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Molecular and Cellular Biochemistry 232: 133–141, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Influence of 50 Hz electromagnetic fields incombination with a tumour promoting phorbolester on protein kinase C and cell cycle in humancells

Doreen Richard,1 Sandra Lange,1 Torsten Viergutz,2 Ralf Kriehuber,1

Dieter G. Weiss1 and Myrtill Simkó1

1University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology,Rostock; 2Research Institute for the Biology of Farm Animals, Dummerstorf, Germany

Received 18 June 2001; accepted 20 December 2001

Abstract

It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogen-esis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the familyof protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levelsincluding the activation of transcription factors, we evaluated in the present study the effects of exposure of human amnioticfluid cells (AFC) to 50 Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbolester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity,and on the cell cycle distribution.

Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the mem-brane fraction after exposure to 10, 50, 100 nM, and 1 µM TPA. EMF exposure alone showed no effect on PKC translocation.Co-exposure to 10, 50, and 100 nM TPA and 1 mT EMF revealed a significant additive effect (25 ± 50, 66 ± 29, 22 ± 50%,respectively) with the most prominent increase at the concentration of 50 nM TPA. At the highest concentration of TPA used(1 µM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity,showing a significant increase of 10 ± 16% in total PKC activity after co-exposure to 50 nM TPA and 1 mT EMF when com-pared to 50 nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G

0/G

1-phase followed by a delayed

transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that inAFC cells TPA at lower concentrations (≤ 100 nM) induces a less than maximum effect on the PKC pathway, which can beenhanced by the applied EMF. (Mol Cell Biochem 232: 133–141, 2002)

Key words: electromagnetic fields, protein kinase C, phorbol ester, cell cycle, amniotic fluid cells

Abbreviations: EMF – electromagnetic fields; PKC – protein kinase C; TPA – 12-O-tetradecanoylphorbol 13-acetate; AFC –amniotic fluid cells; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis

Address for offprints: M. Simkó, University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology, Albert-Einstein-Strasse 3, D-18051 Rostock, Germany

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Introduction

Electromagnetic fields (EMF) with a frequency of 50/60 Hzhave been reported to exert a wide range of biological effects[1, 2]. A number of epidemiological studies [3–6] have indi-cated that an increased risk of several cancers such as braintumours, leukemia, and breast cancer is associated with expo-sure to electromagnetic fields from overhead power-lines orother electromagnetic sources. The NIEHS Working Group [2]concluded that power-line frequency EMF are ‘possibly car-cinogenic to humans’, however, the cellular and molecularmechanisms underlying these effects remain to be elucidated.

While there are a relatively large number of negative re-ports on the genotoxic capacity of EMF, mutagenic effectsare described in several studies, including increased fre-quency of chromosomal aberrations [7], increased micronu-cleus formation [8] or an increase in DNA strand breaks [9].Despite the fact that the energy associated with environmentalEMF is too low to cause direct structural changes of DNA[10, 11], there are explanations for these effects, e.g. thatcertain cellular processes altered by exposure to EMF, suchas the formation and the stability of free radicals, indirectlyaffect the structure of DNA [12]. Furthermore, if EMF ex-posure is connected to tumourigenesis, it is generally sug-gested that it acts as a tumour promoter rather than an initiator[13, 14].

One of the hypotheses to explain the cellular effects ofEMF focuses on its interaction with signal transduction sys-tems, involving a cascade of phosphorylation reactions cat-alyzed by specific protein kinases including protein kinaseC (PKC). PKC is a family of serine/threonine protein kinasesthat are directly involved in the transmission of a wide vari-ety of extracellular mitogenic signals from the cell membraneto the nucleus [15]. PKC is reversibly activated by diacyl-glycerol and intracellular Ca2+ and requires acidic phospho-lipids for full activation. It is stimulated by phorbol esters,such as 12-O-tetradecanoylphorbol 13-acetate (TPA), whichmimic diacylglycerol. Upon activation PKC is translocatedfrom cytosolic to membrane compartments and can be recov-ered in the particulate fraction. Membrane association ortranslocation is, therefore, commonly used to assess the ac-tivation state of PKC in vitro.

