Original article

J. KASZUBA-ZWOINSKA1, K. WOJCIK2, M. BERETA3, A. ZIOMBER1, P. PIERZCHALSKI5,
E. ROKITA2, J. MARCINKIEWICZ3, W. ZARASKA4, P. THOR1


PULSATING ELECTROMAGNETIC FIELD STIMULATION PREVENTS
CELL DEATH OF PUROMYCIN TREATED U937 CELL LINE



1Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland; 2Department of Biophysics, Jagiellonian University Medical College, Cracow, Poland; 3Department of Immunology, Jagiellonian University Medical College, Cracow, Poland; 4Institute of Electron Technology, Cracow, Poland; 5Department of Physiology, Jagiellonian University Medical College, Cracow, Poland


  Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1x106 cells/ml to 0.0625x106 cells/ml, were exposed to a pulsating magnetic field 50Hz, 45±5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1x106cells/ml and 0.5x106 cells/ml density. To eliminate density effect on cell death, for further studies density 0.25x106 cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 µg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.

Key words: U937 cell line, electromagnetic field, pulsating electromagnetic field (PEMF), cell viability, apoptosis, necrosis, puromycin, flow cytometry



INTRODUCTION

Numerous laboratory studies suggest that PEMF affects almost all cellular processes resulting in cell proliferation, changes in signal transduction pathways, DNA damage, changes in function and viability of immune competent cells or has tumoricidal/anti-tumoricidal effects (1-11). Epidemiological studies on populations living within EMF revealed possible correlation between exposure and neoplastic diseases, particularly leukemia and brain tumors (12, 13). However the results of these studies are difficult to interpret due to many variables and the multi-factorial nature of the neoplasia itself. The most widely held hypothesis is that EMF promotes carcinogenesis by facilitating the proliferation of genetically altered cells (14, 15). Currently researches conclude that there is limited evidence that magnetic fields from power line cause childhood leukemia or other cancer in children (12, 16).

Experimental data with electromagnetic field interaction have shown diminished expression of c-kit protein in nervous ganglia, caused by electromagnetic field induced apoptosis (17, 18). On the other hand, among variety of results obtained involving biological effects of EMF, there also has been found by several authors that EMF has no significant effects on cancer cells (19, 20). Thus data from experimental studies are also not univocal. The discrepancies in cited findings prompted us to investigate EMF effect on viability and death human U937 lymphoid cells.


MATERIALS AND METHODS

Cell culture

Human U937 lymphoid cell line was obtained from American Cell Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 medium (Gibco-BRL, USA), supplemented with 10% (v/v) fetal calf serum (Gibco-BRL, USA) heat inactivated, L-glutamine 0.2 M and gentamicin 50 mg/ml (Sigma-Aldrich, Germany) at 37°C in a 5%CO2 incubator of 90% humidity. Cells viability was monitored by trypan blue exclusion method and counted with a haemocytometer (Burker chamber). The experiments were performed on cells in the logarithmic phase of growth under condition of 98% viability, as assessed by trypan blue exclusion. U937 cells were passed every four days.

For experiments U937 cells were seeded into 96-well (Nunck, Denmark) culture plates and grown at several densities, starting from 1x106 cells/ml through 0.5; 0.25; 0.125 to 0.062 x106 cells/ml. After four days of experiment cells were harvested by centrifugation at 1300 rpm for 10 min and used for feather analysis. Each density point has always been done as fourfold repetition and each value represented a mean from the pulled density experimental points.

Cell death induction

To induce cell death a high dose of puromycin 100 µg/ml (Sigma-Aldrich, Germany) was added to U937 cells cultured at density 0.25 x106 cells/ml excluding density induced apoptosis/ necrosis. Cells were stimulated with puromycin simultaneously with exposure to PEMF for 3 h. Than harvested, washed three times with fresh medium, resuspended at initial volume and cultivated for 24 h, afterwards PEMF exposure was repeated in 24 h intervals to obtain three PEMF doses, 3 h each one (50 Hz, 45±5 mT). Following third PEMF dose cells were analyzed for cell death evaluation. Cells without puromycin treatment were non stimulated (NS), cells not undergone PEMF were control group (K).

Puromycin induced cell death was carried out simultaneously with PEMF stimulation 3 h in 24 h intervals. After last puromycin and PEMF treatment cells were harvested, washed three times with fresh medium, resuspended at initial volume, cultivated for 24 h and analysed for cell death. Each experimental cell culture point was done at four repetitions.

