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| TITLE: | Changes in Gene Expression Accompanying Exposure to EMF | ||
| Principal Investigator |
Gayle E. Woloschak | Argonne National Laboratory | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cellular Function | Gene Expression | Cell Cycle |
| FY95 Funds | R01ES07141 $ 151,975 | Start Date 09/28/94 | End Date 08/31/97 |
| Rationale and Summary |
Recent reports have described exposure to electric and magnetic fields (EMF) as a possible risk factor in the development of several forms of cancer including leukemia, breast cancer, and malignant glioma. Laboratory based studies have yet to find a definitive mechanism that would explain cancer development. We intend to test the hypothesis that specific genes modulated in response to low-frequency EMF will determine specific intracellular pathways affected by the exposure. In the experiments done to date, we have documented few changes in gene expression associated with EMF exposure. Exposures were set up using continuous sinusoidal 60-Hz magnetic fields at an intensity of 100 µT (1000 mG) for lengths of time up to 72 h. We applied the technique of differential display for detection of mRNA induced in cells following EMF exposure. We failed to find any bands on gels reproducibly induced at early times of EMF exposure (6 h). One band, was found to be induced at later times (24-72 h) and was selected for further study. Several other bands proved to be artifactual or not reproducibly affected by EMF exposure when tested by repeated Northern blots. Hybridization of Northern blots (RNA derived from cells exposed to 60- Hz EMF) demonstrated induction of the band of interest between 24 and 72 h of exposure. Several transcripts were detected, though one predominant RNA was evident in HeLa cells. A partial sequence of this band and a search through Genbank revealed identity with several known genes, all of which had long interspersed element (LINE) repeats in introns (including the human -globulin and human Rb genes). A check of the sequence of the band against LINE showed high homology. Considering the vast divergence and high copy number of LINE sequences in the genome, we could conclude that the EMF-induced band was a LINE sequence. The portion we identified is part of an ORF (open reading frame) found near the 3' end of an expressed LINE sequence; this is the correct region for detection using RT-PCR with a dT primer and this arbitrary primer. Southern blots of this band to restriction-enzyme-digested mouse and human DNA have revealed hybridization to repetitive elements consistent with LINE sequences. | ||
| Experimental Design and Exposure Conditions |
Exposures are done using continuous sinusoidal 60-Hz magnetic fields at intensities from 0-100µT (0-1000 mG) for lengths of time up to 72 h. We are currently using the human cell line HeLa, though we plan to test other cell lines as well. Cell cycle changes will be monitored by flow cytometry. RNA changes will be determined by a new technique called differential display. In this technique, RNA is harvested and cDNA is synthesized using reverse transcriptase and then amplified. Identified bands are then tested by dot blots and Northern blots to ensure that levels of the transcript are changed in response to EMF. Later experiments will examine how these genes are regulated. | ||
| Quality Assurance Measures |
Magnetic fields are slowly turned on and off over 2 seconds to produce no induced current. All experiments will be replicated at least three times prior to reporting of data. We test all bands by dot blot and Northern blot hybridizations to uniform results. We have set up several negative controls, such as monitoring expression of genes encoding ornithine decarboxylase and proliferating cell nuclear antigens. These genes are not affected by EMF exposure. We have also included some positive controls, particularly the use of ionizing radiation to induce known positive responses. | ||
| Results and Discussion |
We have determined that very few genes are affected by EMF exposure. In fact, at early time points (within the first 6 h following exposure) we have not yet identified a reproducible change in expression either at the level of total transcription of when specific genes are studied. Recent preliminary studies have suggested that 24-72 h exposure times may lead to the induction of LINE in response to EMF exposure. This was identified by the technique of differential display which resulted in a new band. A partial sequence of this band and a search through Genbank revealed identity with several known genes, all of which had LINE element repeats in introns (including the human -globulin and human Rb genes). A check of the sequence of the band against LINE showed high homology. Considering the vast divergence and high copy number of LINE sequences in the genome, we could conclude that the EMF-induced band was a LINE sequence. The portion we identified is part of an ORF found near the 3' end of an expressed LINE sequence; this is the correct region for detection using RT-PCR with a dT primer and this arbitrary primer. Southern blots of this band to restriction-enzyme-digested mouse and human DNA have revealed hybridization to repetitive elements consistent with LINE sequences. The significance of the induction of LINEs following EMF exposure is unknown. It is interesting that this is a relatively late event, detectable following 24-72 h of exposure. This means induction is not likely part of an immediate early response pathway but rather may be associated with more chronic exposures. It has been suggested that expression of LINE may be associated with reintegration of LINE into the human genome, especially since expressed LINE frequently have associated reverse transcriptase activity. This retrotransposon-like activity has been documented predominantly in LINE which have been introduced at new sites in the genome. | ||
| Recent Publications |
Libertin, C.R., Panozzo, J., Groh, K., Chang-Liu, C-M., Schreck, S., and Woloschak, G.E. Effects
of gamma rays, ultraviolet radiation, sunlight, microwaves, and electromagnetic fields on gene
expression mediated by human immunodeficiency virus promoter. Radiation Research, 140:91-96,
1994.
Paunesku, T. And Woloschaak, G.E. Use of labeled primers for differential display. Mammalian Genome, in revision, 1995. Woloschak, G.E., Paunesku, T., Chang-Liu, C.-M., and Grdina, D.J. Changes in gene expression associated with radiation exposure. In press, Radiation Research 1895-1995: Congress Proceedings. (Ed. By Hu. Hagen, J. Jung, and C. Streffer). Woloschak, G.E., Chang-Liu, C-M., Panozzo, J., Groh, K., Hull, J., and Grdina, D. (Abstract) Changes in gene expression associated with EMF exposure. Presented at DOE Contractor’s Review, 1994. Woloschak, G.E., Panozzo, J., Paunesku, T., Chang-Liu, C.-M., Groh, K.R., and Hull, J. (Abstract) 60-Hz EMF expsoure induces expression of LINEs. The 1995 Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery & Use of Electricity, Submitted, 1995. |
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