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Similarities in the proteins synthesized by Sciara salivary gland cells in response to electromagnetic fields and to heat shock
27
Bioelectrochemistry and BioeneRetics, 31 (1993) 27-38 Elsevier Sequoia S.A., Lausanne
JEC BB 01577
Similarities in the proteins synthesized by Sciaru salivary gland cells in response to electromagnetic fields and to heat shock M. Blank ‘, 0.
Khorkova ’ and R. Goodman 2
Departmentsof Physiology I and Pathology ‘, Columbia University, 630 West 168th St., New York, NY 10032 (USA)
(Received 17 June 1992; in revised form 11 August 1992)
We have studied changes in the biosynthetic responses of salivary gland cells from Sciara coprophila to two stresses, electromagnetic (EM) fields and heat shock (HS). To characterize the entire field of proteins synthesized, we converted the data from two-dimensional gels into protein distribution curves (%mass vs. MW, %mass vs. ~11. The responses to the two different stimuli, EM and HS, are quite similar to each other and different from those of the control. There are new peaks at MW = 30 kD and pI = 7.1 for both EM and HS. There is also a shift of the peak in pI distribution from 5.8 to 6.3 for both EM and HS. A plot of the changes in the two pl peaks as a function of EM field strength defines a dose-response curve as well as the range of the effect. These changes appear to be characteristic of the cellular response to stress.
INTRODUCTION
In the debate on the biological effects of environmental non-ionizing electromagnetic (EM) waves, probably the strongest experimental evidence has come from studies of changes in biosynthesis. EM waves in the range below 300 Hz (and including 60 Hz) have been shown to affect both transcription and translation in cell and organ cultures [l-53. The changes in the proteins are rather complicated and difficult to analyze, but there appear to be similarities in the patterns of the responses of cells to two physical stresses, EM fields, and heat shock (HS). Focusing on the 53 major proteins newly synthesized following stimulation (out of the several hundred that could be detected), several general properties of the biosynthetic response of the salivary gland cells of Sciaru were noted [6,7]: l EM-stimulated cells show relative changes in the MW distribution (e.g. increases in the 20-50 kD range, with decreases outside this range). HS-stimulated 0302-4598/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights reserved
28
cells show similar patterns after subtraction of the major HS protein band around 70 kD. l New proteins (not present in control gels) tend to be of lower molecular weight and to have a different isoelectric point distribution from the control proteins. The smaller new proteins are more highly charged, both positively and negatively, than control proteins. l Missing proteins tend to have higher molecular weights, but there is no one-to-one relation between missing and new proteins. These changes suggest that EM fields may stimulate a pathway that is similar to the one used by cells in response to HS and other kinds of physical stress. This does not preclude the possibility of specific responses to each stimulus, as suggested by the observation that the new proteins have a distribution of charges (i.e. smaller proteins are more highly charged) that would be expected if they arose from an interruption (i.e. early termination) of biosynthesis on the ribosome. In this paper we have examined these issues in greater detail, and present evidence for patterns in biosynthetic responses. EXPERIMENTAL
Procedures Experimental groups
The experimental samples included cells maintained at the normal growth temperature of 20°C and isolated from known EM field sources (control), exposed to HS at 375°C and exposed to a 60 Hz sinusoidal field at magnetic fields that varied by factor of 10 increments from 0.8 to 800 FT. Characterktics of electromagnetic fields
Four different magnetic field strengths were used with a 60 Hz sinusoidal EM field. The field strengths used were 0.8, 8, 80 and 800 u,T. Signal generation /cellular
exposure
Cells are exposed within a pair of 13 X 14 cm Helmholtz coils with an 8 cm spacing. The.coils are constructed of wire bundles approximately 1 cm in diameter wound around a square form. The coils are positioned vertically so that the oscillating magnetic field is generated in a horizontal plane, thus inducing a relatively uniform electric field in the conductive medium. The value of the electric field is dependent on the depth of the medium, in this case ca. 0.5 cm. Petri dishes (15 mm X 50 mm> containing the salivary glands were placed horizontally on a Plexiglas stand in an area between the coils shown to have a uniform magnetic field. The Helmholtz coils are shielded in a mu metal container within the incubator. Continuous sine waves were generated by a Wavetek function generator (Wavetek model 21,ll MHz) connected to a power regulator. The power regulator (Electra-Biology Inc.> produces amplitudes of 0.5, and 50 u,V. To produce 500 p,V
29
(800 u,T), an amplifier (Realistik, RadioShack, 800 W) was used in conjunction with the function generator. The function generator, power regulator and amplifier were outside the incubator. All exposure took place within a 20°C tissue culture incubator. Control cultures were placed in an identical incubator within a sham apparatus. Exposure times were 45 min for all field strengths tested. The 45 min time-point was used for two-dimensional gel electrophoretic analyses to ensure adequate incorporation of the isotope for analysis. No thermal changes (*O.lC) were measured during the exposure periods. Preparation of cells
Late fourth instar Scziwa coprophila female larvae were used for these experi-. ments. Fifty pairs of salivary glands (1.8 x lo4 cells) attached to the larval bodies were placed in 2 ml of Schneider’s Drosophila medium (SDM) minus methionine (GIBCO) and exposed to selected EM field strengths, isolated from the field or heat shocked at 37°C. The salivary glands were then dissected free of the larval body in ice-cold SDM minus methionine. For these experiments 35S-methionine at 100 @i/ml was added to each dish. Sample preparations (using a Kendrick Laboratories Inc. sample preparation kit) were performed in the cold. Anulysis of polypeptides