Cell death evaluation

Annexin V - APC labeled (BD Biosciences, USA) was used to quantitatively determine the percentage of cells within population that were undergoing apoptosis. 7-amino-actinomycin D (BD Biosciences, USA) as standard flow cytometric viability probe was used to distinguish viable from non-viable cells. Annexin V-APC positive cells were analyzed as apoptotic and both Annexin V-APC and 7-AAD were either in the end stage of apoptosis or undergoing necrosis analysed as already dead. For staining, U 937 cells were washed twice with cold PBS and resuspended in 1x binding buffer (BD Biosciences, USA) at concentration 1x106 cells/ml. Than 100 µl of solution was transferred to 5 ml culture tube and 5 µl of Annexin V-APC and 5 µl of 7-AAD were added. Cells have been gently vortexed and incubated in dark for 15 min at RT. Prior to flow cytometric analysis 400 µl of 1x binding buffer was added and cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) using Cell-Quest software. Suggested controls to set up compensation and quadrants encompassed unstained cells, cells stained with AnnexinV-APC alone (for FL-4 fluorescence)and cells stained with 7-AAD alone (detected in FL-3). A minimum 10.000 events were collected on each sample.

Magnetic stimulation

The generator produced pulsating field 50 Hz, 45±5 mT inside the cell culture incubator. Rationale for choosing such frequency of PEMF was related to the following reasons: frequency of magnetic stimulation is higher then the range, which directly depolarizes autonomic fibers, heating effect minimal and all power devices generate EMF with such frequency. The 96-well plate with cells was placed in the generator pocket. The field was applied for 3 h per each stimulation with 24 h intervals between stimulations if more than one has been used. The control samples were in the same incubator but in a distance of 35 cm from the generator.

Statistical analysis

Date were expressed as mean and (±) standard deviation (SD) and compared using the Student t-test considering P<0.05 defined as significantly different.


RESULTS

Cell density induced apoptosis/necrosis

Human lymphoid cell line U937 was cultivated at different range of culture densities, from 1x106-0.062 x106 cells/ml, for 4 days, subsequently cell density was calculated. U937 grown at initial density 1x106 cells/ml and 0.5x106 cells/ml achieved after 4 days of cultivation 3.4x106 cells/ml and 2.1x106 cells/ml density, respectively. These cultures treated with PEMF three times for 3 h per each stimulation reached 2x106 cells/ml and 1.751x106 cells/ml abundance, Fig. 1.

Fig. 1. Influence of PEMF stimulation on number of cells during 4 days of cultivation. K - cells not treated with PEMF, PEMF - cells PEMF exposed. Data are expressed as mean±SD of 3 experiments performed in tetra fold repetition; statistical significance was determined by Student t-test analysis as *P<0.05.

Increase in amount of the cultivated U937 cells (1x106 cells/ml and 0.5x106 cells/ml) induced cell apoptosis and necrosis by itself. PEMF potentiated effect of density and induced cell death.

PEMF caused apoptosis, which reached at densities 1x106 cells/ml; 0.5x106 cells/ml; 0.25x106 cells/ml 21±2.6%, 14±0.38%, 6.8±0.45%, respectively. Lower cell densities did not show measurable effects of PEMF on apoptosis, Fig. 2.

Fig. 2. Apoptosis of U937 cells cultivated at different densities during 4 day culture by PEMF stimulation. K - cells not treated with PEMF; PEMF - cells exposed to PEMF three fold per 3h, with 24h intervals. Data are expressed as mean±SD of 3 experiments performed in tetra fold repetition; statistical significance was determined by the Student t-test analysis as *P<0.05.

PEMF death induced was strongest and reached at concentrations of 1x106 and 0.5x106 cells/ml, about 15.9±3.7% and 14.6±2.7%, respectively Fig. 3.

Fig. 3. Percentage of the dead cells induced in U937 cell line cultivated at different densities by PEMF stimulation during 4 day culture. K - U937 cells not stimulated with PEMF; PEMF - U937 cells stimulated with PEMF three fold per 3h, with 24h intervals. Data are expressed as mean±SD of 3 experiments performed in tetra fold repetitions; statistical significance was determined by the Student t-test analysis as *P<0.05.

Effect of PEMF on puromycin treated U937cells

In order to exclude density induced apoptosis and necrosis, we used 0.25x106 mln cells/ml density to investigate influence of PEMF on cell viability. Puromycin, as an known unselective antitumor drug inducing apoptosis was chosen for studying effects of PEMF. Experiments with applied puromycin have found, that puromycin treated control cells undergone apoptotic changes in 8.9±1.6%. PEMF stimulated cells and treated with puromycin were apoptotic at 6.6±1.4%, Fig. 4. Likewise three doses of puromycin itself caused cell death of 59.6±8.8% and puromycin combined with PEMF 51.8±5.8% necrotic cells, Fig.5.

