NEW BIOPHYSICAL FIELD ATTACKS CANCER CELLS

Abstract

The article is devoted to the results of biophysical experiments by which the existence of the field of so-called pulse vector magnetic potential was successfully proved. The decision to carry out these tests was motivated by a favourable coincidence of pieces of knowledge obtained in the solution of other biophysical-research projects. The aim of the original experiment was only to check whether the field of pulse vector magnetic potential was detectable via a chosen biophysical way, when the live cells by their life manifestation would play the role of sensors of the presence of this field. After the discovery of the surprising behaviour of live cells irradiated by the pulse vector magnetic potential (with the presence of exciting pulse magnetic field excluded), specific research was started into both its influence on various biological materials, using further in vitro methods, and in the direction of understanding the physical nature of this biophysical phenomenon. The research is only at the beginning but interesting results can already be reported that are worthy of the attention of wider professional public.

Keywords: pulse vector magnetic potential; cell metabolism; chemiluminescence; bioluminescence; oncogenically transformed cells

Abbreviations: A-vector magnetic potential; B-magnetic induction; B_imp-pulse magnetic induction; PVMP-pulse vector magnetic potential; BL-bioluminescence; CL-chemiluminescence; RLU*s-relative luminescence units; HBSS-Hank’s balanced salt solution; ROM-reactive oxygen metabolite; NADPH-nicotinamide adenine dinucleotide phosphate; NME-normal mammary epithelium; WST-1-(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate); MTT-(3-[4,5-dimethylthiazol-2-yl] - 2,5-diphenyltetrazolium bromid); XTT-(sodium 3´-[1-(phenylaminocarbonyl) - 3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonicacid hydrate; MTS-(3-(4,5-dimethylthiazol-2-yl) - 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium salt

1. Introduction

Vector magnetic potential

A magnetic field is not conservative and so there is no possibility of simplifying both its fundamental equations (Fushchich, Nikitin, 1987) as in the case of the electrostatic field by introducing a real scalar potential. Therefore, in the second half of the 19^th century, the authors of electric and magnetic field theory used an "auxiliary" physical quantity, of course without assuming its real existence, so-called vector magnetic potential A, which would allow simplifying the mathematical description of the magnetic field in a similar way.

The definitional relation of this "auxiliary" quantity is the equation

where B is the vector of magnetic induction [T] and A is the vector magnetic potential [Tm or Wb/m]. This is a partial differential equation for a given quantity B and a desired unknown quantity A, and its solution is not unique, i.e. for a given magnetic field B there are infinitely many vector potentials A.

In 1956 Aharonov and Bohm (Peshin, Tonomura, 1989) published a report on an experiment that dealt with the surprising property of this quantity and disproved the former notions about its merely auxiliary function. The result of this experiment was verifiable interferential phenomena, produced by influencing one of the rays of a split beam of electrons moving in vacuum by the presence of a vector magnetic potential. The sensor of the action of the field of force of vector magnetic potential A on the resulting measurable quantity was the interference of wave-fronts of electrons detected by a special interferometer.

Pulse vector magnetic potential

The presence of a static vector magnetic potential A could manifest itself only by a change in the kinetic moment of electrons and the success of the two scientists was conditional on a complicated experimental facility for registering this change. After the above experiment, the attempts to examine the physical nature of the vector magnetic potential A were discontinued. We have chosen another way of proving the existence of vector magnetic potential A, namely the influence of varying or, to be more precise, pulse vector magnetic potential (PVMP) on elementary particles of the size of atoms. Our hypothesis concerning in the force effect on moving elementary particles starts from the presumption that the PVMP field generates secondarily excited pulse electric and magnetic fields and thus influences the formation and motion of charge carriers (ions) through the cell membrane.

A live cell serving as an indicating element between the applied PVMP field and the measurable change in its life manifestation was chosen as the sensor. The essence of the experimental method is the presumption that the effect of PVMP field is multiplied by the number of objects hit so that it will be possible to determine the relations between influenced and non-influenced cells by means of statistical methods. Three different biophysical in vitro methods were chosen, which are used in our specialized laboratories. For the envisaged experiments a numerically controlled facility with pulse power generators and special toroidal applicators was prepared (experimental system Toroid; ENJOY Ltd., Czech Republic). An unexpected result of these experiments was the findings that in most of the cases investigated the PVMP field had a negative influence on cell metabolism.

