OBJECTIVETo investigate the changes of gene expression in rat neuron induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) to screen for RF EMF-responsive genes and the effect of different exposure times and modes on the gene expression in neuron.

METHODSTotal RNA was extracted immediately and purified from the primary culture of neurons after intermittent exposed or sham-exposed to a frequency of 1.8 GHz RF EMF for 24 hours at an average special absorption rate (SAR) of 2 W/kg. Affymetrix Rat Neurobiology U34 array was applied to investigate the changes of gene expression in rat neuron. Differentially expressed genes (Egr-1, Mbp and Plp) were further confirmed by semi-quantitative revere transcription polymerase chain reaction (RT PCR). The expression levels of Egr-1, Mbp and Plp were observed at different exposure times (6, 24 h) and modes (intermittent and continuous exposure).

RESULTSAmong 1200 candidate genes, 24 up-regulated and 10 down-regulated genes were found by using Affymetrix microarray suite software 5.0 which are associated with multiple cellular functions (cytoskeleton, signal transduction pathway, metabolism, etc.) after functional classification. Under 24 h and 6 h intermittent exposure, Egr-1 and Plp in experiment groups showed statistic significance (P < 0.05) compared with the control groups, while expression of Mbp did not change significantly (P > 0.05). After 24 h continuous exposure, Egr-1 and Mbp in experiment groups showed statistic significance (P < 0.05) compared with the control group, while expression of Plp did not change significantly (P > 0.05). Under the same exposure mode 6 h, expression of all the 3 genes did not change significantly. Different times (6, 24 h) and modes (intermittent and continuous exposure) of exposure exerted remarkable different influences on the expression of Egr-1, Mbp, Plp genes (P < 0.01).

CONCLUSIONThe changes of many genes transcription were involved in the effect of 1.8 GHz RF EMF on rat neurons; Down-regulation of Egr-1 and up-regulation of Mbp, Plp indicated the negative effects of RF EMF on neurons; The effect of RF intermittent exposure on gene expression was more obvious than that of continuous exposure; The effect of 24 h RF exposure (both intermittent and continuous) on gene expression was more obvious than that of 6 h (both intermittent and continuous).

Animals ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Down-Regulation ; radiation effects ; Electromagnetic Fields ; Neurons ; metabolism ; radiation effects ; Rats ; Up-Regulation ; radiation effects

Animals ; Down-Regulation ; radiation effects ; Heavy Ion Radiotherapy ; adverse effects ; Infertility, Male ; etiology ; Male ; Mice ; Random Allocation ; Sperm Motility ; radiation effects ; Spermatozoa ; metabolism ; radiation effects ; Tubulin ; metabolism

OBJECTIVETo investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms.

METHODSNK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126.

RESULTSMicrowave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01).

CONCLUSIONMicrowave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression.

Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Cell Line ; Dose-Response Relationship, Radiation ; Down-Regulation ; Humans ; Killer Cells, Natural ; radiation effects ; MAP Kinase Signaling System ; Microwaves ; adverse effects ; NK Cell Lectin-Like Receptor Subfamily K ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo explore the changes in the expressions of the tight junction related protein occludin and junctional adhesion molecule-1 (JAM-1) of the blood-testis barrier and their significance in rats after microwave radiation.

METHODSEighty male Wistar rats were exposed to microwave radiation with average power density of 0, 10, 30 and 100 mW/cm2 for five minutes, and dynamic changes in the expressions of testicular occludin and JAM-1 were observed by Western blot and image analysis at 6 h, 1 d, 3 d, 7 d and 14 d after the radiation.

RESULTSThere was a significant down-regulation in the expression of the occludin protein at 3 - 7 d, 6 h - 7 d and 6 h - 14 d (P < 0. 05), as well as in that of JAM-1 at 3 - 7 d, 1 - 7 d and 1-14 d (P < 0.05) after exposure to 10, 30 and 100 mW/cm2 microwave radiation.

CONCLUSIONThe decreased protein expressions of occludin and JAM-1 may play an important role in the microwave radiation induced-damage to the blood-testis barrier.

Animals ; Blood-Testis Barrier ; metabolism ; radiation effects ; Cell Adhesion Molecules ; metabolism ; Down-Regulation ; Male ; Membrane Proteins ; metabolism ; Microwaves ; Occludin ; Rats ; Rats, Wistar ; Testis ; metabolism ; radiation effects

OBJECTIVETo investigate the changes of gene expression in rat neurons induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) and to screen for the RF EMF-responsive genes.

