FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WOODS, M.
Right arrow Articles by ALEXANDER, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WOODS, M.
Right arrow Articles by ALEXANDER, D. R.
(The FASEB Journal. 2000;14:2284-2290.)
© 2000 FASEB

Lyn and Syk tyrosine kinases are not activated in B-lineage lymphoid cells exposed to low-energy electromagnetic fields

MARGARET WOODS*, FEDJA BOBANOVIC{dagger}, DAVID BROWN{ddagger} and DENIS R. ALEXANDER*1

* Laboratory of Lymphocyte Signalling and Development,
{dagger} Laboratory of Molecular Signalling,
{ddagger} Laboratory of Computational Neuroscience, The Babraham Institute, Cambridge CB2 4AT, United Kingdom

1Correspondence: Laboratory of Lymphocyte Signalling and Development, Molecular Immunology Programme, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, U.K. E-mail: Denis.Alexander{at}BBSRC.AC.UK


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Exposure of B-lineage lymphoid cells to a 100 µT 60 Hz AC magnetic field has been reported to stimulate the rapid activation of Lyn and Syk tyrosine kinases and the induction of protein tyrosine phosphorylation. These findings are significant because of the critical role played by these B cell signaling events in the control of growth and differentiation, and therefore the potential of electromagnetic field (EMF) exposure to induce cancer. We report the first study carried out with the aim of reproducing the reported EMF effects on Lyn and Syk tyrosine kinases. The system used enabled EMF exposure conditions to be carefully controlled and also allowed experiments to be performed blind. The effects of a 100 µT 60 Hz AC magnetic field on protein tyrosine phosphorylation and on Lyn and Syk tyrosine kinase activities were investigated in Nalm-6 and DT40 B cells in the absence and presence of a 46 µT DC magnetic field. However, no significant effects of low-energy electromagnetic fields on tyrosine kinase activities or protein phosphorylation were observed.—Woods, M., Bobanovic, F., Brown, D., Alexander, D. R. Lyn and Syk tyrosine kinases are not activated in B-lineage lymphoid cells exposed to low-energy electromagnetic fields.


Key Words: neoplasia • cancer • protein tyrosine phosphorylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
EPIDEMIOLOGICAL STUDIES HAVE implicated an association between electromagnetic field (EMF) radiation from residentially proximate power lines and household electrical wiring and the development of B-lineage acute lymphoblastic leukemia (ALL), the most common form of childhood cancer (1 2 3 4 5 6 7) . A study by the Swedish National Institute for Occupational Health reported a threefold increase in the incidence of leukemia in children chronically exposed to power line EMFs (8 , 9) . Other epidemiological studies, however, have failed to demonstrate a correlation between exposure to EMFs and the development of childhood cancers (10) . Despite the putative association between exposure to EMF and the development of childhood leukemia suggested by the positive epidemiological data, the molecular mechanisms underlying the possible development of B-lineage ALL after EMF exposure remain unknown, prompting investigations into the effects of EMF on signaling events in B cells (11 , 12) .

B cell signaling pathways are mediated by the B cell antigen receptor (BCR) expressed on the surface of immunocompetent B cells (reviewed in refs 13 14 15 ). The BCR consists of the membrane immunoglobulin mIgM, which binds specific antigen noncovalently associated with disulfide-linked Ig{alpha}/Igß heterodimers that mediate signal transduction. After engagement of the BCR, the first biochemical signal to be detected is the increased phosphorylation of multiple protein substrates on tyrosine residues. This involves recruitment and/or activation of three families of nonreceptor tyrosine kinases: src family kinases such as Blk, Fyn, and Fgr, the Syk kinase, and the tec family kinases such as Btk. Upon phosphorylation of the immunoreceptor tyrosine-based activation motifs of the Ig{alpha}/Igß heterodimers, the BCR couples to downstream signaling pathways, including activation of phospholipase C{gamma}2 and the Ras/MAP kinase pathway, culminating in transcription factor regulation and the induction of B cell proliferation and differentiation.