Two-dimensional electrophoresis was performed by Kendrick Laboratories Inc. (Madison, WI) according to the method of O’Farrell. Isoelectric focusing was carried out in glass tubes of inner diameter 2.0 mm, using 2.0% certified Resolytes pH 4-8 ampholines (BDH from Hoefer Scientific Instruments, San Francisco, CA> for 9600 V h. The first centimeter of the acid end of the tube gel was cut off to remove the SDS bulb that forms there. This area contains no protein. The final tube gel pH gradient extended from about pH 4.0 to pH 8.0 as measured by a surface pH electrode and colored acetylated cytrochrome p1 markers (Calbiochem-Behring, La Jolla, CA). After equilibration for 10 min in SDS sample buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.81, the tube gels were sealed to the top of 10% acrylamide slab gels (0.75 mm thick) and SDS slab gel electrophoresis was carried out for about 4 h at 12.5 mA/gel. The slab gels were prepared after the bromophenol dye front had run off the bottom of the gel for 45 min. The slab gels were fixed in a solution of 10% acetic acid + 50% methanol overnight. Calibration strips containing serial dilutions of i4C-bovine serum albumin (which had been stored at - 20°C in 10% acetic acid + 50% methanol) were added, and both gels and calibration strips were treated with ENHANCE (New England Nuclear, Boston, MA) for 1 h and rehydrated in water for 30 min. The gels and calibration strips were dried on filter paper with the acid end to the left. Fluorography was carried out using Kodak X-OMAT AR film with an exposure of 2 and 4 days at -70°C. The films were developed using Kodak developer and fixer. Figure 1 shows three gels; a control, an EM-stimulated gel and an HS-stimu-
30
Fig. 1. Comparison of autoradiographic patterns following two-dimensional gel electrophoresis (10.0% gels) of 35S-labeled polypeptides from Sciaru salivary gland. Part of each radiograph is shown. Isoelectric points for the gels were in the range 4.0-8.0, and a scale of p1 values is given on the x axis. The y axis shows the molecular weight scale in kilodaltons. The polypeptide patterns characteristic of (a), unexposed control cells for 45 min at 20°C (b) cells exposed to 60 Hz EM field for 45 min at 20°C and 8 uT, and (cl cells exposed for 45 min to heat shock at 37°C are shown.
lated gel. Gels were scanned using a laser densitometer (Newark, DE), Digital Instruments, and images of the gels along with the calibration strips were stored as IMG files on diskettes. Data analysis
The two-dimensional gel electrophoresis technique provides information about the distribution of proteins with regard to MW and p1, and, when coupled with the use of radioactive precursors, it provides information about the amount synthesized. It is very powerful in enabling ,the resolution of hundreds of different proteins, but there are problems of gel uniformity, loading etc. and hence problems of reproducibility from gel to gel, particularly when comparing gels obtained under different conditions. To improve the analysis we have normalized the amounts of protein and report them as a percentage of the total on each gel. Also, when matching sets of gels, instead of focusing on particular proteins, we have looked at the average distribution of proteins in particular ranges of MW and PI. Assuming that a control cell will synthesize not only the same proteins (i.e. the basis of matching spots) but also in the same proportions (i.e. the same distribution of mass), we have used the mass distribution as the basis for the analysis of the diskettes containing images of the gels. The software used in the analysis is QGEL (Kendrick Laboratories) and QuattroPro (Borland). Steps in the analysis are as follows.
31
A4W distribution Computer images of the two-dimensional protein gels are loaded into the QGEL \ FILE directory. In QGEL a grid consisting of ten rows and one column is created on the gel image. A table containing average MWs, pIs, densities and calibrated densities for each block of this grid is printed out. Average MWs and calibrated densities from this table are entered manually into a QuattroPro file. In QuattroPro, calibrated densities of all rows are added to obtain the total density of the gel. Relative film densities as a percentage of the total are calculated for each row. Average MWs are plotted against relative calibrated densities for each individual gel, using the same scale for all gels, and the resulting graphs for the gels belonging to one set are compared. Average MWs, average relative calibrated densities and standard deviations for this set of gels are calculated. These numbers are used to create an average graph for comparison with the average graphs obtained under different conditions. pI distribution Computer images of the two-dimensional protein gels are loaded into QGEL\ FILE directory. In QGEL a grid consisting of three rows and eight columns is created on a gel image. A table containing average MWs, pIs, total densities and total calibrated densities for each block of this grid is printed out. Average pIs and calibrated densities from this table are entered manually into a QuattroPro file. In QuattroPro, calibrated densities for each of the three rows are summed in each column to obtain a graph representing the whole gel. (Each row can also be used separately to obtain a graph for a particular MW region.) The total calibrated density of the gel or of a particular MW region is calculated. (The density of the column on the far right is subtracted because the very high film density usually present in this column is most probably an artefact.) Relative film densities as a percentage of the total are calculated for each column or each block. Average pIs are plotted against relative calibrated densities for each gel or each block of a gel, using the same scale for all gels, and the resulting graphs for the gels belonging to one set are compared. Average pIs, average relative calibrated densities and standard deviations for this set of gels are calculated. These numbers are used to create an, average graph for comparison with average graphs obtained under different conditions.