METHODSNewly-born SD rats in 24 hours were sacrificed to obtain cortex and hippocampus neurons. The cells were divided randomly into two groups: the experiment group (the irradiation group) and the control group (the false irradiation group). In the irradiation group, after twelve days' culture, neurons were exposed to 1.8 GHz RF EMF modulated by 217 Hz at a specific absorption rate (SAR) of 2 W/kg for 24 hours (5 minutes on/10 minutes off) while in the false control group, the neurons were put in the same waveguide as in the irradiation group, but were not exposed to any irradiation. The total RNA was isolated and purified immediately after exposure. The affymetrix rat neurobiology U34 assay was used for detecting the changes in gene expression profile according to the manufacturer's instruction. RF EMF-responsive candidate gene was confirmed by using ribonuclease protection assay (RPA).

RESULTSAmong 1200 candidate genes, the expression levels of 34 genes were up or down regulated. Microtubule associated protein 2 (Map2) gene was selected as the candidate and subjected to further analysis. RPA data clearly revealed that Map2 was statistically significantly up-regulated after neurons were exposed to the RF EMF (P < 0.05).

CONCLUSIONThe modulation of gene expression and function of Map2 as a neuron specific cytoskeleton protein is crucial to maintain the normal framework and function of neurons. The finding that 1.8 GHz RF EMF exposure increases the expression of Map2 might indicate some unknown effects of RF EMF on neurons.

Animals ; Animals, Newborn ; Cell Phone ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Down-Regulation ; Electromagnetic Fields ; Female ; Gene Expression ; radiation effects ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neurons ; metabolism ; radiation effects ; Radio Waves ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest.

METHODSProtein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker.

RESULTSAccompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells.

CONCLUSIONOur results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

Cell Cycle Checkpoints ; radiation effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; DNA Damage ; Down-Regulation ; Fibroblasts ; metabolism ; radiation effects ; Gene Expression Regulation ; radiation effects ; Humans ; Mitosis ; radiation effects ; RNA Interference ; RNA, Small Interfering ; Radiation, Ionizing ; Up-Regulation

OBJECTIVETo explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action.

METHODSAsynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry.

RESULTSApoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P < 0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN.

CONCLUSIONThe radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.

Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; Cobalt Radioisotopes ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; Protein Kinases ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Transfection

This study is to investigate the effect and mechanism of puerarin on DNA damage of HaCaT cells induced by UVB. Puerarin pre-treated cells were irradiated with UVB at 30 mJ x cm(-2). Twenty four hours after irradiation, DNA damage was detected by comet assay, ceramide was measured by thin layer chromatography and gas chromatography, intracellular free calcium ion was analyzed by flow cytometry, the phosphorylation level of p38 protein was examined by Western blotting method. Levels of DNA damage, ceramide, free calcium ion and p-p38 protein were elevated in UVB model cells. Contrary to the model group, all indicators above were reduced in all groups pre-treated by puerarin. Puerarin restrains the ceramide accumulation to block downstream p38 MAPK pathway and calcium ion rising, therefore reduces DNA damage in HaCaT cells induced by UVB.
Calcium ; metabolism ; Cell Line ; Ceramides ; metabolism ; DNA Damage ; drug effects ; radiation effects ; Down-Regulation ; Humans ; Isoflavones ; pharmacology ; Keratinocytes ; cytology ; metabolism ; Phosphorylation ; Signal Transduction ; drug effects ; Ultraviolet Rays ; adverse effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo study the effect of dihydroartemisinin (DHA) combined irradiation on the apoptosis of human lung cancer GLC-82 cells and to study its mechanism.

METHODSThe growth inhibition rate of GLC-82 cells acted by different concentrations DHA was detected using MTT assay at 24, 48, and 72 h, respectively. Clone forming test was used. With multi-target single-hit model, the radiosensitization effect was assessed by calculating sensitizing enhancement ratio (SER).The effect of DHA combined irradiation on the apoptosis of GLC-82 cell cycle distribution and apoptosis were measured by flow cytometry. The protein expression of p53, p21, Bcl-2, and Bax were detected by Western blot.

