From the Biotherapy Program, University of Minnesota,
Minneapolis, Minnesota 55417, ** Division of Biomedical Sciences,
University of California, Riverside, California 92521,
Wayne
Hughes Institute, Roseville, Minnesota 55113, and ¶ Department of
Molecular Genetics, Institute for Hepatic Research, Kansai Medical
University, Moriguchi, Osaka 570, Japan
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ABSTRACT |
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Here, we present evidence that
exposure of DT40 lymphoma B cells to low energy electromagnetic field
(EMF) results in a tyrosine kinase-dependent activation of
phospholipase C2 (PLC-
2) leading to increased inositol
phospholipid turnover. B cells rendered PLC-
2-deficient by targeted
disruption of the PLC-
2 gene as well as
PLC-
2-deficient cells reconstituted with Src homology domain 2 (SH2)
domain mutant PLC-
2 did not show any increase in
inositol-1,4,5-trisphosphate levels after EMF exposure, providing direct evidence that PLC-
2 is responsible for EMF-induced
stimulation of inositol phospholipid turnover, and its SH2 domains are
essential for this function. B cells rendered SYK-deficient by targeted disruption of the syk gene did not show PLC-
2 activation
in response to EMF exposure. The C-terminal SH2 domain of SYK kinase is
essential for its ability to activate PLC-
2. SYK-deficient cells
reconstituted with a C-terminal SH2 domain mutant syk gene
failed to elicit increased inositol phospholipid turnover after EMF
exposure, whereas SYK-deficient cells reconstituted with an N-terminal
SH2 domain mutant syk gene showed a normal EMF response.
LYN kinase is essential for the initiation of this biochemical
signaling cascade. Lymphoma B cells rendered LYN-deficient through
targeted disruption of the lyn gene did not elicit enhanced
inositol phospholipid turnover after EMF exposure. Introduction of the
wild-type (but not a kinase domain mutant) mouse fyn gene
into LYN-deficient B cells restored their EMF responsiveness. B cells
reconstituted with a SH2 domain mutant fyn gene showed a
normal EMF response, whereas no increase in inositol phospholipid
turnover in response to EMF was noticed in LYN-deficient cells
reconstituted with a SH3 domain mutant fyn gene. Taken
together, these results indicate that EMF-induced PLC-
2 activation
is mediated by LYN-regulated stimulation of SYK, which acts downstream
of LYN kinase and upstream of PLC-
2.
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INTRODUCTION |
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A number of epidemiologic studies suggested the possibility that electromagnetic field (EMF)1 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 (6-9).
The Src protooncogene family protein-tyrosine kinase (PTK) LYN plays a
pivotal role in ligand-induced signal transduction events in B-lineage
lymphoid cells (10-17). In a recent study, we discovered that exposure
of B-lineage lymphoid cells to low energy EMF stimulates LYN as well as
its downstream substrate SYK (18). These results prompted the
hypothesis that a delicate growth regulatory balance might be altered
in B-lineage lymphoid cells by EMF-induced activation of a biochemical
signaling cascade intimately linked to LYN kinase. The purpose of the
present study was to further characterize this signaling cascade using
a EMF-responsive lymphoma B cell model and targeted disruption of the
genes for known or suspected participants such as LYN, SYK, and
PLC-2 by homologous recombination knockout.
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EXPERIMENTAL PROCEDURES |
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Cell Lines--
The establishment and characterization of
wild-type and LYN-deficient, SYK-deficient, PLC-2-deficient clones
of DT40 chicken lymphoma B cells were previously reported (19-23).
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.
Western Blot Analysis of Protein Expression and Immune Complex
Kinase Assays--
The expression levels of wild-type and mutant
enzyme proteins in the various cell lines were measured by Western blot
analysis using appropriate monoclonal antibodies, as described
previously (24). In brief, cells (5 × 106
cells/sample) were lysed in 150 µl of SDS lysis buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 µM sodium orthovanadate, 25 mM
dithiothreitol) and boiled for 5 min. The DNA was sheared by several
passages through a 28-gauge needle, and 40-µl amounts of the whole
cell lysate protein samples in SDS-reducing sample buffer were
fractionated on reducing SDS-polyacrylamide gels by overnight
electrophoresis at 4 mA. The proteins were transferred to a 0.45-µm
Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford,
MA) for 1 h at 130 mA using a semidry transfer apparatus (Hoefer
Scientific Instruments, San Francisco, CA). The polyvinylidene
difluoride membranes were incubated for 1 h at room temperature in
blocking solution (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% bovine serum albumin), washed in rinsing
buffer, and incubated for 1 h with the appropriate primary
antibody (i.e. polyclonal rabbit anti-FYN serum at 1:2,000
final dilution, monoclonal 4D10 IgG2a anti-SYK antibody
from Santa Cruz Biotechnology, Inc. at 10 ng/ml final concentration, or
polyclonal rabbit anti-PLC-2 serum at 1:1,000 final dilution mixed
with monoclonal 4D10 IgG2a anti-SYK antibody (for
comparison of SYK protein levels in the lanes as an internal control)
from Santa Cruz Biotechnology, Inc. at 10 ng/ml final concentration) in
blocking solution followed by three 10-min washes in rinsing buffer.
