OBJECTIVETo study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of stress-activated protein kinase(SAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.

METHODSChinese hamster lung(CHL) cell line was exposed to power-frequency magnetic fields with two intensities for different exposure durations. The cytoplasmic protein was extracted and the phosphorylated portion of SAPK and SEK1/MKK4 was measured with Western blotting analysis. The SAPK enzymatic activity was measured by the solid-phase kinase assay in cells exposed to power-frequency magnetic fields for 15 min.

RESULTSBoth 0.4 mT and 0.8 mT power-frequency magnetic fields could enhance the phosphorylation of SAPK in a time-relative course manner, and reached the maximum extent at 15 min, with an increase of 20% and 17% respectively. The solid-phase kinase assay showed that the enzymatic activities of SAPK were also increased, which were 2.9 +/- 0.4 and 2.1 +/- 0.9 times of control respectively. However, the duration of SAPK phosphorylation induced by 0.8 mT was longer than that of 0.4 mT, while the duration and extent of SAPK dephosphorylation was remarkably shorter than that of 0.4 mT. The power-frequency magnetic fields under equal conditions could not phosphorylate(activate) the SEK1/MKK4.

CONCLUSIONPower-frequency magnetic fields could activate the SAPK, but not SEK1/MKK4. It is suggested that power-frequency magnetic fields may activate SAPK signal transduction pathway through a kinase other than SEK1/MKK4. The activation mechanism of SAPK of power-frequency magnetic fields needs to be identified in more detail.

Animals ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Activation ; radiation effects ; Lung ; metabolism ; radiation effects ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; physiology ; radiation effects ; Magnetics ; Phosphorylation

OBJECTIVETo investigate the role of Caspase-3 in retinal damage caused by light exposure in rats.

METHODSLight injury to retina was induced by persistent exposure to illumination (intensity: 30 000 +/- 50 lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1, 3, 7, and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis.

RESULTSThe examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, especially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pigment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day.

CONCLUSIONApoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

Animals ; Apoptosis ; radiation effects ; Caspase 3 ; genetics ; metabolism ; radiation effects ; Dose-Response Relationship, Radiation ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; radiation effects ; Light ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Retina ; enzymology ; pathology ; radiation effects ; ultrastructure

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H2O2. This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H2O2 failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H2O2 treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H2O2, but elevated cellular levels of p21Cip/WAF1, a p53 target that can suppress H2O2-induced cell death by blocking JNK activation. Consistently, H2O2-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.
Antioxidants/pharmacology ; Cell Death ; Cells, Cultured ; Enzyme Activation/radiation effects ; Fibroblasts/enzymology/radiation effects ; *Gamma Rays ; Heat-Shock Proteins/metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases/*antagonists & inhibitors ; Oxidative Stress/*radiation effects ; Research Support, Non-U.S. Gov't ; Water/pharmacology

OBJECTIVETo study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.

METHODSChinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies.

RESULTSPower-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK.

CONCLUSIONPower-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.

Animals ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Activation ; radiation effects ; Lung ; enzymology ; radiation effects ; MAP Kinase Kinase 3 ; metabolism ; MAP Kinase Kinase 6 ; metabolism ; Magnetics ; p38 Mitogen-Activated Protein Kinases ; metabolism ; radiation effects

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.
Breast Neoplasms/*drug therapy/*enzymology/pathology ; Cell Death/drug effects/radiation effects ; Cell Line, Tumor ; Cell Proliferation/drug effects/radiation effects ; DNA Damage ; Enzyme Activation/drug effects/radiation effects ; Enzyme Inhibitors/*pharmacology/*therapeutic use ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Phospholipase D/*antagonists & inhibitors/metabolism ; Radiation Tolerance/*drug effects ; Radiation, Ionizing ; p38 Mitogen-Activated Protein Kinases/metabolism

BACKGROUNDRadiation is a promising treatment for in stent restenosis and restenosis following percutaneous transluminal coronary angioplasty, which has troubled interventional cardiologists for a long time. It inhibits neointima hyperplasia, vascular remodeling, and increases the mean luminal diameter. The mechanism of intracoronary brachytherapy for restenosis is not well understood. Endogenous gaseous transmitters including nitric oxide and carbon monoxide are closely related to restenosis. Hydrogen sulfide, a new endogenous gaseous transmitter, is able to inhibit the proliferation of vascular smooth muscle cells and vascular remodeling. This study aimed to clarify the effect of radiation on cystathionine-gamma-lyase/hydrogen sulfide pathway in rat smooth muscle cells.