Generation of pulse vector magnetic potential

The vector magnetic potential A is connected with magnetic induction B by relation (1), from which the characteristic linkage of the two physical quantities follows, i.e. at a given point the direction of vector A is always perpendicular to the direction of the vector of magnetic induction B. The source of static vector magnetic potential A is, for example, a very long coil (l/d >> 1, l is the length and d the diameter of the coil) with the magnetic field concentrated in its core. Aharonov and Bohm used such an inductor already in the experiment mentioned above. Another example of a possible source of vector magnetic potential A is the so-called toroidal coil (toroid), which can be thought of as a very long coil with its beginning and end connected to form a torus of mean diameter "r" and surface "S" of the core cross-section. Magnetic energy resulting from the flow of electric current through the coil winding is concentrated only in its core. When a unipolar or bipolar pulse signal produced by pulse power generator supplied the toroid winding, a pulse magnetic field of magnetic induction B_imp is generated in the toroid core, accompanied by PVMP field. The basic layout of the measuring system is given in Fig. 1. This system consists of the pulse power generator, toroid, and one well of the 96-well microplate with indicated main direction of PVMP field at the moment a magnetic field B_imp is generated by a unipolar current pulse (Rampl, 2006), (Rampl, 2007).

Theoretically, the pulse magnetic field outside the toroid is zero because it is completely closed inside the toroid. In real toroidal coils, however, in particular due to the inaccuracies in the execution of their windings this pulse magnetic field can be measured, ranging within a negligible level of B_imp ≈ (10^-6 to 10^-5) T. Magnetic shield can be used to remove even this small field without affecting the existence of the PVMP field.

2. Goals of experiments with pulse vector magnetic potential

Two lines were followed in the research into the biophysical action of the PVMP field:

1. Confirmation of the existence of the PVMP field. Within the initial idea of proving the effect of the PVMP field of toroidal coils on the viability of biological material, the primary goal was to find and confirm by various biophysical methods the effect of the field on cell growth, and to compare this effect with the action of pulse magnetic field of conventional air-cored coils, for different electrical and design parameters of the exciters of the two types of field.
2. Exploitation of the effect of the PVMP field. In the case of a verifiable effect of the PVMP field, the follow-up goal was to establish the nature of the PVMP field effect, i.e. establishing the extent of growth induction or growth inhibition in different types of cell cultures, and their dependence on the spatial layout of the measuring system and on exciting signal parameters. This goal implies both investigating the properties of this field and the possibilities of its utilization.

3. Methods and results

In the first part of the study the model organism chosen was the bacterium Photorhabdus (=Xenorhabdus) luminescens, which was acted on by exciters of pulse magnetic field and PVMP field. It is a gram-negative rod-shaped bacterium, between 2 x 0.5 and 10 x 2 μm in size, belonging to the family Enterobacteriaceae. The bacterium lives freely in soil or is found as symbiont in the gut of entomopathogenic nematodes of the family Heterorhabditidae. Upon invasion of an insect host by the nematode, the bacteria are released into the hemocoel, where they multiply and help in killing the insect host. This species is the only terrestrial bioluminescence (BL) bacterium, all the other species with own BL are marine bacteria. The BL characteristic of bacteria is very similar to that of marine bacteria (maximum at 490 nm). The cultures can grow exponentially (to double the number of bacteria at 30ºC 1.5 hours are necessary); the largest luminescence is in the stationary growth phase (Hyršl, et al., 2004). This reaction is highly sensitive to the chemical, physical and biological factors of external environment, which may affect cellular breathing, synthesis of proteins and lipids, cellular integrity, and a correct functioning of biological membranes. BL bacteria are therefore employed for a rapid detection of the presence of pollutants and for a targeted study of biological effects of external factors. A special applicator, adapted to the shape of wells of the 96-well microplate, allowed concentrating irradiated energy almost immediately into the medium tested. Results of these experiments indicated that in all types of the exciter of pulse magnetic field and PVMP field the cellular material is affected to a statistically verifiable degree. Exciters of the pulse magnetic field are air-cored coils and those of the PVMP field are toroidal coils with ferrite cores.

