Bio-Medical Materials and Engineering 24 (2014) 1391–1397 DOI 10.3233/BME-130943 IOS Press
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Biochemical kinetics of cell proliferation regulated by extremely low frequency electromagnetic field D. Y. Geng*, C. H. Li, X. W. Wan and G. Z. Xu Province-Ministry Joint Key Laboratory of Electromagnetic Field and Electrical Apparatus Reliability, Hebei University of Technology, Tianjin, China
Abstract. To study the mechanism of cells subjected to external electromagnetic fields, the expression of cyclin kinase inhibitor p27 is analyzed in the four cell cycle phases. The regulatory functions are investigated in gap phase1 to synthesis, gap phase 2 to mitotic phase and post mitotic phase transition in the mammalian cell cycle processes. A mathematical model is developed to meet the general cell cycle regulatory network based on the molecular dynamics method. Phase plane analysis results show that the p27 over-expression can lead to the hysteresis effect of cell cycle processes and phase transition delay. It is an universal approach to predict the key regulatory gene in signal transduction pathway. Keywords: Electromagnetic fields, Mathematical model, Molecular dynamics method, Cell cycle, Phase plane analysis
1. Introduction Biological effects of extremely low frequency electromagnetic fields (ELF-EMFs) and their possible consequences upon human health began to arise more and more scientific attention and became a recurrent subject of public debate [1]. There have been a lot of discussions about the response of biological system to ELF-EMFs exposure [2-5]. Although a large number of in vivo and in vitro studies had been done, the results were inconsistent [6,7]. Data on the effects of ELF-EMFs are inconsistent probably due to the differences in exposure levels, exposure durations, genotoxicity endpoints and other experimental variables. Therefore, in vivo and in vitro studies have concentrated on responses to ELF-EMFs at molecular level such as cell proliferation and other processes required for cell growth and division [8, 9]. In previous works, studies have been focused on whether the electromagnetic field at ground level beneath ultra high voltage power lines could effect on testis tissue in mice at a molecular level. The results indicated that long-term exposure to controllable ELFEMFs environment produced by actual power transmission lines would decrease the S phase fraction and the Proliferation Index of testis germ cells in BALB/c mice [10]. Conversely, Trillo et al [11] and Martinez et al [12] have reported that intermittent exposure to weak 50Hz electromagnetic fields at the intensity of 10T and 100T respectively enhances proliferation in human neuroblastoma NB69 cells. *
Corresponding author. E-mail: [email protected].
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1392
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
It is well known that these fields can neither denature proteins nor damage cellular macromolecules directly. Therefore, the mechanism of ELF-EMFs induced biological reactions is likely to involve new molecular mechanisms which may change the current view on ELF-EMFs. A number of studies [13, 14] have reported a potential relationship between the exposure to ELFEMFs and the expression of signal transduction molecules. The cell cycle proteins related to cell proliferation are investigated to identify and characterize the cellular processes involved in the proliferative effects of ELF-EMFs [15, 16]. The regulation of cell proliferation is a central issue in the life of complex multicellular organisms. Nevertheless, due in part to the complexity of molecular control networks dictating the division, metabolic or genotoxic stress of cells in particular tissues, it is still unclear which regulation factors and pathways could be related to the changes of cell proliferation. Any changes of the activity of biomolecules in cell cycle regulation would lead to different cell result, so how to verify key components of the signal pathways induced by ELF-EMFs become a common issue. The aim of this study is to develop a numerical simulation for conveniently determining the key components to regulate cell proliferation. It will be contributable to understand the processes of cell proliferation inhibition and its molecular mechanisms. 2. Molecular biological mechanisms Cell cycle is the period of time during which a cell doubles its genetic content and distributes itself to two daughter cells. In most cells, this period is coupled to the duplication of other cell contents and cells can divide only after doubling their cell mass, which creates special phases into the cell cycle. Cell proliferation is the process when cells reproduce themselves by growing and then dividing into two equal copies. Growth factors employ a range of growth factor signal pathways to activate cells to enter the cell cycle. To understand how growth factors control cell proliferation, both the nature of the signal mechanisms and how they impinge upon the cell cycle machine that regulates cell growth and cell division should be considered. Cell cycle signal is responsible for controlling the orderly sequence of events occurring when a cell is stimulated to grow and divide. The interface between the growth factor signal pathways and the cell cycle signal system is critical in controlling cell proliferation. The main features of the signal system are the dynamics of cyclin and its dependent kinase (Cyclin-CDK) expression that occurs during the course of the cell cycle, as shown in Fig. 1 [17].
