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. 2013;8(1):e54775.
doi: 10.1371/journal.pone.0054775. Epub 2013 Jan 23.

Magnetic shielding accelerates the proliferation of human neuroblastoma cell by promoting G1-phase progression

Wei-chuan Mo  1 , Zi-jian Zhang, Ying Liu, Perry F Bartlett, Rong-qiao He
Affiliations

Magnetic shielding accelerates the proliferation of human neuroblastoma cell by promoting G1-phase progression

Wei-chuan Mo et al. PLoS One. 2013.

Abstract

Organisms have been exposed to the geomagnetic field (GMF) throughout evolutionary history. Exposure to the hypomagnetic field (HMF) by deep magnetic shielding has recently been suggested to have a negative effect on the structure and function of the central nervous system, particularly during early development. Although changes in cell growth and differentiation have been observed in the HMF, the effects of the HMF on cell cycle progression still remain unclear. Here we show that continuous HMF exposure significantly increases the proliferation of human neuroblastoma (SH-SY5Y) cells. The acceleration of proliferation results from a forward shift of the cell cycle in G1-phase. The G2/M-phase progression is not affected in the HMF. Our data is the first to demonstrate that the HMF can stimulate the proliferation of SH-SY5Y cells by promoting cell cycle progression in the G1-phase. This provides a novel way to study the mechanism of cells in response to changes of environmental magnetic field including the GMF.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The geomagnetic shielding system for cell culture.
A magnetic shielding box was contained in a cell culture incubator. GMF control cells were incubated on the bottom layer of a steel shelf beside the magnetic shielding box. Two fans were installed on the top of the box to facilitate the exchange of gas and temperature between the chambers of the magnetic shielded box and the cell culture incubator.
Figure 2
Figure 2. The morphology and density of cells in the HMF.
Bright-field images of SH-SY5Y cells seeded at densities of 1.0×104/cm2, 2.0×104/cm2, and 3.0×104/cm2 in 96-well plate after 48 h incubation in the HMF and GMF. Bar = 100 µm.
Figure 3
Figure 3. The proliferation of SH-SY5Y cells was accelerated in the HMF.
A: Cell proliferation assay by CCK-8 kit corresponding to the treatments in (Figure 2) (n = 6). B: Cells were seeded at 2.0×104/cm2 in 6-well plates and cell proliferation was measured by crystal violet staining after 48 h incubation in the GMF and HMF (n = 6). C: Cells were seeded at 2.0×104/cm2 in 60 mm petri dishes and incubated for 48 h in the GMF and HMF. The numbers of SH-SY5Y cells were measured at day 1, day 2, and day 3 by hematocytometery (n = 3). D: Cells were seeded at 1.5×104 cells/cm2 in 96-well plates. Cell proliferation was measured after 48 h incubation in the reference field (GMF’), in the GMF control shelf (GMF), and in the HMF (n = 6). Error bar = s.d.; n = 3; *p<0.05; **p<0.01.
Figure 4
Figure 4. Cell division increases after 48 h HMF exposure.
SH-SY5Y cells were stained with 25 µM CFSE and incubated for 48 h in the HMF and GMF. A: The fluorescence intensities measured by flow cytometry. CFSE-unlabeled cells were the blank control (grey). CFSE-labeled cells collected immediately after staining (0 h) were the positive control (blue). CFSE-labeled cells incubated in DMEM with 0.5% FBS were the low-proliferation control (pink). B: The geometry means of the CFSE fluorescence. Error bar = s.d.; n = 3; **p<0.01.
Figure 5
Figure 5. The growth curves of SH-SY5Y cells at different seeding densities.
SH-SY5Y cells were seeded in 96-well plates at densities of (A) 0.15×104 cells/cm2, (B) 0.3×104 cells/cm2, (C) 1.5×104 cells/cm2, and (D) 3.0×104 cells/cm2. Cell proliferation was measured by CCK-8 assay each day throughout the incubation period. Error bar = s.d.; n = 6; *, p<0.05; **, p<0.01.
Figure 6
Figure 6. The effect of HMF on cell proliferation depends on the concentration of FBS.
SH-SY5Y cells were seeded in 96-well plates at a density of 1.0×104 cells/cm2. Cells were incubated in cell culture medium containing different concentrations of FBS (0%, 0.1%, 0.25%, 0.5%, 1%, 2.5%, 5%, and 10%). Cell proliferation was measured using the CCK-8 assay after 48 h incubation in the HMF and GMF. Error bar = s.d.; n = 6; *, p<0.05; **, p<0.01.
Figure 7
Figure 7. Cell cycle progression of SH-SY5Y cells was altered in the HMF.
Cell samples were collected at 4 h intervals from 8 h to 52 h. The DNA content of SH-SY5Y cells was determined by flow cytometry with propidium iodide (PI) staining. The percentage of cells at (A) G1-phase, (B) S-phase, and (C) G2/M-phase was measured. Error bar = s.d.; n = 3; *p<0.05; **p<0.01.
Figure 8
Figure 8. G1-phase progression of SH-SY5Y cells was stimulated in the HMF. A–B:
Cells were synchronized at G1-phase by serum starvation before being harvested and seeded in 60 mm petri dishes at a density of 3.5×104 cells/cm2. In panel A, the percentage of G1-phase cells was plotted for G1-arrested cells released from 0 h to 18 h. In panel B, G1-phase SH-SY5Y cells were released under four incubation modes: GMF (16 h), HMF (4 h)+GMF (12 h), HMF (8 h)+GMF (8 h), or HMF (16 h). Black blocks indicate incubation periods in the GMF; white blocks indicate incubation periods in the HMF. C: Cells were synchronized at the G1/S border phase by thymidine double block. Blocked G1/S border cells were released for 4 h. Cells were harvested at 2 h and 4 h. D: Cells were synchronized at M-phase by nocodazol treatment before being seeded and released in either the HMF or GMF in 6-well plates at a density of 1.0×104 cells/cm2. Cells were harvested after 4 h incubation. DNA content was determined by flow cytometry with PI staining. Error bar = s.d.; n = 3; *p<0.05; **p<0.01.
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Grants and funding

This work was supported by the Queensland-Chinese Academy of Sciences Biotechnology Fund (Grant No. GJHZ1131), the project of Chinese Academy of Sciences for the development of major scientific research equipment (Grant No. YZ201148), and the National Nature Science Foundation of China (Grant No. 31200628). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.