As the further experimental objects there were the professional phagocytes which represent a key protection mechanism against invading microbial pathogens. Neutrophils are the first cells that infiltrate the inflammatory area after infection. They are later followed by activated monocytes, macrophages and eosinophils. Prior to being swallowed by phagocytes, the bacteria are opsonized by serum immunoglobulins and/or complement components. After opsonization, the bacteria adhere to specific surface receptors of phagocytes and are enclosed inside the intracellular membrane body referred to as phagosome. Later the phagosome becomes fused with lysosomes (present in phagocytes) and the bacteria are killed and degraded by various enzymatic and non-enzymatic substances stemming from lysosomes. The highly toxic and most effective killer factors of phagocytes include reactive oxygen metabolites (ROM) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals formed on the inner surface of phagolysosome. These ROM are produced from molecular oxygen in a process called respiratory burst, characterized by a dramatic increase in the activity of hexosemonophosphate shunt. Activated phagocytes reduce molecular oxygen to superoxide anion via the electron transport system of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase. The superoxide anion yields hydrogen peroxide either by spontaneous dismutation or in a reaction catalysed by superoxide dismutases. Hydrogen peroxide serves here as a substrate for a reaction catalysed by myeloperoxidase, in which a number of highly toxic metabolites are formed, inclusive of hypochlorous acid. When ROM are released, there appear electron-excited states, which emit photons during the return to ground state. This emission is referred to as chemiluminescence (CL). Since the natural CL of phagocytes is very weak, it is usually enhanced by luminophores (luminol or lucigenin). It is supposed that lucigenin is highly specific to superoxide anion and that it thus reflects the activity of NADPH oxidase. On the contrary, CL derived from luminol depends on the myeloperoxidase activity. Neutrophils, eosinophils, and monocytes exhibit the activity of both NADPH oxidase and myeloperoxidase and thus generate CL that is dependent on both luminol and lucigenin. Mature macrophages have a reduced content of myeloperoxidase and consequently also a reduced CL derived from luminol (Lojek et al. 1997), (Li et al. 1998), (Rost et al. 1998).

3.1 Material and method for measuring BL of light-emitting bacteria

Bacteria P. luminescens subsp. akhurstii CCM 7075 (model culture) were cultured for 48 hours in a liquid medium (Brain Heart Infusion, Pepton, NaCl, H₂O) at 28ºC. Their concentration was established on a Spekol 11 spectrometer (Carl Zeiss, Germany) at a wavelength of 400 nm to optical density = 1.0; where liquid medium was used as the background control. For the irradiation and subsequent measurement of BL, identical volumes of 50 μl of bacterial suspension were pipetted into all wells of the 96-well microplate. Using the so-called Hank’s balanced salt solution (HBBS), the content of all wells was subsequently filled to 250 μl. In the following graphs, the full line denotes a non-irradiated reference suspension of bacteria while the dotted line denotes the irradiated suspension under testing. The BL of bacteria was measured using an LM 01T luminometer (Immunotech, Czech Republic) at laboratory temperature at hourly intervals until 8 hours after the irradiation. The results represent average values from at least two iterations. The BL measured is given as integral of BL curves in relative luminescence units (RLU*s) and the values measured were used to plot kinetic curves. In all cases, the biological material was irradiated for 15 minutes before the beginning of experiment. The first point in the following graphs gives BL measurement at time 0 (i.e. immediately following the action of the magnetic or the PVMP field). In the course of experiments, this method was also used in testing other concentrations of bacteria.

Results

Study of the effect of pulse magnetic field. The measurement resulted in a graph giving the dependence of the luminescent surface of Photorhabdus luminescens bacteria on time. Data: Magnitude of pulse magnetic induction B_imp ≈ 10 mT, pulse frequency f = 15 Hz, pulse width T = 1 ms, coil resistance R = 10 Ω, Iimp = 0.7 A, exciters in close proximity to the suspension of bacteria under testing (Rampl 2003).

Study of the effect of PVMP field. The measurement resulted in a graph giving the dependence of the luminescent surface of Photorhabdus luminescens bacteria on time. Data: Pulse frequency f = 15 Hz, pulse width T = 1 ms, toroidal coil resistance R = 2 Ω, I_imp = 0.7 A, exciters in close proximity to the suspension of bacteria under testing.