Fig. 1. Changes in the level of different cyclin isoforms and p27 during the course of the cell cycle
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
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Although Cyclin-CDK expression data have extremely high dimensionality [18] and the signal transduction pathways affected by ELF-EMF are more than one, the final result can be attributed to the dynamics of growth factors which depend upon the up-regulation of the cyclin-dependent kinase (CDK) inhibitor p27 which inhibits cyclin E and cyclin A. 3. Mathematical model and simulation 3.1. Wiring diagram of cell cycle regulatory network Early in 1981, Goldberer et al. [19] studied the behavior of reversible covalent modification system at transient and steady state through mathematical method. After more than 20 years, Attila et al. developed a model which described protein activation and inactivation of general cell cycle transition regulation network based on the more complex regulatory factor, as shown in Fig. 2 [20].
Fig. 2. Wiring diagram of the generic cell cycle regulatory network
Here, all major regulatory interactions have been proposed for different eukaryotic organisms. Solid lines represent reactions, dashed lines regulatory interactions, and a protein sitting on a reaction arrow
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D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
represents activation. Regulatory modules of the system are distinguished by colored backgrounds, in which numbers 1 to 13 describe the exits of M module, Cdh1 module, CycB transcription factor, CycB synthesis/degradation, G2 module, CycB inhibiton by CKI, CKI transcription factor, CKI synthesis/degradation, CycE inhibition by CKI, CycE synthesis/degradation, CycE/A transcription factor, CycA inhibition by CKI, CycA synthesis/degradation respectively . 3.2. Mathematical model of cell cycle engine The molecular mechanism on the wiring diagram Fig. 2, is the representation of the suggested biochemical reactions among the regulatory proteins of a general cell cycle engine. The chemical reactions of the interacting molecules can be translated into differential equations by using the principles of biochemical kinetics. All the parameters and values are described in [17].
dactCycA = (k sap + k sapp ⋅ TFE ) ⋅ mass + (Vdi + k dia ) ⋅ Tri A − (Vda + k asa ⋅ freeCKI ) ⋅ actCycA (1) dt dactCycB = Vsb ⋅ mass + V25 ⋅ (CycB − TriB − actCycB ) + (2) dt (k dib + Vdi ) ⋅ (CycB − preMPF − actCycB ) − (Vdb + Vwee + k asb ⋅ freeCKI ) ⋅ actCycB
dactCycE = (k sep + k sepp ⋅ TFE ) ⋅ mass + (Vdi + k die ) ⋅ TriE − (Vde + Vase ⋅ freeCKI ) ⋅ actCycE (3) dt dCycA = (k sap + k sapp ⋅ TFE ) ⋅ mass − Vda ⋅ CycA dt
(4)
dCycB = Vsb ⋅ mass − Vdb ⋅ CycB dt
(5)
dCycE = (k sep + k sepp ⋅ TFE ) ⋅ mass − Vde ⋅ CycE dt
(6)
dmass = μ ⋅ mass dt
(7)
3.3. Phase-plane analysis and numerical analysis Arbitrary units (au) are used for concentration unit in all equations while the actual concentrations of most of the regulatory proteins in the cell are still unknown. The simulation analysis of the differential equation can be simulated through the Winpp software. The mammalian cell cycle trajectory in Fig. 3 is computed for cells proliferation with mass doubling time 24 hours.
