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Research Communications |
a Dipartimento di Biologia, Università di Roma `Tor Vergata', via della Ricerca Scientifica, 00133, Rome, Italy
b DABAC, Università della Tuscia, Viterbo, Italy
c Istituto di Medicina Sperimentale, CNR, Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: MFs • endoplasmic reticulum • viable cells • etoposide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fewer, scattered studies investigated the interference of MFs on cell metabolism, pointing to altered rates of transcription of c-myc (10) and other genes (11), to a slight decrease in the rate of spontaneous cell death in culture (12), and to changes in plasma membrane (13); consistent data report that MFs influence Ca2+ fluxes, particularly Ca2+ entry from the extracellular environment through the plasma membrane (ref 14 and references therein).
Multicellular organisms eliminate unnecessary cells by apoptosis, a cell-intrinsic mechanism that leads healthy cells to choose self-elimination. This occurs under physiological conditions (i.e., regression of fetal structures) as well as in response to damage. In this latest instance, severely hit cells die passively by necrosis, whereas those that have been only mildly damaged die by apoptosis (15), via a mechanism involving p53 action (16, 17) and probably poly(ADP-ribosyl)polymerase activation (15). Under these circumstances, apoptosis might free the organism from retention of potentially dangerous mutated or transformed cells; indeed, treatments/situations impairing the cell's ability to respond to mild DNA damage by apoptosis result in a higher rate of survival of the damaged cells (15). This increases the mutation frequency among surviving cells, as observed in cells lacking wild-type p53 induced to apoptosis by X-rays (18).
Ca2+ ions as mediators of intracellular signaling are crucial for the development of apoptosis. An increase in cytosolic Ca2+ concentration ([Ca2+]i) of apoptosing cells (19), due to emptying of intracellular Ca2+ stores and to Ca2+ influx from the extracellular medium (20), is a general phenomenon, independent of the apoptogenic stimulus. However, the role of [Ca2+]i in the development of apoptosis is ambiguous. A detailed analysis of the specific literature shows that apoptotic [Ca2+]i elevation has different `meanings' in different cell systems, as pointed out in ref 21. It may act as a trigger for cell death in some well-studied cells such as freshly explanted rat thymocytes (22). On the contrary, it could be a defense mechanism against apoptosis in many other cell systems that are perhaps less studied as far as apoptosis is concerned, such as the nerve cells described in ref 23.
In the present study we show that MFs' ability to modulate Ca2+ fluxes causes a reduction of the rate of stress-induced apoptosis, increasing the survival of cells hit by damaging agents. This perhaps explains MFs' paradoxical ability to increase the effect of mutagenic agents without being mutagenic per se.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of apoptosis
Apoptosis was induced with 10 µg/ml puromycin (PMC); 100 µM etoposide (VP16) (24); 1 mM H2O2 for 1 h, followed by recovery in fresh medium (15); heat shock at 43°C for 1 h, followed by recovery in fresh medium (15); aging of the culture (25); and 2 µM dexametazone.
Other treatments
The following compounds were added 15 min before induction of apoptosis:10 nM thapsigargin; 500 ng/ml ionomycin; 10 µM nifedipine; 1 mM EGTA.
Analysis of apoptosis
DNA digestion was analyzed by conventional electrophoresis, as described (15), or by PFGE analysis of DNA high molecular weight (HMW) fragmentation (24). Nuclear fragmentation was detected after staining with the vital dye Hoechst 33342 (1 µg/ml) according to the nuclear morphological features (24).
Quantitation of apoptosis
The fraction of cells with fragmented, crescent-shaped, or shrunken nuclei was evaluated among the Hoechst-stained cells by counting at least 300 cells in at least three randomly selected fields (15).
Analysis of viable cells
Cell viability was assessed by membrane impermeability to trypan blue and propidium iodide, positive staining with vital dyes, normal nuclear shape and texture revealed upon vital staining with Hoechst 33342.
Recovery
Washout experiments were performed as described (26). Briefly, drugs were removed after 4 h of the apoptogenic treatment ±6 millitesla (mT) MFs. U937 were then resuspended in fresh medium, and aliquots of 2 x 105 cells were seeded in quadruplicates in petri dishes for recovery. For each treatment, two petri dishes were placed outside and two within 6 mT MFs. The number of viable cells was estimated in a hemocytometer at the times indicated.
Magnetic field application
MFs were produced by metal magnetic disks of known intensities; magnets produce static MFs without producing alternating MFs. Unlike electric field-driven, solenoid-generated MFs, static MFs do not induce any temperature increase. MFs intensity is given in millitesla (1 T=104 G).
Magnets were placed under the culture petri dish. Since MFs intensity decreases according to the square of the distance, the actual field intensity on the cells was calculated considering the thickness of the bottom of the petri dish (1.2 mm). MFs were applied concomitantly with the apoptogenic treatments, unless otherwise specified.