In previous studies it was suggested that PKC or othercomponents of the signal transduction cascade are affectedby electric or magnetic fields [16–18]. Luben [19] and Montiet al. [20] reported that exposure to EMF causes an increasein PKC activity. Some studies indicate a synergistic interac-tion between EMF and tumour promoters [21–24]. If the re-ported effects of EMF on signal transduction processes arebiologically significant, downstream effects, like changes orperturbations in cell cycle distribution, should be observed.

To further understand the response of signal transductionprocesses to EMF exposure, we examined in the present study

the subcellular localization and the enzymatic activity of PKCin human amniotic fluid cells (AFC) after exposure to hori-zontally polarized 50 Hz electromagnetic field (1 mT) aloneor in combination with TPA. Perturbations of the cell cycleprogression were investigated, because our previous resultson EMF-induced micronucleus formation [8, 25] indicatedthat the observed genetic instability is based on indirect (sec-ondary) cellular mechanisms, e.g. modulation of signal trans-duction pathways.

Materials and methods

Chemicals and antibodies

Unless otherwise noted all supplies were obtained from SigmaChemical Co. (St. Louis, MO, USA). 12-O-tetradecanoyl-phorbol 13-acetate (TPA) stock solution (1 mg/ml) was pre-pared in dimethylsulfoxide (DMSO), stored at –20°C anddiluted with cell culture medium before use. Propidium io-dide stock solution was prepared in HEPES buffered saline(HBS; 154 mM NaCl, 14 mM HEPES, pH 7.4) at 1 mg/mland stored at 4°C in the dark. Ribonuclease A (100 Kunitzunits/mg solid) was dissolved in HBS directly prior to use andpreincubated for 3 h at 37°C. Mouse monoclonal anti-pro-tein kinase C (clone MC5) and anti-α-tubulin (clone B-5-1-2)antibodies were used at 1:5000 dilutions. Alkaline phosphatase(AP)-conjugated goat anti-mouse IgG secondary antibody(sc-2058) was obtained from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA, USA) and was used at a dilution of 1:3000.

Cell culture

Human diploid amniotic fluid cells (AFC, Coriell Institutefor Medical Research, Camden, NJ, USA, Repository No.GM 00472) are normal, non-transformed, primary cells,which are established from the amniotic fluid. The adherentlygrowing cells have a spindle-shaped fibroblast-like morphol-ogy. The cell doubling time of the AFC cells is about 24 h.Cells were grown as a monolayer in RPMI-1640 medium(Gibco BRL, Grand Island, NY, USA) supplemented with20% heat-inactivated fetal calf serum (Biochrom KG, Ber-lin, Germany). Cells were maintained in cell culture flasksat 37°C in a humidified atmosphere (5% CO

2/95% air). Cell

culture medium was replaced every 2–3 days. Cells weregrown to subconfluence and then detached by trypsinization(0.05% trypsin/0.53 mM EDTA, Gibco BRL, Grand Island,NY, USA). Synchronization of AFC cells for cell cycle analy-ses was achieved by contact inhibition maintaining cells forprolonged periods of time in confluent cultures until they restin G

0-phase.

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EMF exposure conditions

For exposure to EMF, we used a Helmholtz coil system [25],which consists of a pair of coils with 400 mm in diameter anda distance between the coils of 200 mm (Phywe Systeme,Göttingen, Germany). The coils were wound with a magneticwire 154 times around a frame with 2.1 Ω. A function gen-erator (OS 9020G, Kepco Model BOP 100-1 M OperationalAmplifier) and a Precision Oscilloscope (OS-9020, Goldstar)were used as a power source and for monitoring the sinusoi-dal electric and magnetic field, respectively. The magneticflux density was adjusted to 1 mT and the field uniformityand consistency over the exposure period was measuredusing a F.W. Bell Gauss/Tesla Meter model 6010 (Bell, Or-lando, FL, USA) with integrated temperature sensor. Tem-perature inside the coils was kept at 37 ± 0.2°C and monitoredcontinuously for 24 h before starting the experiments. In theorientation used, the exposure system generated horizontallypolarized magnetic fields in respect to the culture medium.Cells with a maximal confluence of 75% during experimentswere exposed to 50 Hz EMF by placing the plastic cultureflasks (25 cm2, TPP) containing a volume of 6 ml culturemedium during exposure, into the centre of the exposuresystem, which is contained in a standard tissue culture incu-bator. Control groups were incubated at the same time insidean identical, separate incubator without EMF exposure sys-tem.

