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Bioelectromagnetics 27:354^ 364 (2006)
Real-Time Measurement of Cytosolic Free
Calcium Concentration in Jurkat Cells During
ELF Magnetic Field Exposure and Evaluation
of the Role of Cell Cycle
Cheryl R. McCreary,
1,4
S. Jeffrey Dixon,
5
Laurence J. Fraher,
3,6
Jeffrey J.L. Carson,
1,4
and Frank S. Prato
1,2,4
*
1
Imaging Program, Lawson Health Research Institute, London, Ontario, Canada
2
Department of Nuclear Medicine, St. Joseph’s Health Care, London, Ontario, Canada
3
Departments of MedicalandBiochemistry, University of Western Ontario,
London, Ontario, Canada
4
Medical Biophysics, University of Western Ontario, London, Ontario, Canada
5
Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
6
Metabolism and Diabetes Program, Lawson Health Research Institute, London,
Ontario, Canada
Extremely low frequency magnetic fields (ELF MF) have been reported to alter a number of cell
signaling pathways, including those involved in proliferation, differentiation and apoptosis where
cytosolic free calcium ([Ca
2þ
]
c
) plays an important role. Tobetter understand the biological conditions
under which ELF MF exposure might alter [Ca
2þ
]
c
, we measured [Ca
2þ
]
c
by ratiometric fluorescence
spectrophotometry during exposure to ELF MF in Jurkat E6.1 cells synchronized to different phases of
the cell cycle. Suspensions of cells were exposed either to a near zero MF (Null) or a 60 Hz, 100 mT
sinusoidal MF superimposed upon a collinear 78.1 mT static MF (AC þDC). An initial series of
experiments indicated that the maximum increase in [Ca
2þ
]
c
above baseline after stimulation with
anti-CD3 was significantly higher in samples exposed to AC þDC (n¼30) compared to Null (n¼30)
with the largest difference in G2-M enriched samples. However, in a second study with G2-M enriched
cells, samples treated with AC þDC (n¼17) were not statistically different from Null-treated
samples (n¼27). Detailed analysis revealed that the dynamics in [Ca
2þ
]
c
before and after stimulation
with anti-CD3 were dissimilar between Null samples from each study. From the results, we concluded
(i) that the ELF MF increased [Ca
2þ
]
c
during an antibody-induced signaling event, (ii) that the ELF
MF effect did not depend to a large degree on cell cycle, and (iii) that a field-related change in [Ca
2þ
]
c
signaling appeared to correlate with features in the [Ca
2þ
]
c
dynamics. Future work could evaluate
[Ca
2þ
]
c
dynamics in relation to the phase of the cell cycle and inter-study variation, which may reveal
factors important for the observation of real-time effects of ELF MF on [Ca
2þ
]
c
. Bioelectromagnetics
27:354– 364, 2006. 2006 Wiley-Liss, Inc.
Key words: 60 Hz AC; DC; fluorescence; ratiometric measurement; indo-1
INTRODUCTION
At the cellular level, weak (<1 mT), extremely
low frequency (<300 Hz) magnetic fields (ELF MF)
have been shown to alter immune function, cell
proliferation, gene expression, DNA repair, and
second messenger pathways such as calcium signaling
(reviewed in Lacy-Hulbert et al., 1998; Loscher and
Liburdy, 1998). Calcium is a ubiquitous second
messenger that is involved in the regulation of many
physiological functions. Measurement of cytosolic free
calcium concentration ([Ca
2þ
]
c
) after or during ELF
MF exposure led investigators to suggest that field-
induced alterations in [Ca
2þ
]
c
might lead to down-
stream physiological effects. For example, Carson et al.
[1990] were the first to report an increase in [Ca
2þ
]
c
in
HL60 cells during ELF MF exposure. Lindstrom et al.
[1993, 1995b] found that exposure of quiescent Jurkat
2006 Wiley-Liss, Inc.
——————
Grant sponsor: Canadian Institutes of Health Research (CIHR).
*Correspondence to: Frank S. Prato, Imaging Program, Lawson
Health Research Institute, 268 Grosvenor Street, London, Ont.,
Canada. E-mail: prato@lawsonimaging.ca
Received for review 12 September 2005; Final revision received
10 March 2006
DOI 10.1002/bem.20248
Published online 19 May 2006 in Wiley InterScience
(www.interscience.wiley.com).
E6.1 cells to a 50 Hz MF induced [Ca
2þ
]
c
oscillations.