Evaluation: Although the pulse magnetic field of air-cored coil is in principle accompanied by a field of pulse vector magnetic potential, the favourable effect of magnetic field on the bioactivity of the bacteria used is evidently predominant. Eight hours after irradiation, the bacteria exhibited a BL increased by 17500 RLU*s. When under the same surrounding conditions, the same concentrations of bacteria were irradiated only by a PVMP field generated by toroid with ferrite cores (external magnetic field not measurable), their life functions were slowed down: eight hours after irradiation they exhibited a BL that was ca. 5000 RLU*s lower than in the non-irradiated reference sample. These initial findings led to the experiments being extended to other types of cellular material, using further biophysical methods.

3.2 Material and method of measuring CL accompanying the respiratory burst of phagocytes

In the experiments, plasma was used that had been obtained from peripheral blood of laboratory Wistar rats and enriched with leucocytes (buffy coat). Plasma with leucocytes (50 μl) was pipetted into the 96-well microplate. This biological material was always irradiated for 15 minutes, after which 150 μl of HBBS and 25 μl of spare luminol solution (final concentration 1 mM) were added. Finally, the leucocytes were stimulated with 25 μl of zymosan from Saccharomyces cerevisiae yeast, which was opsonized with the Wistar rat complement. The respiratory burst of phagocytes was measured by the luminescence method. Luminescence was measured by an LM 01T luminometer (Immunotech, Czech Republic) in the microplate wells at 37ºC for a period of 60 minutes. The values measured were plotted to yield curves representing the kinetics of respiratory burst of non-affected phagocytes in comparison with phagocytes irradiated by exciters of pulse magnetic field and PVMP field.

Results

Study of the effect of pulse magnetic field and PVMP field. The results represent average values from five iterations. Data: Magnitude of pulse magnetic induction B_imp ≈ 10 mT, pulse frequency f = 15 Hz, pulse width T = 2 ms, electrical properties of both types of coil the same as in the preceding case, exciters in close proximity to the medium under testing.

Evaluation: As has already been said, when a pulse magnetic field is acting, the PVMP field forms an integral part of it. But in this case, the maximum of pulse vector magnetic potential is in the plane of the flow direction of electric current (i.e. in the plane of air-cored coil) and thus the effect on respiratory burst of phagocytes is evidently less than the concentrated effect of the PVMP field in the toroid axis. Moreover, the input of toroidal coils was, because of their electrical properties, only 20% of the input power into air-cored coils. At the curve maximum the decrease in luminescence after irradiation by pulse magnetic field was 40% while after irradiation by PVMP it was as much as 80%.

3.3 Method of measuring growth inhibition and determination of cellular apoptosis/necrosis

Growth inhibition assay (WST-1 colourimetric test) principle. In recent years different tetrazolium salts like MTT (Mosmann 1983), XTT (Scudiero et al. 1988) and MTS (Cory et al. 1991) have been described which can be used for the measurement of cell proliferation and viability. The tetrazolium salts are cleaved to insoluble formazan by cellular enzymes (Berridge et al. 1996). An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active (viable) cells in the culture. Quantification of the formazan dye produced by metabolically active cells by a scanning multiwell spectrophotometer (ELISA reader). The absorbance of the dye solution is measured at appropriate wavelengths.

Advantages of WST-1 compared to other cell proliferation agents. Cell proliferation reagent WST-1 (Ishiyama et al. 1995) has several advantages compared to the below mentioned compounds:

• In contrast to MTT that is cleaved to water-insoluble formazan crystal and therefore has to be solubilized after cleavage, WST-1 yields water-soluble cleavage products like XTT and MTS which can be measured without an additional solubilization step.
• In contrast to XTT and MTS, WST-1 is more stable. Therefore, WST-1 can be used as a ready-to-use solution and can be stored at +2 to +8ºC for several weeks without significant degradation.
• WST-1 has a wider linear range and shows accelerated colour development compared to XTT.

The results are a comparison of the absorbance of control cells without irradiation and the absorbance of cells irradiated by one of the fields according to the relation

(2)

where
           A (V24) is the absorbance of irradiated sample incubated for a period t = 24 h (or t = 48 or 72h),
           A (K0) is the absorbance of reference sample at time t = 0 h (at the time of experiment beginning),
           A (K24) is the absorbance of reference sample at time t = 24 h (or t = 48 or 72 h),

           with % of inhibition = 100 - % of growth [%].

