Here we present evidence that exposure of DT40
lymphoma B-cells to low energy electromagnetic fields (EMF) results in
activation of phospholipase C-
2 (PLC-
2), leading to increased
inositol phospholipid turnover. PLC-
2 activation in EMF-stimulated
cells is mediated by stimulation of the Bruton's tyrosine kinase
(BTK), a member of the Src-related TEC family of protein tyrosine
kinases, which acts downstream of LYN kinase and upstream of PLC-
2.
B-cells rendered BTK-deficient by targeted disruption of the
btk gene did not show enhanced PLC-
2 activation in
response to EMF exposure. Introduction of the wild-type (but not a
kinase domain mutant) human btk gene into BTK-deficient
B-cells restored their EMF responsiveness. Thus, BTK exerts a pivotal
and mandatory function in initiation of EMF-induced signaling cascades
in B-cells.
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INTRODUCTION |
A number of epidemiologic studies suggested the possibility that
EMF1 radiation from
residentially proximate power lines, household electrical wiring, and
appliance usage may contribute to the risk of childhood acute
lymphoblastic leukemia (1-5). A recent study by Linet et
al. (6) showed that living in homes characterized by high measured
time-weighted average magnetic field levels or by the highest wire code
category does not increase the risk of acute lymphoblastic leukemia in
children. However, concerns regarding other forms of EMF exposure
remain. Since no directly genotoxic effects are exerted by EMF, it is
thought that EMF may participate in leukemogenesis of childhood acute
lymphoblastic leukemia by influencing their proliferation, survival,
and/or differentiation programs (7-9).
In a recent study, we discovered that exposure of B-lineage lymphoid
cells to low energy EMF stimulates the Src protooncogene family protein
tyrosine kinase, LYN, leading to downstream activation of protein
kinase C (10). These results prompted the hypothesis that a delicate
growth regulatory balance might be altered in B-lineage lymphoid cells
by EMF-induced activation of LYN kinase. In a subsequent study, we
examined the molecular mechanism of enhanced inositol phospholipid
turnover in lymphoma B-cells exposed to low energy EMF (11). Our
findings were consistent with a sequential activation model according
to which EMF exposure first leads to activation of Src family protein
tyrosine kinases. Src family protein tyrosine kinases domains interact
with and phosphorylate as yet unidentified immunoreceptor
tyrosine-based activation motifs, leading to recruitment of spleen
tyrosine kinase (SYK) as well as PLC-
2 via their Src
homology (SH2) domains to the phosphorylated immunoreceptor
tyrosine-based activation motifs. Subsequently, SYK is activated by Src
family protein tyrosine kinases and phosphorylates PLC-
2 leading to
its activation. Activation of PLC-
2 results in increased inositol
phospholipid turnover, production of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) and protein kinase C activation (11).
BTK is a member of the Src-related TEC family of PTKs (12, 13), and its
enzymatic activity is regulated by LYN kinase (14, 15). Mutations in
the btk gene have been linked to severe developmental blocks
in human B-cell ontogeny, resulting in human X-linked
agammaglobulinemia (16-18). BTK was also identified as the mediator of
apoptosis in B-lineage lymphoid cells exposed to ionizing radiation
(19). Recent evidence suggests an important role for BTK in the
regulation of PLC-
2 activity level (20). The concerted actions of
both BTK and SYK are required for the B-cell antigen receptor-induced
PLC-
2 activation (20). Therefore, we decided to examine the
potential participation of BTK in EMF-induced activation of PLC-
2.
Here, we show that low energy EMF exposure initiates a biochemical
signaling cascade intimately linked to BTK. Our study provides
unprecedented experimental evidence that BTK is the mediator of
EMF-induced enhanced inositol phospholipid turnover in lymphoma
B-cells.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
The establishment and characterization of
wild-type and BTK-deficient, LYN-deficient, and SYK-deficient clones of
DT40 chicken lymphoma B-cells were reported previously (19-25).
Wild-type and mutant cells were maintained in suspension cultures at
37 °C, 5% CO2 in a humidified incubator. The culture
medium was RPMI 1640 (Life Technologies, Inc.), supplemented with 10%
fetal calf serum, 2.5% chicken serum, 10 mM
L-glutamine, and 50 mM 2-mercaptoethanol as
described previously (11, 19).
EMF Exposure--
A homogeneous vertical magnetic field was set
up by using a Merritt's coil-based in vitro low frequency
EMF exposure system (11). Merritt's four square coil system is known
to produce a large volume of uniform magnetic field. Unless otherwise
indicated, the applied vertical sinusoidal 60-Hz field was 0.1 millitesla (1 gauss). The current needed to obtain 1 gauss was 0.7 A. The magnetic field was parallel to the coil axis and was uniform near the axis and the center of the coil system. Cells were maintained at
all times in a low AC (8 milligauss) environment except during a single
centrifugation step. This was achieved by using a 2-pole motor tissue
incubator (CEDCO model IRE 93) with low AC fields for routine cell
culture and by defining the lowest field regions within the incubator.