In another study with Jurkat cells, the power spectral
density of [Ca
2þ
]
c
oscillations was reduced in cells
exposed to ELF MF [Galvanovskis et al., 1999]. While
the literature contains many positive findings, negative
results also have been reported. In one case, [Ca
2þ
]
c
oscillations recorded in Jurkat cells were unaffected
during exposure to 50 Hz MF [Wey et al., 2000]. Other
laboratories have observed calcium signaling events in
a number of cell types and found that exposure to ELF
MF had no effect [Lyle et al., 1997; Sisken and
DeRemer, 2000; Shahidain et al., 2001; Craviso et al.,
2002; Madec et al., 2003]. Differences in study outcome
may be related to a number of methodological differ-
ences between laboratories: (i) MF exposure conditions
were often different between studies and between
laboratories, which complicated interpretation of
results even on the same biological endpoint. (ii) There
were often inconsistencies in the biological protocol.
For instance, two laboratories used different criteria
to select individual cells for inclusion during [Ca
2þ
]
c
measurements [Lindstrom et al., 1993; Wey et al.,
2000]. (iii) Differences in biochemical conditions could
lead to different experimental outcomes. For example,
intracellular and extracellular calcium levels were
shown to influence cellular functions such as apoptosis,
differentiation, and secretion during MF exposure
[Karabakhtsian et al., 1994; Morgado-Valle et al.,
1998; Fanelli et al., 1999; Zhou et al., 2002; Gobba et al.,
2003]. (iv) Finally, the physiological state of the cell at
the time of MF exposure may have been important
[Walleczek and Liburdy, 1990; Walleczek and
Budinger, 1992; Nindl et al., 1997; Diniz et al., 2002].
Data from our lab also support this last possibility. In
previous work, we identified a number of factors that
obscured the effect of ELF MF exposure on [Ca
2þ
]
c
in
Jurkat cells, one of which included the distribution of
cells within the cell cycle [McCreary et al., 2002].
The purpose of the work described here was to
extend our previous findings with Jurkat E6.1 cells by
measurement of [Ca
2þ
]
c
dynamics during ELF MF
exposure of samples enriched at one of three phases of
the cell cycle. We exposed cell samples enriched in G0/
G1, S, and G2-M phases to either a 60 Hz, 100 mT
1
sinusoidal MF collinear with a 78.1 mT static MF
(AC þDC) or a control condition in which the ambient
static MF was zeroed to a tolerance of 0.5 mT (Null).
These MF conditions were selected since in our
laboratory they have been associated with effects on
[Ca
2þ
]
c
in vitro [McCreary et al., 2002] and on the
response to a thermal stimulus in vivo [Prato et al.,
1995, 2000]. In addition, the combination of time-
varying and static MF satisfied the theoretical predic-
tions of the ion resonance model and its variants
[Lednev, 1991; Blanchard and Blackman, 1994], which
have shown consistency with experimental results from
other laboratories [Blackman et al., 1995; Baureus
Koch et al., 2003; Sarimov et al., 2005].
MATERIALS
The Jurkat E6.1 clonal cell line (TIB-152) was
purchased from the American Type Culture Collection
(ATCC, Manassas, VA). Fetal bovine serum and
antibiotic/antimycotic solution (10 000 units/ml pen-
icillin, 10 000 mg/ml streptomycin, and 25 mg/ml
amphotericin B) were purchased from Invitrogen
(Burlington, ON). The acetoxymethyl ester form of
the fluorescent calcium indicator indo-1 (indo-1 AM)
was purchased from Molecular Probes (Eugene, CA,
USA). The monoclonal antibody, anti-CD3 (UCHT-1
mouse anti-human), was purchased from ID Labs
(London, ON, Canada). Modified RPMI 1640 medium
(with L-glutamine and without phenol red or sodium
bicarbonate) and all other chemicals were obtained
from Sigma-Aldrich Canada (Oakville, ON, Canada).
Buffered saline solution (BSS) was prepared in our
laboratory and contained (in mM): 135 NaCl, 5 KCl,
1 MgCl
2
, 1 CaCl
2
, 10 glucose, and 10 HEPES (pH 7.30).
Conditioned RPMI 1640 medium (cRPMI) was pre-
pared in our laboratory by culturing Jurkat E6.1 cells
(6–7 10
5
cells per ml) in modified RPMI 1640 with
10% fetal bovine serum, 1% antibiotic/antimycotic
solution, 10 mM HEPES, and 23.8 mM sodium
bicarbonate (supplemented RPMI) for 24 h. The cells
were pelleted by centrifugation (300gfor 5 min), the
supernatant collected and the conditioned medium was
stored at 70 8C.
METHODS
Overview
The potential effect of exposure to ACþDC MF
on [Ca
2þ
]
c
homeostasis and [Ca
2þ
]
c
signaling was
examined by comparing MF-exposed samples to
samples treated with a Null MF condition, which
represented our standard control.
2
Normalized ratios
——————
1
AC magnetic field strength here and throughout the text reported
as peak amplitude, that is, one half of the peak to trough difference
in the waveform.
——————
2
In the natural environment biological systems are exposed to the
earth’s magnetic field. For this reason, it could be argued that a
Null MF may not be a suitable control condition; however,
calcium signaling measurements performed in Jurkat cells in our
lab indicated that there was no difference between samples
exposed to a DC MF similar to that of the earth’s MF and samples
exposed to a Null MF.
