donnellan et al., 1997) and with humanastrocytomas (French et al., 1997), in a doubleblind assessment of FITC–phalloidin-stainedHMC-1 cells there was no significant morphologi-cal difference between the control (unexposed)HMC-1 cells and the exposed cells at any timepoint in the exposure period. The assessment ofmorphology is more difficult in these cells as theyare polymorphic and less adherent than the RBL2H3 cells, and this combined with the smallnumber of cells available for assessment at lowconfluence may have resulted in changes inducedby exposure being missed. We therefore attemptedto collect representative confocal microscopeimages from at least 10 different fields for eachcondition. Double-blind analysis of these images

744 Cell Biology International, Vol. 23, No. 11, 1999

showed no consistent significant morphologicaldifference between the two treatments.

Protein kinase C

In a series of four separate experiments, there was aconsistent trend for an increase in the amount ofimmunoreactive protein kinase C in the membranefraction of the exposed cells and a concomitantdecrease in the cytosolic fraction. A representativeWestern blot is shown in Fig. 3A and theaverage densitometry of the bands from the fourexperiments is shown in Fig. 3B.

Gene expression

In two separate experiments, reproducible and sig-nificant (>25%) changes in expression between theexposed and the control HMC-1 cells were seen inonly three genes out of a total of 588 genes screened(0.5%) (Table 1): the proto-oncogene c-kit; thetranscription factor Nucleoside diphosphate kinaseB, and the apoptosis-associated gene DAD-1. Thisindicates that such exposure does not result in abroad effect on gene expression, and indeed theeffects on specific genes are moderate rather thansubstantial.

There was some variability between the exper-iments. Some genes were altered in one experiment

but not the other (e.g. Hsp 86 and 27), and somegenes were altered in different directions betweenexperiments (e.g. thymosin beta 10). This may havebeen due to differences in cell passage number,stage of the cell cycle, or physical variations withinthe exposure chamber.

Fig. 2. Confocal micrography of F-actin distribution in HMC-1 cells mock-exposed (A) or exposed (B) to 864.3 MHz asdescribed in Materials and Methods. On day 7, cells were fixed and permeabilized and F-actin distribution visualized withphalloidin–FITC. Scale bar represents 10 µm.

DISCUSSION

Mast cells are useful targets to study the effectsof microwave exposure on signal transductionbecause they have well-characterized signallingpathways and provide a ready biological read-out.Our earlier studies demonstrated effects on a ratmast cell line, RBL 2H3, including enhancement ofcalcium ionophore-induced secretion, and morpho-logical changes both similar to the effects seen withthe addition of the phorbol ester PMA (Donnellanet al., 1997). In unpublished further workusing differential display and Northern blotting,we detected alterations in mRNA levels ofapproximately five genes (data not shown).

In this study we exposed a human mast cell lineto the same field. The HMC-1 cell line, derivedfrom a human mast cell leukaemia, is the onlyestablished cell line with a phenotype similar tothat of human mast cells. HMC-1 cells differ fromRBL 2H3 cells in a number of ways. HMC-1 cells

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Fig. 3. Protein kinase C localization is altered in HMC-1 cells following exposure to an 864.3 MHz field. A: Western blot of PKCin HMC-1 cells exposed (e) or mock exposed (c) to the microwave field as per Materials and Methods. Cells were lysed and theproteins separated into membrane associated and non-membrane associated fractions. Samples were analysed by SDS–PAGEand the proteins transferred to PMDF membranes. The membranes were probed with an anti-pan PKC antibody and the resultsvisualized by chemiluminescence. B: PKC western blots were analysed by densitometry and relative expression determined. Theaverage of results from four experiments are shown as percentage change in PKC expression in exposed cells relative to mockexposed cells.

do not express an IgE receptor, and they expresslow levels of tryptase, indicating that they areimmature mast cells (Nilsson et al., 1994). Beingimmature makes them a suitable target for studyingmast cell differentiation and activation. HMC-1cells have a functional mutation of the stem cellfactor receptor (c-kit) (Furitsu et al., 1993). c-kitencodes a trans-membrane tyrosine kinase recep-tor. In HMC-1 cells, c-kit is mutated so that it isconstitutively activated in a ligand-independentmanner. Furitsu et al. (1993) postulate that thisactivating mutation is involved in oncogenesis ofsome cell types. Intriguingly, we have shown thatone effect of microwave exposure was to upregulatethe expression of c-kit by approximately 36% inHMC-1 cells. It is possible that this wouldresult in greater ligand independent activation ofc-kit, leading to an increase in the oncogenicphenotype.

A similar degree of upregulation (40–100%) of aproto-oncogene by microwave exposure hasrecently been reported for c-Fos in mouse fibro-blasts (Goswami et al., 1999), indicating that someproto-oncogenes might be particularly sensitive tomicrowave and radiofrequency exposure.

