Decreased c‐Myc expression and its involvement in X‐ray‐induced apoptotic cell death of human T‐cell leukaemia cell line MOLT‐4

Purpose: To investigate the possible involvement of c‐Myc and ceramide‐c‐Jun N‐terminal kinase (JNK) pathway in X‐ray‐induced apoptotic cell death of MOLT‐4 cells.

Materials and methods: The expressions of c‐Myc protein and c‐myc mRNA after X‐irradiation were analysed by Western blotting and RT‐PCR between radiosensitive MOLT‐4 and radioresistant variant Rh‐1a cells with less JNK activation than the parental cells. Apoptotic cell death was determined by a dye exclusion test, the appearance of chromatin condensation and DNA fragmentation. The effect of a JNK activator anisomycin or c‐Myc inhibitor peptides (Int‐H1‐S6A, F8A) on the amount of c‐Myc protein and on the induction of apoptosis was investigated, respectively.

Results: In X‐irradiated MOLT‐4 cells, amounts of both c‐myc mRNA and c‐Myc protein rapidly decreased, which was followed by apoptotic cell death, while little change or limited reduction of c‐Myc protein was observed in X‐irradiated Rh‐1a cells with accompanying higher cell viability. Exposure of MOLT‐4 and Rh‐1a cells to c‐Myc inhibitor peptides similarly induced apoptotic cell death with decreases of c‐Myc protein. Anisomycin rapidly induced JNK activation and a subsequent decrease of c‐Myc protein, causing cell death in MOLT‐4 cells. On the other hand, Rh‐1a cells were more resistant to anisomycin than parental MOLT‐4 cells, showing less JNK activation and a delayed decrease of c‐Myc protein.

Conclusion: A decrease of c‐Myc protein was considered important in X‐ray‐induced apoptotic cell death of MOLT‐4 cells; activation of the JNK pathway caused reduction in the amounts of c‐myc mRNA and c‐Myc protein, and finally induced apoptotic cell death.     

Inhibition of cyclooxygenase‐2 with NS‐398 and the prevention of radiation‐induced transformation,micronuclei formation and clonogenic cell death in C3H 10T1/2 cells

Purpose: Abnormally high levels of the cyclooxygenase (COX)‐2 isozyme as well as the prostaglandin metabolites produced by the COX pathway have been observed in a variety of malignancies, including cancers of the skin, pancreas, colon, breast, cervix, prostate, and head and neck. Furthermore, exogenous genotoxic agents, including ionizing radiation (IR), have been shown to induce cellular transformation and to elevate COX‐2 activity, whereas exposure to agents that specifically inhibit COX‐2 activity have been shown to inhibit transformation. These data suggest a possible role of COX‐2 both in IR‐mediated cellular transformation processes and cell death.

Materials and methods: C3H 10T1/2 and/or HeLa cells were treated with N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]‐methanesulfonamide (NS‐398) and/or exposed to IR. Following treatment, cells were assayed for neoplastic transformation, clonogenicity, growth rates, cell cycle distribution, micronuclei formation and DNA damage by established methodologies. Statistical tests were performed on data as described.

Results: In the present study, experiments in normal murine fibroblast C3H 10T1/2 cells demonstrated that the chemical inhibition of COX‐2 activity with moderate doses of NS‐398 abrogated IR‐induced transformation events by fourfold and protected irradiated C3H 10T1/2 cells from clonogenic cell death. Considering that these doses of NS‐398 had no significant effect on cellular proliferation or cell cycle distribution in C3H 10T1/2 cells, the results suggest that inhibition of COX‐2 either increases DNA repair or prevents the accumulation of DNA damage. In supplemental experiments, treatment with NS‐398 caused a 1.5‐fold reduction in IR‐induced micronuclei formation and a significant decrease in DNA damage.

Conclusions: These results suggest a role for COX‐2 inhibitors in the normal tissue response to IR when administered at therapeutically achievable doses and therefore may have clinical implications for radiation oncology patients in the prevention of IR‐induced malignancy.     

Chromatin‐ and temperature‐dependent modulation of radiation‐induced double‐strand breaks

Purpose: To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double‐strand breaks (DSB) in structures derived from human diploid fibroblasts.

