(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
,
DAVID BROWN
and
DENIS R. ALEXANDER*1
* Laboratory of Lymphocyte Signalling and Development,
Laboratory of Molecular Signalling,
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
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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
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INTRODUCTION
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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
/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/Igß heterodimers, the BCR couples to downstream
signaling pathways, including activation of phospholipase C
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 PLC2 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.
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MATERIALS AND METHODS
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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
[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
[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.
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Table 1. No significant effect of a 100 µT 60 Hz AC magnetic field on protein
tyrosine phosphorylation in Nalm-6 or DT40 cellsa
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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
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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
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RESULTS AND DISCUSSION
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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.
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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.
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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.
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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.
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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.
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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.
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|
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.
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ACKNOWLEDGMENTS
|
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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.
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M. J Crumpton
The Bernal Lecture 2004 Are low-frequency electromagnetic fields a health hazard?
Phil Trans R Soc B,
June 29, 2005;
360(1458):
1223 - 1230.
[Abstract]
[Full Text]
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ABSTRACT
INTRODUCTION