We also demonstrated a consistent and signifi-cant decrease in the expression of two otherimportant regulatory genes, DAD-1, andnucleoside diphosphate kinase B.

DAD1, the defender against apoptotic cell death,is related to Bcl-2 and functions as a cell deathrepressor. It was initially identified as a negativeregulator of programmed cell death in BHK21 cells(Kelleher et al., 1997). Therefore, we can postulatethat a decrease by almost 50% of the expression ofDAD-1 following microwave exposure wouldresult in an upregulation of apoptotic activity.Future studies in this area should consider theeffect of microwave exposure on apoptosis.

There is evidence that the other gene of interest,nucleoside diphosphate kinase (NDPK), acts as atumour metastasis suppressor and c-myc transcrip-tion factor (Heo et al., 1997). There are two NDPKisoforms—alpha and beta. We demonstrated adecrease in the expression of the beta isoformfollowing microwave exposure. Using rat mam-mary adenocarcinoma cell lines, Fukuda et al.(1996), demonstrated that expression of both iso-forms was reduced in highly metastatic cells com-pared with poorly metastatic cells. This reduced

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Table 1.HMC-1 genes exhibiting consistent changes in levels ofexpression in cells exposed to 864.3 MHz compared to

mock exposed cells in two separate experiments

Name of protein/gene Average% change

c-kit +36 (�1)Defender against death (DAD) �47 (�1)Nucleoside diphosphate kinase B �38 (�2)

expression correlated with diminished NDPK-enzyme activity. However, transfection of bothisoforms into highly metastatic cells resulted inreduced lung metastasis only in those clones trans-fected with NDPK-alpha but not NDPK-beta.Therefore the role of NDPK-beta in metastasis isunclear.

In one of the experiments we observed an up-regulation of two heat shock response genes, indi-cating the possibility that microwave exposure mayact as a cell stress. This cannot be attributed to ageneral heating effect, for three reasons. First, thetemperature of the culture medium in the exposedflasks was not significantly warmer than that in thesham flasks at any time point during the exposure(Fig. 1). Second, the overall temperature of thecultures fell from 37�C to approximately 26.5�Cduring the 20-min exposure, and third, the generalresponse of these cells does not accord with athermal stress.

A second possibility is that localized ‘hot spots’within the culture vessels gave rise to the effectsobserved. In Telstra’s analysis (Anderson et al.,1998) a temperature rise of 1.8�C was observed inone area of the flask after 20 min. In the exposurescenario in these experiments, with the temperaturesteadily falling from 37�C during the 20 min expo-sure, a temperature rise of less than 2�C in someareas of the flask is not likely to be biologicallysignificant.

It is possible therefore that the exposure maypose another form of stress on the cells. When anenvironmental, physiological or pathological stressis imposed on a cell, there is a rapid influx of Ca2+

(Ding et al., 1998), which activates and translocatesprotein kinase C to the membrane (Haneda et al.,1995), and results in an increase in the synthesis ofheat shock proteins (Hsps) (Wu, 1995). Hsps act as‘molecular chaperones’, binding to and stabilizingunfolding proteins, thus providing the cell withprotection from imposed environmental stresses.Daniells et al. (1998) have reported that using a

pulsed microwave exposure, Hsp genes were acti-vated at levels comparable to those observed withmoderate concentrations of Zn2+ and Cu2+,known to induce the stress response. Importantly,they showed that lower power levels tended, ingeneral, to induce larger responses, which is con-trary to the results expected if the effect were duesimply to heating. They concluded that athermalmicrowave exposure causes the activation of cellu-lar stress responses (Hsp gene induction), presum-ably reflecting increased levels of protein structuralalteration within cells (the common signal leadingto Hsp gene induction). Lin et al. (1998) haveshown that athermal exposure of cells to 60 Hzelectromagnetic fields also results in an increase ofHsp 70 RNA and protein, mediated via mycprotein activation.

Our results for protein kinase C (PKC) providesome support for microwave exposure acting asan athermal stress, as a significant proportion ofcytosolic PKC was translocated to the membranein the exposed cells (Fig. 3). This is the first reportof microwave exposure affecting PKC directly,although Adey (1990), reported that 450-MHz ex-posure at athermal levels acted synergistically withPMA to affect the function of gap junctions inCHO cells. In addition, there have been somereports of extremely low frequencies (ELF—60 Hz)affecting PKC activity (Holian et al., 1996) andPKC-associated signalling pathways (Uckun et al.,1995; Dibirdik et al., 1998). Furthermore, ELFshave been demonstrated to potentiate PMA in theactivation of ornithine decarboxylase activity (Byuset al., 1988) and in the formation of foci in cells(Cain et al., 1993). Translocation of PKC is associ-ated with its activation (McLaughlin and Breen,1999), and can be induced by known physiologicalstresses (Haneda et al., 1995). In addition, recentunpublished studies from our laboratory show anupregulation of some heat shock proteins in humancells following microwave exposure.