Materials and methods: Agarose plugs with different chromatin structures (intact cells±wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X‐rays at 2 or 37°C and lysed using two different lysis protocols (new ice‐cold lysis or standard lysis at 37°C). Induction of DSB was determined by constant‐field gel electrophoresis.

Results: The dose‐modifying factor (DMF_temp) for irradiation at 37 compared with 2°C was 0.92 in intact cells (i.e. more DSB induced at 2°C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMF_temp was influenced by the presence of 0.1?M DMSO or 30?mM glutathione, but not by post‐irradiation temperature.

Conclusion: The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post‐irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.     

Comparison of DNA damage and repair following radiation challenge in buccal cells and lymphocytes using single‐cell gel electrophoresis

Purpose: To develop a reproducible single‐cell gel electrophoresis assay for DNA damage and repair in buccal mucosa and sublingual exfoliated cells.

Materials and methods: Buccal mucosa and sublingual cells and lymphocytes from six individuals (three males, three females, aged 34–45 years) were challenged with increasing doses of gamma‐rays. DNA strand breaks and DNA repair were measured using the single‐cell gel electrophoresis assay.

Results: Baseline DNA strand breaks were significantly greater in buccal mucosa and sublingual cells compared with lymphocytes. Buccal mucosa and sublingual cells did not differ from each other with respect to induction of DNA strand breaks by 2 or 4?Gy gamma‐rays. However, they showed a smaller increase in gamma‐ray‐induced DNA strand breaks compared with lymphocytes (32–53% less than lymphocytes; ANOVA p<0.0001). Unlike lymphocytes, which repaired 83% of DNA strand breaks, buccal mucosa and sublingual cells exhibited only a minimal capacity for DNA repair (approximately 0–14% of the level in lymphocytes).

Conclusions: Buccal mucosa and sublingual cells exhibit an apparent resistance to the expression of radiation‐induced DNA strand breaks in vitro and an apparent lack of DNA strand break repair in the single‐cell gel electrophoresis assay.     

Cellular origin of ionizing radiation‐induced NF‐κB activation in vivo and role of NF‐κB in ionizing radiation‐induced lymphocyte apoptosis

Purpose: To investigate the cellular origin of ionizing radiation (IR)‐induced NF‐κB activation in vivo and the role of NF‐κB in IR‐induced lymphocyte apoptosis.

Materials and methods: NF‐κB activities were analysed by gel shift/supershift assay in isolated murine T‐ and B‐cells, macrophages (M?) and tissues from normal and T‐ and B‐cell‐deficient Rag1 mice with or without exposure to IR. IR‐induced lymphocyte apoptosis was determined by analysis of 3,3′‐dihexyloxacarbocyanine iodide (DiOC₆) uptake, annexin‐V staining and the sub‐G0/1 population, or by TUNEL assay.

Results: The results showed that IR activated NF‐κB in lymphocytes, including both T‐ and B‐cells, but failed to do so in M?. Furthermore, T‐ and B‐cell‐deficient Rag1 mice exposed to IR exhibited a significant reduction in NF‐κB activation as compared with normal mice. Although NF‐κB1 (p50) gene knockout or NF‐κB decoy oligonucleotide treatment specifically inhibited IR‐induced lymphocyte NF‐κB activation, they had no significant effect on IR‐induced lymphocyte apoptosis.

Conclusions: This finding suggests that lymphocytes are the main cellular origin of IR‐induced NF‐κB activation in vivo. However, NF‐κB activation has no significant effect on IR‐induced lymphocyte apoptosis.     

Post‐irradiation approaches to treatment of radiation injuries in the context of radiological terrorism and radiation accidents: a review

Purpose: Events of the recent past have focused attention on the possibility of radiological (nuclear) terrorism and on the implications of such terrorist threats for radiation accident preparedness. This review discusses recent advances in the knowledge about how radiation injuries from such events might be treated pharmacologically, and the practical barriers to clinical utilization of these approaches.

