ABSTRACT
Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.
ABSTRACT
Objective:To identify the differentially expressed snoRNAs in the carcinogenesis of cells induced by α-particles radiation and predict the targeted genes and RNA-co-expression networks.Methods:Full transcriptome expression microarray biochips were employed to screen the differentially expressed snoRNAs between human bronchial epithelial BEP2D cell line and its derivative malignantly transformed cell line BERP35T-4 established by α-particle irradiation. The expression changes of snoRNAs and their derived sdRNAs were confirmed by qRT-PCR. The functional domains, targets and co-expression networks of snoRNA were predicted by bioinformatics analysis.Results:Consistent with the result of microarray assay, the expression changes of the screened snoRNAs were confirmed by qRT-PCR. The expressions of sno116 family decreased in BERP35T-4, which was 0.105% ( t=26.60, P<0.01) of BEP2D, and they were generally down-regulated in radiation-induced carcinogenic BERP35T-4 cells and the human lung cancer cell lines A549 and H1299. It was also found that the expression level of the sdRNAs derived from sno116-14 was significantly different in the same cells. It was speculated that these less expressed sdRNAs of sno116-14 could be due to degradation as the consequence of interaction with their targets. The co-expression networks of sno116 family with other types of RNA were established, and the predicted targets of sno116-14 included ZNF280D, TFDP1, CCDC28B, RPS6KA3, CANX, RUNX1 and KALRN, which were related to the functions of cell proliferation and cytoskeletal structure. Conclusions:Some differentially expressed snoRNAs related to α-particle induced carcinogenesis have been identified. It is predicted that the target gene of sno116-14 is involved in the biological processes such as cell proliferation, cytoskeletal structure and the signaling pathways for function regulation, providing new information for the function model of C/D box snoRNAs and the mechanism of radiation carcinogenesis.
ABSTRACT
Objective@# To identify the differentially expressed miRNAs in the exosomes secreted from γ-ray irradiated cells and provide new clues in disclosing the mechanisms of radiation-induced bystander effects.@*Methods@#The human bronchial epithelial cells (BEP2D) were irradiated with 60Co γ-rays, and the exosomes were collected by ultracentrifugation from the culture medium of 2 Gy-irradiated cells and non-irradiated control. The exosomes were identified by an electron microscopy. The miRNA microarray technique was used to analyze the miRNA expression profiles in the exosomes. qRT-PCR was used to verify the miRNAs expression. The functional pathways of miRNAs targeting genes were predicted by informatic analysis using the databases of TargetScan, miRanda, GO and KEGG.@*Results@#Sixteen miRNA with significantly increased expression (P<0.05) were identified in the exosomes of BEP2D cells at 4 h post-2 Gy irradiation as compared with the non-irradiated control cells, among which miR-100-5p, miR-1246, miR-29b-3p, and miR-7-5p were further confirmed to be unregulated by qRT-PCR assay (P<0.05). Meanwhile, the expression changes of above-mentioned four miRNAs were also investigated in the irradiated cells. The data indicated the expression was significantly increased at 2 h post-2 Gy irradiation for the miR-100-5p, miR-1246, miR-29b-3p in addition to miR-7-5p. However, all these four miRNAs were downregulated in the cells at 4 h post-irradiation and then gradually recovered. Bioinformatics analysis showed that the targeted genes of these differentially expressed miRNAs might participate in the biological processes and signal pathways of cell adhesion, mTOR signal pathway, chromatin modification, HR and NHEJ pathways of DNA repair and so on.@*Conclusions@# Radiation-inducible miRNAs have been identified in the exosomes from the irradiated BEP2D cells. The target genes of these miRNAs play roles in a series of important biological processes and functional pathways, which provides new clues in elucidating the mechanisms of radiation-induced bystander effects.