32
RESULTS
Figure 1 shows three gels: a control, an EM-stimulated gel and an HS-stimulated gel. In this paper we shall focus on the properties of the distribution of proteins rather than on individual proteins. However, we should note that the synthesis of actin (44 kD) and hsp70 (70 kD) on the gels is increased by exposure of cells to the magnetic field at 60 Hz. This result confirms our previous observations [l]. The increased synthesis of hsp70 and actin also supports recent results from analyses of transcription autoradiography on chromosomes from Drosophila melanoguster salivary gland cells exposed to EM fields. These experiments showed that transcription is increased in specific chromosomal regions, including those that contain the genes for hsp70 and actin [8]. The distribution of protein synthesis in the different MW ranges is shown in Fig. 2 for control conditions and also for EM stimulation at the lowest field level tested (0.8 PT). The main differences due to EM are the slight reduction of % mass around 20 kD, the pronounced peak at 30 kD and the depressions around 35 kD and 70 kD. Increasing the strength of the magnetic field to 8 ~.LTmaintains the changes at 20 kD and 30 kD but reverses the change at 70 kD to an increase in mass. At 800 bT, the highest field strength studied, the effects appear smaller but are still present. The changes seen in the p1 distribution are more pronounced. Figure 3 compares the distribution under control conditions with the lowest field tested (0.8
21 0
10
20
30
40
60
60
70
60
60
I 100
MW/kD
Fig. 2. Differences in the distribution of mass between EM-stimulated protein synthesis and the control. %Mass is plotted vs. MW for the control (0 ) and for the EM-stimulated sample (0.8 FT for 45 min) (W ). The control data are the average of nine gels, and the EM data are the average of three; standard errors are indicated.
33
81 4.5
L~ 5
5.5
8
6.5
7
7.5
Fig. 3. Differences in the distribution of isoelectric points between EM-stimulated protein synthesis and the control. %Mass is plotted vs. pI for the control (0) and the EM-stimulated sample (0.8 pT for 45 min) (~1. The control data are the average of nine gels, and the EM data are the average of three; standard errors are indicated.
~L.T)and we see two main features-a shift of the peak in the distribution to more neutral p1 (from 5.8 to 6.3) and the development of a peak at ~17.1. Both features can be seen in graphs for all levels of stimulation studied; the effect was weakest at 800 p,T, the highest field intensity studied. Figure 4 shows the effect of increasing EM field intensity on the magnitudes of the two peaks in the p1 distribution, and can be thought of as a dose-response curve. It also defines the apparent range of effectiveness of EM stimulation. The distribution patterns that result for HS show many similarities to those for EM stimulation. Figure 5 indicates that in the MW distribution, there are depressions at about 20 kD, 35 kD and 80 kD. (There is a relative peak at 30 kD, as well as an increase at MWs above 100 kD.) Figure 6 shows a similar shift of the p1 peak to about 6.3 and a new peak at 7.1. The changes in the p1 distribution can be compared in the different MW ranges, and the distributions in the ranges of MW 16 kD (8-23 kD), 30 kD (23-43 kD1 and 65 kD (43-250 kD) are shown in Figs. 7, 8 and 9 respectively. Several points can be made about these graphs: l The EM and HS curves are quite similar and both differ from the control curve. l The peak at p1 7.1 appears to be made up of polypeptides from all MW ranges, but primarily the two lower ranges (16 kD and 30 kD). l The shift in the p1 peak from 5.8 to 6.3 appears to be due primarily to polypeptides in the highest MW range (65 kD).
34
Fig. 4. The effect of increasing EM field intensity shown by plotting the magnitudes of the two peaks in the pI distribution. %Mass is plotted vs. EM field intensity for pI 6.3 (*) and pI 7.1 (X 1.
.~. ._._ __ __.__ _-._-.
0
10
20
30
40
50
50
70
50
90
__
loo
hdW/kD
Fig. 5. Differences in the distribution of mass between HS-stimulated protein synthesis and the control. %Mass is plotted vs. MW for the control (0) and the HS-stimulated sample (at 37°C for 45 min) (A). The control data are the average of nine gels, and the HS data are the average of four; standard errors are indicated.
35
_ ___ _-_
4.5
5
5.5
6
6.5
7
PI Fig. 6. Differences in the distribution of mass between HS-stimulated protein synthesis and the control. %Mass is plotted vs. pI for the control ( 0 ) and the HS-exposed sample (at 37°C for 45 mitt) (A 1. The control data are the average of nine gels, and the HS data are the average of four; standard errors are indicated.