Establishment of cellular apoptosis and necrosis. The method is based on the quantitative sandwich immunotest. It establishes mononucleosomes and oligonucleosomes in cellular lysates and in the culture medium subsequent to cell growth. Thus, it enables determining the cells undergoing the late stage of apoptosis (cellular lysates) and cells broken by necrosis (culture medium). The amount of mononucleosomes and oligonucleosomes is expressed by the so-called enrichment factor EF, which is calculated from the following formula

(3)

           where EF up to 1.3 being considered negative.

Material and method

WST-1 colourimetric assay. When monitoring growth inhibition, the antiproliferative effect of the PVMP field was tested by seeding the selected cell cultures in the 96-well microplates in the amount of 3000, 2000 and 1000 cells per well (i.e. three different concentrations in one experiment, a minimum of 5 wells for each concentration). The cells were cultured at 37oC in the incubator with 5% CO₂ in humidified atmosphere. The following day after seeding, the medium was replaced by fresh medium and the cells were irradiated by the PVMP field for a period of 15 minutes. After 24, 48 and 72 h intervals of the treatment, WST-1 colourimetric assay (Cell Proliferation Reagent WST-1; Roche, Mannheim, Germany) was performed according to the manufacturer’s protocol. The absorbance was measured in three parallel wells. The growth inhibition (cytotoxicity) was evaluated in at least three independent experiments and expressed as the cell viability normalized to untreated controls A microplate with the same number of cells served as the non-irradiated control.

Establishment of cellular apoptosis and necrosis. The cells were seeded in two 96-well microplates with a concentration of 5000 cells per well. The cells were cultured at 37oC in the incubator with 5% CO₂ in humidified atmosphere One day after seeding, the medium was replaced by fresh medium and the cells were irradiated for a period of 15 minutes. In control 96-well microplate, the cells remained non-irradiated. After incubation of 4 or 16 hours, cellular apoptosis and necrosis were established using the commercially available Cell Death Detection ELISA Kit (Roche, Mannheim, Germany).

Study of the effect of PVMP field on human malignant melanoma cell lines. Eight human stabilized malignant melanoma cell lines, denoted WM1158, WM852, WM9, WM1617, WM164, WM35, WM902B and WM983A, were used. All of them are a donation from Prof. M. Herlyn from the Wistar Institute (Philadelphia, PA, USA). The cell cultures were grown in the DMEM medium (GIBCO, USA) supplemented with 2 g/l of sodium bicarbonate, L-glutamine, insulin, antibiotics (penicillin, streptomycin) and 10% fetal bovine serum (FBS; GIBCO, USA) at a temperature of 37ºC in an atmosphere with 5% CO₂ (Boudný et al. 2005), (Kovařík et al. 2005), (Lauerová et al. 2006), (Fojtová et al. 2007).

Three parallel experiments were run, in which average values of the growth of malignant melanoma cells were determined. The experiments were repeated three times, for a period of 3 days. The reproducibility of all the experiments was excellent. In the WM852 and WM1617 lines, there was repeatedly a faster cell growth after irradiation than with the non-irradiated control sample (data not shown). On the contrary, in the other 6 cell lines, there was a growth inhibition of up to 31%. But it should be mentioned that growth induction or growth inhibition of 10-20% can be considered an error of the experiments or the fluctuation within the biological variability.

The cells exhibit a maximum growth inhibition 24 h after the irradiation (growth inhibition up to 31%). Later at 37ºC (48-72 h after the irradiation), their regeneration and growth renewal are probably set in (growth inhibition up to 17% only). For this reason, an experiment was carried out with irradiating two cell lines for a period of 15 minutes each day (every 24 hours) for three days altogether. In randomly selected lines, there was some growth inhibition (2-9%) only in the third day, i.e. after a total of 3 irradiations. In order to find out whether the growth inhibition after irradiation was due to the execution of apoptosis, as is the case for example of ionising irradiation or whether the cells simply broke due to necrosis, two cell lines (WM164 and WM35) were irradiated. After the incubation at 37ºC for 4 and 6 hours, apoptosis and necrosis were examined in the cells. In the two lines, apoptosis was not detected in any interval but in the WM164 cell line a slight necrosis manifested itself after 4 hours already. Data: Pulse frequency f = 15 Hz, pulse width T = 1 ms, toroidal coil resistance R = 2 Ω, I_imp = 0.7 A, exciters in close proximity to the medium under testing. The results of irradiation are given in the following figure.

