We have shown that ELF magnetic fields do not alter the expression of the c-myc proto-oncogene in HL60 cells, a human leukemia line, as previously reported by others. To clarify further the limits of gene expression changes induced by magnetic fields in HL60 cells, differential display PCR, a method capable of examining thousands of genes, was used to determine whether any genes are field-responsive in those cells. No indication of gene expression changes was observed.

The failure to find an effect in HL60 cells does not shed light on the capability, or lack thereof, for ELF magnetic fields to contribute to neoplastic transformation since those cells are already transformed. The relevant processes may already be disrupted in those cells so that no further effect could be discerned. Therefore, current work in this project focuses on cells that are not fully cancerous. Specifically, since evidence suggests that magnetic fields are not capable of initiating cancer, but only in promoting transformation, we are using a cell line, JB6, that is initiated but not fully transformed.

We are now proceeding to determine whether magnetic fields can indeed contribute to the promotional steps in carcinogenesis. Experiments are being carried out to measure transformation rates of JB6 cells and to define the specific magnetic field conditions, if any, that give a maximal response. Then specific gene expression changes that occur under those conditions will be investigated. That work will focus on the transcription factor AP-1, known to be important in JB6 cell transformation.

The magnetic field exposure system is a double-wound square 4-coil configuration (field variation within culture area < 0.5%) generating a vertical 60 Hz magnetic field with two identical chambers and coil sets that can be randomly assigned for exposure and sham. The static geomagnetic field measured with a gaussmeter with a Hall effect probe was 30.7 µT, angle of inclination 30 in one chamber and 32.8 µT, angle of inclination 12 in the other. The 60 Hz fields present at each culture dish location were measured before and after exposure using a power frequency field meter. For sham exposures, background fields were 0.1 µT.

HL60 cells (batch F-9085), a human promyelocytic leukemia line, were obtained from ATCC and grown in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin. JB6 cells (clone 41a) were grown in DMEM with 5% fetal bovine serum and penicillin/streptomycin. Both cell lines were immediately expanded in order to freeze 100 vials each to provide a uniform stock for subsequent experimentation.

For each experiment with HL60 cells, a new vial of frozen cells was thawed, expanded for 11 days and used at the same passage number. Cell growth was monitored daily throughout the expansion. Sixteen hours prior to exposure, cells were centrifuged (1000 rpm, 5 min) and resuspended in fresh medium at a concentration to give the appropriate density at time of exposure and plated in the outer ring of organ culture dishes. Dishes of cells were randomly picked and placed alternately into the two exposure chambers and allowed to equilibrate for 20 minutes prior to exposure. After exposure, dishes were removed from the chambers simultaneously and randomly coded by an investigator who did not know the field conditions in the chambers. Subsequent manipulations of cells were alternated between dishes from each chamber to avoid handling artifacts. Samples remained coded until all data analysis was completed. Similar handling is being used for JB6 cell experiments.

Steady-state RNA levels for specific genes are being assessed using ribonuclease protection assays. Conditions have been chosen to provide probe excess and a linear response. Differential display analysis was accomplished using RNAmap kits from GenHunter.

Transformation of JB6 cells is being measured by standard soft agar growth assays. Anchorage- independent growth is measured in the presence or absence of various field conditions. The number and size distribution of resulting colonies and any possible shift in TPA dose-response is assessed in each assay.

As a positive control to define the resolution of the protection assay as well as to indicate that the cells respond normally to other agents, HL60 cells were treated with 3 nM 12-o-tetradecanoyl- phorbol-13 acetate (TPA), which induces a biphasic response in MYC mRNA levels. In contrast, -2 microglobulin was not responsive to TPA treatment. We observed an increase in MYC to 1.60 ± 0.37 (n=21) times the initial level within 30 min and a decrease to 0.29 ± 0.21 (n=21) after 90 min, consistent with previous observations.

For the differential display, each RNA sample was tested for genomic DNA contamination by performing a control reaction without reverse transcriptase. PCR products were analyzed on denaturing sequencing gels. Undried gels were exposed to film for 16-48 hours. Since ddPCR is characterized by some variability, PCR reactions were performed and analyzed in duplicate to differentiate between real differential expression and the random variation inherent in this system. In addition, any potential difference was assessed in an independent set of reactions.

For JB6 transformation assays, a series of assays were carried out to choose a suitable lot of serum, as defined by low background in the absence of TPA and a high transformation efficiency in the presence of the tumor promoter.

The biological effects of each magnetic field exposure condition are evaluated in a series of six experiments, two each sham (left chamber) vs. exposed (right chamber), exposed (left) vs. sham (right), and sham vs. sham. The particular experimental set-up as well as the choice of exposed chamber is unknown to the investigator. Actual fields in both chambers are measured at the position of each culture dish.

Since the above results were obtained with already transformed cells, they do not necessarily reflect the potential or lack thereof for magnetic fields to contribute to carcinogenesis. To address this directly, we have begun a series of experiments to determine whether magnetic fields can act as a tumor promoter. A first series of experiments has been completed looking at one field condition (60 Hz, 1 mT vertical field). Organ culture dishes with an inner well and an outer annulus were used in these assays to assess the potential role of the induced electric current. For each experiment 30 independent dishes of cells are used. In addition to the data shown, some cells were not exposed to TPA; in these cases no colonies were formed, regardless of field conditions. In this single set of experiments, the data do not indicate any effect of the magnetic field on transformation. Since this is only one field condition, it is too early to draw any conclusions regarding magnetic fields and tumor promotion.

Persengiev SP, Saffer JD, Kilpatrick DL (in press) "An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain." Proc Natl Acad Sci USA