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
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Fig. 3. Numerical simulation of mammalian cell cycle model
The cell cycle is a process in which one cell becomes two cells. Any abnormal expression of some regulation factors could cause cell proliferation arrest. For example, Patruno A et al [21] reported that the exposure of HaCaT cells to ELF-EMF could increase iNOS and eNOS expression levels, and eNOS gene inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis. In fact, ELFEMF is a mild oxidative stressor and DNA damage inducer [22]. Exposure to ELF-EMF may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentration which down-regulates the levels of p21 and p27 [23]. As a result, the expression of protein p27 in a cell cycle phase can be investigated to obtain the quantitative relationship between the levels of p27 and cell proliferation based on biochemical kinetics, as shown in Fig. 4.
Fig. 4. P27 over-expression on the cell cycle
Different levels of p27 expression will lead to the different changes of cell proliferation and the course of time phase, as shown in Table 1 which indicates that, over-expression of p27 could prolong the time of each cell cycle phase, especially G1 and S phase induced by Cyclin E-CDK, when the interphase transfer is slowing down or inhibited.
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D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
Table 1 P27 over-expression on change rates of cyclin-CDK dimers Cyclin-CDK dimers Cyclin A-CDK Cyclin B-CDK Cyclin D-CDK Cyclin E-CDK
P27 expression 1.698 3.439 0.117 1.130
P27 over-expression (100%) 1.784 3.482 0.124 1.532
Change rates (%) 5.064 1.250 5.983 35.575
4. Conclusion In this paper a simulation method based on biochemical kinetics has been developed for study on the signal transduction mechanism for changes of cell proliferation activity induced by ELF-EMF. The method combines with molecular biology to predict the key regulatory factors affected by external electromagnetic fields, which can help simplify the experimental data processing in vivo and in vitro. Although a regulation factor p27 inhibiting cell proliferation is investigated, other factors regulated cell proliferation can be screened out in accordance with the method. Moreover, the method can also be incorporated into further studies on the signal transduction pathways for drug intervention or EMF exposure, so as to simplify the experimental process in the way of immunocyte chemistry, Western blot and proteomic analysis, etc. 5. Acknowledgement Project supported by the Natural Science Foundation of Hebei Province (No. E2012202012) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20121317120003). References [1] [2]
[3] [4] [5] [6]
[7] [8]
G. Chen and Z. Xu, Global protein expression in response to extremely low frequency magnetic fields, Adv Exp Med Biol 990(2013), 107–110. S. Das, S. Kumar, S. Jain, V. D. Avelev and R. Mathur, Exposure to ELF- magnetic field promotes restoration of sensori-motor functions in adult rats with hemisection of thoracic spinal cord, Electromagn Biol Med 31(2012), 180– 194. J. kim, C. S. Ha, H. J. Lee and K. Song, Repetitive exposure to a 60-Hz time-varying magnetic field induces DNA double-strand breaks and apoptosis in human cells, Biochem Biophys Res Commun 400 (2010), 739–744. P. D. Bayat, A. Ghanbari, B. Saeid, M. Khazaei, R. Ghorbani and M. Ayubian, Effect of exposure to extremely low electro-magnetic field during prenatal period on mice spleen, Indian J Exp Biol 49(2011), 634–638. E. Foroozandeh, P. Derakhshan-Barjoei and M. Jadidi, Toxic effects of 50 Hz electromagnetic field on memory consolidation in male and female mice, Electromagn Biol Med 29(2013), 293–299. M. Zhang, X. Li, L. Bai, K. Uchida, W. Bai, B. Wu, W. Xu, H. Zhu and H. Huang, Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study, Bioelectromagnetics 34(2013), 74–80. I. Salehi, K. G. Sani and A. Zamani, Exposure of rats to extremely low-frequency electromagnetic fields (ELF-EMF) alters cytokines production, Electromagn Biol Med 32(2013), 1–8. M. Bouwens, S. de Kleij, G. Ferwerda, J. J. Cuppen, H. F. Savelkoul and B. M. Kemenade, Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines, Bioelectromagnetics 33(2012), 226–237.