Real-Time Measurement of [Ca
2þ
]
c
355
from synchronized Jurkat cells loaded with indo-1 were
obtained from fluorescence measurements on different
samples that contained cells at one of three phases of the
cell cycle. This approach resulted in six experimental
groups: three distributions of cells in the cell cycle and
two MF conditions. In each experiment, the dynamics
of [Ca
2þ
]
c
during MF was monitored at three different
stages of activation by anti-CD3: resting or basal
[Ca
2þ
]
c
prior to anti-CD3, the initial [Ca
2þ
]
c
response
after anti-CD3, and the sustained [Ca
2þ
]
c
response after
anti-CD3.
Cell Culture
Cells were grown in RPMI, passaged twice
weekly, and diluted with fresh medium once between
passages. Cultures were kept at 37 8C inside a vented
Mu-metal box within an incubator that contained a
humidified atmosphere of 5% CO
2
and 95% air. The
Mu-metal box (33 38 20 cm) was constructed of
1 mm thick nickel iron alloy with four 2.5 cm diameter
circular vents on both the top and bottom of the box. The
vents were topped with 2.5 cm long Mu-metal cylinders
to improve the degree of ambient MF shielding. This
ensured that the cell cultures were exposed to low static
(<0.3 mT) and ELF (<0.001 mT) MF during culturing
and that the MF exposure prior to the experiments
was standardized. The cell culture from ATCC was
expanded through four passages to form a bank of stock
cells frozen with 7% dimethyl sulfoxide (DMSO) and
kept in a liquid nitrogen cryotank. Stock cells were then
thawed, washed with RPMI and grown for subsequent
experimentation. All of the cells used in these experi-
ments were passaged fewer than 12 times after arrival in
the laboratory.
Synchronization and DNA Analysis
Jurkat E6.1 cells were reversibly arrested in
S phase by treatment with 200 ng/ml aphidicolin for
18–19 h [Merrill, 1998; Kai et al., 1999]. Cell density
(by Neubaum hemocytometer), viability (by trypan
blue exclusion), and DNA content (by flow cytometry)
were determined every 2– 5 h thereafter for 30 h. For
flow cytometry, a sample of approximately 1 10
6
aphidicolin-treated cells was taken from the culture
flask and centrifuged at 500g. The cells were resus-
pended in 250 ml of phosphate-buffered saline (PBS)
with 0.1% bovine albumin and fixed with 750 ml of 70%
cold ethanol. Samples were cooled on ice and stored at
48C for up to 2 weeks. The fixed Jurkat cells were
washed twice with PBS, treated with 1 mg/ml RNase A,
then labeled with propidium iodide (27 mg/ml) for at
least 1 h at 4 8C.
A Coulter XL-MCL (Beckman-Coulter, Miami,
FL, USA) was used for flow cytometry of DNA content
(excitation with 15 mW Argon laser at 488 nm with
fluorescence detection at 620 nm). At least 10 000
events were collected for each determination of the
distribution of cells within the cell cycle. Multicycle
software (Phoenix Flow Systems, San Diego, CA,
USA) was used to fit the ungated data. The signal from
debris and clumping was fit using a non-linear, least
squares fitting algorithm that assumed the probability of
aggregation between any two cells was independent of
phase of the cell cycle. The clumping fraction of the
signal was subtracted from the total signal before the
distribution of cells within the cell cycle was deter-
mined. To allow sufficient time for more than one
experiment to be performed at a given time point, that is,
both Null and AC þDC from the same preparation,
progression of the cell cycle was slowed by incubation
at 4 8C for up to 3.5 h. From the cell density and flow
cytometry data, it was determined that G0/G1, S, and
G2-M phases were maximally enriched 14–18, 1–4,
and 6-8 h after aphidicolin treatment, respectively (data
not shown).
Sample Preparation and
Fluorescence Measurement
Cell culture, sample handling, indo-1 AM loading
and fluorescence measurement have been described
previously [Carson and Prato, 1996; McCreary et al.,
2002]. Briefly, aliquots of cells were taken from the
suspension cultures, washed, and resuspended in 1 ml
BSS. A sample of 1.5 10
6
cells was treated with 6 ml
DMSO. Three more samples each of 1.5 10
6
cells was
taken from the same flask and treated with 1 mM indo-1
AM in 6 ml DMSO. The four samples were incubated in
an open circulating water bath at 37 8C for 30 min the
pump motor was 2 m distant from the bath to minimize
possibility of stray MF. After incubation, the cells were
washed three times to remove residual extracellular
indo-1 AM. Both the DMSO and the indo-1 AM treated
samples were resuspended in cRPMI with a final cell
density of 2.5 10
5
cells/ml.