For detection of the target enzyme proteins, horseradish
peroxidase-conjugated sheep anti-mouse/rabbit IgG (Signal Transduction
Laboratories; 1:2500 dilution, 45-min incubation) and the ECL
chemiluminescence detection system (Amersham Life Sciences) were used
according to the manufacturers' recommendations. The membranes
immunoblotted with anti-FYN or anti-SYK antibodies were stripped and
reblotted with the mouse monoclonal anti-actin antibody (Sigma, catalog
no. A-4700; 1:50,000 final dilution) to compare the protein levels in
the individual lanes. In other experiments, wild-type and mutant SYK
proteins were immunoprecipitated, and in vitro immune
complex kinase assays were performed, as described previously (25,
26).
EMF Exposure--
A homogenous vertical magnetic field was set
up by using a Merritt's coil-based in vitro low frequency
EMF exposure system (18). Merritt's four square coil system is known
to produce a large volume of uniform magnetic field. The applied
vertical sinusoidal 60-Hz field was 0.1 mT (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 mG)
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 -minimum essential medium
in 1.5-ml capacity microcentrifuge tubes) were exposed to a 1-Gauss,
60-Hz EMF by placing the tubes at the center of the four-coil field
generator, which was contained in an 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 placement of the
cells, to avoid fluctuations of the EMF during activation of the
apparatus. The coils were turned off only after the cells were taken
out of the exposure system. Ice-cold perchloric acid (20%) was added
after EMF exposure to the cell suspensions to stop further response.
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, the pH was neutralized to 7.5 with ice-cold 10 mM KOH, and the solution was centrifuged again. The
supernatant was collected and stored at
20 °C for subsequent
measurement of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3)
levels.
Analysis of Stimulation of Inositol Phospholipid Turnover-- Ins-1,4,5-P3 levels were measured by using a D-myo-[3H]Ins-1,4,5-P3 assay system purchased from Amersham Corp., as reported elsewhere (26-29). This highly sensitive assay is based on the competition between nonradiolabeled 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 an Ins-1,4,5-P3-specific and sensitive bovine adrenal binding protein (26-29). In some experiments, cells were preincubated for 1 h at 37 °C with the PTK inhibitor genistein (ICN Biomedicals, Costa Mesa, CA) at 100 µg/mL (370 µM) or for 24 h at 37 °C with the PTK inhibitor herbimycin A (Sigma) at 7 µg/ml (12 µM), as previously reported (26-29). In some experiments, cells were stimulated with 3 µg/ml anti-chicken IgM monoclonal antibody M4 at 37 °C for the indicated periods of time in the figures.
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RESULTS AND DISCUSSION |
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Activation of PLC-2 in DT40 Lymphoma B Cells Exposed to Low
Energy EMF--
The catalytic activity of PLC-
2 is regulated
through tyrosine phosphorylation by receptor- and non-receptor-type
PTKs, and biochemical signals that trigger tyrosine-specific protein
phosphorylation have been shown to precede the activation of PLC-
2
and stimulation of inositol phospholipid turnover in many experimental
systems (10, 12-17, 26). Therefore, we examined the effects of EMF on
inositol phospholipid turnover in DT40 lymphoma B cells using a highly
specific and quantitative
D-myo-[3H]Ins-1,4,5-P3
assay system. Exposure of DT40 cells to EMF stimulated a rapid and
biphasic increase in the production of Ins-1,4,5-P3 leading
to markedly elevated Ins-1,4,5-P3 levels at 1 min after the
start of EMF exposure, which were 9.9-fold higher than the Ins-1,4,5-P3 levels in the sham-treated controls (21.8 ± 9 pmol/106 cells versus 2.2 ± 0.6 pmol/106 cells, p < 0.05). Thereafter, the
level of Ins-1,4,5-P3 rapidly declined but at 5 min it was
still higher than the baseline (Fig. 1A). The magnitude and
kinetics of EMF-induced Ins-1,4,5-P3 response were similar
to those of the Ins-1,4,5-P3 response triggered by engagement of the B cell antigen receptor with an anti-IgM monoclonal antibody (Fig. 1A). These experiments provided evidence that
the biochemical signal triggered in DT40 lymphoma B cells by low energy EMF is intimately linked to signal transduction pathways that stimulate
inositol phospholipid turnover, producing Ins-1,4,5-P3 as a
second messenger. The PTK inhibitors herbimycin A and genistein attenuated the EMF-triggered Ins-1,4,5-P3 signals (Fig.