EMF exposure of B-lineage cells was reported by Uckun et al. to stimulate up to nine- and threefold increases in Lyn and Syk kinases, respectively, resulting in a marked increase in the tyrosine phosphorylation of multiple electrophoretically distinct substrates and a twofold activation of PKC (11) . By using mutant DT40 cells, the authors concluded that activation of Lyn kinase is sufficient and necessary for EMF-induced cell signaling events in B-lineage lymphoid cells. This study has been followed by two more recent publications by the same authors that give further evidence for the roles of Syk and Btk in the initiation of EMF-induced signal transduction in B cells (12 , 16) .The first of these studies reported that exposure of B cells to EMF results in a tyrosine kinase-dependent activation of PLC{gamma}2 and an increase in inositol phospholipid turnover (12) . Since the signal transduction pathways mediated by protein tyrosine kinases play a critical role in the control of cell proliferation, survival, and differentiation, the authors suggested that the evidence in these three papers supports a sequential activation model by which EMF-induced activation of Lyn kinase could alter the balance of growth regulation in lymphoid cells (12) . This series of studies (11 , 12 , 16) is significant in that it has provided a body of evidence that includes the most robust effects of EMF reported to date. The effects were large and consistent between the studies, and the observations being made in B cells suggest a plausible link between exposure to EMFs and the development of B-lineage ALL. Since these findings may play a key role in contributing to our understanding of the mechanisms underlying biological EMF effects, rigorous replication of the original study (11) on which the latter studies are based is of the utmost importance.

In a recently published study, Miller and Furniss (17) failed to replicate the EMF-induced activation of Btk and inositol phosphate production reported by Uckun et al. (12 , 16) . Here, we report the first study that attempts to replicate the original findings of Uckun et al. Using the human pre-B cell line Nalm-6 (18) and the chicken B cell line DT40 (19 , 20) , as in the original study (11) , we have investigated the effects of exposure to a 100 µT 60 Hz magnetic field on the induction of protein tyrosine phosphorylation and on the activities of Lyn and Syk tyrosine kinases. The details of the experimental protocols used in this study were established with the cooperation of the Uckun laboratory and were followed closely. However, some additional modifications were incorporated in order to ensure as great a degree of control over experimental and EMF exposure conditions as possible. Under the rigorously controlled experimental conditions used in this study to reproduce the field exposure conditions used by Uckun et al., we did not observe any significant effect of a 100 µT 60 Hz magnetic field on the induction of protein tyrosine phosphorylation or on the activities of Lyn and Syk protein tyrosine kinases in B-lineage lymphoid cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cell lines and culture reagents
Nalm-6 human pre-B cells (kindly provided by Professor P. Beverley, Compton, U.K.) were cultured in RPMI 1640 supplemented with 5% fetal calf serum (FCS), 10 mM L-glutamine, and antibiotics, 100 IU/ml penicillin, and 100 µg/ml streptomycin. DT40 cells (kindly provided by Dr. S. Miller (SRI, Menlo Park, Calif.) from a stock culture originally provided by Dr. F. Uckun, Hughes Institute, St. Paul, Minn.) were cultured in RPMI 1640 supplemented with 5% fetal bovine serum (FBS), 1% chicken serum, 10 mM glutamine, 1 mM pyruvate, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Both cell lines were incubated at 37°C, 5% CO2 in a humidified incubator. Cells were maintained in log growth phase at a density between 5 x 105/ml and 1.5 x 106/ml by passaging three times a week. FBS, FCS, chicken serum, penicillin, and streptomycin were from Sigma Co. (St. Louis, Mo.). Pyruvate was from Gibco BRL (Grand Island, N.Y.).

Antibodies and reagents
CD19 monoclonal antibody homoconjugate was kindly provided by Dr. F. Uckun (Hughes Institute). Mouse mAb were specific for chicken IgM (Southern Biotechnology Associates, Birmingham, Ala.), Lyn and Grb-2 (both from Transduction Laboratories, Lexington, Ky.), Syk (4D10) (Santa Cruz Biotechnology, Santa Cruz, Calif.), and phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, N.Y.). Rabbit antibodies used were specific for Lyn (sc-15), Syk (sc-C20) (Santa Cruz Biotechnology), and Bcl-xL (Ab-1) (Calbiochem-Novabiochem Corporation, La Jolla, Calif.). Alkaline phosphatase-conjugated goat anti-mouse Ig was from DAKO (Glostrup, Denmark). Other reagents used were enolase (from rabbit muscle) (Sigma), tubulin (kindly provided by Dr. T. Crabbe, Celltech, U.K.), CDP-Star (Tropix, Bedford, Mass.), and [{gamma}32P] ATP (NEN Life Science Products, Boston, Mass.).