Fig. 4. Percentage of the apoptotic cells induced in U937 cell line culture upon, one and three doses of puromycin treatment and simultaneous three fold PEMF stimulation. K - cells not stimulated with PEMF; PEMF - cells exposed to PEMF, NS - cells not stimulated with puromycin, PUR 1x and PUR 3x - cells treated with one and three doses of puromycin, respectively. Data are expressed as mean±SD of 3 experiments performed in tetra fold repetitions; statistical significance was determined by the Student t-test analysis as *P<0.05.

Fig. 5. Percentage of the dead cells induced in U937 cell line culture upon one and three doses of puromycin treatment and simultaneous threefold PEMF stimulation. K - cells not stimulated with PEMF; PEMF - cells exposed to PEMF, NS - cells not stimulated with puromycin, PUR 1x and PUR 3x - cells treated with one and three doses of puromycin, respectively. Data are expressed as mean±SD of 3 experiments performed in tetra fold repetitions; statistical significance was determined by the Student t-test analysis as *P<0.05.


DISCUSSION

The two best characterized cell death processes are: programmed - apoptosis and incidental - necrosis, that is induced by extracellular conditions such as hypoxia or cytotoxic agents (21-23).

Apoptosis is regulated by two major independent pathways the death receptor induced - extrinsic and the stress mediated (EMF) - intrinsic or mitochondrial (24). The stress-induced pathway is mediated by pro-apoptotic members of Bcl-2 family that are associated with mitochondria. The receptor induced physiological pathway depends on the activation of death receptors like Fas-R or TNF-R. Intrinsic pathway is triggered under inconvenient environmental conditions involving drugs, irradiation or growth factors withdrawal (25, 26).

Previously, we have described that PEMF caused apoptosis of the peripheral blood mononuclear cells (PBMC) originated from patients with Crohn's disease and changed secretion of cytokines. We found out, that native proliferating leukocytes or in vitro mitogens treated under PEMF influence, are more susceptible for apoptosis than non-dividing cells and the cell death depends upon dose of PEMF (27). In these study, we utilized U937 lymphoid cell line to investigate PEMF-induced cellular interaction.

Firstly, we tested the importance of cell density in a culture as an agent determining PEMF influence on proliferating activity of the cells. This PEMF interaction corresponds to density of culture. Maximal PEMF induced death effect on cells occurred at highest densities. These findings are consistent with results obtained by other authors. Hisamitsu et al., in studies with human myelogenous leukemic cell lines HL-60 and ML-1, found that under PEMF intensity of 1 µT for 3 h, viability of cells decreased to 87,8% despite low intensity of magnetic field used (15). The slight differences in viability between their and our results are probably related to intensity of EMF.

However there are also some reports which could not find any effects exerted by magnetic field of 50 Hz and 1,5 mT intensity on expression of CD4+ cell surface receptor on human PBMCs. Other studies investigating different magnetic fields structures at 50-60Hz with flux densities 500, 100 20 and 2 µT also have shown no effects on human PBMCs (28, 29). Reported discrepancies are probably related to native form of used PBMCs with low proliferating activity and lower intensity of the magnetic field.

In order to discriminate and clarify mechanisms responsible for cell death after PEMF we stimulated U937 cells with puromycin prior to PEMF exposition. Puromycin treatment combined with simultaneous single PEMF exposition reduced the rate death cells rate. Three fold PEMF exposition increased viability about 25%. Our data are in agreement with Grassi et al. (30). They found that 50 Hz EMF exposure enhanced proliferation and inhibited puromycin induced death in human neuroblastoma and rat neuroendocrine cells (30, 31). Explanations of observed protective PEMF effect is possibly action of the PEMF on the expression of Bcl-2 family mitochondrial member genes (32, 33). Another possibility was presented by Dolhi et al. (33), who suggested that apoptosis might be inhibited by cytosolic survivin. PEMF acting on this protein, may improve viability of the cells (34-36). These findings are consistent with our results, that PEMF protects U937 cells against puromycin - induced cell death.

Acknowledgements: The authors thank Ms. Daisy MacCallum, student of Jagiellonian University Medical College for her helpful suggestions, support and English guidance.

Conflict of interests: None declared.



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R e c e i v e d : July 9, 2009
A c c e p t e d : March 19, 2010

Author’s address: Dr. Jolanta Kaszuba-Zwoinska, Ph.D., Department of Pathophysiology, Jagiellonian Univesity Medical College, 18 Czysta Str., 31-125 Cracow, Poland; Phone: +48 12 6333947; Fax: +48 12 6329056; e-mail: jkaszuba@cm-uj.krakow.pl