ABSTRACT
Objective To study the changes in miRNAs expression in the exosomes of human umbilical vein endothelial cells(HUVECs) after 60 Co γ-rays expose using microRNA(miRNA) chips and bioinformatics techniques so as to provide new clues to the mechanism of radiation-induced vascular tissue injury and its bystander effects.Methods HUVECs exosomes were collected in the control and 4 Gy irradiated cells by ultra-high-speed centrifugation,and further confirmed using transmission electron microscopy (TEM) and Western blotting of exosomes biomarkers.miRNA microarray was used to analyze miRNA expression profiles of exosomes and cells.Also,real-time quantitative PCR(qRT-PCR) was used to verify differentially expressed miRNAs,and the miRDB and TargetScan were performed to predict the target genes of the differentially expressed miRNAs.Bioinformatics analysis was performed using DAVID,KEGG and other online tools.Results Compared with the control exosomes from non-irradiated HUVECs,miRNA microarray analysis revealed that 5 up-regulated,and 13 down-regulated miRNAs were identified in the exosomes from HUVECs at 0.5 h after 4 Gy-irradiation,and 16 up-regulated and 5 down-regulated miRNAs at 2 h after 4 Gy-irradiation.Moreover,38 and 85 miRNAs were differentially expressed respectively in the HUVECs at 0.5 h and 2 h after radiation.The difference was statistically significant(P<0.01).The results of bioinformatics showed that these miRNAs might exert the radiation-induced bystander effect (RIBE) by regulating MAPK signal pathways,RAS and PI3K-Akt signal pathways.Conclusion The ionizing radiation injury significantly alters the components and expression levels of exosomal miRNAs,which play important roles in regulating the signal pathways in response to radiation.
ABSTRACT
Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.
ABSTRACT
Objective To investigate the expression of radiation-induced IER5 protein and screen its potential interaction proteins that may participate in DNA repair process.Methods HeLa cells were irradiated with 4 Gy ionizing radiation.IER5 protein expression in whole cell lysate and in nuclear fraction were detected by Western blot at different timepoints after irradiation.3 × Flag-IER5 pCMV plasmid was constrcuted and the Flag tagged-IER5 expression was verified by Western blot.293T cells were transfected with 3 × Flag-IER5 pCMV plasmid.After irradiation the cells were collected and proteins were extracted.The IER5 interaction proteins were purified using immunoprecipitation and separated by 12％ SDS-polyacrylamide gel electrophoresis.Then the binding proteins were cut from the gel and analyzed by Mass spectrometry.Results The expression of IER5 protein began to increase 4 hour post-irradiation and its peak level was observed at 12 hour post-irradiation,and it lasted until 48 hour after irradiation.The expression level of IER5 protein in whole cell lysate and nuclear fraction were both increased.With the mass spectrometry analysis,a total of 347 proteins and 256 proteins were identified in irradiated and nonirradiated groups,respectively.Fourty one differential proteins were obtained,where 10 proteins were associated with DNA metabolic process and DNA rapair in the irradiated group and the poly(ADP-ribose) polymerase 1 (PARP1) protein was further confirmed by Western blot.Conclusions IER5 protein is an DNA damage related protein,and it may participate in DNA repair process.
ABSTRACT
Objective To determine the Golgi dispersal in radiation damaged cells and the protective effect of vanillin derivatives.Methods Immunofluorescence, cell cycle analysis of flow-cytometry,Western blot,and clone formation were used.Results Immunofluorescence observation showed that the Golgi dispersal caused by 2 Gy 60 Coγ-ray was significantly increased in a dose-dependent manner in the range of 4-10 Gy as was demonstrated by the fact that the Golgi area was significantly increased. When the irradiated cells were treated with the radioprotective agent VND3207, a vanillin derivative,the Golgi dispersal induced by radiation was significantly reduced.The radiation-induced Golgi dispersal was also displayed in a pattern of time-course after irradiation in the HeLa cells, and persisted at least to 36 h post-irradiation. Cell cycle test results indicated that the Golgi dispersal was not associated with the G2/M arrest triggered by radiation-induced DNA damage response.VND3207 could promote cell survival by plate colony formation assay.Conclusion The Golgi dispersal can be caused byγ-ray irradiation in a dose-and time-dependent manner, and VND3207 can provide a good protection against radiation injury associated with inhibited Golgi dispersal.
ABSTRACT
Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.
