The goal of the proposed research is the elucidation of the molecular mechanism by which low energy electromagnetic fields initiate a cascade of cytoplasmic and nuclear signaling events in human lymphohematopoietic cells. Our research plan involves a) immune complex kinase assays to examine the effects of EMF on the enzymatic activity of specific Src-family and non-Src family protein tyrosine kinases (PTK) in normal and leukemic B-cell precursors corresponding to discrete developmental stages of human B-cell ontogeny, b) detailed biochemical and biologic studies of CD19 receptor function in B-cell precursors following EMF exposure, c) use of CD19 receptor directed tyrosine kinase inhibitors to elucidate the role of CD19-receptor associated tyrosine kinases in the pleiotropic effects of EMF including activation of protein kinase C and c-jun protooncogene, d) use of two distinct model systems (viz., K562-CD19 and UPN1lyn models) to examine the importance of CD19 receptor and LYN kinase in EMF induced biochemical signals, e) correlative studies to explore the relationship between EMF responses of leukemic cells and various biomarkers which are routinely measured as well as f) statistical analyses to examine the relationship between various biomarkers, clinical/laboratory data and residential EMF measurements. From each newly diagnosed acute lymphoblastic leukemia case, we routinely obtain several hundred million leukemic cells. Therefore, more than sufficient cells will be available for the proposed experiments which will require 50-100 million cells. In addition, our data will be interfaced with the residential EMF data generated in the CCG-E15-epidemiology study. We therefore believe that our proposed research will significantly contribute to the current knowledge of the biologic effects of EMF and their possible molecular mechanisms.

We will use the Merritt four-coil array system (ratio of windings 26:11:11:26), with further modifications as suggested by Kirschvink (M/K coil system). In this system the antiparallel vs parallel (control vs experimental) current flow can be selected by a pari of independently operating double-pole, double-throw switches. This allows for double-blind experiments in which the two switches (concealed in separate boxes) are set by different individuals, so that neither the investigator nor the laboratory personnel know which coil is experimental and which is control until the data are processed and the switch positions are decoded. Alternatively, a computer randomization algorithm with computer-controlled relays can be used to eliminate any investigator participation in choosing the coil activation direction.

We will use two identical incubators and two identical M/K coil systems. This will allow control and experimental exposures to be carried out under identical conditions simultaneously.. Both incubators will be fitted with magnetoabsorptive steel (mu metal) shields placed so as to shield the exposure system from fields produced by the warming and temperature circuitry in the top of the incubator. One set of coils will be energized with the current running in parallel (i.e. producing magnetic field) and the other with the current flowing antiparallel (i.e. zero induced magnetic field_), using double blind randomization as described above for all experiments. These coils will be energized by a power amplifier (Kepco BOP 20-10M) driven by a function generator (Beckman) producing 60-Hz sine wave AC current in the coils. The AC magnetic field intensity within the exposure volume will be calibrated using a Hall-effect transverse probe (FG Bell) and a gaussmeter (Bell model 610). Waveforms will be monitored continuously using a digital oscilloscope with leads for both the electrical power supply and the gauss meter. Field strength will be recorded with a strip chart recorder attached to the output of the gauss meter. Static geomagnetic fields and their orientation will be recorded daily before and after each experiment. If findings by us or others indicate it is desirable, the geomagnetic field can be canceled or modified using properly oriented DC "bucking" coils; we do not intend at this time to investigate the geomagnetic field as high priority experimental variable but this can be incorporated in the experiments easily if future findings by others confirm its importance.

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. Recent studies indicate that stimulation of protein kinases in B-lymphocyte precursors is a requisite step in the generation of the pleiotropic effects of ionizing radiation, including the activation of PKC and PKC-dependent serine kinases, nuclear factor kappa B (NKfB), as well as c-jun protooncogene expression

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. Our studies provide direct 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.