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
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D. Hao, L. Yang, S. Chen, J. Tong, Y. Tian, B. Su, S. Wu and Y. Zeng, Effects of long-term electromagnetic field exposure on spatial learning and memory in rats, Neurol Sci 34(2013), 157–164. D. Y. Geng, G. Z. Xu, X. H. Zhang, L.X. Xing, H. Y. Guo, W. L. Yan and Y. H. Wang, Effects of Electromagnetic Field of UHV Transmission Lines Exposure on Testis Tissue in Mice, IEEE Transactions on Magnetics 45(2009), 4853 – 4856. María Ángeles Trillo, María Antonia Martínez, María Antonia Cid, Jocelyne Leal and Alejandro Úbeda, Influence of a 50 Hz magnetic field and of all-trans̻retinol on the proliferation of human cancer cell lines, International Journal of Oncology 40(2012), 1405–1413. Trillo MÁ, Martínez MA, Cid MA and Úbeda A, Retinoic acid inhibits the cytoproliferative response to weak 50̻Hz magnetic fields in neuroblastoma cells, ONCOLOGY REPORTS 29(2013), 885–894. M. Sulpizio, S. Falone, F. Amicarelli, M. Marchisio, F. Di Giuseppe, E. Eleuterio, C. Di Ilio and S. Angelucci, Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells, J Cell Biochem 112(2011), 3797–3806. L. Potenza, R. Saltarelli, E. Polidori, P. Ceccaroli, A. Amicucci, S. Zeppa, A, Zambonelli and V. Stocchi, Effect of 300 mT static and 50 Hz 0.1 mT extremely low frequency magnetic fields on Tuber borchii mycelium, Can J Microbio 58(2012), 1174–1182. V. Martirosyan, N. Baghdasaryan and S. Ayrapetyan, Bidirectional frequency-dependent effect of extremely lowfrequency electromagnetic field on E. coli K-12, Electromagn Biol Med 2012. A. Inhan-Garip, B. Aksu, Z. Akan, D. Akakin, A. N. Ozaydin and T. San, Effect of extremely low frequency electromagnetic fields on growth rate and morphology of bacteria, Int J Radiat Biol 87(2011), 1155–1161. B. Novak and J. J. Tyson, A model for restriction point control of the mammalian cell cycle, Journal of Theoretical Biology 230(2004), 563–579. Zahra Shaeiri and Reza Ghaderi, Genetic diagnosis of cancer by fuzzy–rough gene selection and the complementary hierarchical fuzzy classifier, Biomed Mater Eng 21(2011), 37–52. A. Goldbeter and D. E. Jr Koshland, An amplified sensitivity arising from covalent modification in biological systems, Proc. Natl. Acad. Sci. USA 78(1981), 6840–6844. Attila Csika´ sz-Nagy, Dorjsuren Battogtokh, Katherine C. Chen, Be´ la Nova´ k and John J. Tyson, Analysis of a Generic Model of Eukaryotic Cell-Cycle Regulation 90(2006), 4361–4379. A Patruno, M Pesce, G Vianale, S Di Luzio, A Tulli, S Franceschelli, A Grilli, R Muraro and M Reale, Extremely low frequency electromagnetic fields modulate expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and cyclooxygenase-2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing, Br J Dermatol 162(2010), 258–266. RJ Budak, R Polaniak, L Budak, K Zwirska-Korczala, M Skonieczna, A Monsiol, M Kukla, A Duawa-Budak and E Birkner, Short-term exposure to 50ԜHz ELF-EMF alters the cisplatin-induced oxidative response in AT478 murine squamous cell carcinoma cells, Bioelectromagnetics 33(2012), 641–651. JE Park, YK Seo, HH Yoon, CW Kim, JK Park and S Jeon, Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation, Neurochem Int 62(2013), 418–424.
