(Received for publication, April 13, 1995; and in revised form, July 18, 1995)
From the
Here, we present evidence that exposure of B-lineage lymphoid cells to low energy electromagnetic fields (EMF) stimulates the protein tyrosine kinases Lyn and Syk, results in tyrosine phosphorylation of multiple electrophoretically distinct substrates, and leads to downstream activation of protein kinase C (PKC). EMF exposure enhances protein tyrosine phosphorylation in Syk deficient but not in Lyn-deficient B-lineage lymphoid cells and stimulates Lyn kinase activity in wild-type as well as Syk-deficient B-lineage lymphoid cells. These results indicate that activation of Lyn kinase is sufficient and mandatory for EMF-induced tyrosine phosphorylation in B-lineage lymphoid cells. The PKC activity increases later than the Lyn activity and pretreatment with the PTK inhibitors genistein or herbimycin A abrogates the EMF-induced PKC signal. Thus, stimulation of Lyn is a proximal and mandatory step in EMF-induced activation of PKC in B-lineage lymphoid cells. Our observations prompt the hypothesis that a delicate growth regulatory balance might be altered in B-lineage lymphoid cells by EMF-induced activation of Lyn.
B-lineage acute lymphoblastic leukemia (ALL) ()is the
most common form of childhood cancer(1, 2) . B-lineage
ALL is a heterogeneous group of diseases that are thought to originate
from putative developmental lesions in normal B-lymphocyte precursor
clones during early phases of ontogeny, which allegedly lead to
maturational arrest at discrete stages of lymphopoiesis. It is
generally believed that such developmental lesions lead to an
uncoupling of proliferation and differentiation in target lymphocyte
precursor clones(1, 2) . The stabilization and
subsequent clonal expansion of usually transient B-lymphocyte precursor
cell phenotypes have been proposed as critical proximal events in the
leukemogenesis of B-lineage ALL(1, 2, 3) .
Recently, concern has emerged about the possibility that EMF radiation from residentially proximate power lines, household electrical wiring, and appliance usage may contribute to the risk of childhood ALL (4, 5, 6, 7, 8, 9, 10, 11, 12) . Several studies of associations between childhood leukemia and residential exposure to EMF from electric power lines yielded epidemiologic evidence for a link between EMF exposure and ALL(4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . A recent study by the Swedish National Institute for Occupational Health indicated that children in Sweden chronically exposed to powerline frequency magnetic fields exhibited a 3-fold increase in their incidence of leukemia(14) , and similar data have been reported by other investigators(14, 15, 16, 17, 18, 19, 20) .
The molecular mechanism by which EMF exposure could participate in the complex process leading from changes in cell properties to the actual development of B-lineage ALL in vivo has not been deciphered. No directly genotoxic actions of low energy EMF are known, and it is widely agreed that exposure of cells to nonionizing EMF does not produce mutations or chromosome damage(17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . Thus, any participation by EMF in leukemogenesis of B-lineage ALL is likely to be by means of influencing survival, proliferation, and/or differentiation of B-lineage lymphoid cells rather than by producing the primary mutational or ``initiation'' event. A point of exceptional experimental focus in current EMF research is the investigation of EMF effects on signal transduction pathways(28, 29, 30, 31, 32, 33, 34) . Several investigators noted that EMF exposure activates a protein kinase C (PKC)-linked signaling cascade(34, 35) . However, EMF-induced signaling events proximal to PKC activation have not been evaluated.
Protein tyrosine kinases (PTK) participate and
play pivotal and myriad roles in initiation of signal cascades that
affect proliferation and survival of human B-lineage lymphoid
cells(36, 37, 38, 39, 40, 41, 42, 43) .
Notably, recent studies indicate that stimulation of protein tyrosine
kinases in B-lymphocyte precursors is a requisite step in the
generation of the pleiotropic effects of ionizing radiation, including
the activation of protein kinase C (PKC) and PKC-dependent serine
kinases, nuclear factor B (NF-
B), as well as c-jun proto-oncogene expression(36, 37, 38) .
A delicate balance of oncogenic versus tumor-suppressive
proteins might be altered when a tyrosine kinase regulatory pathway is
activated(44) .
The purpose of this study was to examine the role of PTK in EMF-induced activation of the PKC pathway in B-lineage lymphoid cells. Here, we present evidence that stimulation of PTK is an important and mandatory proximal step in EMF-induced activation of the PKC signaling cascade in B-lineage lymphoid cells.
Figure 1:
EMF exposure induces tyrosine
phosphorylation of multiple electrophoretically distinct substrates in
B-lineage lymphoid cells. NALM-6 human pre-B or DT-40 chicken B cells
were exposed to 60-Hz EMF for the indicated periods. After EMF
exposure, cells were lysed with SDS lysis buffer and boiled. Equivalent
amounts of protein were fractionated on 10.5% polyacrylamide gels,
transferred to Immobilon-PVDF membranes, and immunoblotted with an
anti-phosphotyrosine antibody followed by incubation with I-protein A (1 µCi/ml) and autoradiography for
detection of phosphotyrosyl proteins. Controls included unstimulated
samples (CON, negative control) as well as samples stimulated
with an anti-CD19
CD19 monoclonal antibody homoconjugate (1
µg/ml
10 min)(39) . Molecular masses (in kDa) of
the phosphotyrosyl protein substrates were calculated from prestained
molecular size markers run as standards.