Ca2+ measurements
Fluo3 staining
Cells (1x107/ml) were washed twice in Hank's balanced salt solution (HBSS), then loaded with 1 µM Fluo3-AM at 20°C for 40 min. After dye removal, cells were resuspended in fresh HBSS ± 0.65 mM CaCl2 at the concentration of 2 x 106/ml. Cells were stored at 20°C until use and warmed at 37°C for 2 min before measurements.
Flow cytometry analysis
[Ca2+]isoi measurements could not be performed in a fluorometer as the cuvette-fitted chamber did not allow the use of the metal magnetic disks necessary for creating MFs. This problem was circumvented by determining [Ca2+]isoi-related fluorescence by means of flow cytometry analysis as described in ref 27, using a FACScan (Becton Dickinson, San Jose, Calif.) tuned at 488 nm, using the FL1 photomultiplier (bandpass 530 nm, bandwidth 30 nm). Data were recorded in list mode for further analysis with the lysis II software (Becton Dickinson). The mean fluorescence value was determined by counting 5000 cells. Fluorescence values were converted in [Ca2+] values according to the equation of Grynkiewicz et al. (28) (Fluo3 Kd=390 nM; Fmax was measured in the presence of 
10 µM ionomycin and 0.65 mM CaCl2).
Measurement of cytosolic calcium levels
Three different Ca2+ pools were measured: 1) basal [Ca2+]i; 2) [Ca2+]i upon addition of 10 nM thapsigargin in cells kept in the absence of free Ca2+; and 4) [Ca2+]isoi upon further addition of 0.6 mM Ca2+. In this instance, a further increase in [Ca2+]isoi is observed, due to Ca2+ influx from the extracellular medium triggered by stores emptying (29).
Ca2+ influx modulation
The effect of MFs on thapsigargin-mediated [Ca2+]isoi increase was determined by placing magnetic disks close to the flow cytometry sample tubes in order to obtain the indicated MF intensity. Cells were placed in MFs simultaneously with the measurement, unless otherwise indicated, and kept within MFs for the time required for the measurement. Nifedipine (10 µM) was added 10 min before measurement.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MFs, applied concomitantly with the topoisomerase II inhibitor etoposide, a well-known apoptogenic agent, decreased the extent of apoptosis. The effect was already detectable after 1 h of treatment and was maintained thereafter (
Fig. 1A, B).
The antiapoptotic effect of MFs could be modulated by increasing field intensities. The minimal intensity required to detect an antiapoptotic effect was 0.6 mT, about 15-fold higher than terrestrial MF, the effect increasing almost linearly up to about 6 mT ( Fig. 1C). Higher MFs intensities failed to further reduce apoptosis. For this type of analysis, no shielding against the natural variations of terrestrial MFs was required, their values (ranging at about ± 0.75 G, 0.075 mT) being negligible with respect to the MF intensities applied.
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Cells were placed in MFs at the moment of apoptosis induction. Preincubation of cells in MFs for 1, 4, or 7 days did not increase MFs' antiapoptotic effect (not shown) nor could preincubation by itself reduce apoptosis if cells were placed outside MFs during the apoptogenic treatment. This indicates that the antiapoptotic effect of MFs is immediate and immediately reversed, thus not implying any long-term changes such as an altered pattern of gene expression.
MFs reduce apoptosis by interfering with the apoptotic process and not with the inducer
MFs were proved to be able to reduce apoptosis induced by the protein synthesis inhibitor puromycin ( Fig. 2A),
which suggests that their antiapoptotic effect is due to an interference with the apoptotic signaling and not with the mechanism of action of etoposide. We also excluded the involvement of a possible generalized MF-dependent reduction of drug uptake through plasma membrane, as suggested by their failure to affect the uptake rate of a reporter fluorescent drug (rhodamine 123) at any concentration (from 10 ng to 10 µg/ml) in U937 cells (flow cytometric analysis, not shown). As a complementary experiment, MFs were tested for their ability to reduce the apoptosis induced by agents that bypass drug uptake, such as hydrogen peroxide (15), which freely cross cell membranes, heat shock (15), and spontaneous aging of the culture (25). We observed that MFs affected drug-induced and drug-independent apoptosis ( Fig. 2B) to the same extent. Overall, these results indicate that MFs' antiapoptotic effect on U937 does not depend on the inducer, but is related to an interference with apoptotic intracellular signaling.
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MFs operate a rescue to full viability on cells hit by apoptogenic agents
The decrease of apoptosis was always paralleled by a corresponding increase in the fraction of viable cells, thus excluding the possibility that it might merely reflect a change in the mode of cell death from apoptosis to necrosis. In fact, the increase in cell survival due to MFs was maintained during the subsequent culture of treated cells after removal of the apoptogenic agents in washout experiments (recovery), independent of the presence of MFs during the recovery period.