Aliquots of cell suspension (3 ml each) were
transferred to one of four quartz cuvettes: one for
autofluorescence measurement (i.e., DMSO-treated
only; cuvette 1) and three for indo-1-loaded cells
(cuvettes 2, 3, and 4). Fluorescence excitation was at
352 nm (6 nm slit) with emission detection at 398 nm
(10 nm slit) and 490 nm (10 nm slit). For each
time point t(j), fluorescence was recorded from each
cuvette at 398 nm (F
398,i
(j)) and 490 nm (F
490,i
(j)),
where irepresented the cuvette number {1, 2, 3, 4}
and jthe time index {1, 2, 3,...,N}. These data were
used to form three ratios by the relation R
k
(j)¼
(F
398,kþ1
(j)F
398,1
(j))/(F
490,kþ1
(j)F
490,1
(j)), where
k¼{1, 2, 3}. The ratios were normalized to form three
356 McCreary et al.
normalized ratios by the relation NR
k
(j)¼
R
k
(j)/R
k
(t(j)¼300), where R
k
(t(j)¼300) was estimated
from linear regression of the ratio data acquired during
first 5 min (indicated as Pre in Fig. 1B). Although
sufficient data was collected to permit calibration of the
ratios into [Ca
2þ
]
c
, averaged results based on values
from the calculated [Ca
2þ
]
c
showed greater variability
than results based on normalized ratios. Therefore,
results have been presented as normalized ratios instead
of [Ca
2þ
]
c
where appropriate. If excessive scatter from
dust particles was observed or the agonist injection
failed to reach the sample directly, for example, it hit the
inside surface of the cuvette, then the data from the
affected sample was excluded from the triplicate
average. Triplicate determinations were obtained in
92% of the experiments. Duplicate averages were
obtained in 8% of the experiments.
Magnetic Field Exposure System
Stray MF artifacts were minimized by a periscopic
optical system attached to the fluorescence spectropho-
tometer. This modification allowed the four samples to
be monitored and isolated from stray MF associated
with electronic equipment. A static MF was generated
by a triaxial mutually orthogonal square Helmholtz coil
system (with diameters of 0.8, 0.9, and 1.0 m). The
static field was monitored with a digital magnetometer
(Model DM 2220, Schonstedt Instrument Company,
Reston, VA, USA) and the input current was adjusted on
each of the three DC power supplies to achieve the
desired static field magnitude and direction. The ELF
MF was produced by a Helmholtz coil (0.208 m
diameter) and a computer controlled waveform gen-
erator (Model 75, Wavetek, San Diego, CA, USA). The
signal from the waveform generator was amplified
(Model 7570, Techron Inc., Elkhart, IN, USA) and then
fed to the coil. The ELF MF exposure system was able
to generate an ELF waveform on top of a DC offset,
which permitted exposure of samples to a collinear
AC þDC MF combination. Details of the apparatus
have been described elsewhere [Carson and Prato, 1996].
Time Course for Each Experiment
Samples were allowed to equilibrate to 37.0 8C for
5 min before fluorescence measurements were started.
Temperature variation within and between experiments
was less than 0.1 8C. During the first 5 min of
fluorescence data collection, that is, t(j)¼0–300 s, a
Null MF was applied. The ELF MF exposure started at
t(j)¼300 s, which consisted of either a Null condition
(ambient static fields 0 0.5 mT) or an AC þDC
condition (78.1 0.5 mT static MF superimposed
collinearly with a 100 0.5 mT 60 Hz sinusoidal MF).
The ELF MF was applied for the duration of the
Fig.1. Time course fluorescence data showing raw and derived
data fromcuvettes1 and 2 for a typicalexperiment.Data from cuv-
ettes 3 and 4 are omitted for clarity, but resemble results
from cuvette 2. A: Raw PMT voltage representing the fluores-
cence signal from DMSO-treated Jurkat cells and indo-1-loaded
Jurkat cells. B: Autofluorescence-corrected fluorescence ratio
derived from the data in panel A. Monoclonal antibody (anti-
CD3, 0.67 mg/ml final concentration), digitonin, and EGTA were
added at 1200, 2400, and 30 00 s, respectively. C:Normalized
ratio derived fromdata in panel B. See methods for notation and
analysis.
Real-Time Measurement of [Ca
2þ
]
c
357
experiment. The sequence of Null versus ELF MF
exposure was randomized.
At t¼1200 s, anti-CD3 at the final concentration
of 0.67 mg/ml, was injected simultaneously into each
cuvette in a 50 ml saline vehicle via an automated
injection system. The calcium response was followed
for 20 min (t(j)¼1200–2400 s). Thereafter, samples
were treated in succession with digitonin (50 mM),
which permeabilized the cell membrane and saturated
indo-1 with Ca
2þ
(maximum fluorescence signal), and
EGTA (13 mM), which chelated Ca
2þ
(minimum
fluorescence signal; Fig. 1A). Throughout each experi-
ment a gas mixture containing 5% CO
2
and 95% air was
blown through a diffuser over the samples to maintain
the pH of the conditioned medium. The data acquis-
ition, agonist injection system, non-magnetic mixing
system, and AC MF generation were computer
controlled.