Design of EMF exposure system
The EMF exposure system used for all exposure studies was kindly donated by Dr. Robin Hesketh and his colleagues at the laboratory of Dr. J. Metcalfe (Department of Biochemistry, University of Cambridge, U.K.). With the exception of the lysis buffer injection system, which was designed in our laboratory, and the water-jacketed incubator, which was replaced with a new one of identical design to the original, the system was used as described previously (21) . Briefly, the exposure system consisted of a water-jacketed incubator containing a mu-metal box, within which the residual geomagnetic field was <1 µT and the background AC magnetic field was <5 nT r.m.s. Cells were exposed to EMF by placing each sample of a cell suspension in a 35 mm petri dish (Costar, Cambridge, Mass.) in the center of a circular Helmholtz coil that generated a vertical magnetic field. The electrical signal was provided by a 12 MHz synthesized function generator (Wavetek, San Diego, Calif.) driving an audio amplifier (Sony, Tokyo, Japan), and magnetic fields were measured with a three-axis fluxgate sensor and power supply unit (Bartington, Oxford, U.K.). Cells were exposed either to an AC magnetic field alone or to a combination of AC and DC magnetic fields. Simultaneous experimentation with field-exposed samples, incubated in three energized coils, and control samples, incubated in three nonenergized coils, was achieved by separating the two sets of coils within the incubator with a mu-metal baffle placed vertically within the mu-metal box. At field strengths up to 100 µT, the background AC magnetic field detected at control coils was <50 nT. Computer monitoring allowed continuous logging of EMF exposure conditions and temperature throughout experiments. The lysis buffer injection system designed in our laboratory was used to introduce lysis buffer into the petri dishes via an inlet at the top of the incubator thus allowing experiments to be terminated by cell lysis in preparation for biochemical analysis without the need to open the incubator door. This prevented any possible perturbations in EMF exposure conditions. The design of the EMF exposure system also allowed experiments to be conducted in a ‘blinded’ fashion. The connections supplying current to the Helmholtz coils could be altered so as to activate either of the two sets of coils. The individual responsible for the biochemical analysis was unaware of the magnetic field exposure conditions, and the identity of the field-exposed and control samples was kept concealed until data analysis had been completed. The individual responsible for randomization of the exposure conditions ensured that an equal number of experiments were carried out in which cells were exposed to EMF on the right- and left-hand sides of the incubator in order to eliminate any bias within the incubator itself.

EMF exposure and preparation of samples for biochemical analysis
Protocols for cell culture and biochemical analysis were defined with the assistance of the original investigators during a visit to Dr. Uckun’s laboratory with the aim of reproducing the assays and field exposure conditions as closely as possible. Cells in log growth phase were washed three times in serum-free 50 mM HEPES-buffered RPMI 1640 and resuspended at 1 x 108 cells/800 µl. They were then aliquotted at 800 µl/petri dish and preincubated in the EMF exposure system in a low EMF environment for 1 h prior to exposure. In experiments where cells were to be exposed to the combination of AC and DC magnetic fields, all cells were preincubated for an additional 45 min in the presence of a 46 µT DC magnetic field alone. During each experiment, cells were exposed either to a 100 µT 60 Hz AC magnetic field alone or to the combination of this field with a 46 µT DC magnetic field, chosen to mimic the geomagnetic field, applied in parallel. Cells were exposed for the times shown. Whole cell lysates were then prepared from triplicate control and field-exposed samples by the addition of concentrated Nonidet P-40 lysis buffer (200 µl/petri dish) prior to opening the incubator door. Lysis buffer contained a final concentration of 1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA and 10 mM EDTA, supplemented with phosphatase inhibitors, 20 mM Na4P2O7, 1 mM Na3VO4, and 50 mM NaF and protease inhibitors 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM (4-(2-aminoethyl)-benzenesulfonylfluoride). Cell lysates were then incubated on ice for 10 min, centrifuged at 10,000 g for 10 min at 4°C, and aliquotted for biochemical analysis. Alongside each EMF experiment, whole cell lysates prepared from NALM-6 cells stimulated with either CD19 monoclonal homoconjugate reagent (1 µg/ml) or the protein tyrosine phosphatase inhibitor, pervanadate, or from DT40 cells stimulated with either anti-IgM (10 µg/ml) or pervanadate, were used as positive controls for the induction of protein tyrosine phosphorylation. Stimulations were carried out for 1 min with antibodies or for 10 min with pervanadate in a final volume of 800 µl. Reactions were terminated by the addition of 200 µl concentrated lysis buffer to give a final concentration of 1% Nonidet P-40. Pervanadate was freshly prepared prior to use by adding 25 µl of 0.2 M Na3VO4 to 500 µl of 3.4 mM H2O2. This mixture was incubated in a foil-wrapped Eppendorf tube at room temperature for 15 min, and 3 µl catalase was added 30 s before the addition of 100 µl to each cell suspension.