In conclusion, the observations reported aboveprovide some evidence that low-level microwaveexposure can affect both gene expression and mol-ecules involved in important cell regulatory func-tions such as growth and death. However, theeffects, especially on gene expression, are quitesmall with reproducible changes in only 0.5% ofgenes examined. This indicates that for this expo-sure set up, the effect of athermal exposure is quitelimited. It is important, nonetheless, to define themechanism by which such effects are observed. Forreasons outlined above, it seems unlikely that theeffects could be explained by a general thermaleffect, and temperature rises in hot spots within the

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exposure vessel at the power used (2 W) are notsufficient to induce a heat-shock response (forwhich exposures at temperatures at or greater than40�C are needed). Therefore another explanation isneeded for the changes observed. It is clear that thiswork should be extended using an exposure systemwhich gives a homogeneous exposure, as the varia-bility produced by the resonant chamber makes itdifficult to draw definite conclusions. Therefore,we plan to use a TEM (Transverse Electric andMagnetic) cell, a standard design which producesa spatially uniform exposure over the sample vol-ume. The fields in the TEM cell have transverseelectric and magnetic fields in a variant of thecoaxial geometry used in transmission lines.

Clearly, the approach taken of using a cDNAarray to study biological effects of microwaveexposure offers many advantages over previousstudies. A large number of genes can be screened atthe same time and compared directly to a controlgroup. Identification of specific genes which arealtered in their expression levels, supported byNorthern blotting data, will assist in determiningwhich signal transduction pathway(s) is (are) likelyto be affected by microwave exposure, and what, ifany, is the likely relevant health outcome.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the assistance ofDr Russell Ludowyke for provision of the HMC-1mast cell line and for ongoing helpful discussionsand advice. CH was supported by a University ofNew South Wales School of Medicine project grantand the project was partially funded from a NewSouth Wales Health research and developmentinfrastructure grant.

REFERENCES

A WR, 1990. Joint actions of environmental nonionizingelectromagnetic fields and chemical pollution in cancerpromotion. Environ Health Perspect 86: 297–305.

A V, R J, L J, 1998. Limitedcharacterisation of an in vitro radiofrequency exposurefacility at the Centre for Immunology, St Vincent’s Hospital,Sydney. Telstra Research Laboratories, Melbourne,Australia, 29 April 1998.

B CV, L RL, F RM, A WR, 1984.Alteration in protein kinase activity following exposure ofcultured lymphocytes to modulated microwave fields. Bio-electromagnetics 5: 34–51.

B CV, K K, P S, A WR, 1988. Increasedornithine decarboxylase activity in cultured cells exposed to

low energy modulated microwave fields and phorbol estertumor promoters. Cancer Res 48: 4222–4226.

C CD, T DL, A WR, 1993. 60 Hz magnetic fieldacts as co-promoter in focus formation of C3H/10T1/2 cells.Carcinogenesis 14: 955–960.

C GH, L LM, C SF, 1995. Cell cycle alterationsinduced by isothermal 27 MHz radio-frequency radiationexposure. Bioelectrochem Bioenerget 37: 131–140.

C SF, L LM, C G, 1992. Effects of RF powerabsorption in mammalian cells. Ann NY Acad Sci 649:166–175.

D C, D I, T D, S P, T J,D P D, 1998. Transgenic nematodes asbiomonitors of microwave-induced stress. Mutat Res399: 55–64.

D I, K D, K T, T-A L,C A, P D, T D, L R, U FM, 1998.Stimulation of src family protein-tyrosine kinases as aproximal and mandatory step for SYK kinase-dependentphospholipase C�2 activation in lymphoma B cells exposedto low energy electromagnetic fields. J Biol Chem 273:4035–4039.

D XZ, T GC, K JG, 1998. Overexpression ofHSP-70 inhibits the phosphorylation of HSF-1 by activatingprotein phosphatase and inhibiting protein kinase C activity.FASEB J 12: 451–459.

D M, MK DR, F PW, 1997. Effectsof exposure to electro-magnetic radiation at 835 MHzon growth, morphology and secretory characteristicsof a mast cell analogue, RBL-2H3. Cell Biol Int 21: 427–439.

F PW, D M, MK DR, 1997. Electro-magnetic radiation at 835 MHz changes the morphologyand inhibits proliferation of a human astrocytoma cell line.Bioelectrochem Bioenerget 43: 13–18.

F M, I A, Y Y, S N, I N,H N, N N, I T, N GL, KN, 1996. Decreased expression of nucleoside diphosphatekinase alpha isoform, an nm23-H2 gene homolog, is associ-ated with metastatic potential of rat mammary-adenocarcinoma cells. Int J Cancer 65: 531–537.