Conclusions: A wide range of pharmacological approaches are being developed in the laboratory that could greatly expand the ability to treat acute and chronic radiation injuries. However, there are currently a variety of practical and legal barriers that would prevent the actual clinical use of most of the approaches. There are also the potential weaknesses in most of the current programmes for dealing with the consequences of radiation accidents or nuclear terrorism, including the absence of widespread radiation biodosimetry capabilities and the resulting inability to triage. If a major radiation accident or terrorist event occurs, the lack of biodosimetry and treatment capabilities will be compounded by widespread public fear of ‘radiation’.     

‘In‐field’ and ‘out‐of‐field’ functional impairment during subacute and chronic phases of experimental radiation enteropathy in the rat

Purpose: To investigate subacute and chronic functional consequences of localized irradiation of rat small intestine on exposed and shielded segments (proximal and distal).

Materials and methods: The surgical model of a scrotal hernia was used. The ileal loop was exposed to single doses of 18, 21 or 29.6?Gy X‐irradiation. Epithelial structure and transport capacity were followed 2 and 26 weeks post‐exposure.

Results: Irradiated segments showed mucosal ulceration followed by transmural fibrosis. Transport capacity was impaired from 2 to 26 weeks. Subacute functional impairment was noticed in the proximal segment, without either morphological alteration or neutrophil influx. At 26 weeks, both proximal and distal segments showed impaired epithelial transport capacity, with neutrophil influx in the submucosa in cases of 21‐Gy exposure and in the submucosa and muscularis propria after 29.6?Gy.

Conclusions: Radiation enteritis was characterized by functional impairment, within as well as outside, the irradiation field. During the subacute phase, the irradiated segment may be a source of mediators which might influence intestinal function outside the site of injury via the blood stream and/or enteric nervous system. The development of an intestinal occlusion syndrome during the chronic phase might be responsible for intestinal dysfunction but it does not rule out a possible inflammatory process developing in the shielded parts of the small intestine.     

Dosimetry and risk from low‐ versus high‐LET radiation of Auger events and the role of nuclide carriers

Purpose: To analyse the lethality to mammalian cells of ¹²⁵I‐decays in DNA, in antipyrine in the whole cell and in oligodeoxynucleotides in the nucleus outside DNA as a function of Auger event‐site and number.

Materials and methods: Auger events cause both low‐ and high‐linear energy transfer energy depositions including charge neutralization at the daughter nuclide. Microdosimetry allows the expression of absorbed dose to a defined micromass and the number of such events at given sites. Published data were used to relate micromass dose and event number to the dose to reduce survival to 37% of the initial survival (D₃₇).

Results: The D₃₇ of ¹²⁵I‐decays in DNA was 0.1?Gy in terms of absorbed dose to the cell nucleus and about 30 in terms of average decays per nucleus or whole cell. The D₃₇ of ¹²⁵I‐decays in antipyrine was 1.5?Gy for absorbed dose to the cell nucleus, about 250 in terms of average decays per nucleus and about 2×10³ for average decays per whole cell. ¹²⁵I‐decays in oligodeoxynucleotides were much less toxic than ¹²⁵I‐decays in antipyrine by a factor of about 25 in terms of average absorbed dose to the cell nucleus, by a factor or about 40 in terms of average decays per cell nucleus and by a factor of six in terms of average decays per whole cell.

Conclusion: The unexpected low toxicity of ¹²⁵I‐decays in nuclear oligodeoxynucleotides outside the DNA in comparison with ¹²⁵I‐decays in antipyrine in the nucleus or the whole cell demands further attention on the role of oligodeoxynucleotides in altering cellular radiation sensitivity.     

Lack of inverse dose‐rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T‐47D cells arrested in G2 by ionizing radiation

Purpose: To investigate the radiosensitivity of human breast cancer cells, T‐47D, irradiated with low dose‐rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation.

Materials and methods: Cells were irradiated with ⁶⁰Co γ‐rays with dose‐rates of 0.37 and 0.94?Gy?h^?1. Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear‐bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3‐245 and Hoechst 33258 was used for additional staining of DNA. Two‐parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTAR^PLUS flow cytometer.

Results: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94?Gy?h^?1 resulted in exponential dose–survival curves with nearly equal α values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2.

Conclusions: T47‐D cells accumulate in G2 during low dose irradiation, but no inverse dose‐rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose‐rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation‐induced cell death in G2.     