Treatment protocol

For the PKC experiments, exponentially growing cells weresubjected to: (1) no treatment (control), (2) EMF exposureover a time course of 15 min to 24 h (50 Hz, 1 mT), (3) treat-ment with different concentrations of TPA (1, 10, 50, 100, and1000 nM) for 1 h, (4) 45 min treatment with different con-centrations of TPA (1, 10, 50, 100, and 1000 nM) followedby simultaneous exposure to EMF (50 Hz, 1 mT) and TPAfor 15 min.

Cell cycle analyses were performed with synchronizedAFC cells. After allowing to settle and attach for 10 h, cul-ture medium was completely changed, and cells were treated/co-treated continuously over a 36 h-period with 50 Hz EMF(1 mT) alone or in combination with 1, 10, or 50 nM TPA,respectively.

Subcellular fractionation

Exponentially growing cells were rinsed twice with ice-coldPBS and harvested by cell scraping. After pelleting at 194 × gfor 5 min in a refrigerated centrifuge, cells were lysed in500 µl of ice-cold suspension buffer (50 mM Tris–HCl buffer,

pH 7.4, containing 2 mM EDTA, 2 mM EGTA, 2 mM di-thiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF),25 µg/ml of each, leupeptin, aprotinin, and pepstatin) fol-lowed by passing several times through a 21 gauge needle.Lysates were centrifuged in a minifuge at 20,800 × g for 1 hat 4°C to yield the cytosolic fraction. The pellet was resus-pended in 200 µl suspension buffer containing 1% TritonX-100, homogenized with a Wheaton homogenizer and in-cubated on ice for 30 min. Homogenates were centrifuged asabove and the resulting supernatant was designed as the mem-brane (particulate) fraction. Cytosolic and membrane fractionswere boiled for 5 min in sodiumdodecylsulfate (SDS)-samplebuffer (0.76% Tris, 2% SDS, 10% glycerol, 20% β-mercapto-ethanol, 0.005% bromphenol blue) and subjected to SDS-PAGE and Western blot analysis.

Western blot analysis

Following determination of protein concentration accordingto the method of Bradford [26], cell lysates were separatedon SDS-PAGE (10% polyacrylamide gel) using the MightySmall II gel electrophoresis system (SE 250; Hoefer, San Fran-cisco, CA, USA). Proteins were transferred to polyvinylidenedifluoride (PVDF) membrane (Biorad Laboratories, Hercules,CA, USA) and stained with Ponceau-S to ensure uniform load-ing and blotting. The membranes were blocked in Tris-buff-ered saline (TBS; 20 mM Tris-HCl, 500 mM NaCl, pH 7.5)containing 5% non-fat dry milk (TBS/milk) overnight at 4°C.After washing with TTBS (TBS containing 0.1 % Tween-20),membranes were probed for 2 h at RT with primary antibod-ies in TTBS containing 1% milk (TTBS/milk). After repeatedwashing with TTBS, blots were incubated for 1 h at RT insecondary AP-conjugated antibody in TTBS/milk, finally fol-lowed by three 15 min washes in TTBS. Proteins were visu-alized with the Immun-Star™ Chemiluminescent DetectionSystem according to the manufacturer’s instructions (BioradLaboratories, Hercules, CA, USA). The chemiluminescenceintensity was detected with the Fluor-S™ MultiImager, andrelative protein amounts were quantified using the QuantityOne software (Biorad Laboratories, Hercules, CA, USA).