Anti-phosphotyrosine immunoblot analysis
Whole cell lysate proteins (5x106 cell equivalents) were resolved on 10.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels and electroblotted onto 0.45 µm Immobilon-PVDF membranes (Millipore, Bedford, Mass.) using a Semi-phore transfer unit (Hoeffer Scientific Instruments, San Francisco, Calif.). They were then immunoblotted either for phosphotyrosine using the 4G10 mAb or for Grb-2 in order to normalize phosphotyrosine signals for protein loading. Proteins were detected using alkaline phosphatase-conjugated goat anti-mouse IgG and were visualized by fluorography using a chemiluminescence detection system, CDP-Star (Tropix). Immunoblots were analyzed first by exposure to X-ray film and then by using a PhosphorImager (Bio-Rad Laboratories, Hercules, Calif.) for quantitation of appropriate bands using the Molecular Analyst software package (Bio-Rad Laboratories). Tyrosine phosphorylation was quantitated by analyzing total bands in each lane on the immunoblot. Results did not materially differ when individual bands were chosen for comparison.

Immunoprecipitations and immune complex kinase assays
Whole cell lysates (5x107 cell equivalents/sample) were precleared with Omnisorb (Calbiochem) (100 µl/500 µl lysate) for 30 min at 4°C. Antibodies specific for Lyn (sc-15), Syk (sc-C20), or Bcl-xL (Ab-1), used as an isotype control, were coupled to protein G Sepharose 4 Fast Flow beads (Pharmacia Biotech, Uppsala, Sweden) (10 µl antibody/20 µl beads). Lyn, Syk, or Bcl-xL were immunoprecipitated by incubation of lysates with the antibody-coupled beads (20 µl beads/500 µl lysate) for 1 h at 4°C, and immune complexes were then washed three times with 1% Nonidet P-40 lysis buffer and once with kinase buffer [25 mM HEPES, pH 7.4, 25 mM MgCl2, 2.5 mM MnCl2, 2 mM dithiothreitol (DTT), and 0.1 mM Na3VO4 for Lyn or 20 mM HEPES, pH 7.4, 3 mM MnCl2, and 0.05% Brij-35 for Syk]. Immune complex kinase assays were performed for 20 min at room temperature in 20 µl kinase buffer supplemented with 5 µCi [{gamma}32P]ATP (3000 Ci/mmol) and either 5 µM ATP and 3 µg enolase for Lyn or 1 µm ATP and 80 ng tubulin for Syk. Kinase reactions were terminated by the addition of 10 µl hot 3x SDS-polyacrylamide gel electrophoresis sample buffer (containing Tris-HCl, pH 6.8, final concentrations of 2% SDS, 10% glycerol, and 100 mM DTT), and samples were boiled for 5 min. Immunoprecipitates were electroblotted as described above. Kinase assays were analyzed by autoradiography, first by exposing the membranes to film (BioMax MS-1 film) (Eastman Kodak Company, Rochester, N.Y.) and then by using the PhosphorImager for quantitation. Protein levels of Lyn or Syk were detected on the same membrane by chemiluminescence and quantitated by PhosphorImager analysis. Kinase values were normalized according to the amount of kinase protein measured in each immunoprecipitate.

Statistical analysis
Statistical analysis of log to the base 10 of the density values generated by PhosphorImager analysis (which were linear in relation to increasing signal intensities) was performed by ‘randomized block analysis’ with and without log protein as a covariate using the statistical package ‘Genstat’. The results shown in Tables 1 2 3 are for these log transformed values with the covariance adjustment, although this adjustment did not significantly change the conclusions. Differences between control and field-exposed samples were accepted as significant at P < 0.05.