Exponentially growing cells (5 × 106 cells/ml) in
serum-free medium in 1.5-ml capacity microcentrifuge tubes were exposed
to the 1-gauss, 60-Hz EMF by placing the tubes at the center of the
four-coil field generator contained in the incubator with shielding
sheets of metal alloy at the bottom of the chambers. Control tubes were
simultaneously placed inside a duplicate incubator without the exposure
apparatus. To measure the fields in the incubators, the laminar flow
hood, centrifuge, and nearby areas, a gaussmeter (MAG model 25, Magnetic Sciences International) was used. EMF strength was constantly
monitored with the gaussmeter and adjusted manually if needed. In all
experiments, the coils were activated before the cells had been placed
in, to avoid fluctuations of the EMF during turning on the apparatus. The coils were turned off only after the cells were taken out of the
exposure system.
Immune Complex Kinase Assays of BTK--
To evaluate the effects
of EMF on the enzymatic activity of BTK, exponentially growing cells
were exposed to EMF and, at the indicated time points after EMF
exposure, the test tubes were immediately immersed in ice water for
30 s to 1 min. Cells were spun down at 3,000 × g
for 5 min at 0 °C and lysed in a 1% Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA) containing 1 mM
Na3VO4 and 1 mM sodium molybdate as
phosphatase inhibitors, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and
1 mM phenylmethylsulfonyl fluoride as protease inhibitors. Lysates were spun twice at 12,000 × g for 10 min at
4 °C prior to immunoprecipitation. 500-µg samples of the cell
lysates were immunoprecipitated with a polyclonal rabbit anti-BTK
antibody (3 µl/500 µg of lysate) and immune complex kinase assays
(26-29), as well as anti-BTK Western blot analyses, were performed as
described (14, 19). All BTK kinase and Western blot autoradiograms were subjected to densitometric scanning using an automated AMBIS system (Automated Microbiology System, Inc., San Diego, CA), and for each time
point a stimulation index was determined by comparing the density
ratios of the kinase and protein bands to those of the base-line sample
and using the formula: stimulation index (SI) = (density of kinase
band/density of BTK protein band)test sample:(density of
kinase band/density of BTK protein band)base-line control
sample. The expression levels of PLC-
2 and actin in whole
cell lysates of wild-type and mutant DT40 B-cells were examined by
Western blot analysis using an enhanced chemiluminescence (ECL)
detection system (Amersham Pharmacia Biotech) as reported previously
(11).
Analysis of Stimulation of Inositol Phospholipid
Turnover--
Ice-cold perchloric acid (20%) was added after EMF
exposure to the cell suspensions to stop further reaction. The test
tubes were kept on ice for 20 min and then sedimented at 2,000 × g for 15 min at 4 °C. The supernatant was collected and
the pH was neutralized to 7.5 with ice-cold 10 mM KOH and
centrifuged again. The supernatant was then collected and stored at
20 °C for subsequent measurement of Ins-1,4,5-P3
levels.
D-myo-[3H]inositol-1,4,5-trisphosphate
assay system purchased from Amersham Pharmacia Biotech was used to
measure Ins-1,4,5-P3 levels as reported (11, 26, 28, 29).
This highly sensitive assay is based on the competition between
unlabeled Ins-1,4,5-P3 in the cellular extracts and a fixed
quantity of a high specific activity
[3H]Ins-1,4,5-P3 tracer for a limited number
of binding sites on a bovine adrenal binding protein specific and
sensitive to Ins-1,4,5-P3 (26, 28, 29).
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RESULTS AND DISCUSSION |
Activation of BTK in DT40 Lymphoma B-cells Exposed to Low Energy
Electromagnetic Fields--
LYN kinase plays a pivotal role in
ligand-induced signal transduction events in B-lineage lymphoid cells
and is thought to mediate its downstream effects (e.g.
activation of PLC-
2 and inositol phospholipid turnover) by
activating the tyrosine kinases SYK and BTK (13-15, 20, 22, 24). We
previously reported that exposure of B-lineage lymphoid cells to low
energy EMF stimulates the protein tyrosine kinase LYN, and activation
of LYN kinase was sufficient and mandatory for EMF-induced tyrosine
phosphorylation in B-lineage lymphoid cells (10). To further elucidate
the EMF-induced signal transduction events in B-lineage lymphoid cells,
we decided to examine the enzymatic activity of BTK in DT40 lymphoma
B-cells after EMF exposure using immune complex kinase assays. We first exposed DT40 lymphoma B-cells to low energy EMF at a constant frequency
(60 Hz), but increasing intensities (1-10 gauss), and examined the
enzymatic activity of BTK at various time points after the initiate of
the EMF exposure. As shown in Fig. 1,
60-Hz EMF exposure at a field intensity of 1 gauss or 3 gauss induced rapid activation of BTK (p77BTK) kinase activity, as
reflected by increased autophosphorylation. This rapid activation of
BTK was observed in four consecutive independent experiments. The
magnitude of activation was less at higher field intensities.