Androgen and the blocking of radiation‐induced sensitization to Fas‐mediated apoptosis through c‐jun induction in prostate cancer cells

Purpose: To clarify the key mechanism by which androgen makes prostate cancer cells highly resistant to Fas‐mediated apoptosis.

Materials and methods: The role of c‐jun induction by 10?nM dihydrotestosterone (DHT) in 5?Gy radiation‐induced up‐regulation of Fas and sensitization to the apoptosis was studied by using the human prostate cancer cell line LNCaP.

Results: On exposure to 5?Gy radiation, LNCaP cells demonstrated high sensitization to Fas‐mediated apoptosis through increased Fas expression, stabilized p53 expression and binding to p53 response elements within the promoter and first intronic region of the Fas gene. Following treatment with DHT, in vivo binding of p53 to its response elements was strongly inhibited. In addition, DHT significantly up‐regulated c‐jun expression through extracellular stress‐regulated kinase (ERK) activation, and transfection of an antisense oligonucleotide for c‐jun or ERK inhibition by PD98059 cancelled DHT‐mediated suppression of radiation‐induced transactivation of Fas gene and sensitization to Fas‐mediated apoptosis.

Conclusions: Radiation‐induced Fas sensitization in prostate cancer cell was mediated through p53‐dependent transactivation of the Fas gene, which can be blocked by androgen stimulation mainly through induction of c‐jun.     

Influence of high‐frequency electromagnetic fields on different modes of cell death and gene expression

Purpose: International thresholds for exposure to non‐ionizing radiation leading to non‐thermal effects were conservatively set by the International Commission on Non‐Ionizing Radiation Protection (ICNIRP). The aim of this study was to examine whether biological effects such as different modes of cell death and gene expression modifications related to tumorgenesis are detectable above the threshold defined.

Materials and methods: Human leukaemia cells (HL‐60) grown in vitro were exposed to electromagnetic fields (EMF; t_½r about 1?ns; field strength about 25 times higher than the ICNIRP reference levels for occupational exposure) leading to non‐thermal effects using a high‐voltage‐improved GTEM cell 5302 (EMCO) connected to a pulse generator NP20 (C=1?nF, U_Load=20?kV). HL‐60?cells were harvested at 0, 24, 48 and 72?h after radiation exposure. Micronuclei, apoptosis and abnormal cells (e.g. necrosis) were determined using morphological criteria. In parallel, the expression of 1176 genes was measured using Atlas^TM Human 1.2. Array. Based on high data reproducibility calculated from two independent experiments (>99%), array analysis was performed.

Results: No significant change in apoptosis, micronucleation, abnormal cells and differential gene expression was found.

Conclusions: Exposure of HL‐60 cells to EMFs 25 times higher than the ICNIRP reference levels for occupational exposure failed to induce any changes in apoptosis, micronucleation, abnormal morphologies and gene expression. Further experiments using EMFs above the conservatively defined reference level set by the ICNIRP may be desirable.     

Bowman–Birk protease inhibitor activates DNA‐dependent protein kinase and reduces formation of radiation‐induced dicentric chromosomes

Purpose: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes.

Materials and methods: An effect of BBI upon DNA repair was investigated by quantification of radiation‐induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA‐dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53‐derived peptide.

Results: The formation of radiation‐induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI‐mediated reduction of dicentric chromosome formation depended on the presence of wild‐type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA‐dependent protein kinase (DNA‐PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA‐PK) activity. In SCID mouse fibroblasts deficient in DNA‐PK activity, BBI failed to reduce the amount of radiation‐induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI.

Conclusions: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA‐PK activity.     

Intramural neovascularization and haemorrhages are major long‐term effects of intravascular γ‐radiation after stenting

Structural changes that might influence the structural integrity of the vessel in response to intravascular brachytherapy (IVB) and stenting were examined, focus being on the importance of neovascularization in rabbit stented arteries. Stents were implanted in the infrarenal aortas of rabbits, immediately followed by gamma IVB or a sham radiation procedure, and the arteries harvested at 6 months. Labelling for von Willebrand factor showed an increase in adventitial and medial neovascularization in irradiated versus control arteries group (5.04±0.89 versus 1.51±0.23?mm^?2, respectively; p=0.004). Moreover, intramedial haemorrhages (free hemosiderin deposition) and inflammation (macrophages) were only observed in irradiated arteries. No significant change in expression of matrix metalloproteinase 1, 2 or 3 was observed between the irradiated and control group while collagen content decreased in the irradiated versus the control group (10.05%±1.48% versus 31.92%±3.12%, respectively; p<0.001). The study supports the hypothesis that IVB associated with stenting induces late deleterious effects on the medial layer, characterized by formation of intramural neovessels, haemorrhages and a decrease in collagen content.     