81 4.5
5
5.5
6
6.5
7
7.5
PI Fig. 7. The pI distribution in the range of low MWs around 16 kD (8-23 kD). %Mass is plotted vs. pI for the control ( •I ), the EM-stimulated sample (B ) and the HS-stimulated sample (A 1. The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 16% of the total protein.
61 4.5
5
5.5
6
6.5
7
7.
PI Fig. 8. The pI distribution in the mid-range of MWs around 30 kD (23-43 kD). %Mass is plotted vs. pI for the control (01, the EM-stimulated sample (W) and the HS-stimulated sample (A). The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 42% of the total protein.
20.18161412. lo-
64 4.5
6 6.5 7 ; PI Fig. 9. The pI distribution in the high range of MWs around 65 kD (43-250 kD). %Mass is plotted vs. pI for the control (0 1, the EM-stimulated sample (m) and the HS-stimulated sample (A ). The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 40% of the total protein. 5
5.5
37 DISCUSSION
The cellular response to EM stimulation Our results indicate that the pattern of proteins synthesized as a result of EM stimulation differs from the control pattern and is similar to the pattern seen in response to heat shock. The main features of the response are as follows: new peak at 30 kD for both EM and HS; new peak at pl 7.1 for both EM and HS; shift of peak in p1 distribution from 5.8 to 6.3 for both EM and HS. Figure 1 shows that the prominent spot at 30 kD and pl 7 shifts to more acidic p1 in both EM and HS gels. This is probably due to the introduction of phosphate and sialic acid groups post-translationally through the activation of kinases and glycosylases. It should be noted that, despite this shift, there is an overall shift in the opposite direction. The lowest intensity studied (0.8 uT) is well above ambient conditions (about 0.1 p,T in the mu metal box) and clearly above the threshold. The responses follow a similar pattern at all field strengths, but there is a decrease at 800 PT. If the three orders of magnitude encompass the effective range of this stimulus, higher field strengths could be ineffective or inhibitory. Stress response The two stimuli, EM and HS, are quite different. The temperature rise of 17°C during HS causes many changes in the cells, including large accelerations of enzymatic reactions, while even the largest EM stimuli are very weak and do not appear to affect many systems. The initial reactions in signal transduction are also undoubtedly different for the two stimuli. Despite these differences, there is a similar biosynthetic response. Both stimuli affect a final common pathway, and the cells respond in a largely undifferentiated way to what they see as a stress, regardless of modality. The data of Fig. 4, showing essentially no variations in the response to different EM intensities above threshold, suggest an all-or-none type of response. The small differences between the two responses may indicate that EM stirnulation causes some specific effect, as suggested in our earlier paper [7]. For example, in Fig. 7, the EM stimulus results in somewhat greater amounts at the two extremes of the p1 distribution for the low MW polypeptides (i.e. smaller proteins are more highly charged, both positively and negatively). However, other suggested specific effects of EM, for example that the highest MW polypeptides appear to concentrate at the center of the p1 distribution and that the p1 distribution shifts to more neutral pH, also occur in response to HS. Over all, both EM and HS appear to be tapping into the same stress response pathway.
38
Signal transduction and mechanism
The present study provides some information about the signal transduction mechanism. Our observation that the response to EM occurs at very low field intensity suggests a rather specific response, in contrast with the response to the HS stimulus (a 17°C increase) which is a large perturbation that is bound to affect virtually every system in the cell. The relatively flat dose-response curve (Fig. 4) also suggests that once EM triggers a system in the cell, further stimulation does not increase the response. The kinds of changes that can occur at very low intensities are suggested by other studies‘where alternating currents induced by EM fields affect ATP splitting by the membrane Na,K-ATPase at very low electric field strengths [91. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Since magnetic fields penetrate cells virtually unattenuated, such fields may affect ion (e.g. promoters, repressors) binding in the nucleus directly as well as via induced currents. ACKNOWLEDGMENT
We thank the Electric Power Research Institute (EPRI) and the Office of Naval Research (ONR) for their support, and Alun Uluc for technical assistance. REFERENCES 1 2 3 4 5 6 7 8 9
R. Goodman and A.S. Henderson, Proc. Natl. Acad. Sci. USA, 85 (1988) 3928. R. Goodman and A.S. Henderson, Bioelectrochem. Bioenerg., 25 (1991) 335. K.J. McLeod, R.C. Lee and H.P. Ehrlich, Science, 236 (1987) 1465. R.A. Luben, C.D. Cain, M.Y. Chen, D.M. Rosen and W.R. Adey, Proc. Natl. Acad. Sci. USA, 79 (1982) 4180. C.V. Byus, K. Kartun, S. Pieper and W.R. Adey, Cancer Res., 48 (1988) 4222. M. Blank and R. Goodman, Bioelectrochem. Bioenerg., 19 (1988) 569. M. Blank and R. Goodman, Bioelectrochem. Bioenerg., 21 (1989) 307. R. Goodman, D. Weisbrot, A. Uluc and A.S. Henderson, Bioelectromagnetics, 13 (1992) 111. M. Blank, M. Faseb J., 6 (1992) 2434.