Statistical Analysis
Before statistical analysis was performed,
the normalized ratios within each experiment
were averaged according to the relation NR(j)¼
(NR
1
(j)þNR
2
(j)þNR
3
(j))/3. Each time course of the
average normalized ratio was then characterized by a
number of descriptive statistics. These included the
slope, intercept and average over the 15 min prior the
introduction of anti-CD3 (basal); the slope, intercept
and average over the 15 min starting 5 min after the
introduction of anti-CD3 (active); and the rate of initial
increase (rise), rate of recovery (recovery) to a new
steady state level, the maximum gain in the normalized
ratio above baseline (peak), and the time to reach the
peak after the introduction of anti-CD3 (as shown in
Fig. 1C).
In the first study (Study 1), cell samples for
experiments conducted on the same day came from the
same parent culture and each combination of exposure
condition and cell cycle distribution was performed on a
single day. Repeated measures multivariate analysis of
variance was used for statistical comparison. The ten
descriptive statistics of the average normalized ratios
were compared within the three cell cycle distributions
and two exposure conditions. The distribution (skew
and kurtosis) and variance of the descriptive statistics
were examined for each group to establish if the
assumptions of normality and variance were met.
Where significant main effects were found, post-hoc
comparisons were performed with a paired t-test
between exposure groups. Multivariate analysis of
variance was used to analyze the descriptive data from
the second study (Study 2). It was also used to compare
the results between Study 1 and 2. Differences
were considered statistically significant at the P.05
level.
RESULTS
Synchronization
The cell distributions within the cell cycle after
aphidicolin treatment were summarized in Table 1. For
S phase enrichment, the number of cells in S phase was
82% compared to 31% in an asynchronous culture. For
experiments with G2-M phases enriched, the propor-
tion of cells in G2-M in comparison to the asynchronous
culture increased from 12% to 50%, whereas for G0/G1
enrichment, the distribution of G0/G1 cells increased
from 56% to 65%. The cell viability was 93–97% for up
to 30 h after aphidicolin treatment. The doubling time of
the population was approximately 24 h, similar to
untreated, asynchronous Jurkat E6.1 cultures. There
were no significant differences in cell cycle distribution
between samples that were exposed to the Null or the
AC þDC condition.
Dependence of Descriptive Statistics
on Cell Cycle
The average normalized ratios for G0/G1, S, and
G2-M enriched distributions are shown in Figure 2. The
corresponding descriptive statistics describing the
normalized ratios are given in Table 2. Dependence of
the descriptive statistics on cell cycle distribution was
observed primarily after the introduction of anti-CD3
and was independent of magnetic field condition. The
active slope and intercept were significantly greater in
TABLE 1. Distributions of Synchronized Jurkat E6.1 Cells for G0/G1, S, and G2-M Enriched
Phases (Average SE)
Enriched phase
G0/G1 (%) S (%) G2-M (%)
Null AC þDC Null AC þDC Null AC þDC
G0/G1 (n¼10) 65 2642232222122143
S(n¼10) 9 11018228139192
G2-M (n¼10) 31 2313192202503492
358 McCreary et al.
G0/G1 enriched cultures than in G2-M enriched
cultures (P<.03 for each descriptive statistic). The
active intercept and active average were significantly
greater in G0/G1 enriched cultures than in S enriched
cultures (P<.05 for each descriptive statistic). No
significant interaction between magnetic field condition
and phase of the cell cycle was found.
ELF MF Effects
In Study 1, an effect of ELF MF exposure was
observed in the peak transient response to anti-CD3 in
aphidicolin-treated cells. There was a significant effect
of MF exposure on the peak increase in normalized
ratio above baseline after anti-CD3 [F
(1,9)
¼11.68,
P¼.008], which indicated that the peak [Ca
2þ
]
c
in cells
exposed to AC þDC was greater than the peak [Ca
2þ
]
c
in cells exposed to the Null condition over all cell cycle
distributions. Although, no significant interaction
between cell cycle distribution and magnetic field
exposure was detected, it was decided that investigative
post-hoc analyses should be performed to compare
exposure groups that represented each enriched phase
of the cell cycle. It was determined that the largest
difference in peak normalized ratio between exposure
groups was observed for G2-M enriched samples.
However, the difference was not statistically significant
(P¼.057, Fig. 2C). Likewise, comparison of the
descriptive statistics between the Null and AC þDC
groups representative of each cell cycle distribution
revealed no statistically significant differences
(Table 2).