View this table:
[in this window]
[in a new window]
  		Table 1. No significant effect of a 100 µT 60 Hz AC magnetic field on protein tyrosine phosphorylation in Nalm-6 or DT40 cellsa


View this table:
[in this window]
[in a new window]
  		Table 2. No significant effect of the combination of 100 µT AC and 46 µT DC magnetic fields on protein tyrosine phosphorylation in Nalm-6 or DT40 B cellsa


View this table:
[in this window]
[in a new window]
  		Table 3. No significant effect of the combination of 100 µT AC and 46 µT DC magnetic fields on the activities of Lyn and Syk kinases in Nalm-6 cellsa


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
No effect of a 100 µT 60 Hz AC magnetic field on protein tyrosine phosphorylation in B-lineage lymphoid cells
In our initial study, the effect of EMF exposure on protein tyrosine phosphorylation in B-lineage cells was investigated by exposing either Nalm-6 or DT40 B cells to a 100 µT 60 Hz magnetic field for a fixed period. Nalm-6 cells were exposed for 10 min and DT40 cells were exposed for 1 min exposure times indicated by the results of Uckun et al. to be optimal for the induction of tyrosine phosphorylation in these cells (11) . Alongside each EMF experiment, positive controls for the induction of tyrosine phosphorylation were generated in order to demonstrate the normal functioning of the B cell signaling machinery. Nalm-6 cells were stimulated either with the tyrosine phosphatase inhibitor pervanadate or with a CD19 monoclonal antibody homoconjugate, which contains the minimal binding sites for the B cell coreceptor CD19, whereas DT40 cells were stimulated either with pervanadate or with anti-IgM. A series of nine experiments were performed for each cell line; of these, some were carried out in a ‘blinded’ fashion: seven experiments for Nalm-6 and five for DT40. In these experiments the identity of control and field-exposed samples was kept concealed until biochemical analysis had been completed. Results from a representative experiment using DT40 cells are shown in Fig. 1 , although similar results were obtained using Nalm-6 cells (data not shown). A summary of the results obtained in this series of experiments (Table 1 ) shows that, in contrast with the findings of Uckun et al., there was no significant effect of a 100 µT 60 Hz AC magnetic field on protein tyrosine phosphorylation after exposure of Nalm-6 cells for 10 min or DT40 cells for 1 min. Similar results were obtained after exposure of Nalm-6 cells under the same EMF conditions for either 1 min or 20 min (data not shown). Comparison of data from the different experiments in this series showed that similar results were obtained whether experiments were performed in a ‘blinded’ fashion or not.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 1. EMF exposure does not induce protein tyrosine phosphorylation in DT40 B cells. DT40 B cells were either incubated in nonenergized coils (controls) or exposed to a 100 µT 60 Hz AC magnetic field for 1 min, and whole cell lysates were prepared from triplicate control (lanes 4–6) or field-exposed (lanes 1–3) samples by the addition of lysis buffer via the lysis buffer injection system prior to opening the incubator door. Positive controls were generated by preparing whole cell lysates from DT40 cells that had been stimulated with anti-IgM (10 µg/ml) for 1 min. Lysates were immunoblotted using antibodies for phosphotyrosine (4G10) or Grb-2 as described in Materials and Methods.

The experimental protocol used for the experiments summarized in Table 1 incorporated a preincubation period of 1 h prior to EMF exposure as compared with at least 2 h in the protocol previously used (11) . However, an investigation into the effect of the length of this preincubation period showed that similar results were obtained whether cells were preincubated for 1 or 2 h. We also made the decision to omit the 30 s–1 min immersion of samples in ice water after EMF exposure as described in the Uckun study (11) . Tyrosine phosphatases may be more sensitive to incubation at 4°C than tyrosine kinases, leading to a possible nonspecific increase in protein tyrosine phosphorylation on cooling. In our hands, a 5 min incubation of unstimulated cells did not influence the outcome of EMF experiments (data not shown), and therefore does not seem to have contributed to the increased tyrosine phosphorylation detected in the Uckun study. Nevertheless, we omitted this step in order to avoid any possibility of artifactual activation of B cells during EMF experiments.