Bioelectrochemistry and BioeneRetics, 31 (1993) 27-38 Elsevier Sequoia S.A., Lausanne
JEC BB 01577
Similarities in the proteins synthesized by Sciaru salivary gland cells in response to electromagnetic fields and to heat shock M. Blank ‘, 0.
Khorkova ’ and R. Goodman 2
Departmentsof Physiology I and Pathology ‘, Columbia University, 630 West 168th St., New York, NY 10032 (USA)
(Received 17 June 1992; in revised form 11 August 1992)
We have studied changes in the biosynthetic responses of salivary gland cells from Sciara coprophila to two stresses, electromagnetic (EM) fields and heat shock (HS). To characterize the entire field of proteins synthesized, we converted the data from two-dimensional gels into protein distribution curves (%mass vs. MW, %mass vs. ~11. The responses to the two different stimuli, EM and HS, are quite similar to each other and different from those of the control. There are new peaks at MW = 30 kD and pI = 7.1 for both EM and HS. There is also a shift of the peak in pI distribution from 5.8 to 6.3 for both EM and HS. A plot of the changes in the two pl peaks as a function of EM field strength defines a dose-response curve as well as the range of the effect. These changes appear to be characteristic of the cellular response to stress.
INTRODUCTION
In the debate on the biological effects of environmental non-ionizing electromagnetic (EM) waves, probably the strongest experimental evidence has come from studies of changes in biosynthesis. EM waves in the range below 300 Hz (and including 60 Hz) have been shown to affect both transcription and translation in cell and organ cultures [l-53. The changes in the proteins are rather complicated and difficult to analyze, but there appear to be similarities in the patterns of the responses of cells to two physical stresses, EM fields, and heat shock (HS). Focusing on the 53 major proteins newly synthesized following stimulation (out of the several hundred that could be detected), several general properties of the biosynthetic response of the salivary gland cells of Sciaru were noted [6,7]: l EM-stimulated cells show relative changes in the MW distribution (e.g. increases in the 20-50 kD range, with decreases outside this range). HS-stimulated 0302-4598/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights reserved
28
cells show similar patterns after subtraction of the major HS protein band around 70 kD. l New proteins (not present in control gels) tend to be of lower molecular weight and to have a different isoelectric point distribution from the control proteins. The smaller new proteins are more highly charged, both positively and negatively, than control proteins. l Missing proteins tend to have higher molecular weights, but there is no one-to-one relation between missing and new proteins. These changes suggest that EM fields may stimulate a pathway that is similar to the one used by cells in response to HS and other kinds of physical stress. This does not preclude the possibility of specific responses to each stimulus, as suggested by the observation that the new proteins have a distribution of charges (i.e. smaller proteins are more highly charged) that would be expected if they arose from an interruption (i.e. early termination) of biosynthesis on the ribosome. In this paper we have examined these issues in greater detail, and present evidence for patterns in biosynthetic responses. EXPERIMENTAL
Procedures Experimental groups
The experimental samples included cells maintained at the normal growth temperature of 20°C and isolated from known EM field sources (control), exposed to HS at 375°C and exposed to a 60 Hz sinusoidal field at magnetic fields that varied by factor of 10 increments from 0.8 to 800 FT. Characterktics of electromagnetic fields
Four different magnetic field strengths were used with a 60 Hz sinusoidal EM field. The field strengths used were 0.8, 8, 80 and 800 u,T. Signal generation /cellular
exposure
Cells are exposed within a pair of 13 X 14 cm Helmholtz coils with an 8 cm spacing. The.coils are constructed of wire bundles approximately 1 cm in diameter wound around a square form. The coils are positioned vertically so that the oscillating magnetic field is generated in a horizontal plane, thus inducing a relatively uniform electric field in the conductive medium. The value of the electric field is dependent on the depth of the medium, in this case ca. 0.5 cm. Petri dishes (15 mm X 50 mm> containing the salivary glands were placed horizontally on a Plexiglas stand in an area between the coils shown to have a uniform magnetic field. The Helmholtz coils are shielded in a mu metal container within the incubator. Continuous sine waves were generated by a Wavetek function generator (Wavetek model 21,ll MHz) connected to a power regulator. The power regulator (Electra-Biology Inc.> produces amplitudes of 0.5, and 50 u,V. To produce 500 p,V
29
(800 u,T), an amplifier (Realistik, RadioShack, 800 W) was used in conjunction with the function generator. The function generator, power regulator and amplifier were outside the incubator. All exposure took place within a 20°C tissue culture incubator. Control cultures were placed in an identical incubator within a sham apparatus. Exposure times were 45 min for all field strengths tested. The 45 min time-point was used for two-dimensional gel electrophoretic analyses to ensure adequate incorporation of the isotope for analysis. No thermal changes (*O.lC) were measured during the exposure periods. Preparation of cells
Late fourth instar Scziwa coprophila female larvae were used for these experi-. ments. Fifty pairs of salivary glands (1.8 x lo4 cells) attached to the larval bodies were placed in 2 ml of Schneider’s Drosophila medium (SDM) minus methionine (GIBCO) and exposed to selected EM field strengths, isolated from the field or heat shocked at 37°C. The salivary glands were then dissected free of the larval body in ice-cold SDM minus methionine. For these experiments 35S-methionine at 100 @i/ml was added to each dish. Sample preparations (using a Kendrick Laboratories Inc. sample preparation kit) were performed in the cold. Anulysis of polypeptides