PKC assay

PKC enzyme activity in cytosolic and membrane fractionswas assayed by using SignaTECT™ Protein Kinase C (PKC)Assay System (Promega Corp., Madison, WI, USA) accord-ing to the manufacturer’s instructions. Briefly, PKC activitywas assayed by measuring the phosphorylation of the spe-cific substrate for the PKC isoforms α and β, the biotinylatedpeptide Neurogranin

(28–43) (AAKIQAS*FRGHMARKK). The

[γ-32P]-labeled substrate is recovered from the reaction mix-

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ture (100 mM Tris-HCl, pH 7.5, 1.25 mM EGTA, 2 mM CaCl2,

0.5 mg/ml BSA, 50 mM MgCl2, 1.6 mg/ml phosphatidyl-

serine, 0.16 mg/ml diacylglycerol, 10 µCi/µl [γ-32P]ATP(3.000 Ci/mmol) by spotting onto the SAM2™ membrane.After removing of unhydrolysed ATP by repeated rinsing in1% H

3PO

4, samples were analysed for [γ-32P] radioactivity

by liquid scintillation counting.

Flow cytometric analysis

The percentage of cells in each phase of the cell cycle wasquantified by flow cytometry using propidium iodide-stainedcells. After treatments, cells were washed in PBS, harvestedby trypsinization, and fixed in cold 70% (v/v) ethanol for atleast 30 min. After centrifugation at 328 × g for 12 min cellswere resuspended in 0.5 ml of a pre-warmed solution of1 mg/ml RNase and incubated at 37°C for 30 min. CellularDNA was stained with 50 µl of 0.5 mg/ml propidium iodidefor additional 30 min at 37°C in the dark. DNA content wasanalysed in a flow cytometer (Epics Elite, Coulter Electron-ics, Hialeah, FL, USA). Typically 10,000 cells were countedper sample. The percentage of cells in each phase of the cellcycle was determined from the histograms using the Multi-cycle Software Package (Phoenix Flow System, San Diego,CA, USA). The experiments were repeated 5 times.

Statistics

The statistical significance of differences between control andexperimental groups in Western blot analyses was assessedby the Wilcoxon matched pairs signed rank test. The quanti-fied raw data of band intensities of controls and experimentswere analysed using α-tubulin as an internal standard. TheWilcoxon matched pairs signed rank test was used to com-pare PKC activity values in subcellular fractions of cellsexposed to TPA and EMF with those of the correspondingcontrol cells. For data analysis of flow cytometry the two-tailed Student’s t-test was used to determine the statisticalsignificance. Generally, a p-value of less than 0.05 was con-sidered to be statistically significant.

Results

Subcellular localization of PKC in response to combinedexposure to TPA and EMF

In untreated control cells PKC resides in the cytosol only. Notranslocation of PKC from the cytosolic to the membranefraction could be observed in exponentially growing AFCcells after exposure to 50 Hz, 1 mT EMF for 15 min up to

24 h (data not shown). PKC translocation to the membranefraction after exposure to 10, 50, 100, and 1000 nM TPA for1 h could be observed. Figure 1 displays a representativeimmunoblot showing the membrane-bound 80 kDa band ofPKC protein after chemiluminescence detection. Co-expo-sure to TPA and EMF (45 min pre-exposure to TPA followedby 15 min exposure to TPA and 1 mT EMF) revealed a sig-nificant additive effect of EMF at 10, 50, and 100 nM TPA(25 ± 50, 66 ± 29, and 22 ± 50%, respectively) with the mostprominent increase at the concentration of 50 nM TPA. Noadditive effect could be observed at the highest concentra-tion of 1 µM TPA (Fig. 1).

