Magnetic elds increase cell survival by inhibiting apoptosis via modulation of Ca2/ inux

C. FANELLI,* S. COPPOLA,* R. BARONE,* C. COLUSSI,* G. GUALANDI, P. VOLPE,*, AND L. GHIBELLI*,1 *Dipartimento di Biologia, Universita di Roma Tor Vergata, via della Ricerca Scientica, 00133, ` ` Rome, Italy; DABAC, Universita della Tuscia, Viterbo, Italy; and Istituto di Medicina Sperimentale, CNR, Rome, Italy

Static magnetic elds with intensities starting from 6 gauss (611004 tesla, T) were found to decrease in an intensity-dependent fashion, reaching a plateau at 6 1 1003 T, the extent of cell death by apoptosis induced by several agents in different human cell systems. This is not due to a change in the mode of cell death (i.e., to necrosis) or to a delay of the process itself; rather, the presence of magnetic elds allows the indenite survival and replication of the cells hit by apoptogenic agents. The protective effect was found to be mediated by the ability of the elds to enhance Ca2/ inux from the extracellular medium; accordingly, it was limited to those cell systems where Ca2/ inux was shown to have an antiapoptotic effect. Magnetic elds thus might interfere with human health by altering/restoring the equilibrium between cell death and proliferation; indeed, the rescue of damaged cells may be the mechanism explaining why magnetic elds that are not mutagenic per se are often able to increase mutation and tumor frequencies.Fanelli, C., Coppola, S., Barone, R., Colussi, C., Gualandi, G., Volpe, P., Ghibelli, L. Magnetic elds increase cell survival by inhibiting apoptosis via modulation of Ca2/ inux. FASEB J. 13, 95102 (1999)
ABSTRACT

Key Words: MFs endoplasmic reticulum viable cells etoposide

RECENT CONCERN ABOUT the possible harmful effects of magnetic elds (MFs)2 on human health has led to a growing interest in the inuence of MFs on life processes. Although the data reported in the literature are quite heterogeneous with regard to MF intensity (from 1007 to 10 T), type of eld (static or oscillatory), and subjects exposed to MFs (i.e., from in vitro cultured cells to humans), they nonetheless suggest a link between MFs and tumorigenicity (1). MFs are claimed to enhance the mutation rates of cells exposed to mutagens (2, 3), increase tumor cell survival after various cytocidal therapies (4), and increase tumor rate in cancer-prone mice strains (5). However, no direct tumorigenic or mutagenic effect has ever been attributed to MFs (6): no DNA damage has been detected after exposure to MFs (7) or did
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MFs interfere with the rate of DNA break formation/ repair due to clastogenic treatments (8). The mechanism explaining the putative cocarcinogenic and comutagenetic effect of MFs, still unknown, has been hypothesized to be nongenetic (9). 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 inuence Ca2/ uxes, 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 cells 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/ inux from the extracellular medium (20),
Correspondence: Dipartimento di Biologia, Universita di ` Roma Tor Vergata, via della Ricerca Scientica, 00133 Roma, Italy. E-mail: lina.ghibelli@seneca.uniroma2.it 2 Abbreviations: ER, endoplasmic reticulum; FCS, fetal calf serum; HBSS, Hanks balanced salt solution; HMW, high molecular weight; MFs, magnetic elds; mT, millitesla; PBL, peripheral blood leukocytes; PMC, puromycin.
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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 specic 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/ uxes 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.

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 elds (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). Briey, 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 1 105 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 eld application MFs were produced by metal magnetic disks of known intensities; magnets produce static MFs without producing alternating MFs. Unlike electric eld-driven, solenoid-generated MFs, static MFs do not induce any temperature increase. MFs intensity is given in millitesla (1 T104 G). Magnets were placed under the culture petri dish. Since MFs intensity decreases according to the square of the distance, the actual eld 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 specied. Ca2/ measurements Fluo3 staining