No effect of the combination of 100 µT AC and 46 µT DC magnetic fields on protein tyrosine phosphorylation in B-lineage lymphoid cells
Our initial study (Table 1) involved exposure of B cells to a 100 µT 60 Hz AC magnetic field in the absence of the static geomagnetic field, since experiments were protected from this field by the mu-metal box within which the experiments were carried out. However, since the experiments reported by Uckun et al. were performed in the presence of the static geomagnetic field, a second study was carried out in order to determine whether the combination of the 100 µT AC magnetic field and a static DC magnetic field, equivalent in magnitude and direction to the geomagnetic field, was critical to the induction of protein tyrosine phosphorylation in Nalm-6 and DT40 B cells. Experiments were carried out as in the first study except that cells were exposed to the combination of a 100 µT 60 Hz AC magnetic field and a 46 µT DC magnetic field applied in parallel. In these experiments, control samples were exposed solely to the DC magnetic field (as described in Materials and Methods). Four experiments were performed for each cell line, and the results (Fig. 2 and Table 2 ) show that there was no significant effect of the combination of the AC and DC magnetic fields on protein tyrosine phosphorylation after exposure of Nalm-6 cells or DT40 cells for 10 min or 1 min, respectively.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 2. Protein tyrosine phosphorylation is not induced in DT40 B cells exposed to a combination of AC and DC magnetic fields. DT40 cells were exposed for 1 min either to a 100 µT 60 Hz AC magnetic field in combination with a 46 µT DC magnetic field, applied in parallel (‘EMF’, lanes 1–3), or to a 46 µT DC magnetic field alone (control, lanes 4–6). After EMF exposure, whole cell lysates were prepared from control or field-exposed samples and these were subject to immunoblot analysis for phosphotyrosine and Grb-2 as described in Materials and Methods.

Lyn and Syk kinases are not activated after exposure of Nalm-6 cells to the combination of 100 µT AC and 46 µT DC magnetic fields
In our final study, in order to investigate more directly the effects of EMF exposure on the activation of B cell protein tyrosine kinases, we assessed the effects of EMF exposure on the activities of Lyn and Syk kinases. Nalm-6 cells were exposed to the combination of 100 µT AC and 46 µT DC fields for 10 min, and the activities of Lyn and Syk kinases were determined by immune complex kinase assays using enolase and tubulin, respectively, as exogenous substrates. In the original study (11) , kinase activity and protein loading were determined from separate blots. However, in the current study, in order to implement rigorous control for differences in protein loading, kinase activities were normalized for amounts of Lyn or Syk protein measured on the same blot. Figure 3 and Table 3 show that, under the experimental conditions used, there was no significant effect of EMF exposure on the activity of either kinase.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Lyn and Syk kinases are not activated after exposure of Nalm-6 cells to EMF. Nalm-6 cells were exposed either to the combination of 100 µT AC and 46 µT DC magnetic fields (‘EMF’, lanes 1–3) or to a 46 µT DC magnetic field alone (controls, lanes 4–6) for 10 min. Whole cell lysates were prepared by the injection of lysis buffer via the lysis buffer injection system prior to opening the incubator door and immune complex assays were performed as described in Materials and Methods. Autoradiograms of kinase assays and immunoblots of the same membranes probed with antibodies for A) Lyn and B) Syk are shown. Anti-Bcl-xL was used as a matched isotype control mAb for immunoprecipitations.

In summary, we examined the effect of a 100 µT 60 Hz AC magnetic field on tyrosine phosphorylation and on the activities of Lyn and Syk tyrosine kinases in B-lineage lymphoid cells in the first reported attempt to reproduce the findings of a study by Uckun et al. (11) . The protocols used were established with the cooperation of the Uckun laboratory, although some modifications were incorporated in order to exert greater control of the EMF exposure conditions. Under the rigorously controlled conditions applied in our experiments, however, in contrast with the findings of Uckun et al., we did not detect any significant effect of EMF exposure on tyrosine phosphorylation or on the activities of Lyn and Syk kinases in Nalm-6 or DT40 B cells. Therefore, we conclude that low-energy EMFs do not activate B cells.

