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%CO
2
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 1x10
6
cells/ml through 0.5; 0.25; 0.125 to 0.062 x10
6
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 x10
6
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 1x10
6
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 de
viation (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 1x10
6-0.062 x10
6
cells/ml, for 4 days, subsequently cell density was calculated. U937 grown at
initial density 1x10
6 cells/ml and 0.5x10
6
cells/ml achieved after 4 days of cultivation 3.4x10
6
cells/ml and 2.1x10
6 cells/ml density, respectively.
These cultures treated with PEMF three times for 3 h per each stimulation reached
2x10
6 cells/ml and 1.751x10
6
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 (1x10
6 cells/ml and 0.5x10
6 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 1x10
6
cells/ml; 0.5x10
6 cells/ml; 0.25x10
6
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 1x10
6
and 0.5x10
6 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 0.25x10
6
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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