Therefore, a field intensity of 1 gauss was used in subsequent
experiments.

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Fig. 1.
EMF-induced BTK activation in DT40 lymphoma
B-cells. DT40 cells were exposed to 60-Hz EMF at field intensities
of 1 gauss (G), 3G, 5G, 7G, or 10 G. Controls
(CON) were not exposed to EMF. At 10 and 30 min, EMF
exposure was stopped with a 1% Nonidet P-40 lysis buffer. The lysates
were immunoprecipitated with a polyclonal anti-BTK antibody and then
subjected to immune complex kinase assays (see "Experimental
Procedures"). The radioactivity (in counts/min) of the
autophosphorylated BTK bands was determined as a measure of the BTK
enzymatic activity.
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Different domains of BTK are important for its physiologic functions
(13, 30). BTK has a pleckstrin homology (PH) domain, a TEC homology
domain, a single SH3 domain, a single SH2 domain, and a catalytic
kinase domain (12, 13, 17, 30). Mutations in the SH2 domain as well as
PH domain of the BTK result in defective B-cell development, leading to
human X-linked agammaglobulinemia (16, 31-34). The PH domain is
responsible for interactions with various isoforms of protein kinase
C,
subunits of heterotrimeric GTP-binding proteins and
phosphatidylinositol-4,5-bisphosphate, the precursor to
Ins-1,4,5-P3 (35-38). The SH3 domain is responsible for
interactions with proline-rich sequences such as TEC domains, whereas
the SH2 domain facilitates the interactions with
tyrosine-phosphorylated proteins (13, 38). It has been shown that the
SH2 and PH domains of BTK are required for the activation of PLC-
2
in B-cell antigen receptor-mediated B-cell activation (20), but the
same domains are not essential for the activation of BTK induced by
ionizing radiation (19).
Therefore, we decided to determine if the SH2 and PH domains of BTK are
required for its activation following EMF exposure. Exposure of control
wild-type DT40 cells to EMF resulted in a time-dependent
activation of BTK, as measured by enhanced autophosphorylation. While
the autophosphorylation showed a 7.9-fold increase at 30 min by
densitometric scanning of the autoradiogram, the abundance of the
enzyme, as determined by anti-BTK Western blot analysis, showed only a
6% increase during the course of the experiment, suggesting altered
specific activity. The BTK-protein adjusted stimulation indices (SI)
were 1.9 at 2.5 min, 4.6 at 5 min, 6.4 at 15 min, and 7.4 at 30 min. As
shown in Fig. 2, A and
B, the magnitude and time course of BTK activation in
BTK-deficient DT40 cells reconstituted with the wild-type human
btk gene were very similar to those of wild-type DT40 cells
(BTK-protein adjusted SI at 30 min = 9.6). By comparison,
introduction of a SH2 domain-mutant or a PH domain-mutant human
btk gene resulted in substantially attenuated EMF responses,
as evidenced by the 3-4-fold lower maximum SI than in wild-type DT40
cells, suggesting important roles for the SH2 and PH domains in BTK
activation after EMF exposure (Fig. 2, C and
D).

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Fig. 2.
EMF-induced BTK activation of BTK-deficient
DT40 cells reconstituted with wild-type or mutant forms of the human
btk gene. DT40 cells were exposed to 1-gauss low
frequency EMF, lysed, and immunoprecipitated with the anti-BTK
polyclonal antibody as described under "Experimental Procedures."
The immune complexes were subjected to the immune complex kinase assays
and anti-BTK Western blot analyses. Autoradiograms of the kinase assay
(KA) and Western blots (WB) using anti-BTK
antibody are shown in each panel. A, wild-type
(WT) DT40 cells. BTK-deficient DT40 cells were reconstituted
with wild-type human btk gene
(BTK ,rBTK(WT)) (B) or a
mutation in the SH2 domain
(BTK ,rBTK(mSH2)
(C) or in the PH domain
(BTK ,rBTK(mPH)) (D). The
establishment and characterization of these DT40 clones were described
previously in detail (19, 20).