The reasons for the differences in the observations made between the two laboratories is not immediately apparent. Although the Nalm-6 cells used in our study were obtained from a different source to that used by the original investigators, this is unlikely to be the explanation, particularly since the DT40 cells used were from the same source and responded normally to anti-IgM. It seems reasonable to expect that the marked stimulation of the B cell signaling events reported, which were of the order of magnitude induced by immunological reagents (11) , should be reproducible in cells of the same cell line from different sources. Furthermore, we took particular care to reproduce the exposure conditions of the original study. For example, since the mu-metal box incorporated into our exposure system shielded experimental samples from the static geomagnetic field, we mimicked this field by applying a 46 µT DC magnetic field in parallel to the 100 µT AC magnetic field. It is thought that the resonance conditions created by the superimposition of AC and DC magnetic fields may be critical to the induction of biological effects (22) , although this is still a matter of controversy (23) . However, the combined field effects did not appear to be critical in the present work, since no significant differences were noted when B cells were exposed to the combination of the two fields in parallel. A trend to increased tyrosine phosphorylation and tyrosine kinase activities on EMF exposure in Nalm-6 cells failed to reach the significance level (Tables 2 and 3) . In the original study by Uckun et al. (11) , no statistical analysis was reported. However, the reported increase in Lyn kinase activity on EMF exposure in this previous study was > ninefold and the marked increases in tyrosine phosphorylation were similar to those observed on CD19 ligation (11) . It should be emphasized that in the present study the trend toward increased tyrosine phosphorylation in EMF-exposed Nalm-6 cells not only failed to reach statistical significance, but reflected differences that were trivial in biological terms (viz. increases of 16–32%) in comparison with these previously reported values.

Our findings are consistent with a recently published study in which the reported EMF-induced activation of Btk and inositol phosphate production in DT40 cells (12) could not be replicated (17) . It remains possible that the stimulation of Lyn and Syk kinase activities and protein tyrosine phosphorylation may be induced by certain EMF exposure conditions not investigated in the current study or that any changes induced under the conditions used were too small to be detected by the analytical techniques used. However, this study highlights the necessity for establishing experimental models of biological EMF effects that can be readily reproduced in independent laboratories so that the mechanisms underlying such putative effects can be satisfactorily investigated.


   ACKNOWLEDGMENTS
 
We thank Dr. F. Uckun and his colleagues for arranging a visit to their laboratory and for advice concerning details of experimental protocols. We would also like to thank Dr. A. Lacy-Hulbert for his help in setting up the EMF exposure system and Dr. Steven Miller for his advice. We are indebted to the EMF Biological Research Trust for their financial support.