MATERIALS AND METHODS

Cells and treatments Cells and culture U937 and CEM cells were kept in log phase in RPMI 1640 supplemented with 10% or 5% inactivated fetal calf serum (FCS, respectively; experiments were performed at a concentration of 106 cells/ml. Peripheral blood leukocytes were puried through Ficoll gradient, placed in RPMI 1640 with 10% FCS, and either kept quiescent (0) or activated with 2.5 mg/ ml phytohemagglutinin, 2.5 mg/ml pokeweed, or both; cells were used on the third day of explant/activation. Rat thymocytes were explanted according to ref 22, seeded in RPMI supplemented with 10% FCS at the concentration of 106 cells/ml at 37 C. The experiments were performed immediately. Induction of apoptosis Apoptosis was induced with 10 mg/ml puromycin (PMC); 100 mM 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 mM dexametazone. Other treatments The following compounds were added 15 min before induction of apoptosis:10 nM thapsigargin; 500 ng/ml ionomycin; 10 mM 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 mg/ml) according to the nuclear morphological features (24).
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Cells (11107/ml) were washed twice in Hanks balanced salt solution (HBSS), then loaded with 1 mM 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 1 106/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 uorometer as the cuvette-tted chamber did not allow the use of the metal magnetic disks necessary for creating MFs. This problem was circumvented by determining [Ca2/]isoi-related uorescence by means of ow 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 uorescence value was determined by counting 5000 cells. Fluorescence values were converted in [Ca2/] values according to the equation of Grynkiewicz et al. (28) (Fluo3 Kd390 nM; Fmax was measured in the presence of 10 mM ionomycin and 0.65 mM CaCl2).
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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/ inux from the extracellular medium triggered by stores emptying (29). Ca2/ inux modulation The effect of MFs on thapsigargin-mediated [Ca2/]isoi increase was determined by placing magnetic disks close to the ow 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 mM) was added 10 min before measurement.

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 MFdependent reduction of drug uptake through plasma membrane, as suggested by their failure to affect the uptake rate of a reporter uorescent drug (rhodamine 123) at any concentration (from 10 ng to 10 mg/ml) in U937 cells (ow 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. 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 reect 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). MFs increase capacitative Ca2/ inux in U937 cells To understand the mechanism responsible for the antiapoptotic effect of MFs, we analyzed Ca2/ uxes, which are known to be modulated by MFs and are implied in the onset of apoptosis. Thus, MFs effects on Ca2/ uxes of healthy, untreated U937 were analyzed by ow cytometry measurement of 1) basal
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RESULTS Static MFs decrease the extent of etoposideinduced apoptosis in U937 cells The experiments were performed by applying static MFs to the cells by placing metal magnetic disks, producing MFs of known intensities, under the culture petri dish. MFs were analyzed in the range of intensities between 1.5 G (.15 mT) and 660 G (66 mT; terrestrial MF ranges between 0.15 and 0.7 G, with an average of 0.3 G). Our experiments showed that MFs, applied for up to 1 wk to U937 monocytic cells, did not exert any toxic or apoptogenic effect nor did they affect the rate of cell growth. 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 eld 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. 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.
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Figure 1. Magnetic elds reduce etoposide-induced apoptosis on U937 cells in an intensity-dependent way. 6 mT MFs reduce VP16-induced apoptosis on U937 cells, evaluated as fraction of cells with apoptotic nuclei (A) (average values {SEM; n25) or as the extent of apoptotic HMW DNA fragmentation (1 h treatment with VP16) (B), blocking DNA processing from the Mb range (direct VP16 cuts) to the 50 kb range (apoptotic degradation). C) Apoptosis is affected by MFs in an intensity-dependent way; the values (average values from three complete experiments) were measured after 4 h of treatment with VP16 {MFs; eld intensities higher than 6 mT failed to further reduce apoptosis.

