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| TITLE: | The Effect of 60 Hz EMF on Tyrosine Phosphorylation | ||
| Principal Investigator |
Jau-Shyong Hong, Ph.D. | Laboratory of Environmental Neurosciences, NIEHS | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cellular Function | Cellular Processes | Signal Transduction |
| FY95 Funds | DIR-5 $ 46,000 | Start Date 10/94 | End Date 9/96 |
| Rationale and Summary |
Biological and epidemiological studies have suggested that low-frequency electromagnetic fields (EMF) may adversely affect human health. However, the mode of action of EMF on cells is largely unknown. On the other hand, DC electric fields have long been known to be an effective stimulus in cell activation. For example, DC fields cause polarized cell movement, neurite outgrowth and clustering of integral membrane proteins. We found recently that some of its effect may be mediated by the activation of receptor tyrosine kinases. These kinases are normally activated by binding of specific ligands such as peptide growth factors. It now appears that DC electric fields can activate these kinases directly in the absence of ligands. We are now interested in applying this new concept to study the effect of EMF in cell activation. As a first step, we plan to examine the effect of 60 Hz EMF on cultured human epidermoid carcinoma A431 cells. These cells have an abundance of receptors for epidermal growth factor (EGF) on their surface. The EGF receptor is a prototype of the receptor for tyrosine kinase, which is normally activated by EGF. Upon ligand binding, these receptors become dimerized and tyrosine becomes phosphorylated. By probing the Western blot of the lysate of A431 cells with an antibody to phosphotyrosine, we are able to demonstrate the tyrosine phosphorylation of the EGF receptor. Control cultures not treated with the ligand showed very low level of tyrosine phosphorylation. We are now in a position to test whether EMF can mimic the ligand causing tyrosine phosphorylation of EGF receptors in A431 cells. Since tyrosine phosphorylation of receptor tyrosine kinases is an integral step involved in stimulating cell proliferation and transformation, these studies may shed light on the molecular mechanisms of EMF-mediated cellular responses. | ||
| Experimental Design and Exposure Conditions |
A431 cells are grown in tissue culture flasks. Before exposure, the cells are serum-starved to avoid the activation of receptor tyrosine kinases by factors in the serum. The cultures are exposed to EMF in a modified tissue culture incubator equipped with a double-turn Helmholtz coil which is 44.2 cm in diameter and is wound with 0.82 mm wire. The two coils are connected in series and are placed with their planes perpendicular to the ground. The culture flasks are placed with their bottom parallel to the axis of the coil. The AC current is delivered by a Wavetek function generator. After EMF exposure for a duration ranging from several hours to several days, the cultures are processed for Western blots to probe the tyrosine phosphorylation of the EGF receptor. | ||
| Quality Assurance Measures |
Two identical incubator setups are available for this study. Thus, a test and a control exposure can be conducted in parallel. These setups were assembled and calibrated by the US EPA. Since cells are placed in enclosed, grounded incubators, stray fields are avoided. | ||
| Results and Discussion |
To date, we have obtained background information on cultured primary cells exposed to DC
electric fields. Our results have shown that these fields cause the redistribution of nicotinic
acetylcholine receptors to the cathodal edge of cultured muscle cells. This redistribution process
can be blocked by a tyrosine kinase inhibitor. The receptor clusters induced by these fields are
colocalized with phosphotyrosine immunocytochemical staining. We have now set up the
exposure chamber and are in the process of performing calibration. In addition, we have
examined cell lines for their potential for tyrosine phosphorylation in response to ligands. The
A431 cell line has been found to be the most appropriate for the initial phase of this study. We plan
to expose these cells to EMF and to study the tyrosine phosphorylation in the near future.
These studies have not been completed. This study is a collaboration between the University of North Carolina; Dr. H. B. Peng, and the Laboratory of Environmental Neuroscience, NIEHS. |
||
| Recent Publications |
Peng, H. B., L. P. Baker, and Z. Dai. 1993. A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells. J. Cell Biol. 120:197-204. | ||