To further investigate the possibility that [Ca
2þ
]
c
signaling was altered by exposure to an ACþDC MF
during the G2-M phase of the cell cycle, the MF
condition was repeated in Study 2 using a protocol
similar to Study 1. Twenty-seven samples were exposed
to the Null condition and seventeen samples were
exposed to the AC þDC condition. The distribution of
cells within the cell cycle for each MF exposure
condition in Study 2 was similar to the distribution of
cells for G2-M enriched samples in Study 1 (Compare
Table 3 to Table 1). In Study 2, the average normalized
ratios for the Null and AC þDC exposure groups were
similar with significant overlap of the SE at each time
point (Fig. 3). Analysis of the descriptive data revealed
no statistically significant difference between the two
exposure groups (Table 4).
However, when the normalized ratios between
Studies 1 and 2 were compared, differences were
found (Fig. 4). Qualitative differences between the rate
of increase before anti-CD3 and the shape of the
recovery to the steady state level after anti-CD3
between Study 1 and 2 were apparent. Statistical
analysis of the descriptive data validated the qualitative
findings (Table 4). Statistically significant differences
between the Null groups from each study were obtained
for the basal slope, basal intercept, basal average,
time to peak, recovery rate, and active average.
Statistically significant differences between AC þDC
groups from Studies 1 and 2 were also found and
indicated in Table 4.
Fig. 2. The effect of MF exposure on normalized ratios in
synchronized cells from Study1.The average and standard error
ofthe normalizedratioat eachtimepoint wascalculatedforindivid-
ualexperimentalgroupsandplottedasindicatedinthelegend.Null
and AC þDC exposed groups for G 0/G1, S, and G2 -M enriched
samplesareshownin panels A,B,andC,respectively.The number
of experimentsin eachgroupappearsin the legend.
Real-Time Measurement of [Ca
2þ
]
c
359
DISCUSSION
ELF MF Effects
In Study 1, we examined the dependence of
calcium signaling on ELF MF exposure in Jurkat cells
synchronized with aphidicolin. Analysis of the pooled
data revealed a statistically significant increase in the
peak-normalized ratio after anti-CD3 stimulation dur-
ing ELF MF exposure. Post-hoc analysis of ELF MF
experiments with samples enriched to different phases
of the cell cycle indicated that the largest change in
[Ca
2þ
]
c
signaling occurred in G2-M enriched samples.
However, the increase in the peak normalized ratio after
anti-CD3 in G2-M enriched samples failed to achieve
statistical significance. The result suggested that ELF
MF influenced calcium signaling in Jurkat cells, but in a
manner that was only weakly dependent on the phase of
the cell cycle.
It is unlikely that the difference obtained with the
pooled data could be attributed to selection bias or
experimental artifact. In both studies, large numbers of
replicate experiments were done, groups were properly
randomized on each day, and treatment conditions were
balanced between each day of experiments. Addition-
ally, experimental artifact could be ruled out since the
apparatus was thoroughly characterized for artifacts
that could have arisen from vibration, heating effects,
electronic interference, magnetic interference, and
optical issues [Carson and Prato, 1996].
Possible Mechanisms Underlying
ELF MF Effect
It is now well understood in many cell types,
including Jurkat, that calcium signaling involves
calcium release from intracellular calcium stores
followed by capacitative calcium entry via channels in
the plasma membrane [Putney, 2001, 2005]. Given
that the peak normalized ratio after stimulation in ELF
MF-exposed samples was the only descriptive statistic
to be measurably different from Null-exposed samples,
we concluded that ELF MF exposure affected the
calcium-release dependent pathway. Although the
exact molecular target could not be determined
from our data, several candidates were hypothesized,
which were supported by data from other laboratories.
TABLE 2. Descriptive Statistics from Study 1 (Average SE)
Descriptive statistic
G0/G1 S G2-M
Null AC þDC Null AC þDC Null AC þDC
Basal slope (10
4
/s)
b
1.2 0.1 1.2 0.1 0.9 0.2 0.8 0.2 1.0 0.2 1.0 0.1
Basal intercept 0.969 0.004 0.976 0.006 0.982 0.008 0.989 0.004 0.976 0.006 0.969 0.005
Basal average 1.06 0.01 1.06 0.01 1.05 0.01 1.05 0.01 1.05 0.01 1.05 0.01
Rise (10
2
/s) 1.5 0.1 1.5 0.1 1.3 0.1 1.5 0.1 1.2 0.1 1.4 0.1
Peak
a
1.04 0.05 1.07 0.05 0.92 0.06 1.03 0.04 0.88 0.04 1.00 0.05
Time to peak (s) 80.9 4.7 83.7 3.6 86.5 4.4 80.9 3.7 85.1 4.5 88.0 5.9
Recovery (10
3
/s) 4.2 0.4 4.5 0.6 4.3 0.6 3.6 0.5 3.3 0.4 3.8 0.4
Active slope
a
(10
5
/s) 10 210 210 2924272
Active intercept
a,b
2.04 0.06 2.01 0.03 1.83 0.06 1.89 0.08 1.81 0.05 1.89 0.07
Active average
b
1.82 0.03 1.82 0.03 1.65 0.04 1.71 0.05 1.73 0.03 1.75 0.06
a
Post-hoc comparison of samples grouped by cell cycle show a significant difference between G0/G1 and G2-M enriched phases (indicated
by bolded text).
b
Post-hoc comparison of samples grouped by cell cycle show a significant difference between G0/G1 and S enriched phases (indicated by
bolded text).