Evaluation: No clear conclusion that this kind of irradiation particularly inhibits the cell growth can be drawn from the experiments with human malignant melanoma cells. A wider range of experiments on a larger set of cell lines is necessary. After the irradiation of malignant skin melanoma, growth inhibition of up to 31% occurred in 70% of cell lines. In 67% of these lines, the growth inhibition was low (up to 20% at the maximum), which can be considered an error of the experiments and the fluctuation within biological variability. An average post-irradiation growth inhibition of 41% was observed in 25% of cell lines of malignant melanoma (data not shown here). In particular, the fact that the cells of human malignant melanoma are resistant to both radiotherapy and chemotherapy is a strong stimulus to further experimental research.

From the viewpoint of achieving the basic goal, proving the existence of the PVMP field, we started from the assumption that cell lines from other tumours might respond more markedly to the action of the PVMP field, i.e. by a higher degree of growth inhibition. Breast carcinoma lines were therefore chosen for the verification of the influence of this field. Two cell lines of the breast carcinoma, denoted ZR 75-1 and PMC 42, were used. In this paper, only the results are given that document the most significant differences between the influence of the two fields of force and the irradiation of normal and oncogenically transformed cells (Součková et al. 2006).

Study of the effect of PVMP field on human breast carcinoma cell lines. A plot of the time dependence of growth inhibition of the breast carcinoma lines, ZR 75-1 and PMC 42, after irradiation by the PVMP field is given in Fig. 6. Data: Irradiation time 15 minutes, pulse frequency f = 15 Hz, pulse width T = 1 ms, toroidal coil resistance R = 2 Ω, I_imp = 0.7 A, exciters in close proximity to the medium under testing.

Study of the effect of PVMP field on normal mammary epithelium (NME). A plot of the time dependence of growth inhibition of a control normal mammary epithelium after irradiation by the PVMP field is given in Fig. 7. Data: Irradiation time 15 minutes, pulse frequency f = 15 Hz, pulse width T = 1 ms, toroidal coil resistance R = 2 Ω, I_imp = 0.7 A, exciters in close proximity to the medium under testing.

Study of the effect of pulse magnetic field on breast carcinoma cell lines. To verify whether the effect of pulse magnetic field on cancer cells is or is not similar to the effect of the PVMP field, irradiation by magnetic field was carried out under the same conditions as in the preceding cases. Data: Irradiation time 15 minutes, magnitude of pulse magnetic induction B_imp ≈ 10 mT, pulse frequency f = 15 Hz, pulse width T = 1 ms, coil resistance R = 10 Ω, I_imp = 0.7 A, exciters in close proximity to the medium under testing. The plot of growth inhibition is given in Fig. 8.

Evaluation: The results of irradiating the breast carcinoma are encouraging for further research. In particular, the growth inhibition effect of irradiation by the PVMP (Fig. 6) field is obvious in comparison with the influence of pulse magnetic field (Fig. 8). An unexpected but highly positive result is almost negligible effect of the PVMP field on healthy cells (Fig.7).

4. Discussion and conclusion

Assessing the results of the influence of the PVMP field, which were obtained using different biophysical methods, has led to the following conclusions:

The experiments described have confirmed with much probability the existence of the PVMP field and its probably different nature with respect to the pulse magnetic field. Conclusions regarding the practical exploitation of the established growth inhibition of oncogenically transformed cells cannot, as yet, be formulated unambiguously because only a limited number of experiments were carried out. Both directions of the research, i.e. seeking methods for establishing the existence of the PVMP field and its effects, should be continued using the above methods as well as other suitable in vitro methods. Simultaneously, however, technical and technological conditions should be prepared for experiments carried out by in vivo methods on animal models, possibly in the presence of chemotherapeutics or immunotherapeutics (such as cytostatics or cytokines). A true advance in the research will only occur when the physical nature of the PVMP field has been established. After some experiments (not documented here), it was found that our working hypothesis given in chapter 1, which is necessary for a precise targeting of experiments, has some shortcomings that cannot be overcome even if the idea of the effect of secondarily excited magnetic and electric fields in cellular material is accepted. In addition to the different effects of the two types of field mentioned above (Fig. 2 and Fig. 3), the PVMP field exhibits a slower than expected drop in effectiveness in dependence on the distance from the wells containing the medium under testing. Theoretical work and experiments are therefore currently being organized that should help in the solution of this problem. Unfortunately, live cells still serve as the only applicable "measuring device", with all the known drawbacks. In any case, it can be said in conclusion that the possibility of highly promising findings is obviously opening up.