Effects of TPA and EMF exposure on PKC activity

Total PKC activity showed a significant increase of 10 ± 16%after co-exposure to 50 nM TPA and EMF compared with50 nM TPA alone (Fig. 2A). After exposure of AFC cells to50 Hz, 1 mT EMF for 15 min, an increase of 15 ± 22% inPKC activity in the membrane fraction was observed, no al-teration occurred in the cytosol (Fig. 2B). A significant in-crease in the membrane-associated PKC activity resulted aftertreatment of AFC cells with 10, 50, 100, and 1000 nM TPAfor 1 h (57 ± 25, 60 ± 25, 45 ± 23, and 68 ± 25%, respec-

Fig. 1. Analysis of protein expression of membrane-associated PKC in ex-ponentially growing AFC cells after combined treatment with TPA and 50 HzEMF (1 mT). Cells were pre-exposed to 10, 50, 100, and 1000 nM TPA, re-spectively, for 45 min, followed by a 15 min exposure to both TPA andEMF. After subcellular fractionation, protein extracts were separated by 10%SDS-PAGE, transferred to PVDF membrane, immunoblotted with mono-clonal anti-PKC antibody against PKC α and βI isozymes and detected withECL. A representative immunoblot of PKC expression in membrane fractionsshows the 80 kDa PKC band (upper). PKC band intensities were quantifiedby optical scanning on a Fluor-S-MultiImager and data of quantitative analysisare shown in the diagram (lower) (n = 9). Data indicate an additive effect ofEMF at lower concentrations of TPA (≤ 100 nM). Data points marked withan asterisk are statistically significant (Wilcoxon, *p < 0.05).

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tively) referring to untreated control cells, whereas a con-comitant decrease in cytosolic activity was exclusively ob-served at 50 nM TPA by 23 ± 9% and 1 µM TPA by 29 ± 9%(Fig. 2B). The data indicate further that 45 min pre-exposureto 50 nM TPA, followed by co-exposure to 1 mT EMF for15 min was associated with a significant increase in enzy-matic activity in the cytosolic fraction by 16 ± 9% and in themembrane fraction by 8 ± 22%. At the highest TPA concen-tration used (1 µM) additional EMF exposure resulted in adecrease of membrane-associated PKC activity by 8 ± 22%,whereas cytosolic activity is unchanged.

Effects of TPA and EMF exposure on cell cycle distribution

The influence of a continuous exposure to different concen-trations of phorbol ester (1, 10, and 50 nM TPA) and 50 HzEMF on cell cycle progression was determined in synchro-nized AFC cells by flow cytometric analyses over a 36 h-period. As shown in Fig. 3A, treatment of cells with EMF

alone did not alter the cell cycle distribution compared tountreated control cells. Data demonstrate further that treat-ment with TPA resulted in a dose-dependent transient cellcycle arrest in G

0/G

1-phase (Fig. 3B) followed by a delayed

Fig. 2. PKC activity of exponentially growing AFC cells after combinedtreatment with TPA and 50 Hz EMF (1 mT). Cells were pre-exposed to 10,50, 100, and 1000 nM TPA, respectively, for 45 min, followed by a 15 minexposure to both TPA and EMF. (A) Total PKC activity. (B) PKC activ-ity after subcellular fractionation expressed as mean ± S.E.M. (n = 6)(C = control). Data points marked with an asterisk show statistically sig-nificant differences between treatment and the corresponding control(Wilcoxon, *p < 0.05).

Fig. 3. Cell cycle distribution in synchronized AFC cells after continuousexposure to 1 mT EMF and TPA (1, 10, and 50 nM) over a 36 h-period. Atthe indicated time points after exposure cells were harvested, fixed in etha-nol and DNA content was measured by propidium iodide staining and sin-gle parameter flow cytometry. Panel A shows no perturbations of cell cycledistribution due to EMF exposure. Treatment with the PKC agonist TPAresults in a transient dose-dependent cell cycle arrest in G0/G1-phase (B)followed by a delayed transit through S-phase (C), whereas G2/M-phase isnot influenced (D); (C = control).