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Biochemical kinetics of cell proliferation regulated by extremely low frequency electromagnetic field D. Y. Geng*, C. H. Li, X. W. Wan and G. Z. Xu Province-Ministry Joint Key Laboratory of Electromagnetic Field and Electrical Apparatus Reliability, Hebei University of Technology, Tianjin, China
Abstract. To study the mechanism of cells subjected to external electromagnetic fields, the expression of cyclin kinase inhibitor p27 is analyzed in the four cell cycle phases. The regulatory functions are investigated in gap phase1 to synthesis, gap phase 2 to mitotic phase and post mitotic phase transition in the mammalian cell cycle processes. A mathematical model is developed to meet the general cell cycle regulatory network based on the molecular dynamics method. Phase plane analysis results show that the p27 over-expression can lead to the hysteresis effect of cell cycle processes and phase transition delay. It is an universal approach to predict the key regulatory gene in signal transduction pathway. Keywords: Electromagnetic fields, Mathematical model, Molecular dynamics method, Cell cycle, Phase plane analysis
1. Introduction Biological effects of extremely low frequency electromagnetic fields (ELF-EMFs) and their possible consequences upon human health began to arise more and more scientific attention and became a recurrent subject of public debate [1]. There have been a lot of discussions about the response of biological system to ELF-EMFs exposure [2-5]. Although a large number of in vivo and in vitro studies had been done, the results were inconsistent [6,7]. Data on the effects of ELF-EMFs are inconsistent probably due to the differences in exposure levels, exposure durations, genotoxicity endpoints and other experimental variables. Therefore, in vivo and in vitro studies have concentrated on responses to ELF-EMFs at molecular level such as cell proliferation and other processes required for cell growth and division [8, 9]. In previous works, studies have been focused on whether the electromagnetic field at ground level beneath ultra high voltage power lines could effect on testis tissue in mice at a molecular level. The results indicated that long-term exposure to controllable ELFEMFs environment produced by actual power transmission lines would decrease the S phase fraction and the Proliferation Index of testis germ cells in BALB/c mice [10]. Conversely, Trillo et al [11] and Martinez et al [12] have reported that intermittent exposure to weak 50Hz electromagnetic fields at the intensity of 10T and 100T respectively enhances proliferation in human neuroblastoma NB69 cells. *
Corresponding author. E-mail: [email protected].
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1392
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
It is well known that these fields can neither denature proteins nor damage cellular macromolecules directly. Therefore, the mechanism of ELF-EMFs induced biological reactions is likely to involve new molecular mechanisms which may change the current view on ELF-EMFs. A number of studies [13, 14] have reported a potential relationship between the exposure to ELFEMFs and the expression of signal transduction molecules. The cell cycle proteins related to cell proliferation are investigated to identify and characterize the cellular processes involved in the proliferative effects of ELF-EMFs [15, 16]. The regulation of cell proliferation is a central issue in the life of complex multicellular organisms. Nevertheless, due in part to the complexity of molecular control networks dictating the division, metabolic or genotoxic stress of cells in particular tissues, it is still unclear which regulation factors and pathways could be related to the changes of cell proliferation. Any changes of the activity of biomolecules in cell cycle regulation would lead to different cell result, so how to verify key components of the signal pathways induced by ELF-EMFs become a common issue. The aim of this study is to develop a numerical simulation for conveniently determining the key components to regulate cell proliferation. It will be contributable to understand the processes of cell proliferation inhibition and its molecular mechanisms. 2. Molecular biological mechanisms Cell cycle is the period of time during which a cell doubles its genetic content and distributes itself to two daughter cells. In most cells, this period is coupled to the duplication of other cell contents and cells can divide only after doubling their cell mass, which creates special phases into the cell cycle. Cell proliferation is the process when cells reproduce themselves by growing and then dividing into two equal copies. Growth factors employ a range of growth factor signal pathways to activate cells to enter the cell cycle. To understand how growth factors control cell proliferation, both the nature of the signal mechanisms and how they impinge upon the cell cycle machine that regulates cell growth and cell division should be considered. Cell cycle signal is responsible for controlling the orderly sequence of events occurring when a cell is stimulated to grow and divide. The interface between the growth factor signal pathways and the cell cycle signal system is critical in controlling cell proliferation. The main features of the signal system are the dynamics of cyclin and its dependent kinase (Cyclin-CDK) expression that occurs during the course of the cell cycle, as shown in Fig. 1 [17].