[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/ inux 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/ uxes 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/ inux through the plasma membrane (Fig. 4B). Ca2/ entry was efciently blocked by nifedipine, a dihydropyridine antagonist of plasma membrane L-type Ca2/ channels, both within and outside 6 mT MFs (Fig. 4B). This result conrms 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 voltageinsensitive ICRAC Ca2/ channels of nonexcitable cells. Our results show that MFs do not mobilize Ca2/ per se, but are able to increase the extent of an ongoing Ca2/ inux. 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/ inux occur
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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/ uxes 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/ inux was also found to depend on the intensity of the eld, reaching a maximum of 1.9-fold increase at 6 mT (Fig. 4C). MF alteration of Ca2/ uxes is responsible for MFs antiapoptotic effect The experiments on MFs effects on Ca2/ uxes were performed on healthy, nonapoptotic cells stimulated with thapsigargin. The existence of a capacitative Ca2/ inux during apoptosis has never been denitively 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 uorescence 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 elicFANELLI ET AL.

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Figure 3. Magnetic elds increase the survival of cells hit by the apoptogenic treatments. Cells were induced to apoptosis by puromycin in the presence or absence of 6 mT MFs; puromycin was then washed out and cells were reseeded for recovery as described in Materials and Methods (one of three experiments with similar results is shown). Cells rescued by MFs from PMC-induced apoptosis remained viable and capable of duplication. MFs also increased survival of U937 treated with 10 mM or 100 nM VP16 in similar recovery experiments (not shown).

Figure 2. Magnetic elds reduce apoptosis induced by all the agents tested on U937. A) 6 mT MFs reduce puromycin-induced apoptosis at all time points (average values {SEM; n19); B) On U937, MFs affect apoptosis induced by all agents tested. Values are the average of at least ve experiments for each treatment {SEM. Apoptosis was measured at 4 h of continuous treatment (PMC and VP16); at 6 h of recovery (H2O2); at 18 h of recovery (heat shock); and at 4 days of aging.

iting 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/ inux might be the cause of MFs antiapoptotic effect. The link between an MF-dependent increase in Ca2/ inux through plasma membrane and rescue from apoptosis was investigated by testing whether MFs may still exert an antiapoptotic action when Ca2/ is inhibited. Indeed, nifedipines effect of abolishing MF-dependent Ca2/ inux alterations (Fig. 4 B) was accompanied by the abolition of MFs antiapoptotic

Figure 4. Magnetic elds increase capacitative Ca2/ inux in U937 cells. Healthy U937 were stained with Fluo3 as described in Materials and Methods. Measurements of 1) basal [Ca2/]isoi; 2) increase in [Ca2/]isoi due to store emptying with thapsigargin; and 3) increase in [Ca2/]isoi due to Ca2/ inux were performed within or outside of MFs (see Materials and Methods). Panel A shows the time course of the changes in uorescence of untreated U937 upon addition of thapsigargin to elicit store emptying and Ca2/ to elicit capacitative Ca2/ inux. B) Peak values of (a) basal, (b) reticular, and (c) capacitative Ca2/ concentrations of healthy U937 within and outside 6 mT MFs are given as nM [Ca2/]isoi (the raw uorescence values being converted according to the equation described in Materials and Methods). MFs increase capacitative Ca2/ inux without affecting basal or reticular Ca2/ values. Nifedipine inhibits capacitative Ca2/ inux within and outside MFs. C) The extent of capacitative Ca2/ inux in the presence of different MFs intensities: MFs increase capacitative Ca2/ inux in a dose-dependent way, with maximal increase at 6 mT. All values of [Ca2/]isoi are the average of at least 20 measurements for each MFs intensity {SEM.
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inux is that MFs must have no protective effect in those cells where Ca2/ inux is, instead, a trigger for apoptosis. This implication was veried by experiments showing that in rat thymocytes, MFs, though increasing an ongoing Ca2/ inux just as they do in U937 (not shown), do not reduce apoptosis independent of the agent used (Fig. 7). This result conrms 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. 6 with Fig. 7). MFs are protective in those systems where Ca2/ plays an antiapoptotic role By modulating Ca2/ inux in other cell types (Fig. 8), we conrmed 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 ts with the observation that MFs are able to enhance activationrelated Ca2/ inux in PBL (14).