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As expected, no BTK bands were detected in immune complex kinase assays
or anti-BTK Western blots of DT40 cells, which were rendered
BTK-deficient through targeted disruption of the btk gene by
homologous recombination knockout, and were used as a negative control
(Fig. 3A). Targeted disruption
of the lyn gene abolished the activation of BTK after EMF
exposure, indicating that LYN kinase acts upstream of BTK in the
EMF-induced signaling cascade (Fig. 3B). By comparison,
targeted disruption of the syk gene did not abolish the BTK
activation. However, the magnitude of the BTK signal seemed markedly
attenuated in SYK-deficient DT40 cells (maximum SI: 2.9 at 15 min),
consistent with the existence of cross-talk between BTK and SYK in
generating an optimal EMF response (Fig. 3C).

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Fig. 3.
Time course of EMF-induced BTK activation in
mutant DT40 lymphoma B cells. Cells were exposed to 1-gauss low
frequency EMF. At the times indicated in the panels, EMF exposure was
stopped with a 1% Nonidet P-40 lysis buffer. The lysates were
immunoprecipitated with a polyclonal anti-BTK antibody and then
subjected to immune complex kinase assay or anti-BTK Western blot
analyses. Phosphorylation in the kinase assay (KA) was
initiated by the addition of radiolabeled ATP and terminated after 10 min. Autoradiograms of the kinase reaction (KA) and Western
blots (WB) are shown in each panel. Mutant DT40 clones were
rendered deficient for specific tyrosine kinases through target
disruption of the respective genes by homologous recombination
knockout. A, BTK-deficient (BTK ) DT40 cells.
B, LYN-deficient (LYN ) mutant DT40
cells. C, SYK-deficient (SYK ) mutant DT40
cells.
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Role of BTK in Activation of PLC-
2 in DT40 Lymphoma B-cells
Exposed to Low Energy EMF--
In accordance with our previous study
(11), exposure of DT40 cells to EMF resulted in enhanced inositol
phospholipid turnover (Fig.
4A). Because BTK is required
for the B-cell antigen receptor-induced PLC-
2 activation in DT40
cells (20), we then examined the role of BTK in EMF-induced inositol
phospholipid turnover. Targeted disruption of the btk gene
abolished the EMF-induced Ins-1,4,5-P3 signal (Fig.
4B). Introduction of wild-type (but not a kinase domain
mutant) human btk gene into the BTK-deficient DT40 cells restored their ability to respond to EMF with enhanced inositol phospholipid turnover (Fig. 4, C and D). The lack
of the Ins-1,4,5-P3 signal in BTK
and
BTK
,rBTK(K
) cells was not due to lower
expression levels of PLC-
2 enzyme in these cells (Fig.
4E). These results demonstrate that BTK is essential for
EMF-induced PLC-
2 activation in DT40 lymphoma B-cells, and its
kinase domain is required for the Ins-1,4,5-P3 response.

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Fig. 4.
Time course of EMF-induced inositol
phospholipid turnover in DT40 lymphoma B cells. Cells (5 × 106/ml) were exposed to low frequency EMF
(diamonds). The control cells (squares) were
sham-treated, under identical conditions except for exposure to EMF. At
the times indicated in the figure, the cells were lysed with ice-cold
20% perchloric acid and then assayed for Ins-1,4,5-P3
levels using a radioligand competition assay (see "Experimental
Procedures"). A, wild-type DT40 cells (WT).
B, BTK-deficient cells (BTK ).
C, BTK-deficient DT40 cells reconstituted with wild-type
(WT) human btk gene
(BTK ,rBTK(WT)). D,
BTK-deficient DT40 cells reconstituted with a human btk gene
that contains a mutation in the kinase domain
(BTK ,rBTK(K ). Results
are the mean (±S.E.) values obtained in replicate experiments each
performed with duplicate measurements at every time point.
n = number of independent experiments. E,
anti-PLC- 2 and anti-actin Western blot analysis of wild-type,
BTK , BTK ,rBTK(WT), and
BTK ,rBTK(K ) DT40 cells.
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In summary, we examined the molecular mechanism of enhanced tyrosine
phosphorylation and increased inositol phospholipid turnover in DT40
lymphoma B-cells exposed to low energy EMF. Our findings provide
unprecedented evidence that EMF exposure initiates a biochemical signaling cascade intimately linked to BTK. Like the LYN kinase, which
functions upstream, BTK plays an important role in initiation and
maintenance of signaling events that control the proliferation and
survival of B-lineage lymphoid cells. Recent studies demonstrated that
BTK regulates apoptotic signals (19, 39, 40). Therefore, this study
further supports the hypothesis that a delicate growth regulatory
balance in B-lineage lymphoid cells might be altered by EMF exposure.
The mechanism by which exposure of lymphoma B-cells to low energy EMF
triggers activation of LYN and BTK remains to be deciphered.