Two-dimensional electrophoresis was performed by Kendrick Laboratories Inc. (Madison, WI) according to the method of O’Farrell. Isoelectric focusing was carried out in glass tubes of inner diameter 2.0 mm, using 2.0% certified Resolytes pH 4-8 ampholines (BDH from Hoefer Scientific Instruments, San Francisco, CA> for 9600 V h. The first centimeter of the acid end of the tube gel was cut off to remove the SDS bulb that forms there. This area contains no protein. The final tube gel pH gradient extended from about pH 4.0 to pH 8.0 as measured by a surface pH electrode and colored acetylated cytrochrome p1 markers (Calbiochem-Behring, La Jolla, CA). After equilibration for 10 min in SDS sample buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.81, the tube gels were sealed to the top of 10% acrylamide slab gels (0.75 mm thick) and SDS slab gel electrophoresis was carried out for about 4 h at 12.5 mA/gel. The slab gels were prepared after the bromophenol dye front had run off the bottom of the gel for 45 min. The slab gels were fixed in a solution of 10% acetic acid + 50% methanol overnight. Calibration strips containing serial dilutions of i4C-bovine serum albumin (which had been stored at - 20°C in 10% acetic acid + 50% methanol) were added, and both gels and calibration strips were treated with ENHANCE (New England Nuclear, Boston, MA) for 1 h and rehydrated in water for 30 min. The gels and calibration strips were dried on filter paper with the acid end to the left. Fluorography was carried out using Kodak X-OMAT AR film with an exposure of 2 and 4 days at -70°C. The films were developed using Kodak developer and fixer. Figure 1 shows three gels; a control, an EM-stimulated gel and an HS-stimu-
30
Fig. 1. Comparison of autoradiographic patterns following two-dimensional gel electrophoresis (10.0% gels) of 35S-labeled polypeptides from Sciaru salivary gland. Part of each radiograph is shown. Isoelectric points for the gels were in the range 4.0-8.0, and a scale of p1 values is given on the x axis. The y axis shows the molecular weight scale in kilodaltons. The polypeptide patterns characteristic of (a), unexposed control cells for 45 min at 20°C (b) cells exposed to 60 Hz EM field for 45 min at 20°C and 8 uT, and (cl cells exposed for 45 min to heat shock at 37°C are shown.
lated gel. Gels were scanned using a laser densitometer (Newark, DE), Digital Instruments, and images of the gels along with the calibration strips were stored as IMG files on diskettes. Data analysis
The two-dimensional gel electrophoresis technique provides information about the distribution of proteins with regard to MW and p1, and, when coupled with the use of radioactive precursors, it provides information about the amount synthesized. It is very powerful in enabling ,the resolution of hundreds of different proteins, but there are problems of gel uniformity, loading etc. and hence problems of reproducibility from gel to gel, particularly when comparing gels obtained under different conditions. To improve the analysis we have normalized the amounts of protein and report them as a percentage of the total on each gel. Also, when matching sets of gels, instead of focusing on particular proteins, we have looked at the average distribution of proteins in particular ranges of MW and PI. Assuming that a control cell will synthesize not only the same proteins (i.e. the basis of matching spots) but also in the same proportions (i.e. the same distribution of mass), we have used the mass distribution as the basis for the analysis of the diskettes containing images of the gels. The software used in the analysis is QGEL (Kendrick Laboratories) and QuattroPro (Borland). Steps in the analysis are as follows.
31
A4W distribution Computer images of the two-dimensional protein gels are loaded into the QGEL \ FILE directory. In QGEL a grid consisting of ten rows and one column is created on the gel image. A table containing average MWs, pIs, densities and calibrated densities for each block of this grid is printed out. Average MWs and calibrated densities from this table are entered manually into a QuattroPro file. In QuattroPro, calibrated densities of all rows are added to obtain the total density of the gel. Relative film densities as a percentage of the total are calculated for each row. Average MWs are plotted against relative calibrated densities for each individual gel, using the same scale for all gels, and the resulting graphs for the gels belonging to one set are compared. Average MWs, average relative calibrated densities and standard deviations for this set of gels are calculated. These numbers are used to create an average graph for comparison with the average graphs obtained under different conditions. pI distribution Computer images of the two-dimensional protein gels are loaded into QGEL\ FILE directory. In QGEL a grid consisting of three rows and eight columns is created on a gel image. A table containing average MWs, pIs, total densities and total calibrated densities for each block of this grid is printed out. Average pIs and calibrated densities from this table are entered manually into a QuattroPro file. In QuattroPro, calibrated densities for each of the three rows are summed in each column to obtain a graph representing the whole gel. (Each row can also be used separately to obtain a graph for a particular MW region.) The total calibrated density of the gel or of a particular MW region is calculated. (The density of the column on the far right is subtracted because the very high film density usually present in this column is most probably an artefact.) Relative film densities as a percentage of the total are calculated for each column or each block. Average pIs are plotted against relative calibrated densities for each gel or each block of a gel, using the same scale for all gels, and the resulting graphs for the gels belonging to one set are compared. Average pIs, average relative calibrated densities and standard deviations for this set of gels are calculated. These numbers are used to create an, average graph for comparison with average graphs obtained under different conditions.