MFs did not just delay apoptosis, but operated a rescue to viability that allowed the rescued cells to reach the normal replicative rate immediately after recovery ( Fig. 3).
The result of this experiment has double value: it is important from the point of view of basic science. On the other hand, it shows that MFs interfere with apoptosis at a step preceding the irreversible commitment to death (26) but also that MFs favor the survival of cells hit by damaging agents, thus pointing to a possible role of MFs in indirectly favoring mutagenesis (and carcinogenesis).
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MFs increase capacitative Ca2+ influx in U937 cells
To understand the mechanism responsible for the antiapoptotic effect of MFs, we analyzed Ca2+ fluxes, which are known to be modulated by MFs and are implied in the onset of apoptosis. Thus, MFs effects on Ca2+ fluxes of healthy, untreated U937 were analyzed by flow cytometry measurement of 1) basal [Ca2+]i; 2) increase in [Ca2+]isoi due to thapsigargin, a drug that mobilizes Ca2+ from the endoplasmic reticulum (ER) store into cytosol, and 3) the further [Ca2+]i increase due to Ca2+ influx through plasma membrane channels from the extracellular medium, triggered by ER emptying, the so-called capacitative Ca2+ entry (29). Figure 4A
shows the kinetic analysis of Ca2+ fluxes on U937 kept outside MFs. We found that 6 mT MFs did not alter basal [Ca2+]isoi or affect the extent of Ca2+ release from ER, but caused a nearly twofold increase in Ca2+ influx through the plasma membrane ( Fig. 4B). Ca2+ entry was efficiently blocked by nifedipine, a dihydropyridine antagonist of plasma membrane L-type Ca2+ channels, both within and outside 6 mT MFs ( Fig. 4B). This result confirms the observation published in ref 30, which showed the unexpected result that dihydropyridines, in addition to the known effect of blocking voltage-sensitive plasma membrane Ca2+ channels of excitable cells, are also effective in inhibiting voltage-insensitive ICRAC Ca2+ channels of nonexcitable cells.
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Our results show that MFs do not mobilize Ca2+ per se, but are able to increase the extent of an ongoing Ca2+ influx. This effect was monitored soon after placing cells in MFs and was not enhanced by preincubations in MFs for 2 min, 5 min, or 2 h, indicating that the MF-induced changes in Ca2+ influx occur within the ~100 s necessary for sample processing. Conversely, preincubation per se did not have any effect when the cells were placed outside MFs during measurement, which shows that the mechanism underlying the disturbance of Ca2+ fluxes by MFs cannot involve any long-term changes such as an altered pattern of gene expression.
As for MFs' antiapoptotic effect, the increase in Ca2+ influx was also found to depend on the intensity of the field, reaching a maximum of 1.9-fold increase at 6 mT (
Fig. 4C).
MF alteration of Ca2+ fluxes is responsible for MFs' antiapoptotic effect
The experiments on MFs' effects on Ca2+ fluxes were performed on healthy, nonapoptotic cells stimulated with thapsigargin. The existence of a capacitative Ca2+ influx during apoptosis has never been definitively shown; however, we have observed that in the course of the apoptogenic treatment with puromycin, the intracellular stores become progressively insensitive to thapsigargin, the increase in cytosolic [Ca2+] due to store emptying passing from 100 fluorescence intensity units (see Materials and Methods) in control cells to 25 at 1 h of treatment, to almost zero at 4 h (experiments in progress). This suggests that a store emptying had occurred that was capable of eliciting a capacitative Ca2+ entry. This observation gave us the logical basis that allowed us to ask the question of whether MFs effect on capacitative Ca2+ influx might be the cause of MFs antiapoptotic effect.
The link between an MF-dependent increase in Ca2+ influx through plasma membrane and rescue from apoptosis was investigated by testing whether MFs may still exert an antiapoptotic action when Ca2+ is inhibited. Indeed, nifedipine's effect of abolishing MF-dependent Ca2+ influx alterations ( Fig. 4B) was accompanied by the abolition of MFs' antiapoptotic effect ( Fig. 5).
Treatments aimed at inhibiting Ca2+ influx by other means, such as extracellular Ca2+ chelation by EGTA, also abrogated MFs' antiapoptotic action ( Fig. 5). These data indicate that MF protection from apoptosis is linked to its ability to increase the extent of Ca2+ influx.
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Agents inducing Ca2+ influx protect U937 from apoptosis
The finding that MFs exert their protective effect by enhancing Ca2+ influx implies that, in U937 cells, an induction of Ca2+ influx must contrast apoptosis. Indeed, U937 cells may belong to that category of cells that, for reasons that do not seem to depend on the histological origin of the cells, Ca2+ ions play an antiapoptotic role.