TABLE 3. Distribution of Synchronized Jurkat E6.1 Cells in
Study 2 (Average SE)
ELF MF exposure % G0-G1 % S % G2-M
Null (n¼27) 27 1191541
AC þDC (n¼17) 26 1191542
Fig. 3. Results from Study 2 for samples of G2- M enrichment.
Each symbolrepresents the normalized ratio obtained by averag-
ing experiments where the exposure condition was similar. The
exposure condition and number of experiments are indicated in
the legend. Error bars represent standard errors of the mean.
360 McCreary et al.
These supporting data included an ELF MF influence
on (i) the binding of anti-CD3 to membrane-
bound receptors [Nindl et al., 2000], (ii) inositol
trisphosphate levels [Korzh-Sleptsova et al., 1995],
and (iii) CD45 phosphatase activity [Lindstrom et al.,
1995a].
Cell Cycle Dependence
We were unable to determine any dependence of
ELF MF modulated calcium signaling on cell cycle.
Our result was consistent with another study where c-
myc transcript levels were measured in G0/G1 enriched
lymphoid cells, but no changes were observed after
exposure to ELF MF [Desjobert et al., 1995]. Other
studies found ELF MF effects on the cell cycle, but only
when the exposure was combined with radiation [Harris
et al., 2002; Tian et al., 2002]. It is largely unknown
how relevantthese reports were to our findings, since it is
unclear if any of the cited reports could be related to
direct or indirect ELF MF-related alterations in [Ca
2þ
]
c
.
Although we could not detect ELF MF-related
changes in [Ca
2þ
]
c
that depend on cell cycle, the basal
and stimulated [Ca
2þ
]
c
response were found to depend
on phase of the cell cycle independent of ELF MF.
Pande et al. [1996] found that [Ca
2þ
]
c
depended on the
phase of the cell cycle with the lowest level at the
beginning of G1, gradually increasing to a maximum at
the G1-S border, then decreasing during S and slightly
increasing again at G2. Although their results provided
plausibility for our findings, our results were not
directly comparable since we only reported relative
changes in fluorescence ratio and not absolute measures
of [Ca
2þ
]
c
. Karas et al. [1999] also measured the
[Ca
2þ
]
c
response to activation of the T-cell receptor
by anti-CD3 monoclonal antibody, OKT-3, in Jurkat
cells with G0/G1, S, and G2-M enriched phases. Phases
of the cell cycle were enriched using counterflow
centrifugal elutriation rather than cell cycle arrest by
chemical treatment. In contrast to our results and those
of Pande and coworkers, they were unable to detect
any cell cycle dependence of the [Ca
2þ
]
c
response.
However, the sample size was low (two independent
samples for each experimental group), which would
make it difficult to detect small changes in a variable
response.
Possible Mechanisms Underlying
[Ca
2þ
]
c
Dynamics
The results from Study 1 indicated the possibility
of a weak dependence of ELF-MF effects on calcium
signaling in G2-M enriched Jurkat cells. We examined
this apparent phenomenon in greater detail in Study 2,
but found no difference between the peak-normalized
ratios after anti-CD3 stimulation from the two exposure
groups. Although this was statistically consistent with
our Study 1 finding, the close proximity to statistical
significance of Study 1 (i.e., P¼.057) was not observed
in Study 2. Therefore, the result of Study 2 might best be
TABLE 4. Descriptive Statistics for G2-M-Enriched Samples
From Study 2 (Average SE)
Descriptive statistic Null (n¼27) AC þDC (n¼17)
Basal slope (10
4
/s) 1.8 0.1
a
1.7 0.1
a
Basal intercept 0.947 0.003
a
0.951 0.006
a
Basal average 1.08 0.01
a
1.08 0.01
a
Rise (10
2
/s) 1.5 0.1 1.6 0.1
Peak 0.91 0.06 0.94 0.05
Time to peak (s) 105 3
a
105 5
Recovery (10
3
/s) 1.1 0.2
a
0.9 0.1
a
Active slope (10
5
/s) 3112
a
Active intercept 1.99 0.06 1.94 0.05
Active average 1.92 0.05
a
1.92 0.04
a
a
Significantly different from same descriptive statistic in Study 1.