Received for publication March 16, 2000. Revision received April 27, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Tomenius, L. (1986) 50-Hz electromagnetic environment and the incidence of childhood tumors in Stockholm County. Bioelectromagnetics 7,191-207[Medline]
  2. Savitz, D. A., Wachtel, H., Barnes, F. A., John, E. M., Tvrdik, J. G. (1988) Case-control study of childhood cancer and exposure to 60-Hz magnetic fields. Am. J. Epidemiol. 128,21-38[Abstract/Free Full Text]
  3. Myers, A., Clayden, A. D., Cartwright, R. A., Cartwright, S. C. (1990) Childhood cancer and overhead powerlines—a case control study. Br. J. Cancer 62,1008-1014[Medline]
  4. London, S. J., Thomas, D. C., Bowman, J. D., Sobel, E., Cheng, T. C., Peters, J. M. (1991) Exposure to residential electric and magnetic fields and risk of childhood leukemia. Am. J. Epidemiol. 134,923-937[Abstract/Free Full Text]
  5. Olsen, J. H., Nielsen, A., Schulgen, G. (1993) Residence near high-voltage facilities and risk of cancer in children. Br. Med. J. 307,891-895
  6. Verkasalo, P. K., Pukkala, E., Hongisto, M. Y., Valjus, J. E., Jarvinen, P., Heikkila, K. V., Koskenvuo, M. (1993) Risk of cancer in Finnish children living close to powerlines. Br. Med. J. 307,895-899
  7. Feychting, M., Schulgen, G., Olsen, J. H., Ahlbom, A. (1995) Magnetic fields and childhood cancer—a pooled analysis of two Scandinavian studies. Eur. J. Cancer 31A,2035-2039
  8. Feychting, M., Ahlbom, A. (1992) Swedish National Institute for Occupational Health, IMM-rapport 6/92 Swedish National Institute for Occupational Health Stockholm.
  9. Feychting, M., Ahlbom, A. (1993) Magnetic fields and cancer in children residing near Swedish high-voltage power-lines. Am. J. Epidemiol. 138,467-481[Abstract/Free Full Text]
  10. Cheng, K. K., Day, N., Cartwright, R., Craft, A., Birch, J. M., en, O. B., McKinney, P. A., Peto, J., Beral, V., Roman, E., Elwood, P., Alexander, F. E., Chilvers, C. E. D., Doll, R., Taylor, C. M., Greaves, M., Goodhead, D., Fry, F. A., Adams, G., Allen, S. G., Maslanyj, M. P., Mee, T. J., Gilman, E., Skinner, J., Williams, D., Deacon, J., Peto, J., Beral, V., Roman, E., Elwood, P., Alexander, F. E., Mott, M., Muir, K., Law, G., Simpson, J. (1999) Exposure to power-frequency magnetic fields and the risk of childhood cancer. Lancet 354,1925-1931[Medline]
  11. Uckun, F. M., Kurosaki, T., Jin, J., Jun, X., Morgan, A., Takata, M., Bolen, J., Luben, R. (1995) Exposure of B-lineage lymphoid-cells to low-energy electromagnetic fields stimulates Lyn kinase. J. Biol. Chem. 270,27666-27670[Abstract/Free Full Text]
  12. Dibirdik, I., Kristupaitis, D., Kurosaki, T., TuelAhlgren, L., Chu, A., Pond, D., Tuong, D., Luben, R., Uckun, F. M. (1998) Stimulation of Src family protein-tyrosine kinases as a proximal and mandatory step for SYK kinase-dependent phospholipase C gamma 2 activation in lymphoma B cells exposed to low energy electromagnetic fields. J. Biol. Chem. 273,4035-4039[Abstract/Free Full Text]
  13. DeFranco, A. L. (1997) The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9,296-308[Medline]
  14. Kurosaki, T. (1999) Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17,555-592[Medline]
  15. Benschop, R. J., Cambier, J. C. (1999) B cell development: signal transduction by antigen receptors and their surrogates. Curr. Opin. Immunol. 11,143-151[Medline]
  16. Kristupaitis, D., Dibirdik, I., Vassilev, A., Mahajan, S., Kurosaki, T., Chu, A., TuelAhlgren, L., Tuong, D., Pond, D., Luben, R., Uckun, F. M. (1998) Electromagnetic field-induced stimulation of Bruton’s tyrosine kinase. J. Biol. Chem. 273,12397-12401[Abstract/Free Full Text]
  17. Miller, S. C., Furniss, M. J. (1998) Bruton’s tyrosine kinase activity and inositol 1,4,5-trisphosphate production are not altered in DT40 lymphoma B cells exposed to power line frequency magnetic fields. J. Biol. Chem. 273,32618-32626[Abstract/Free Full Text]
  18. Uckun, F. M., Burkhardt, A. L., Jarvis, L., Jun, X., Stealey, B., Dibirdik, I., Myers, D. E., Tuelahlgren, L., Bolen, J. B. (1993) Signal-Transduction Through the CD19 receptor during discrete developmental stages of human B-Cell ontogeny. J. Biol. Chem. 268,21172-21184[Abstract/Free Full Text]
  19. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H., Kurosaki, T. (1994) Tyrosine kinases Lyn and Syk regulate B-cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J 13,1341-1349[Medline]
  20. Kurosaki, T., Takata, M., Yamanashi, Y., Inazu, T., Taniguchi, T., Yamamoto, T., Yamamura, H. (1994) Syk activation by the Src-family tyrosine kinase in the B-cell receptor signaling. J. Exp. Med. 179,1725-1729[Abstract/Free Full Text]
  21. Lacy-Hulbert, A., Wilkins, R. C., Hesketh, T. R., Metcalfe, J. C. (1995) No effect of 60 Hz electromagnetic fields on Myc or beta-actin expression in human leukemic cells. Radiation Res 144,9-17[Medline]
  22. Lednev, V. V. (1991) Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics 12,71-75[Medline]
  23. Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. (1998) Biological responses to electromagnetic fields. FASEB J 12,395-420[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WOODS, M.
Right arrow Articles by ALEXANDER, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WOODS, M.
Right arrow Articles by ALEXANDER, D. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS