Expression of heat shock proteins after ultrasound exposure in HL-60 cells.

One of the important cellular defense mechanisms against stress is the induction of heat shock proteins (HSPs). We have recently demonstrated that a low frequency electromagnetic field is unable to induce the heat shock response (HSR). In the present study, we expanded our investigations to the induction of HSPs, particularly Hsp72, by ultrasound (US). Human promyelocytic leukemia HL-60 cells were exposed in suspension to US at 1, 3 and 10 MHz, as well as combinations of two of these frequencies. The ability of US to induce Hsp72 was tested for different frequencies, intensities and exposure times. In addition, the water bath temperature was varied from 30 to 36 degrees C. The Hsp72 protein expression was determined 4 and 24 h after treatment. We found that the amount of Hsp72 increased with increasing US frequency, reaching its highest level of about 1800%, induced by 10 MHz. After increasing the temperature of the water bath, the amount of Hsp72 in the treated cells was also increased, whereas no induction was observed at 30 degrees C. For all treatment conditions, ultrasound of 1 MHz was unable to significantly induce Hsp72. At 10 MHz, the exposure time was varied from 0 to 20 min. We found that the induction of Hsp72 took place after 5 min of exposure. For a fixed level of absorbed US energy, the continuous regime, as well as a pulsation of 1:2 (5 ms on and 5 ms off) induced the same Hsp72 level. Pulsation of 1:5 (2 ms on and 8 ms off) and 1:10 (1 ms on and 9 ms off) did not show any effect. A single sonication of 20 min, as well as a fractionated sonication of two 10 min exposures induced the same level of Hsp72, whereas four exposures of 5 min reduced the Hsp72 level. At the optimum exposure conditions (10 MHz, 10 min), the concentration of other HSPs was also determined. Hsp27 showed no effect but Hsp32, Hsp40 and Hsp72 were induced. Taken together, these results suggest a synergistic interaction between heat and US.

Description of radiation- and ultrasound-induced cell death by a stochastic process.

Cell survival is a stochastic process with the stochastic component being strongly dependent on the irradiation conditions. This process is described by a stochastic model which allows differentiation between the deterministic and stochastic components of survival. The proposed model is tested for four irradiation experiments (2 with ionizing radiation and 2 with ultrasound) and very good agreement with experimental results is demonstrated. It identifies the higher stochasticity of the cell survival for the temporally varying radiation fields and provides the possibility to compare the stochasticity of survival in different radiation fields.

Low-energy electromagnetic fields promote proliferation of vascular smooth muscle cells.

The rationale was to investigate the effects of low-energy electromagnetic fields (EMF) on the proliferation of bovine coronary and murine aortic smooth muscle cells (SMC). EMF were applied to SMC at field frequencies of 25, 50, or 100 Hz, and exposure time was set to 5, 15, or 30 minutes. Significant increases in SMC-counts compared with sham exposed controls were found for all EMF-frequencies tested. The effect was most pronounced for 50 Hz fields with maximum increases of 1.2-fold over controls. Sequential double exposure of mouse aortic SMC to 50 Hz fields revealed significantly enhanced cell proliferation by 1.2 fold compared with single exposure (p < 0.05). Experiments performed on bovine SMC also revealed significant increases in cell proliferation. The results demonstrate that EMF are capable of significantly enhancing the proliferation of vascular SMC. These results rise the question whether EMF would qualify as supportive means to angio-/arteriogenic approaches.

IL-8 release of HL-60 cells treated with electric currents of different wave forms.

Human promyelocytic leukaemia HL-60 cells which have been differentiated by DMSO to granulocytes were used to investigate the effect of different waveforms on the release of interleukine-8 (IL-8). The cells were prestimulated with 100 pM fMLP and subsequently treated for 15 min with different electrical fields and currents. Three hours later the release of IL-8 into the medium was determined by ELISA. Varying the frequency of the sinusoidal electrical current between 0 and 20 Hz resulted in 2 maxima of IL-8 release at 5 and 13 Hz. Prestimulated cells were treated with sine-, triangular-, and rectangular-waveforms at 5 Hz in the current intensity range of 0-3 mA/cm(2). For the three waveforms tested, the IL-8 release was enhanced 1.5 fold. Treatment of the cells with capacitively coupled electric fields of 5 Hz using field strengths between 0 and 10 V(eff)/cm had no effect on the release of IL-8. In comparison to the positive results after sine wave exposure alone, an exposure with sine wave current to which noise had been superimposed had no effect on the HL-60 cells. From these investigations it can be concluded that for electrical current treatment of prestimulated HL-60 cells the release of IL-8 does not depend on the waveform if the waveform information is not destroyed by superimposed noise.

Repetitive transcranial magnetic stimulation does not influence immunological HL-60 cells and neuronal PC12 cells.

BACKGROUND: Transcranial magnetic stimulation (TMS) is a non-invasive method used in medical applications such as brain mapping or as a therapeutic tool in neurological and psychiatric disorders because it can stimulate defined regions of the brain without anaesthesia. METHODS: The action of repetitive transcranial magnetic stimulation (rTMS) on HL-60 and PC12 cells has been investigated. The cells have been stimulated in vitro with different number of pulses (75 - 1250), different intensities (10, 20 and 40%) and different frequencies (0.25, 1 and 10 Hz) by using a double coil (2x70 mm) connected to the 'Magstim rapid'. At selected time points after treatment the following endpoints have been determined: viability, cyclic AMP (cAMP) and heat shock protein 72 (Hsp72) (HL-60 cells), and viability, cAMP, dopamine and noradrenaline (PC12 cells). Viability was measured with the alamarBlue assay, whereas cAMP, Hsp72, dopamine and noradrenaline were determined with enzyme-linked immunosorbent assay (ELISA). RESULTS: In both cell lines viability was not influenced by rTMS treatment, the same was true for the cytosolic cAMP concentration. In HL-60 cells rTMS treatment did not change the Hsp72 content, also a protective effect of rTMS treatment on cell viability before toxic H(2)O(2) treatment was not observed. After high potassium treatment the release of the two neurotransmitters dopamine and noradrenaline in PC12 cells was enhanced 15- and 5-fold, respectively, but after rTMS treatment no change in the release of the two neurotransmitters was observed. CONCLUSIONS: In two mammalian cell lines rTMS treatment in a variety of exposure conditions does not influence any of the measured parameters.

Expression of HSP72 after ELF-EMF exposure in three cell lines.

It has been reported that magnetic fields with flux densities ranging from microT to mT are able to induce heat shock factor, HSP72 mRNA or heat shock proteins in various cells. In this study we investigated changes in the HSP72 mRNA transcription level in three cell lines (HL-60, H9c2, and Girardi heart cells) and in the intracellular HSP72 protein content in two cell lines (HL-60 and Girardi heart cells) after treatment schemes using electromagnetic fields with a flux density of 2 microT to 4 mT, a frequency of 50 Hz and exposure times from 15 to 30 min. None of the treatments or modalities showed any significant effect on the HSP72 protein level, although HSP72 mRNA could be induced, at least to some extent, with one of the parameter combinations in all cell lines tested. Obviously, HSP72 mRNA transcription and translation are not necessarily coupled in certain cells. This leads to the conclusion that electromagnetic field effects on HSP72 mRNA levels are not indicative for downstream effects unless increased mRNA levels can be correlated with increased HSP72 protein levels as well.