5. References

Berridge, M.V, Tan, A.S., McCoy, C.D., Wang, R., 1996. The Biochemical and Cellular Basis of Cell Proliferation Assays That Use Tetrazolium Salts. Biochemica 4, 15-19.

Boudný, V., Dušek, L., Adámková, L., Chumachalová, J., Kocák, I., Fait, V., Lauerová, L., Krejčí, E., Kovařík, J., 2005. Lack of STAT 1 phosphorylation at TYR 701 by IFN gamma correlates with disease outcome in melanoma patients. Neoplasma 4/52, 330-337.

Cory, A.H., Owen, T.C., Barltrop, J.A., Cory, J.G., 1991. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 3/7, 207-212.

Fojtová, M., Boudný, V., Kovařík, A., Lauerová, L., Adámková, L., Součková, K., Jarkovský, J., Kovařík, J., 2007. Development of IFN gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells. British Journal of Cancer 2/97, 231-237.

Fushchich, W.I., Nikitin, A.G., 1987, Symmetries of Maxwell's Equations. Mathematics and its Applications. Springer Pub., Boston, 297 pp.

Hyršl, P., Číž, M., Lojek, A., 2004. Comparison of the bioluminescence of Photorhabdus species and subspecies type strains. Folia Microbiologica 49, 539-542.

Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K., Ohkura, Y., Ueno, K., Watanabe, M., 1995. Novel cell proliferation and cytotoxicity assays using a tetrazolium salt that produces a water-soluble formazan dye. In Vitro Toxicology 8/2, 187-190.

Kovařík, A., Fojtová, M., Boudný, V., Adámková, L., Lauerová, L., Kovařík, J., 2005. Interferon-gamma, but not interferon-alpha, induces SOCS 3 expression in human melanoma cell lines. Melanoma Research 6/15, 481-488.

Lauerová, L., Fojtová, M., Kovařík, A., Adámková, L., Součková, K., Boudný ,V., Jarkovský, J., Kovařík, J., 2006. Inhibitory effect of interferon gamma on cell proliferation is correlated with prolonged induction of SOCS1 and SOCS3 genes in human melanoma lines. Melanoma Research 1/16, p. S38.

Li, Y., Yhu H., Kuppusamy, P., Roubald, V., Zweier, J.L., Trush, M.A., 1998. Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 273 (4), 2015-2023.

Lojek, A., Číž, M., Marnila, P., Dušková, M., Lilius, E., 1997. Measurement of whole blood phagocyte chemiluminuscence in the Wistar rat. Journal of Bioluminiscence and Chemiluminiscence 5/12, 225-231.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65/1-2, 55-63.

Parvez, S.H., Reiss, C., Parvez, S., Labbe, G., 2001. Molecular Response to Xenobiotics. Elsevier Science, Hardbound, 168 pp.

Peshin, M., Tonomura, A., 1989. The Aharonov-Bohm Effect. Technical Physics Letters, 11-12.

Rampl. I., 2003. Research of digitally controlled pulse magnetic-laser field effects and the preparation of development of a new type of therapeutic device. Project of the Ministry of Industry and Trade of the Czech Republic FT-TA/007, Brno, 41 pp.

Rampl, I., 2006. Device for attenuating cellular metabolism. The Czech application for invention PV 2006-379, Brno, 16 pp.

Rampl, I., 2007. Device for attenuating cellular metabolism. The international application for invention PCT/CZ2007/000051, Brno, 12 pp.

Rost, M., Karge, E., Klinger, W., 1998. What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite. J Biolumin Chemilumin 13, 355-363.

Scudiero, D.A., Shoemaker, R.H., Paull, K.D., Monks, A., Tierney, S., Nofziger, T.H., Currens, M.J., Seniff, D., Boyd, M.R., 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48/17, 4827-4833.

Součková, K., Adámková, L., Lauerová, L., Krejčí, E., Kovařík, A., Fojtová, M., Matoušková, E., Buršíková, E., Kovařík, J., Boudný, V., 2006. The effect of interferon treatment on STAT/SOCS status in normal and malignant human breast cells. In: The 31st ESMO Congress, Istanbul, Turkey. Annals of Oncology 17.