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138

transit through the S-phase (Fig. 3C). With rising concentra-tion of TPA the percentage of cells arrested in G

0/G

1-phase

showed a maximum of 77% after 12 h treatment with 50 nMTPA. Concomitant entry into the S-phase was markedly re-duced at this time point. The G

2/M-phase (Fig. 3D) seems to

be influenced by TPA treatment, leading to a weak dose-de-pendent delay in G

2/M-phase in AFC cells. However, these

differences are not statistically significant. The results ofcombined treatment with phorbol ester and EMF indicatedno further effect on cell cycle distribution compared with TPAtreatment alone (Table 1).

Discussion

Numerous reports about detailed effects of EMF exposure onvarious biological systems have been published, but it is still

unknown how such fields initiate a cascade of events lead-ing to the observed biological responses. Most recent inves-tigations have concentrated on possible tumour promoting orco-promoting effects of EMF [27, 28] and it is of raisinginterest to examine whether exposure to power-line frequencyelectromagnetic fields can affect cell signaling processes invitro.

In our experiments, no translocation of PKC could be ob-served in AFC cells by Western blot analysis after exposureto EMF alone. In contrast, we could show that exposure to a50 Hz EMF with a flux density of 1 mT in combination withthe tumour promoting phorbol ester TPA markedly alters thesubcellular localization of PKC. Our study demonstrates thatat lower concentrations of TPA (10, 50, and 100 nM) thetranslocation of PKC from the cytosolic to the membranefraction was significantly increased when cells were exposedadditionally to EMF. Data indicate the most prominent in-

Table 1. Cell cycle distribution of synchronized AFC cells after continuous exposure/co-exposure to horizontally polarized EMF (50 Hz/1 mT) and TPA (1, 10, 50 nM) over a 36 h-period

Exposure time (h)12 16 24 30 36

Control G0/G1 64.60 ± 6.07 54.14 ± 4.75 57.92 ± 3.23 55.12 ± 3.65 59.78 ± 6.69S 23.54 ± 5.31 26.36 ± 4.39 17.00 ± 3.00 21.72 ± 4.07 14.54 ± 4.88G2/M 11.86 ± 3.28 19.44 ± 4.62 25.08 ± 4.60 23.14 ± 1.96 25.66 ± 3.53

1 mT G0/G1 59.92 ± 7.30 51.70 ± 5.14 59.24 ± 3.11 55.34 ± 3.87 61.76 ± 5.37S 28.36 ± 5.49 25.50 ± 4.85 17.46 ± 3.46 21.10 ± 5.41 13.30 ± 4.32G2/M 11.74 ± 3.19 22.84 ± 3.24 23.28 ± 3.87 23.58 ± 3.24 24.98 ± 3.74

1 nM G0/G1 70.62 ± 4.34 61.10 ± 5.45 61.38 ± 5.24 62.26 ± 2.80* 60.64 ± 4.61S 15.32 ± 4.07* 21.20 ± 4.14 12.48 ± 2.32* 15.96 ± 3.03* 15.40 ± 3.58G2/M 14.08 ± 4.14 17.74 ± 2.65 26.14 ± 5.68 21.80 ± 3.86 23.96 ± 2.72

1 nM/1mT G0/G1 68.14 ± 4.58 60.32 ± 6.28 62.30 ± 4.88 62.06 ± 3.47 61.10 ± 5.91S 15.54 ± 3.68 19.42 ± 5.64 12.20 ± 1.90 16.88 ± 2.82 15.70 ± 4.50G2/M 14.36 ± 3.92 18.52 ± 2.16 25.62 ± 4.96 20.92 ± 2.49 24.14 ± 4.02

10 nM G0/G1 74.48 ± 5.49* 67.44 ± 5.11* 58.18 ± 4.52 61.90 ± 3.76* 61.82 ± 4.45S 8.96 ± 3.62* 16.86 ± 4.96* 18.36 ± 3.31 13.22 ± 0.83* 12.32 ± 2.28G2/M 16.58 ± 3.88 15.70 ± 3.76 23.48 ± 3.30 24.84 ± 3.58 25.86 ± 3.00