32
RESULTS
Figure 1 shows three gels: a control, an EM-stimulated gel and an HS-stimulated gel. In this paper we shall focus on the properties of the distribution of proteins rather than on individual proteins. However, we should note that the synthesis of actin (44 kD) and hsp70 (70 kD) on the gels is increased by exposure of cells to the magnetic field at 60 Hz. This result confirms our previous observations [l]. The increased synthesis of hsp70 and actin also supports recent results from analyses of transcription autoradiography on chromosomes from Drosophila melanoguster salivary gland cells exposed to EM fields. These experiments showed that transcription is increased in specific chromosomal regions, including those that contain the genes for hsp70 and actin [8]. The distribution of protein synthesis in the different MW ranges is shown in Fig. 2 for control conditions and also for EM stimulation at the lowest field level tested (0.8 PT). The main differences due to EM are the slight reduction of % mass around 20 kD, the pronounced peak at 30 kD and the depressions around 35 kD and 70 kD. Increasing the strength of the magnetic field to 8 ~.LTmaintains the changes at 20 kD and 30 kD but reverses the change at 70 kD to an increase in mass. At 800 bT, the highest field strength studied, the effects appear smaller but are still present. The changes seen in the p1 distribution are more pronounced. Figure 3 compares the distribution under control conditions with the lowest field tested (0.8
21 0
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30
40
60
60
70
60
60
I 100
MW/kD
Fig. 2. Differences in the distribution of mass between EM-stimulated protein synthesis and the control. %Mass is plotted vs. MW for the control (0 ) and for the EM-stimulated sample (0.8 FT for 45 min) (W ). The control data are the average of nine gels, and the EM data are the average of three; standard errors are indicated.
33
81 4.5
L~ 5
5.5
8
6.5
7
7.5
Fig. 3. Differences in the distribution of isoelectric points between EM-stimulated protein synthesis and the control. %Mass is plotted vs. pI for the control (0) and the EM-stimulated sample (0.8 pT for 45 min) (~1. The control data are the average of nine gels, and the EM data are the average of three; standard errors are indicated.
~L.T)and we see two main features-a shift of the peak in the distribution to more neutral p1 (from 5.8 to 6.3) and the development of a peak at ~17.1. Both features can be seen in graphs for all levels of stimulation studied; the effect was weakest at 800 p,T, the highest field intensity studied. Figure 4 shows the effect of increasing EM field intensity on the magnitudes of the two peaks in the p1 distribution, and can be thought of as a dose-response curve. It also defines the apparent range of effectiveness of EM stimulation. The distribution patterns that result for HS show many similarities to those for EM stimulation. Figure 5 indicates that in the MW distribution, there are depressions at about 20 kD, 35 kD and 80 kD. (There is a relative peak at 30 kD, as well as an increase at MWs above 100 kD.) Figure 6 shows a similar shift of the p1 peak to about 6.3 and a new peak at 7.1. The changes in the p1 distribution can be compared in the different MW ranges, and the distributions in the ranges of MW 16 kD (8-23 kD), 30 kD (23-43 kD1 and 65 kD (43-250 kD) are shown in Figs. 7, 8 and 9 respectively. Several points can be made about these graphs: l The EM and HS curves are quite similar and both differ from the control curve. l The peak at p1 7.1 appears to be made up of polypeptides from all MW ranges, but primarily the two lower ranges (16 kD and 30 kD). l The shift in the p1 peak from 5.8 to 6.3 appears to be due primarily to polypeptides in the highest MW range (65 kD).
34
Fig. 4. The effect of increasing EM field intensity shown by plotting the magnitudes of the two peaks in the pI distribution. %Mass is plotted vs. EM field intensity for pI 6.3 (*) and pI 7.1 (X 1.
.~. ._._ __ __.__ _-._-.
0
10
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50
50
70
50
90
__
loo
hdW/kD
Fig. 5. Differences in the distribution of mass between HS-stimulated protein synthesis and the control. %Mass is plotted vs. MW for the control (0) and the HS-stimulated sample (at 37°C for 45 min) (A). The control data are the average of nine gels, and the HS data are the average of four; standard errors are indicated.
35
_ ___ _-_
4.5
5
5.5
6
6.5
7
PI Fig. 6. Differences in the distribution of mass between HS-stimulated protein synthesis and the control. %Mass is plotted vs. pI for the control ( 0 ) and the HS-exposed sample (at 37°C for 45 mitt) (A 1. The control data are the average of nine gels, and the HS data are the average of four; standard errors are indicated.