This implication was verified by experiments showing that compounds that induce a Ca2+ influx, such as thapsigargin (via the stimulation of a subsequent capacitative) or the Ca2+ ionophore ionomycin (by freely allowing Ca2+ ions to cross plasma membrane), reduced apoptosis on U937 ( Fig. 6,
left).
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Instead, as expected, the opposite behavior was found in freshly explanted rat thymocytes, where thapsigargin and ionomycin were apoptogenic ( Fig. 6, right). It is well known that, in this cell system, Ca2+ influx plays the opposite role—that of triggering apoptosis (22).
MFs do not protect from apoptosis rat thymocytes
A second implication associated with the finding that MFs exert their protective effect by enhancing Ca2+ influx is that MFs must have no protective effect in those cells where Ca2+ influx is, instead, a trigger for apoptosis.
This implication was verified by experiments showing that in rat thymocytes, MFs, though increasing an ongoing Ca2+ influx just as they do in U937 (not shown), do not reduce apoptosis independent of the agent used ( Fig. 7).
This result confirms that the difference in degree of proneness to MFs' antiapoptotic effect does not depend on the inducer used, instead indicating that MFs' antiapoptotic effect depends on the cell type (compare Fig. 6with Fig. 7).
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MFs are protective in those systems where Ca2+ plays an antiapoptotic role
By modulating Ca2+ influx in other cell types ( Fig. 8),
we confirmed the paradigm that MFs are protective in those systems where Ca2+ plays an antiapoptotic role (as we found with the T lymphocytic CEM cell line) and ineffective where Ca2+ plays an apoptogenic role (as we found to occur on EBV+ Burkitt lymphoma B cells). We also observed that MFs protected activated, but not quiescent, peripheral blood leukocytes (PBL) from apoptosis. This fits with the observation that MFs are able to enhance activation-related Ca2+ influx in PBL (14).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data provide a rationale to the still paradoxical finding that MFs might be comutagenic and cocarcinogenic without being mutagenic or carcinogenic by themselves. Their effect of reducing apoptosis, thereby allowing the survival of possibly mutated cells, might be the epigenetic mechanism favoring tumor development postulated by the epidemiologists. Earlier studies published from different laboratories reported no effect of MFs on apoptosis; however, our data do not contradict these studies, which were either performed on cell systems where we did not find any antiapoptotic effect by MFs or with MF intensities that we found not to be effective (31).
We have shown that magnet-generated static magnetic fields enhance capacitative Ca2+ influx with an immediate and reversible effect, without affecting Ca2+ mobilization from intracellular stores. These results agree with previous studies showing Ca2+ uptake alterations by exposure to pulsating electromagnetic fields (4), suggesting that the still unknown mechanism underlying the alteration of Ca2+ fluxes involves changes that both static and pulsating electromagnetic fields are able to induce. The finding that easily available metal magnetic disks may, at least in some instances, substitute for complex apparatuses in order to originate electromagnetic fields might contribute to expanding the study of the cellular effects of magnetic fields, which now is limited to specialized laboratories. Many recent reports from basic and epidemiological sciences, as well as from communication media, suggest that these studies deserve much more attention than is so far believed to be warranted. In particular, the study of the effects exerted by MFs on Ca2+ influx might be of great importance for human health, since Ca2+ fluxes are so crucial in cellular communications that their alterations may negatively affect general homeostasis.
In conclusion, we show here that MFs greater than 0.6 mT (far lower than those present, i.e., at the tip of hand phone antennae) exert a significant effect on the frequency of apoptosis; thus, exposure to fields of this strength may lead to a disturbance of cell homeostasis (32), with clear implications for human health. On the one hand, our data suggest that magnetic fields locally applied to tissues undergoing degeneration (i.e., neuron degeneration, autoimmune diseases) might help to slow down the pathological degenerative processes associated with at least some cell types (actually, `magnetotherapies' belong to a byway of medicine hardly recognized as official since Mesmer's times toward the end of 18th century). On the other hand, the findings described here warn against the presence of magnetic fields where it is required that all cells damaged either accidentally (i.e., sun exposure, mutagens contamination, etc.) or on purpose (cytocidal therapies), die. It will be most important to understand the basis of the various sensitivities of different cell types to Ca2+ fluxes. Indeed, this could be the reason why MF exposure seems to affect only some specific types of tumors, as is revealed by comparing many scattered epidemiological studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: ER, endoplasmic reticulum; FCS, fetal calf serum; HBSS, Hank's balanced salt solution; HMW, high molecular weight; MFs, magnetic fields; mT, millitesla; PBL, peripheral blood leukocytes; PMC, puromycin.
Received for publication June 18, 1998.
Revision received September 2, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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