10 nM/1 mT G0/G1 73.58 ± 5.53 66.28 ± 5.44 57.48 ± 5.60 61.90 ± 4.00 61.92 ± 6.26S 10.16 ± 5.02 19.46 ± 5.65 18.22 ± 4.41 12.60 ± 1.74 11.52 ± 3.41G2/M 16.26 ± 3.61 14.26 ± 3.23 24.26 ± 3.64 25.48 ± 2.60 26.56 ± 3.93

50 nM G0/G1 76.85 ± 4.82* 70.90 ± 5.67* 57.25 ± 3.49 61.90 ± 4.51 62.85 ± 2.92S 6.30 ± 2.84* 13.78 ± 5.84* 21.95 ± 4.09 12.80 ± 1.89* 12.68 ± 1.66G2/M 16.88 ± 4.07 15.38 ± 4.55 20.83 ± 3.35 25.33 ± 2.78 24.53 ± 4.00

50 nM/1 mT G0/G1 77.18 ± 4.62 70.30 ± 5.80 57.98 ± 2.53 62.05 ± 3.86 62.45 ± 2.72S 5.75 ± 3.04 12.80 ± 6.11 22.05 ± 3.86 12.70 ± 0.57 12.53 ± 1.77G2/M 16.63 ± 3.65 15.15 ± 4.52 22.05 ± 5.27 25.90 ± 3.05 24.23 ± 4.10

The percentage of cells in different cell cycle phases was determined by single parameter flow cytometry. The cell cycle distribution of untreated controls att = 0 h is as follows: G0/G1: 79.08 ± 3.84; S: 2.96 ± 0.88; G2/M: 17.94 ± 3.51. Five independent experiments were conducted. Group mean data ± S.D. areshown. *Statistically significant differences between treatment and control (TPA vs. control or 1 mT vs. control). Double treatments were tested against therespective treatment with TPA only (TPA/1 mT vs. TPA). Student’s t-test; two-tailed, p < 0.05.

139

crease in membrane-associated PKC at 50 nM TPA, whereasat higher concentration of TPA (1 µM) this additive effectcould not be observed. We assume therefore that TPA in-duces a less than maximum response at lower concentrations(≤ 100 nM) which is further increased by the applied EMF.Tao and Henderson [24] described an additive effect of 60 Hz,0.1 mT EMF and TPA treatment on differentiation of HL-60cells at low TPA concentrations, whereas higher concentra-tions eliminate this effect because of TPA ‘saturation’. Simi-lar data published by Tuinstra et al. [23] indicate that EMFact in a synergistic manner with low levels of TPA on PKCactivity. The authors observed an EMF effect for cells thatare moderately activated by 50 nM TPA, but not for cells thatare unexposed or treated with a higher concentration (2 µM)of TPA. Therefore, it can be concluded that cells exposed tosuboptimal concentrations of TPA shift from a normal intoan activated state without saturating their physiological ca-pacity to respond to TPA, which makes them susceptible toadditional stimuli such as EMF.

Considering that protein quantity and kinase activity arenot necessarily proportional, we examined the correlationbetween PKC translocation and enzymatic activity. Althoughexposure of AFC cells to EMF alone was not associated withPKC translocation, a significant increase of 15 ± 22% in PKCactivity in the membrane fraction could be demonstrated.However, the total PKC activity is unchanged compared withuntreated control. TPA treatment resulted in both, transloca-tion of PKC from the cytosolic to the membrane fraction anda concomitant increase in membrane-associated enzymaticactivity. However, a concurrent decrease in cytosolic activ-ity could be only observed at 50 and 1000 nM TPA. In addi-tion, our data showed a significant increase in total PKCactivity after co-exposure to 50 nM TPA and EMF comparedwith 50 nM TPA alone. No significant additive effect couldbe observed in total PKC activity at 10 and 100 nM TPA. Asignificant decrease in the membrane-associated PKC activitywas observed after co-exposure with EMF and TPA in highconcentration (1 µM), however, the total enzymatic activitywas not changed. The PKC activation process requires essen-tial cofactors, namely Ca2+, phosphadidylserine, and diacylgly-cerol [15]. Our results show differences between the proteinquantity and PKC activity, which might be explained by thenon-availability of such cofactors.