Figure 2:
Time course of Lyn kinase activation after
EMF exposure. A, unstimulated NALM-6 human pre-B cells were
lysed in a Nonidet P-40-containing buffer, and equal amounts of lysate
(200 µg of protein/reaction mixture) were used for
immunoprecipitation and immune complex kinase assays of the indicated
Src family PTK, as described under ``Experimental
Procedures.'' No primary antibody was added to the lysate of the
control sample (first lane from left). B, NALM-6
cells were exposed to 60-Hz EMF for the times indicated, pelleted,
lysed in Nonidet P-40 buffer. Lyn kinase was immunoprecipitated, and
immune complex kinase assays (39) were performed using half of
the samples, as described under ``Experimental Procedures.''
Autophosphorylation was quantitated by a 4-min liquid scintillation
counting of the incorporated P in the the excised Lyn
kinase bands. C, the other half of the Lyn immunoprecipitates
were fractionated first on 9.5% polyacrylamide gels,
electrophoretically transferred to Immobilon-polyvinylidene difluoride
membranes and immunoblotted for 1 h with anti-Lyn antibody (1:100
dilution) followed by incubation with
I-protein A
(specific activity = 30 µCi/µg; ICN Biomedicals) and
autoradiography for detection of Lyn, as described(39) . Since
the anti-Lyn antibody was generated by using a GST-fusion protein of
the 56-kDa form of Lyn, it preferentially reacts with p56
in Western blot analyses.
Figure 3:
Time course of Syk kinase activation after
EMF exposure. A, NALM-6 cells were exposed to 60-Hz EMF for
the times indicated, pelleted, and lysed in Nonidet P-40 buffer. Syk
kinase was immunoprecipitated and immune complex kinase assays (39) were performed using half of the samples, as described
under ``Experimental Procedures.'' Autophosphorylation was
quantitated by a 4-min liquid scintillation counting of the
incorporated P in the the excised Syk kinase bands. B, the other half of the Syk immunoprecipitates were
fractionated first on 9.5% polyacrylamide gels, electrophoretically
transferred to Immobilon-polyvinylidene difluoride membranes and
immunoblotted for 1 h with anti-Syk antibody (1:100 dilution) followed
by incubation with
I-protein A (specific activity
= 30 µCi/µg; ICN Biomedicals) and autoradiography for
detection of Syk.
DT-40 is a chicken B-cell line that is immunobiologically and biochemically very similar to human B-lineage lymphoid progenitor cells (45, 46) . Like human B-lineage lymphoid cells that are thought to be the progenitors of leukemic cells in children with B-lineage ALL, DT-40 cells express high levels of Lyn and Syk tyrosine kinases but not the other members of the Src- or Syk-PTK families (see (45) and (46) and Fig. 4A). Lyn knock-out and Syk knock-out mutants of the DT-40 cell line were established by targeted gene disruption (see (45) and Fig. 4A). Defects of early signaling events are different between Lyn-deficient and Syk-deficient mutants, demonstrating that Lyn and Syk kinases mediate discrete signaling functions through their enzymatic activities(45) . As shown in Fig. 4B, EMF stimulated Lyn kinase activity in wild-type as well as Syk-deficient DT-40 cells. Furthermore, EMF exposure enhanced protein tyrosine phosphorylation in Syk-deficient but not in Lyn-deficient DT-40 cells (data not shown). Our results indicate that activation of Lyn kinase is sufficient and mandatory for EMF-induced tyrosine phosphorylation in DT-40 cells. However, these experiments in the DT-40 model system failed to provide conclusive information regarding the role of Lyn kinase in EMF-induced activation of Syk kinase because no significant Syk kinase stimulation was observed in wild-type DT-40 cells or Lyn-deficient DT-40 cells (data not shown).
Figure 4:
EMF exposure stimulates Lyn kinase in
wild-type as well as Syk kinase-deficient B-lineage lymphoid cells. A, unstimulated wild-type DT-40 chicken B cells
(DT-40) as well as its Syk kinase deficient
(DT-40
) and Lyn kinase deficient
(DT-40
) mutants were lysed in a Nonidet
P-40-containing buffer, and equal amounts of lysate (200 µg of
protein/reaction mixture) were used for immunoprecipitation and immune
complex kinase assays of Lyn, Blk, and Syk kinases, as described under
``Experimental Procedures.'' B, DT-40
and DT-40
were exposed to
60-Hz EMF for the times indicated, pelleted, and lysed in Nonidet P-40
buffer. Lyn kinase was immunoprecipitated, and immune complex kinase
assays (39) were performed, as described under
``Experimental Procedures.''
Figure 5: PTK-dependent activation of PKC in B-lineage lymphoid cells exposed to EMF. NALM-6 human pre-B cells were exposed to EMF for the times indicated in the presence or absence of the PTK inhibitors herbimycin A (Herb) or genistein (Gen), as described under ``Experimental Procedures.'' Sham-treated controls (Sham) were incubated in the EMF exposure device, with current flowing in antiparallel directions to cancel out the generated magnetic field. Membrane-associated PKC activity was assayed, as detailed under ``Experimental Procedures.''
In summary, we examined the biochemical nature of signaling events in B-lineage lymphoid cells exposed to low energy EMF. This report clarifies the chronological sequence of biochemical events that follow EMF exposure and provides unprecedented evidence that EMF exposure initiates a signaling cascade that is intimately linked to the Src proto-oncogene family PTK Lyn. Lyn kinase participates and plays pivotal and myriad roles in initiation of signal cascades that affect proliferation and survival of B-lineage lymphoid cells(37, 38, 39) . Our observations prompt the hypothesis that a delicate growth-regulatory balance might be altered in B-lineage lymphoid cells by EMF-induced activation of Lyn.