81 4.5
5
5.5
6
6.5
7
7.5
PI Fig. 7. The pI distribution in the range of low MWs around 16 kD (8-23 kD). %Mass is plotted vs. pI for the control ( •I ), the EM-stimulated sample (B ) and the HS-stimulated sample (A 1. The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 16% of the total protein.
61 4.5
5
5.5
6
6.5
7
7.
PI Fig. 8. The pI distribution in the mid-range of MWs around 30 kD (23-43 kD). %Mass is plotted vs. pI for the control (01, the EM-stimulated sample (W) and the HS-stimulated sample (A). The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 42% of the total protein.
20.18161412. lo-
64 4.5
6 6.5 7 ; PI Fig. 9. The pI distribution in the high range of MWs around 65 kD (43-250 kD). %Mass is plotted vs. pI for the control (0 1, the EM-stimulated sample (m) and the HS-stimulated sample (A ). The control data are the average of nine gels, the EM data are the average of three and the HS data are the average of four. The protein in this range represents about 40% of the total protein. 5
5.5
37 DISCUSSION
The cellular response to EM stimulation Our results indicate that the pattern of proteins synthesized as a result of EM stimulation differs from the control pattern and is similar to the pattern seen in response to heat shock. The main features of the response are as follows: new peak at 30 kD for both EM and HS; new peak at pl 7.1 for both EM and HS; shift of peak in p1 distribution from 5.8 to 6.3 for both EM and HS. Figure 1 shows that the prominent spot at 30 kD and pl 7 shifts to more acidic p1 in both EM and HS gels. This is probably due to the introduction of phosphate and sialic acid groups post-translationally through the activation of kinases and glycosylases. It should be noted that, despite this shift, there is an overall shift in the opposite direction. The lowest intensity studied (0.8 uT) is well above ambient conditions (about 0.1 p,T in the mu metal box) and clearly above the threshold. The responses follow a similar pattern at all field strengths, but there is a decrease at 800 PT. If the three orders of magnitude encompass the effective range of this stimulus, higher field strengths could be ineffective or inhibitory. Stress response The two stimuli, EM and HS, are quite different. The temperature rise of 17°C during HS causes many changes in the cells, including large accelerations of enzymatic reactions, while even the largest EM stimuli are very weak and do not appear to affect many systems. The initial reactions in signal transduction are also undoubtedly different for the two stimuli. Despite these differences, there is a similar biosynthetic response. Both stimuli affect a final common pathway, and the cells respond in a largely undifferentiated way to what they see as a stress, regardless of modality. The data of Fig. 4, showing essentially no variations in the response to different EM intensities above threshold, suggest an all-or-none type of response. The small differences between the two responses may indicate that EM stirnulation causes some specific effect, as suggested in our earlier paper [7]. For example, in Fig. 7, the EM stimulus results in somewhat greater amounts at the two extremes of the p1 distribution for the low MW polypeptides (i.e. smaller proteins are more highly charged, both positively and negatively). However, other suggested specific effects of EM, for example that the highest MW polypeptides appear to concentrate at the center of the p1 distribution and that the p1 distribution shifts to more neutral pH, also occur in response to HS. Over all, both EM and HS appear to be tapping into the same stress response pathway.
38
Signal transduction and mechanism
The present study provides some information about the signal transduction mechanism. Our observation that the response to EM occurs at very low field intensity suggests a rather specific response, in contrast with the response to the HS stimulus (a 17°C increase) which is a large perturbation that is bound to affect virtually every system in the cell. The relatively flat dose-response curve (Fig. 4) also suggests that once EM triggers a system in the cell, further stimulation does not increase the response. The kinds of changes that can occur at very low intensities are suggested by other studies‘where alternating currents induced by EM fields affect ATP splitting by the membrane Na,K-ATPase at very low electric field strengths [91. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Since magnetic fields penetrate cells virtually unattenuated, such fields may affect ion (e.g. promoters, repressors) binding in the nucleus directly as well as via induced currents. ACKNOWLEDGMENT
We thank the Electric Power Research Institute (EPRI) and the Office of Naval Research (ONR) for their support, and Alun Uluc for technical assistance. REFERENCES 1 2 3 4 5 6 7 8 9
R. Goodman and A.S. Henderson, Proc. Natl. Acad. Sci. USA, 85 (1988) 3928. R. Goodman and A.S. Henderson, Bioelectrochem. Bioenerg., 25 (1991) 335. K.J. McLeod, R.C. Lee and H.P. Ehrlich, Science, 236 (1987) 1465. R.A. Luben, C.D. Cain, M.Y. Chen, D.M. Rosen and W.R. Adey, Proc. Natl. Acad. Sci. USA, 79 (1982) 4180. C.V. Byus, K. Kartun, S. Pieper and W.R. Adey, Cancer Res., 48 (1988) 4222. M. Blank and R. Goodman, Bioelectrochem. Bioenerg., 19 (1988) 569. M. Blank and R. Goodman, Bioelectrochem. Bioenerg., 21 (1989) 307. R. Goodman, D. Weisbrot, A. Uluc and A.S. Henderson, Bioelectromagnetics, 13 (1992) 111. M. Blank, M. Faseb J., 6 (1992) 2434.