Fig. 1. Changes in the level of different cyclin isoforms and p27 during the course of the cell cycle
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
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Although Cyclin-CDK expression data have extremely high dimensionality [18] and the signal transduction pathways affected by ELF-EMF are more than one, the final result can be attributed to the dynamics of growth factors which depend upon the up-regulation of the cyclin-dependent kinase (CDK) inhibitor p27 which inhibits cyclin E and cyclin A. 3. Mathematical model and simulation 3.1. Wiring diagram of cell cycle regulatory network Early in 1981, Goldberer et al. [19] studied the behavior of reversible covalent modification system at transient and steady state through mathematical method. After more than 20 years, Attila et al. developed a model which described protein activation and inactivation of general cell cycle transition regulation network based on the more complex regulatory factor, as shown in Fig. 2 [20].
Fig. 2. Wiring diagram of the generic cell cycle regulatory network
Here, all major regulatory interactions have been proposed for different eukaryotic organisms. Solid lines represent reactions, dashed lines regulatory interactions, and a protein sitting on a reaction arrow
1394
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
represents activation. Regulatory modules of the system are distinguished by colored backgrounds, in which numbers 1 to 13 describe the exits of M module, Cdh1 module, CycB transcription factor, CycB synthesis/degradation, G2 module, CycB inhibiton by CKI, CKI transcription factor, CKI synthesis/degradation, CycE inhibition by CKI, CycE synthesis/degradation, CycE/A transcription factor, CycA inhibition by CKI, CycA synthesis/degradation respectively . 3.2. Mathematical model of cell cycle engine The molecular mechanism on the wiring diagram Fig. 2, is the representation of the suggested biochemical reactions among the regulatory proteins of a general cell cycle engine. The chemical reactions of the interacting molecules can be translated into differential equations by using the principles of biochemical kinetics. All the parameters and values are described in [17].
dactCycA = (k sap + k sapp ⋅ TFE ) ⋅ mass + (Vdi + k dia ) ⋅ Tri A − (Vda + k asa ⋅ freeCKI ) ⋅ actCycA (1) dt dactCycB = Vsb ⋅ mass + V25 ⋅ (CycB − TriB − actCycB ) + (2) dt (k dib + Vdi ) ⋅ (CycB − preMPF − actCycB ) − (Vdb + Vwee + k asb ⋅ freeCKI ) ⋅ actCycB
dactCycE = (k sep + k sepp ⋅ TFE ) ⋅ mass + (Vdi + k die ) ⋅ TriE − (Vde + Vase ⋅ freeCKI ) ⋅ actCycE (3) dt dCycA = (k sap + k sapp ⋅ TFE ) ⋅ mass − Vda ⋅ CycA dt
(4)
dCycB = Vsb ⋅ mass − Vdb ⋅ CycB dt
(5)
dCycE = (k sep + k sepp ⋅ TFE ) ⋅ mass − Vde ⋅ CycE dt
(6)
dmass = μ ⋅ mass dt
(7)
3.3. Phase-plane analysis and numerical analysis Arbitrary units (au) are used for concentration unit in all equations while the actual concentrations of most of the regulatory proteins in the cell are still unknown. The simulation analysis of the differential equation can be simulated through the Winpp software. The mammalian cell cycle trajectory in Fig. 3 is computed for cells proliferation with mass doubling time 24 hours.