Distribution of radiation‐induced exchange aberrations in all human chromosomes

Purpose: To investigate the DNA‐proportional distribution of radiation‐induced chromosome aberrations for all chromosomes of a male and a female human karyotype.

Materials and methods: Metaphases were prepared from whole blood cultures obtained from two healthy donors and set up after irradiation with 3?Gy 220?kV X‐rays. Single whole‐chromosome FISH painting simultaneously with pancentromeric DNA painting were performed separately for each chromosome of the human karyotype. One thousand exclusively first‐division metaphases were analysed per chromosome and donor. After statistical analysis, the data obtained were compared with theoretically expected values.

Results: All aberration types (translocations, dicentrics) showed deviations from a DNA‐proportional distribution. For both donors, chromosomes 2 and 3 exhibited significantly less and chromosome 4 more symmetrical translocations than expected. Chromosomes 15 and 22 showed more symmetrical translocations than predicted for one of the two donors. Less dicentrics than expected became apparent for chromosomes 2, 3 and 18, while more dicentrics were seen for chromosomes 15, 16 and 17. Moreover, chromosomes 4, 14 and 22 showed a significant deviation from the theoretically expected 1:1 ratio of the yields of symmetrical translocations to the yields of dicentrics.

Conclusion: The results from the whole‐chromosome complement in two different donors confirmed published data from the analysis of single chromosomes that some human chromosomes were not involved in radiation‐induced dicentrics and symmetrical translocations proportional to their DNA content. This must be taken into account if chromosome subsets for dose reconstruction are selected or if whole genomic frequencies have to be calculated from partial genome analysis.     

Time‐ and dose‐dependent changes of intracellular cytokine and cytokine receptor profile of Ewing tumour subpopulations under the influence of ionizing radiation

Purpose: Cytokines and their corresponding cell surface receptors are involved in intercellular signalling pathways and in the radioresistance of normal and malignant cells. The aim was the characterization of the expression of intracellular cytokines, their receptors and apoptosis‐associated markers under the influence of radiation.

Materials and methods: Two Ewing tumours were characterized in vitro before and 4, 24 and 72?h after radiation with 5 and 10?Gy, and in vivo 4, 6 and 15 days after radiation with 5 and 30?Gy by five parameter flow cytometry. Direct fluorescence‐conjugated antibodies directed against intracellular cytokines (interferon‐gamma, tumour necrosis factor [TNF]‐alpha, interleukin 1) and their receptors (CD119, CD120a, CD121a) were used. Annexin V and 7‐amino‐actinomycin D were used to identify radiation‐induced apoptosis.

Results: Inter‐ and intra‐individual heterogeneities were identified by the expression of cytokine receptors and the intracellular cytokine profile before radiation. Time‐ and dose‐dependent up‐regulation of the cytokines TNF‐alpha and interleukin 1 were found in vitro. In vivo, an up‐regulation of CD120a and CD121a was detectable on tumour cell subpopulations. For interferon‐gamma and CD119, no changes were seen.

Conclusions: The observed radiation‐induced changes of cytokine and receptor profile are an indication for complex intercellular interactions in view of radioresistance‐associated mechanisms between cell populations within one individual tumour. The observed heterogeneous response on radiation might have therapeutic implications for an individualized therapy based on combined radiation and cytokine modulation, defined by flow cytometric characterization of markers potentially informative for radioresistance.     

Nucleotide excision repair proteins and their importance for radiation‐enhanced transfection

Purpose: Irradiated cells transfect more efficiently than unirradiated cells because of a radiation‐induced increase in plasmid integration. However, the molecular mechanism is unclear. Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation‐enhanced integration.