ABSTRACT
In the early 1950s,China′s modern industry and national defense developed rapidly. While toxicological research,represented by occupational toxicology,radiological toxicology and military toxicology,was initiated. Along with the expansion of industrial and agricultural activities and production, high- speed economic development,and the changes of the environment and lifestyles,the objects and content of toxicological research have also been expanded. Subdisciplines of toxicology have kept emerging,and an integrated toxicological discipline and research system has been established. Toxi?cological research has become an active area in China. Toxicological publications from China have accounted for about 10% to 20% of total international toxicological research papers in the recent ten years. Toxicologists in China have paid much attention to the toxicities and biological safety of new materials and emerging pollutants,such as nanomaterials and fine particles in the air. Some toxicological research was discussed in this paper.
ABSTRACT
Objective To investigate the effect of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on autophagy induction by ionizing radiation ( IR ) .Methods The cell model of knocking-down DNA-PKcs expression was constructed by transfecting HeLa cells with a pSicoR-based lentivirus vector expressing DNA-PKcs specific shRNA .Cellular growth activity and radiosensitivity were detected by cell countingkit ( CCK)-8 assay.The expression of autophagy related proteins was detected by Western blotting hybridization .Autophagy was also detected by monitoring the autophagic marker green fluorescere protein ( GFP )-light chain 3 ( LC3 ) puncta per cell under an immunofluorescent microscope .Results A cellular model of knocking-down DNA-PKcs expression was successfully generated by transfecting the specific shRNA against DNA-PKcs.Depression of DNA-PKcs significantly decreased the growth activity of HeLa cells and increased the cellular sensitivity to ionizing radiation .Both the expression changes of P 62 and LC3 proteins and immunofluorescent GFP-LC3 puncta observation indicated that knocking-down DNA-PKcs prompted the induction of autophagy by ionizing radiation . Moreover, inactivation of DNA-PKcs led to a decreased phosphorylation of mammalian target of sirolimus ( Rapamycin, RAPA) ( mTOR) at S2481 site.Conclusion Depression of DNA-PKcs expression prompts the induction of autophagy by IR and cellular radiosensitivity .mTOR signaling may be involved in the regulation of autophagy processing by DNA-PKcs.
ABSTRACT
Objective To evaluate the radiosensitization and mechanism of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells.Methods The flow cytometry was used to determine the sub-population of cancer stem cells with the markers CD133/ CD44.The cells were divided into four groups:control group,20 μmol/L NU7926 group,2 Gy irradiation group,and 20 μmol/L NU7026 combined with 2 Gy irradiation group.Cell proliferation and survival were evaluated by colony-formation experiment.Flow cytometry was used to analyze the cell cycle distribution and apoptosis induction.γ-H2AX foci were detected with immune-fluorescence staining analysis by a laser confocal microscopy for investiating the DNA double-strand break repair.Results The flow cytometric data of CD133 +/CD44 + positive cells indicated that the sub-population of cancer stem cell (CSC) took the ratio of (88.14 ± 0.47)％ of the cultured HCT116 cells.The colony-formation efficiency of HCT116 cells was (84.75 ± 1.35) ％ in serum-free mediums in vitro culture.Compared to 2 Gy irradiation alone group,the NU7026 combined with 2 Gy irradiation group had a lower cell colony-forming ratio (t =7.21,P ＜0.01) and a lower survival ratio (t =7.22,P ＜ 0.01).The proportion of CSCs sub-population increased at 48 h post-2 Gy irradiation,suggesting that HCT116 CSCs was more resistant to ionizing radiation.Importantly,NU7026 largely decreased the proportion of CSCs sub-population in 2 Gy-irradiated cells.The difference of CSC proportion between 2 Gy irradiation alone group and 2 Gy combined with NU7026 treatment group was statistically significant (t =9.55,P ＜ 0.01).In addition,the group of NU7026 combined with 2 Gy irradiation had a higher ratio of G2/M arrest 24 h post-irradiation (t =7.67,P ＜ 0.01) and an increased induction of cell early apoptosis (t =8.24,P ＜ 0.05).48 h post irradiation as compared to 2 Gy irradiation alone group.NU7026 treatment significantly inhibited the cellular capacity of repairing DNA double-strand breaks induced by γ-ray irradiation.The γ-H2AX foci of the combined treatments group were much higher than that of 2 Gy irradiation alone group at 2,4,8,24 h postirradiation (t=19.58,11.95,7.01,9.45,P＜0.01).Conclusions DNA-dependent protein kinase catalytic subunit inhibitor NU7026 can significantly sensitize the cancer stem cells of colorectal carcinoma HCT116 cells to γ-ray irradiation.Multiple mechanisms are involved in the radiosensitization effect of NU7026,including DNA repair inhibition,elongation of G2/M arrest,and increase of radiation-induced apoptosis.