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
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Fig. 3. Numerical simulation of mammalian cell cycle model
The cell cycle is a process in which one cell becomes two cells. Any abnormal expression of some regulation factors could cause cell proliferation arrest. For example, Patruno A et al [21] reported that the exposure of HaCaT cells to ELF-EMF could increase iNOS and eNOS expression levels, and eNOS gene inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis. In fact, ELFEMF is a mild oxidative stressor and DNA damage inducer [22]. Exposure to ELF-EMF may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentration which down-regulates the levels of p21 and p27 [23]. As a result, the expression of protein p27 in a cell cycle phase can be investigated to obtain the quantitative relationship between the levels of p27 and cell proliferation based on biochemical kinetics, as shown in Fig. 4.
Fig. 4. P27 over-expression on the cell cycle
Different levels of p27 expression will lead to the different changes of cell proliferation and the course of time phase, as shown in Table 1 which indicates that, over-expression of p27 could prolong the time of each cell cycle phase, especially G1 and S phase induced by Cyclin E-CDK, when the interphase transfer is slowing down or inhibited.
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D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
Table 1 P27 over-expression on change rates of cyclin-CDK dimers Cyclin-CDK dimers Cyclin A-CDK Cyclin B-CDK Cyclin D-CDK Cyclin E-CDK
P27 expression 1.698 3.439 0.117 1.130
P27 over-expression (100%) 1.784 3.482 0.124 1.532
Change rates (%) 5.064 1.250 5.983 35.575
4. Conclusion In this paper a simulation method based on biochemical kinetics has been developed for study on the signal transduction mechanism for changes of cell proliferation activity induced by ELF-EMF. The method combines with molecular biology to predict the key regulatory factors affected by external electromagnetic fields, which can help simplify the experimental data processing in vivo and in vitro. Although a regulation factor p27 inhibiting cell proliferation is investigated, other factors regulated cell proliferation can be screened out in accordance with the method. Moreover, the method can also be incorporated into further studies on the signal transduction pathways for drug intervention or EMF exposure, so as to simplify the experimental process in the way of immunocyte chemistry, Western blot and proteomic analysis, etc. 5. Acknowledgement Project supported by the Natural Science Foundation of Hebei Province (No. E2012202012) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20121317120003). References [1] [2]
[3] [4] [5] [6]
[7] [8]
G. Chen and Z. Xu, Global protein expression in response to extremely low frequency magnetic fields, Adv Exp Med Biol 990(2013), 107–110. S. Das, S. Kumar, S. Jain, V. D. Avelev and R. Mathur, Exposure to ELF- magnetic field promotes restoration of sensori-motor functions in adult rats with hemisection of thoracic spinal cord, Electromagn Biol Med 31(2012), 180– 194. J. kim, C. S. Ha, H. J. Lee and K. Song, Repetitive exposure to a 60-Hz time-varying magnetic field induces DNA double-strand breaks and apoptosis in human cells, Biochem Biophys Res Commun 400 (2010), 739–744. P. D. Bayat, A. Ghanbari, B. Saeid, M. Khazaei, R. Ghorbani and M. Ayubian, Effect of exposure to extremely low electro-magnetic field during prenatal period on mice spleen, Indian J Exp Biol 49(2011), 634–638. E. Foroozandeh, P. Derakhshan-Barjoei and M. Jadidi, Toxic effects of 50 Hz electromagnetic field on memory consolidation in male and female mice, Electromagn Biol Med 29(2013), 293–299. M. Zhang, X. Li, L. Bai, K. Uchida, W. Bai, B. Wu, W. Xu, H. Zhu and H. Huang, Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study, Bioelectromagnetics 34(2013), 74–80. I. Salehi, K. G. Sani and A. Zamani, Exposure of rats to extremely low-frequency electromagnetic fields (ELF-EMF) alters cytokines production, Electromagn Biol Med 32(2013), 1–8. M. Bouwens, S. de Kleij, G. Ferwerda, J. J. Cuppen, H. F. Savelkoul and B. M. Kemenade, Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines, Bioelectromagnetics 33(2012), 226–237.
D.Y. Geng et al. / Biochemical kinetics of cell proliferation regulated by ELF-EMF
[9] [10]
[11]
[12] [13]
[14]
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