There are several reports about the influence of EMF onPKC activity or other kinases involved in signal transductioncascades in different cell systems. It has been shown thattreatment of HL-60 cells with 60 Hz electric fields results indecreased cytosolic but unchanged membrane-associatedPKC activities [29]. Phillips et al. [17] showed an increasein PKC activity upon exposure to EMF. Uckun et al. [16]indicated that 60 Hz, 0.1 mT fields caused activation of ty-rosine kinases and PKC in a human pre-B-leukemia cell line.In a previous study Dibirdik et al. [30] presented that expo-

sure of DT40 lymphoma B cells to EMF results in a tyrosinekinase-dependent activation of phopholipase Cγ2 leading toincreased inositol phospholipid turnover. We suggest fromour results that AFC cells become more susceptible to effectsof EMF exposure because of their sensitivity to TPA. But ithas to be mentioned that such additive EMF effects do notoccur in cells exhibiting maximal activation by TPA.

Several authors discuss different mechanisms for cellularresponses to EMF in dependence on different physiologicalconditions, e.g. the differentiation or activation state of cells[31]. Nindl et al. [32] could demonstrate that the metabolicstate of cells is an important variable in determining the ef-fect of EMF exposure. The authors observed that EMF sen-sitivity of Jurkat cells was highly related to their growth stage.In addition, many effects of EMF on cell signaling are ofrelatively small magnitude and may elicit a biological re-sponse only in the presence of other stimuli [33].

Cell cycle control and, therefore, regulation of prolifera-tion and differentiation processes are mediated via interact-ing signal transduction cascades. Thus, we examined in thepresent study the distribution of the specific phases of the cellcycle of AFC cells in their response to combined treatmentwith TPA and EMF. TPA treatment alone induced an increasednumber of cells in the G

0/G

1-phase with a concomitant de-

crease in S-phase. Our results are consistent with a recentstudy of Slosberg et al. [34] showing that TPA treatment ofHC11 cells induced a transient cell cycle arrest in G

0/G

1-phase

and a corresponding decrease in the percent of cells in S-phase, when compared to untreated cells. Our investigationsshow that additional exposure to EMF did not induce per-turbations of the cell cycle with respect to TPA treated cellsduring the observed time course of 36 h. Activation of PKCis known to cause alterations in the cell cycle progression inseveral cell systems. However, although we showed that EMFinduce additional effects on PKC expression and on enzy-matic activity of PKC, these were not correlated with per-turbations of the cell cycle in AFC cells as observed by flowcytometry. Interestingly, it has also been shown in a recentstudy by Cridland et al. [35] that exposure to EMF alone at20 and 200 µT induced a small increase in the length of G

1-

phase of the cell cycle in normal human fibroblasts, whereasexposure to higher flux densities had no significant effect.Our data showed no effect of EMF exposure alone on cellcycle distribution of AFC cells neither at 1 mT nor at lowerflux densities (20 and 100 µT, data not shown).

In previous studies [8, 25, 36] we have described a sig-nificant increase in micronucleus frequency in human cellsexposed to 50 Hz, 1 mT EMF without affecting cell prolif-eration. Based on these and the results reported here on PKCsubcellular localization and the enzymatic activity, we con-cede that EMF-induced genotoxicity is caused by additionalsignal transduction pathways other than the PKC pathway.We consider that EMF potentiate the cellular response to a

140

chemical agent, such as TPA. We show a significant increasein total PKC activity especially after co-exposure to a mod-erate concentration range of TPA (50 nM) and EMF, whichfit well with the data obtained by Western blot analysis indi-cating the most prominent increase in membrane-associationof PKC at these moderate TPA concentration. In conclusion,we suppose that the PKC pathway is not primarily involvedin EMF induced effects in AFC cells. Further biochemicalinvestigations are needed to elucidate the acting mecha-nisms.

Acknowledgements

This work was supported by the Federal Office for RadiationProtection, Salzgitter, Germany (StSch 4129).

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