METHODSWe studied the effect of radiation on the cystathionine-gamma-lyase/hydrogen sulfide pathway. Rat vascular smooth muscle cells were radiated with (60)Co gamma at doses of 14 Gy and 25 Gy respectively. Then the mRNA level of cystathionine-gamma-lyase was studied by quantitative reverse-transcription competitive polymerase chain reaction. Hydrogen sulfide concentration in culture medium was determined by methylene blue spectrophotometry. Cystathionine-gamma-lyase activity in vascular smooth muscle cells was also studied.

RESULTS(60)Co gamma radiation at a dose of 1 Gy did not affect the cystathionine-gamma-lyase/hydrogen sulfide pathway significantly. However, (60)Co gamma radiation at doses of 14 Gy and 25 Gy decreased the hydrogen sulfide synthesis by 21.9% (P<0.05) and 26.8% (P<0.01) respectively. At the same time, they decreased the cystathionine-gamma-lyase activity by 15.1% (P<0.05) and 20.5% (P<0.01) respectively, and cystathionine-gamma-lyase mRNA expression by 29.3% (P<0.01) and 38.2% (P<0.01) respectively.

CONCLUSIONAppropriate (60)Co gamma radiation inhibits the H(2)S synthesis by inhibiting the gene expression of cystathionine-gamma-lyase and the cystathionine-gamma-lyase activity.

Animals ; Cells, Cultured ; Cobalt Radioisotopes ; Cystathionine gamma-Lyase ; genetics ; metabolism ; Enzyme Activation ; drug effects ; radiation effects ; Gamma Rays ; Hydrogen Sulfide ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; radiation effects ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; radiation effects

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.
Apoptosis/*radiation effects ; Caspases/*metabolism ; Cell Line, Tumor ; DNA Fragmentation/radiation effects ; Dose-Response Relationship, Radiation ; Enzyme Activation/radiation effects ; Flow Cytometry ; Gamma Rays ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Neoplasms/*enzymology/*pathology ; *Protons ; Reactive Oxygen Species/*metabolism ; p38 Mitogen-Activated Protein Kinases/metabolism

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.
Curcumin/*pharmacology ; Enzyme Activation/drug effects/radiation effects ; Enzyme Inhibitors/pharmacology ; Humans ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Keratinocytes/cytology/*drug effects/*radiation effects ; Prostaglandin-Endoperoxide Synthase/*metabolism ; Research Support, Non-U.S. Gov't ; Transcription Factor AP-1/*metabolism ; Ultraviolet Rays ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

BACKGROUNDSolar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.

METHODSFollowing UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCulture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts.

CONCLUSIONSUVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

Cell Line ; Enzyme Activation ; Fibroblasts ; enzymology ; radiation effects ; Humans ; Interleukin-1 ; pharmacology ; Keratinocytes ; physiology ; radiation effects ; Matrix Metalloproteinase 1 ; biosynthesis ; Mitogen-Activated Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; RNA, Messenger ; analysis ; Skin ; radiation effects ; Skin Aging ; Transcription Factor AP-1 ; metabolism ; Ultraviolet Rays

Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the exception of proliferative stem cells, germ cells and activated lymphocytes. Measuring telomerase activity clinically may provide useful diagnostic and prognostic information of cancer. The purpose of this study was to investigate the change in telomerase activity following chemoradiation in rectal cancer, which almost always produces positive enzymatic activity. A total of 24 tumor tissue samples were used in this study, consisting of 12 paired specimens before and 4 week after chemoradiation. Telomerase activity was determined by PCR-based telomeric repeat amplification protocol (TRAP) assay. The telomerase activity was positive in 10 out of 12 patients (83%) in pre-irradiated and post-irradiated states. The levels of telomerase activity was decreased in 8 out of 10 patients after chemoradiation (80%) and two cases showed no change in enzymatic activity. One case showed no activity in either sample. The other case showed no enzymatic activity in the pre-irradiated sample, but showed weak activity in the post-irradiated sample. These data indicate that telomerase activity in rectal cancer is reduced after neoadjuvant chemoradiation therapy, possibly suggesting a mechanism of downstaging following chemoradiation therapy in cancer.
Adult ; Aged ; Antimetabolites, Antineoplastic/administration & dosage ; Antineoplastic Agents/administration & dosage ; Cisplatin/administration & dosage ; Combined Modality Therapy ; Enzyme Activation/radiation effects ; Enzyme Activation/drug effects ; Female ; Fluorouracil/administration & dosage ; Gene Amplification ; Human ; Male ; Middle Age ; Rectal Neoplasms/radiotherapy* ; Rectal Neoplasms/enzymology* ; Rectal Neoplasms/drug therapy ; Telomerase/metabolism*