Fig. 4. Comparison of the average normalized ratios between
Study1 and Study 2.The average normalized ratios from samples
of G2-M enrichment andexposedto the Nullconditionare shownin
panel A. Similarly, results from samples exposed to the AC þDC
MF are shown in panel B.
Real-Time Measurement of [Ca
2þ
]
c
361
explained by an as yet unknown experimental variable
upon which the ELF MF influence on calcium signaling
depended. One explanation may be related to the
mechanism(s) that underlie the differences in calcium
dynamics between the two studies. Perhaps one or more
of these mechanisms rendered cells insensitive to ELF
MF in Study 2.
Mechanistic explanation of the observed [Ca
2þ
]
c
dynamics include the following possibilities.
(i) The increased rate of [Ca
2þ
]
c
rise in resting
cells from Study 2 compared to Study 1 suggested that
indo-1 was leaking from the cells into the extracellular
environment where Ca
2þ
is typically 10 000-fold more
concentrated and leads to higher fluorescence ratios.
Alternatively, the basal permeability of the plasma
membrane and/or intracellular Ca
2þ
store membranes
may have been higher in cells from Study 2 compared
to Study 1.
(ii) Differences in the response to anti-CD3
stimulation may indicate a subtle difference in the
potency of the anti-CD3 between Studies 1 and 2. This
may have been complicated by differences in CD3
receptor expression, co-stimulation by cytokines in
different batches of fetal bovine serum, and extrac-
ellular calcium concentration between batches of
conditioned medium.
(iii) The [Ca
2þ
]
c
response to stimulation in
individual T-lymphocytes and in suspensions of Jurkat
cells was shown to be highly variable [Wulfing et al.,
1997; McCreary et al., 2002]. The proportion of
individual cells that responded to anti-CD3 with an
initial transient increase followed by a small undershoot
and sustained high [Ca
2þ
]
c
may be reduced in Study 2
compared to Study 1. Possibly, a greater proportion of
the cells in Study 2 had a greater response to the same
concentration of anti-CD3. In Study 2, the difference
between the peak [Ca
2þ
]
c
and the elevated steady state
[Ca
2þ
]
c
after activation was smaller and the undershoot
was not observed (Fig. 4).
(iv) Numerous in vitro studies have suggested that
the effects of weak ELF magnetic fields depend on the
metabolic status of the cells [Walleczek and Liburdy,
1990; Muehsam and Pilla, 1999], particularly activation
state [Reinbold and Pollack, 1997; Campbell-Beachler
et al., 1998; Tuinstra et al., 1998; Richard et al., 2002],
differentiation state [Loschinger et al., 1999], and
growth stage [Nindl et al., 1997; Felaco et al., 1999;
Diniz et al., 2002]. Given that the two studies were
separated by over a year it is not unreasonable to suggest
that slight differences in handling and culture con-
ditions could have existed between the two studies, in
turn leading to the differences in [Ca
2þ
]
c
dynamics
between Studies 1 and 2 by one or more of the above-
mentioned mechanisms.
Implications of the Work
We observed an overall 9% increase in the peak-
normalized ratio in ELF-MF exposed Jurkat cells
over and above the response of Null-exposed cells
after stimulation with anti-CD3. This apparent small
increase in the assay metric could have corresponded to
a 40% increase in [Ca
2þ
]
c
after calibration of the
ratiometric data. If all cells in the cuvette responded
with the same magnitude, then it remained debatable
whether such a small change could lead to downstream
biological consequences. However, if only a small
subpopulation of cells responded with a large change in
[Ca
2þ
]
c
with the balance of cells unresponsive to ELF
MF, then large downstream biological effects might be
expected in the subpopulation. At least one report lends
some support to this possibility in Jurkat cells
[Lindstrom et al., 1993].
Future Work
The results from this work led us to suggest
several avenues for future study. First, it would be
worthwhile to explore the reasons for the differences in
[Ca
2þ
]
c
dynamics between the two studies. Specifically,
the roles of anti-CD3 concentration and extracellular
Ca
2þ
should be studied systematically for both Null and
ELF MF exposure conditions. Second, although we
chose ELF MF conditions that followed the predictions
of a theoretical model, the applicability of the model is
still unclear and stronger ELF MF fields might elicit
larger, more reliable changes in [Ca
2þ
]
c
signals after
anti-CD3. Last, ratiometric fluorescence microscopy of
the [Ca
2þ
]
c
response to anti-CD3 in individual Jurkat
cells might reveal the existence of a small population of
cells that are strongly affected by ELF MF.
ACKNOWLEDGMENTS
The authors thank Mr. Mike Keeney and
Ms. Wendy Brown for assistance with the flow
cytometry measurements and analysis; Mr. Larry Stitt,
Drs. Yves Bureau and Alex Thomas for advice on the
statistical analysis of the data; Mr. Lynn Keenliside for
technical assistance with the fluorescence spectropho-
tometer; and Dr. Michelle Belton for assistance with the
manuscript preparation.
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