PROLIFERATIVE ACTIVITY AND MEMBRANE POTENTIAL OF C6 AND HELA CELL LINES IN CULTURE UNDER ELECTRICAL STIMULATION

The influence of long-term electrical stimulation on the functional properties of cells in culture was investigated. C6 rat glioma cells and HeLa human adenocarcinoma cells were used in this work. Stimulation was carried out with uniform electric field for 12 hours with the train of pulses at a frequency of 10 Hz. Measurements of membrane potential were conducted using patch-clamp technique; the proliferative activity was evaluated by cell counting relative to control sample. It was shown that electrical stimulation causes a change in membrane potential of the cells and subsequent changes in their proliferative activity. It was established that membrane depolarisation increases proliferative activity whereas hyperpolarization decreases it. It was established that the effect depends on the parameters of the electric field and the type of cells. The observed effect is associated with activation of potential-dependent ion channels, resulting in changes of the specific phase duration in the cell cycle. The obtained results can be used in development methods of cell engineering.

cell culture, HeLa, C6, electrical stimulation, proliferative activity, membrane potential

1. Funk, R. H. W. Endogenous electric fields as guiding cue for cell migration, Frontiers in Physiology, 2015, vol. 6.

2. Kunitskaya Y., Golubeva L. Resting transmembrane potential of C6, HEp-2c and HEK tumor cell lines during proliferation. Sbornik rabot 70-i nauchnoi konferentsii studentov i aspirantov Belorusskogо gosudarstvennogo universiteta, 15–18 maia 2013 g., g. Minsk [Proceeding of 70th scientific students conference of BSU, 15–18 May 2013, Minsk]. Minsk, 2013, part 1, pp. 134–137. (in Russian).

3. Urrego D., Tomczak A. P., Zahed F., Stühmer W., Pardo L. A. Potassium channels in cell cycle and cell proliferation, Philosophical Transactions of the Royal Society B: Biological Sciences, 2014, vol. 369, pp. 1–10.

4. Llucià-Valldeperas A., Sanchez B., Soler-Botija C., Gálvez-Montón C., Roura S., Prat-Vidal C., Perea-Gil I., Rosell-Ferrer J., Bragos R., Bayes-Genis R. Physiological conditioning by electric field stimulation promotes cardiomyogenic gene expression in human cardiomyocyte progenitor cells. Stem Cell Research & Therapy, 2014, vol. 5, no. 93, pp. 1–5.

5. Lim J. H., McCullen S. D., Piedrahita J. A., Loboa E. G., Olby N. J. Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells. Cell Reprogram, 2013, vol. 15, no. 5, pp. 405–412.

6. Mi Y., Sun C., Yao C., Li C., Mo D., Tang L., Liu H. Effects of steep pulsed electric fields (SPEF) on mitochondrial transmembrane potential of human liver cancer cell. Conference proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2007, pp. 5815–5818.

7. Yuan X., E. Arkonac D., Chao P. G., Vunjak-Novakovic G. Electrical stimulation enhances cell migration and integrative repair in the meniscus. Scientific Reports, 2014, vol. 4, no. 3674, pp. 1–12.

8. Zhao M., Bai H., Wang E., Forrester J. V., McCaig C. D. Electrical stimulation directly induces pre-angiogenic respon- ses in vascular endothelial cells by signaling through VEGF receptors. Journal of Cell Science, 2004, vol. 117, no. 3, pp. 397–405.

9. Titushkin I., Cho M. Regulation of cell cytoskeleton and membrane mechanics by electric field: role of linker proteins. Biophysical Journal, 2009, vol. 96, no. 2, pp. 717–728.

10. Rouzaire-Dubois B., Milandri S., Bostel J. B., Dubois J. M. Control of cell proliferation by cell volume alterations in rat C6 glioma cells. European Journal of Physiology, 2000, vol. 440, pp. 881–888.

11. Sauve R., Bedfer G., RoySingle G. Single Ca++ dependent K+ currents in HeLa cancer cells. The Biophysical Journal, 1983, vol. 45, pp. 66–68.

12. Wang S., Castle N. A., Wang G. K. Identification of RBK1 potassium channels in C6 astrocytoma cells. Glia, 1992, vol. 5, no. 2, pp. 146–153.

13. Negulyaev Y. A., Savokhina G. A., Vedernikova E. A. Calcium-permeable channels in HeLa cells. General Physiology and Biophysics, 1993, vol. 12, no. 1, pp. 19–25.

14. Manor D., Moran N. Modulation of small conductance calcium-activated potassium channels in C6 glioma cells. The Journal of Membrane Biology, 1994, vol. 140, no. 1, pp. 69–79.

15. Sardini A., Amey J. S., Weylandt K. H., Nobles M., Valverde M. A., Higgins C. F. Cell volume regulation and swel- ling-activated chloride channels. Biochimica et Biophysica Acta, 2003, vol. 1618, no. 2, pp. 153–162.

16. Cho M. R., Thatte H. S., Silvia M. T., Golan D. E. Transmembrane calcium influx induced by ac electric fields. The FASEB Journal, 1999, vol. 13, no. 6, pp. 677–683.

17. Huang P., Liu J., Di A., Robinson N. C., Musch M. W., Kaetzel M. A., Nelson D. J. Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase. The Journal of Biological Chemistry, 2001, vol. 276, no. 23, pp. 20093–20100.

18. Olsen M. L., Schade S., Lyons S. A., Amaral M. D., Sontheimer H. Expression of voltage-gated chloride channels in human glioma cells. The Journal of Neuroscience, 2003, vol. 23, no. 13, pp. 5572–5582.

19. Khatib L., Golan D. E., Cho M. Physiologic electrical stimulation provokes intracellular calcium increase mediated by phospholipase C activation in human osteoblasts. The FASEB Journal, 2004, vol. 18, no. 15, pp. 1903–1905.

20. Plonsey R., Barr R. Bioelectricity: a quantitative approach, 3rd ed., New York, Springer Science+Business Media, LLC, 2007. 528 p.