F Y, T T, T T, I H, K H,K U, S H, B JH, A L,K Y, M Y, K Y, K Y,1993. Identification of mutations in the coding sequence ofthe proto-oncogene c-kit in a human mast cell leukemia cellline causing ligand-independent activation of c-kit product.J Clin Invest 92: 1736–1744.

G PC, A LD, P AJ, B JD, M EG,P WF, R R JL, H CR, 1999. Proto-oncogene mRNA levels and activities of multipletranscription factors in C3H10T 1/2 murine embryonicfibroblasts exposed to 835.62 and 847.74 MHz cellularphone communication frequency radiation. Radiat Res 151:300–309.

H M, K R, S T, K D, A S,T M, S Y, 1995. Abnormalities in proteinkinase C and MAP kinase cascade in mesangial cells cul-tured under high glucose conditions. J Diabetes Complicat 9:246–248.

H AW, B A, G V, N D, F J, BML, B MJ, R MH, 1998. A test of lym-phoma induction by long-term exposure of E�-Pim1 trans-genic mice to 50 Hz magnetic fields. Radiation Res 149:300–307.

748 Cell Biology International, Vol. 23, No. 11, 1999

H YJ, K SY, K E, L KJ, 1997. Quaternary structuralanalysis of nucleoside diphosphate kinases using capillaryelectrophoresis. J Chromatog A 781: 251–261.

H O, A RD, L RC, R HM, A BM,W RJ, 1996. Protein kinase C activity is altered inHL60 cells exposed to 60 Hz AC electric fields. Bioelectro-magnetics 17: 504–509.

H FL, Y Y, C M, B MJ, H KP,1989. Differential regulation of protein kinase C isozymes.J Biol Chem 264: 4238–4243.

I OI, J RA, I-J T, H W, AWR, P JL, 1997. Exposure of nerve growth factor-treated PC12 rat pheochromocytoma cells to a modulatedradiofrequency field at 836.55 MHz: effects on c-jun andc-fos expression. Bioelectromagnetics 18: 223–229.

K K, F PW, P R, L RI, 1995.Focal adhesion formation is associated with secretion ofallergic mediators. Cell Motil Cytoskel 3: 215–224.

K DJ, G R, 1997. DAD1, the defender againstapoptotic cell death, is a subunit of the mammalian oligo-saccharyltransferase. Proc Natl Acad Sci USA 94: 4994–4999.

K S, R P, 1998. Changes in cell proliferation due toenvironmental non-ionizing radiation 2. Microwave radia-tion. Bioelectrochem Bioenerget 44: 251–255.

L H, S NP, 1995. Acute low-intensity microwave expo-sure increases DNA single-strand breaks in rat brain cells.Bioelectromagnetics 16: 207–210.

L H, S NP, 1996. Single- and double-strand DNAbreaks in rat brain cells after acute exposure to radio-frequency electromagnetic radiation. Int J Radiat Biol 69:513–521.

L H, H M, B M, J M, G R, 1998.Myc-mediated transactivation of HSP70 expression follow-ing exposure to magnetic fields. J Cell Biochem 69: 181–188.

M RS, A EW, S WL, M EG,P WF, R R JL, 1997. Measurement of DNAdamage after exposure to electromagnetic radiation in thecellular phone communication frequency band (835.62 and847.74 MHz). Radiation Res 148: 618–627.

L RI, K K, F PW, 1994. PMA andcalcium ionophore induce myosin and F-actin rearrange-ment during histamine secretion from RBL-2H3 cells. CellMotil Cytoskel 29: 354–365.

M A, C M, S D, V L, 1995. Cyto-genetic effects of microwaves from mobile communicationfrequencies (954 MHz). Electro Magnetobiol 14: 91–98.

M A, C M, S D, V L, 1996.954 MHz microwaves enhance the mutagenic properties ofmitomycin C. Environ Molec Mutagen 28: 26–30.

ML M, B KC, 1999. Protein kinase C activationpotentiates the rapid secretion of the amyloid precursorprotein from rat cortical synaptosomes. J Neurochem 72:273–281.

N G, B T, K-G M, K L,B JH, S C, N K, H L,1994. Phenotypic characterisation of the human mast cellline HMC-1. Scand J Immunol 39: 489–498.

R M, B A, G V, N D, F J,H AW, 1997. Lymphomas in E�-Pim1 transgenic miceexposed to pulsed 900 MHz electromagnetic fields. Radi-ation Res 147: 631–640.

U FM, K T, J J, J X, M A, TM, B J, L R, 1995. Exposure of B-lineage lym-phoid cells to low energy electromagnetic fields stimulatesLyn kinase. J Biol Chem 270: 27,666–27,670.

W C, 1995. Heat shock transcription factors: structure andregulation. Annu Rev Cell Dev Biol 11: 441–469.