Field exposure to 50 Hz significantly affects wild-type and unfolded p53 expression in NB69 neuroblastoma cells

Abstract

Previous studies have shown that intermittent exposure to a 50 Hz, 100 µT sinusoidal magnetic field (MF) promotes proliferation of human neuroblastoma cells, NB69. This effect is mediated by activation of the epidermal growth factor receptor through a free radical-dependent activation of the p38 pathway. The present study investigated the possibility that the oxidative stress-sensitive protein p53 is a potential target of the MF, and that field exposure can affect the protein expression. To that end, NB69 cells were exposed to short intervals of 30 to 120 min to the aforementioned MF parameters. Two specific anti-p53 antibodies that allow discrimination between the wild and unfolded forms of p53 were used to study the expression and cellular distribution of both isoforms of the protein. The expression of the antiapoptotic protein Bcl-2, whose regulation is mediated by p53, was also analyzed. The obtained results revealed that MF exposure induced increases in p53 gene expression and in protein expression of the wild-type form of p53. Field exposure also caused overexpression of the unfolded form of p53, together with changes in the nuclear/cytoplasmic distribution of both forms of the protein. The expression of protein Bcl-2 was also significantly increased in response to the MF. As a whole, these results indicated that the MF is capable of interacting with the function, distribution and conformation of protein p53. Such interactions could be involved in previously reported MF effects on NB69 proliferation promotion.

Keywords: Bcl-2; NB69; extremely low frequency; magnetic fields; p53.

Figure 1.

Expression of p53 protein at various MF exposure intervals and p53 mRNA after 90-min MF exposure. (A) Western blot quantification of p53. The data, normalized over the corresponding sham-exposed controls, are the means ± SEM of 4 experimental replicates per interval, with 8 samples (4 MF-exposed and 4 sham-exposed controls) per replicate. *P<0.05 and **P<0.01 (unpaired Student's t-test). (B) Representative blots of p53 at the various exposure (MF +) or sham-exposure (MF -) intervals, using β-actin as loading control. (C) RT-qPCR quantification of p53 mRNA expression using the GAPDH housekeeping gene as a reference. The data, normalized over the corresponding controls are the means ± SEM of 3 experimental replicates with 6 samples (3MF- and 3 sham-exposed per replicate). *P<0.05 and **P<0.01 (one-way ANOVA and Bonferroni post hoc test). MF, magnetic field; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

Immunocytochemical analysis of the effects of a MF on the number of cells expressing p53 (p53+). (A) Percentage of p53+ cells after 90-min or 120-min exposure. The data, normalized over the corresponding controls, are the means ± SEM of 4 experimental replicates per exposure interval, with 3 MF- and 3 sham-exposed samples per replicate and 15 microscopic fields analyzed per sample; ****P<0.0001 (unpaired Student's t-test); (****)P<0.0001 (one-way ANOVA and Bonferroni post hoc test). (B) Representative images of p53 expression. Upper panels: Cell nuclei were stained blue with Hoechst (yellow arrow); middle panels: p53 expression (red labeling, white arrow); lower panels: Merged images. Magnification, ×400. MF, magnetic field.

Figure 3.

Effects of 120-min of MF exposure on the number of cells expressing p53. (A) The effects of a MF on the number of wt p53+ or unfolded p53+ cells. (B) The effect of a MF on the nuclear/cytoplasmic distribution of wt p53 labeling. (C) The effect of a MF on the nuclear/cytoplasmic distribution of the specific antigen for unfolded p53. The data, normalized over the corresponding controls, are the means ± SEM of 4 experimental replicates with 3 MF- and 3 Sham-exposed samples per replicate. **P<0.01 and ***P<0.001 (unpaired Student's t-test). (D and E) Representative images of wt p53 and unfolded p53 expression. Upper panels: Cell nuclei were stained blue with Hoechst (yellow arrow); middle panels: p53 expression (red labeling, white arrow); lower panels: Merged images. Magnification, ×400. MF, magnetic field; wt, wild-type.

Figure 4.

Effects of a MF on Bcl-2 expression. (A) Immunoblot analysis of Bcl-2 expression after 30, 60, 90 or 120 min of MF exposure. Data, normalized over controls, are the means ± SEM of 4 experimental replicates per exposure time, with 3 MF- and 3 sham-exposed control samples per replicate. (B) Representative blots for each of the tested intervals; β-actin was used as loading control. (C) Immunocytochemical analysis of the number of Bcl-2+ cells after 30, 60, 90 or 120 min of MF exposure. Each point represents the mean ± SEM of 4 experimental replicates, with 3 MF- and 3 sham-exposed coverslips per replicate. A total of 15 microscopic fields per coverslips were analyzed. (D) Representative images (magnification ×400) of the expression of the antiapoptotic protein Bcl-2 (brown labeling). Nuclei were counterstained with methyl green. *P<0.05, **P<0.01 and ***P<0.001 (unpaired Student's t-test). MF, magnetic field.