RESULTSDifferent concentrations DHA (4, 8, 16, 32, 64, and 128 μg/mL) had cytotoxicity on GLC-82 cells. The IC50 for 24, 48, and 72 h was 38.25,20.58, and 10.36 μg/mL, respectively, in obvious dose- and time-dependent manner. The growth inhibition rate was more significantly increased than that of the blank control group (P < 0.01, P<0.05). DHA had sensitization enhancement effect on GLC-82 cells, with SER of 1.4. DHA combined irradiation could obviously change the structure of GLC-82 cells cell cycle and induce apoptosis (with the apoptosis rate of 21.5%), which was significantly different from that of the blank control group (P < 0.05). Western blot showed the expression of p53 and p21 protein could be increased by DHA combined irradiation, and the expression of Bcl-2 protein down-regulated (P <0.01, P <0. 05).

CONCLUSIONSDHA had stronger cytotoxicity and radiosensitization on GLC-82 cells. Its mechanisms might lie in making the arrest of GLC-82 cells' growth at G0/G1 phase, decreasing the ratio of cells at S phase, restoring the function of p53, decreasing the expression of Bcl-2 protein, and inducing apoptosis in GLC-82 cells.

Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Flow Cytometry ; Humans ; Lung Neoplasms ; metabolism ; Neoplasm Proteins ; metabolism ; Radiation-Sensitizing Agents ; pharmacology ; Tumor Cells, Cultured ; bcl-2-Associated X Protein ; metabolism

BACKGROUND AND OBJECTIVECombined hypoxic cytotoxic drugs and chemoradiotherapy is an important mean of oncotherapy, and Tirapazamine (TPZ) is one of the most remarkable drugs. It has been shown that TPZ has a synergistic effect with radiotherapy on tumor cells, but whether TPZ would down-regulate the expression of the hypoxia-induced genes has not been reported. This study was to investigate the hypoxia-induced mRNA expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and osteopontin (OPN) in human nasopharyngeal carcinoma HNE-1 and CNE-1 cells and the radiosensitization of TPZ, a hypoxia-specific drug, on HNE-1 and CNE-1 cells in vitro.

METHODSThe IC50 values of TPZ for HNE-1 and CNE-1 cells were measured using MTT assay, and the mRNA expressions of HIF-1alpha and OPN in HNE-1 and CNE-1 cells was determined using RT-PCR under aerobic and hypoxic conditions, respectively. The survival rates of HNE-1 and CNE-1 cells treated with or without TPZ at IC10 in the presence or absence of oxygen for 6 h were determined using colony formation assay following exposure to 1-6 Gy of 60Co radiation. The dose-survival curves were plotted and the values of D0, Dq and SER were calculated as a single-hit multitarget model.

RESULTSThe IC50 values of TPZ were 34.81 μmol/L and 35.02 μmol/L in HNE-1 and CNE-1 cells under aerobic condition, and 30.20 μmol/L and 28.48 μmol/L under hypoxic condition, respectively. The expressions of HIF-1alpha and OPN mRNA were reduced by TPZ in HNE-1 cells, but not in CNE-1 cells under hypoxic condition. For the HNE-1 cells, the respective values of D0 and Dq were 0.89 Gy and 0.28 Gy following normoxic irradiation versus 1.47 Gy and 0.44 Gy following hypoxic irradiation. For the CNE-1 cells, the respective values of D0 and Dq were 0.72 Gy and 0.68 Gy following normoxic irradiation versus 0.95 Gy and 0.56 Gy following hypoxic irradiation. The values of D0 and Dq for HNE-1 and CNE-1 cells treated with TPZ under hypoxic condition following irradiation were 0.66 Gy, 0.21 Gy and 0.85 Gy, 0.79 Gy, respectively.

CONCLUSIONTPZ can down-regulate hypoxia-induced expression of HIF-1alpha and OPN mRNA of HNE-1 cells and radiosensitize the HNE-1 cells but not CNE-1 cells, and act as a hypoxia modifier.

Antineoplastic Agents ; pharmacology ; Cell Hypoxia ; Cell Line, Tumor ; Cell Survival ; drug effects ; radiation effects ; Cobalt Radioisotopes ; Down-Regulation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Inhibitory Concentration 50 ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Osteopontin ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Radiation Tolerance ; drug effects ; Radiation-Sensitizing Agents ; pharmacology ; Triazines ; pharmacology