Materials and methods: Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6?Gy and then immediately transfected with a linearized selectable marker plasmid. Transfection‐enhancement ratios (TERs) were calculated as the ratio of the number of drug‐resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation.

Results: Transfection into unirradiated rodent cells was unaffected by NER mutation status. Transfection into unirradiated human cells, however, was increased by NER mutation. The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6?Gy. Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER. Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1.

Conclusions: The mechanism of radiation‐enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.     

Role of Ca2+ in radiation‐induced damage in murine splenocytes

Purpose: To address the links between calcium, peroxidation, cell damage and death and the response of the enzymes involved in free radical metabolism, in splenocytes of mice irradiated with gamma‐rays.

Materials and methods: Splenocytes of Swiss albino mice were irradiated with various doses (0–7?Gy) of gamma‐rays (⁶⁰Co) at a dose‐rate of 0.0575?Gy?s^?1. Membrane peroxidation and fluidity were determined by the thiobarbituric acid‐reactive substances (TBARS) method, and fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), respectively. Apoptosis was analysed by nucleosomal ladder formation and activity of NF‐κB by electrophoretic mobility shift assay (EMSA). The specific activities of the antioxidant enzymes, lactate dehydrogenase (LDH), levels of nitric oxide (NO^?) and glutathione were determined spectrophotometrically. Modulatory effects of Ca²⁺ were examined at 3?Gy using different concentrations (1, 3 and 5?mM) in the presence or absence of the ionophore A23187.

Results: Irradiation of splenocytes resulted in enhanced peroxidative damage, membrane fluidity, apoptosis and DNA binding activity of NF‐κB. The specific activities of LDH and antioxidant enzymes superoxide dismutase (SOD), DT‐diaphorase (DTD), glutathione S‐transferase (GST) and levels of glutathione (GSH) and NO^? were increased with radiation dose up to 4?Gy. Ca²⁺ augmented the radiation‐induced responses. The presence of ionophore A23187 potentiated the modulatory effects of Ca²⁺.

Conclusions: These findings show that Ca²⁺ augments radiation damage and is more effective intracellularly. Ca²⁺, peroxidation, cellular damage and apoptosis are possibly interlinked through signals, as is evident from the increased activity of NF‐κB and generation of NO^?. The enhanced antioxidant status suggests an attempt made by the irradiated cells to maintain their normal functions.     

Extremely low frequency magnetic fields and the promotion of H2O2‐induced cell death in HL‐60 cells

Purpose: To test whether exposure to an extremely low frequency magnetic field (60?Hz, 5?mT) affects hydrogen peroxide (H₂O₂)‐induced cell death in human leukaemia HL‐60 cells.

Materials and methods: Cells were treated with H₂O₂ with or without exposure to an extremely low frequency magnetic fields. Viable cells, apoptotic and necrotic cells were determined by annexin V flow cytometry assay. The levels of apoptosis‐related proteins (caspase‐3, caspase‐7, Bcl‐2 and Bax) and poly(ADP‐ribose) polymerase were detected using Western blotting.

Results: Simultaneous treatment with exposure to the magnetic field and H₂O₂ (85 or 100?µM) for 24?h increased the number of apoptotic and necrotic cells significantly, and significantly decreased the number of viable cells compared with cells treated with H₂O₂ alone. The protein levels of Bax and Bcl‐2 showed no differences between H₂O₂‐treated cells and those treated with both H₂O₂ and an extremely low frequency magnetic field. Exposure to the magnetic field also had no effect on H₂O₂‐induced caspase‐3 activation. However, the protein levels of active caspase‐7 in cells simultaneously exposed to an extremely low frequency magnetic field and H₂O₂ for 2 and 8?h was higher than that of H₂O₂ treatment alone. In addition, simultaneous exposure to an extremely low frequency magnetic field and H₂O₂ caused poly(ADP‐ribose) polymerase cleavage and induced early inactivation at 2?h, while H₂O₂ treatment alone did not produce this effect until 4?h.

Conclusions: The data suggest that although the magnetic field itself cannot induce apoptosis and necrosis, it exerts a promoting effect on H₂O₂‐induced cell death, and it demonstrates that caspase‐7 as well as poly(ADP‐ribose) polymerase might be involved in this process.