1A), providing strong evidence that tyrosine phosphorylation
is a requisite step in the EMF-triggered stimulation of inositol
phospholipid turnover in lymphoma B cells. In contrast to wild-type
DT40 cells, DT40 cells rendered PLC-
2-deficient by targeted
disruption of the PLC-
2 gene did not show any
increase in Ins-1,4,5-P3 levels after EMF exposure (Fig.
1B), confirming that PLC-
2 is required for EMF-induced
stimulation of inositol phospholipid turnover.
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Role of LYN Kinase and Its Downstream Substrate SYK in Activation
of PLC-2 in DT40 Lymphoma B Cells Exposed to Low Energy
EMF--
Because LYN kinase is required for the antigen
receptor-induced PLC-
2 activation in DT40 cells and functions
upstream of SYK (10), we next examined the role of LYN kinase in
EMF-induced inositol phospholipid turnover. Targeted disruption of the
lyn gene abolished the EMF-induced Ins-1,4,5-P3
signal (Fig. 3A). Introduction
of the wild-type (Fig. 3B) or an SH2 domain mutant (Fig.
3C) but not a kinase domain mutant fyn gene (Fig.
3D) or an SH3 domain mutant fyn gene (Fig.
3E) into LYN-deficient cells restored the EMF
responsiveness, even though LYN-deficient cells reconstituted with
these genes expressed similar amounts of FYN protein (Fig.
3F). Thus, the kinase and SH3 domains of FYN are required
for its ability to restore the EMF responsiveness in LYN-deficient
cells. However, the Ins-1,4,5-P3 signal in
FYN(WT)-reconstituted cells was monophasic with a single peak at
45 s (Fig. 3B), whereas the Ins-1,4,5-P3
signal in the parent cell line was biphasic with one early peak at
15 s and a second peak at 1 min (Fig. 1A). Furthermore, the magnitude of the Ins-1,4,5-P3 signal signal in
wild-type FYN-reconstituted cells was smaller than the magnitude of the
biphasic Ins-1,4,5-P3 signal in the parent DT40 cell line.
The reasons for these differences are unknown. Notably, reconstitution
of LYN-deficient cells with a SH2 domain mutant fyn gene
resulted in a biphasic Ins-1,4,5-P3 signal after EMF
exposure, and the kinetics as well as amplitude of this signal were
similar to those of the Ins-1,4,5-P3 signal in the parent
cell line. While these results provide conclusive evidence that the SH2
domain of FYN is not required for the EMF-induced Ins-1,4,5-P3 signal, they also demonstrate that, despite
the apparent similarity of the Ins-1,4,5-P3 signal after
EMF exposure or stimulation with an anti-IgM antibody, significant
differences must exist between EMF-induced signaling events and
signaling events induced by engagement of the B cell antigen receptor
for which the SH2 domain of Src family PTK play a pivotal role.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health, NIEHS Research Grant R01-ES-07175.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Submitted to fulfill in part the requirements for a doctorate of philosophy at the University of Minnesota Graduate School, Minneapolis, MN 55417.
Stohlman Scholar of the Leukemia Society of America. To whom
correspondence and requests for reprints should be addressed: Wayne
Hughes Institute, 2665 Long Lake Rd., Suite 330, St. Paul, MN 55113. Tel.: 612-697-9228; Fax: 612-697-1042; E-mail:
fatih_uckun{at}mercury.ih.org.
1
The abbreviations used are: EMF, electromagnetic
field; PTK, protein-tyrosine kinase; PLC-2, phospholipase C-
2;
SH2, Src homology domain 2; Ins-1,4,5-P3,
inositol-1,4,5-trisphosphate; ITAM, immunoreceptor tyrosine-based
activation motif.
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REFERENCES |
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