Figure 5. Nifedipine or EGTA abrogate the antiapoptotic effect of magnetic elds. The inhibition of Ca2/ ion uptake by nifedipine (nife) or EGTA abrogates the antiapoptotic effect of 6 mT MFs. The values of apoptosis (average from 14 experiments {SEM) were measured after 4 h of treatment with puromycin.

effect (Fig. 5). Treatments aimed at inhibiting Ca2/ inux 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/ inux. Agents inducing Ca2/ inux protect U937 from apoptosis The nding that MFs exert their protective effect by enhancing Ca2/ inux implies that, in U937 cells, an induction of Ca2/ inux 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 veried by experiments showing that compounds that induce a Ca2/ inux, 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). 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/ inux plays the opposite rolethat of triggering apoptosis (22). MFs do not protect from apoptosis rat thymocytes A second implication associated with the nding that MFs exert their protective effect by enhancing Ca2/
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DISCUSSION In this study we show that static MFs exert a strong and reproducible effect of reducing apoptosis in several cell systems. This effect is mediated by MFs ability to increase Ca2/ inux, since its inhibition abro-

Figure 6. Ca2/ modulators exert opposite effects on apoptosis in U937 and thymocytes. The effects compounds that alter Ca2/ uxes exert on apoptosis were probed in U937 (left) and rat thymocytes (right). Apoptosis was measured at 4 h (U937) or 6 h (thymocytes) after treatment with puromycin and/or the Ca2/ inux modulators. The values shown are the average of at least three experiments for each treatment {SEM (thpgthapsigargin; ionoionomycin).
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gated MFs antiapoptotic effect. Other possible effects of MFs, such as an alteration of the pattern of gene expression, were excluded. Our data provide a rationale to the still paradoxical nding 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 nd 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 elds enhance capacitative Ca2/ inux 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 elds (4), suggesting that the still unknown mechanism underlying the alteration of Ca2/ uxes involves changes that both static and pulsating electromagnetic elds are able to induce. The nding that easily available metal magnetic disks may, at least in some instances, substitute for complex apparatuses in order to originate electromagnetic elds might contribute to expanding the study of the cellular effects of magnetic elds, which now is limited to specialized

Figure 7. Magnetic elds do not exert any antiapoptotic effect on rat thymocytes. MFs fail to reduce the extent of apoptosis on rat thymocytes, independent of the apoptogenic agent used; values are the average of at least three experiments for each treatment {SEM. Apoptosis was measured at 6 h of treatment.

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/ inux might be of great importance for human health, since Ca2/ uxes 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

Figure 8. Effect of magnetic elds on puromycin-induced apoptosis in different cell systems. A) Summary of the effect of MFs on puromycin-induced apoptosis in different cell systems is shown; the values of PMC-induced apoptosis (average values from at least ve (U937) or three (thymocytes) experiments {SEM) were measured after 4 (U937 and CEM) or 6 (PBL, rat thymocytes and EBV/ Burkitt lymphoma cells) h of treatment. Basal apoptosis values were 23% for U937, CEM and EBV/ Burkitt lymphoma cells; 35% for quiescent PBL; 6 10% for activated PBL; and 20% for rat thymocytes. B) The effects that Ca2/ modulators and magnetic elds exert on apoptosis are summarized: EBV/ Burkitt lymphoma cells were induced to apoptosis by ionomycin and rescued from puromycin-induced apoptosis by EGTA (not shown); for thymocytes, see Fig. 6 ( right); in these two systems, Ca2/ inux is apoptogenic. On the contrary, in U937, Ca2/ inux is protective (see Fig. 6, left); the same was found in CEM cells, which were rescued by ionomycin from puromycin-induced apoptosis, whereas EGTA was ineffective (data not shown); accordingly, magnetic elds proved to have an antiapoptotic effect on these cells.
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hand phone antennae) exert a signicant effect on the frequency of apoptosis; thus, exposure to elds 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 elds 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 ofcial since Mesmers times toward the end of 18th century). On the other hand, the ndings described here warn against the presence of magnetic elds 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/ uxes. Indeed, this could be the reason why MF exposure seems to affect only some specic types of tumors, as is revealed by comparing many scattered epidemiological studies.
We wish to thank Dr. A. Wyllie, Dr. M. Marini, and Dr. T. Eremenko for helpful discussions and suggestions. This work was partly supported by CNR special project Progetto Strategico Ciclo Cellulare e Apoptosi.

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