In endothelial cells, the effect of electromagnetic fields on several steps of HSP70 gene expression process (i.e. heat-shock transcription factor 1 (HSF1) activation, gene transcription, mRNA accumulation and HSP70 neosynthesis) and the effect of combined exposure to heat shock (for 30 min at 44°C) and electromagnetic fields were also analyzed. Endothelial cells were transfected by electroporation (with phsplacZ and pEGFP-C1 vectors) to evaluate the HSP70 promotor activity of the transfected gene by an assay of beta-galactosidase.
Since HSP70 is a long-lived protein, and since cytoplasmic proteins are mostly degraded through the ubiquitin/proteasome pathway, its potential stabilization was indirectly measured by proteasome avtivity in cell extracts.
|Setup||Four petri dishes were exposed simultaneously by inserting them in the solenoid. The solenoid and the sample holder resulted in a 38 cm high x 10 cm wide plexiglass chamber. The whole setup was placed in an incubator maintained at 37°C. Control samples were not exposed to MF and were incubated in the same incubator in a position where the environmental magnetic field was minimum (2 µT).|
|Additional info||In some experiments, cells were exposed to heat stress for 20 or 30 min at 44°C in a water bath.|
Some cell types (endothelial cells and lymphoma/leukemia cells) showed increased levels of HSP70 protein when exposed for 24 h to 50 Hz, 680 µT electromagnetic fields.
In endothelial cells, electromagnetic fields alone induced only a poor and transient activation of HSF1; however, neither the level of HSP70 mRNA nor the synthesis of HSP70 appeared to be altered significantly.
Transfection experiments involving HSP70 promoter showed that gene transcription was not affected. A marked decrease in proteasome activities in exposed cell extracts was also noted.
The heat-shock-induced levels of HSP70 mRNA and protein were increased by a concomitant weak stressor like electromagnetic fields.
In conslusion the data indicate that in electromagnetic field-exposed endothelial cells, HSP70 gene transcription and translation are unaffected; however, electromagnetic fields alone promoted accumulation of the inducible HSP70 protein, probably by increasing its stability, and it enhanced accumulation and translation of the heat-induced HSP70 mRNA when applied in concert with heat shock.