ABSTRACT
Although metal nano materials have been widely used in various fields, the potential risks of it still could not be neglected. In this paper, the effects and mechanisms of genotoxicity caused by different nano materials were discussed. Human body can be exposed to metal nano materials through multiple pathways, metals nano follow the blood stream in circulatory system and distribute to organs. Metal nano particles are mainly uptaken into cells by endocytosis, and direct or indirect damages to genes can be induced by these particles after metabolism in cells. These damages would affect the course of cell cycle and the stability of the genome, resulting in gene mutation or chromosome aberration, and even leading to the death or malignant transformation of cells.
Subject(s)
Humans , Cell Transformation, Neoplastic , DNA Damage , Metal Nanoparticles , ToxicityABSTRACT
Objective To construct recombinant plasmids of SIK2 cDNA and its truncated mutants and induce its expression in E.coil.Methods We designed primers of SIK2 and its truncated mutants.The gene fragments of SIK2,SIK2-Δ1 (280-926),SIK2-Δ2 (400-926),SIK2-Δ3 (1-400),and SIK2-Δ4 (700-926)were amplified by polymerase chain reaction (PCR)and cloned into pGEX-4T-2 vector to construct recombinant plasmids with GST. The plasmids were transformed into E.coil BL2 1 respectively,and induced with IPTG to express fusion protein. The results were confirmed by Coomassie blue staining and Western blot.Results We successfully constructed recombinant plasmid of SIK2 cDNA and its truncated mutants.Coomassie blue staining and Western blot resutls showed that these plasmids were induced to be expressed in E.coil BL21.Conclusion SIK2 cDNA and its truncated mutants were overexpressed in E.coil BL2 1 ,which lays expereimental foundation for further study on the function of each domain of SIK2 .
ABSTRACT
Objective To quantitatively investigate the effect of low-dose ionizing radiation on the frequencies of chromosome aberrations and micronucleus-containing cells of radiation workers.Methods Nine electronic databases were systematically searched on the basis of the published studies evaluating the effects of low-dose ionizing radiation on the frequencies of chromosome aberrations and micronucleuscontaining cells.Of the 195 studies searched,21 studies were identified with a total of 1 970 626 cells under studying.Cochrane' s Q and I2 statistics were used to evaluate heterogeneity among studies and pooling odds ratio (OR) with 95％ confidence intervals (CI) were calculated using random-effect models or fixed-effect models,and publication bias were also calculated.Meta-analysis was performed using Stata 12.0.Results The pooling OR of chromosome-type aberrations [OR =3.03 (2.59,3.56)],dicentric plus centric rings [OR =4.12 (2.99,5.67)],translocations [OR =2.73 (1.67,4.46)],micronucleuscontaining cells [OR =1.70 (1.40-2.06)] were higher for radiation workers when compared with control group.Conclusions The frequencies of chromosome aberrations and micronucleus cell of peripheral lymphocytes are significantly high in radiation workers who were occupationally exposed to low-dose ionizing radiation.It should be noted that the radiation protection of radiological workers be enhanced.
ABSTRACT
Objective To quantitatively analyze the effects of high background radiation area (HBRA) on the frequencies of translocation and unstable chromosome aberrations in human peripheral blood lymphocytes.Methods Based on the data from 9 published articles retrieved from 7 electronic databases,the Meta-analysis was performed to evaluate the effects of high natural background radiation (HNBR) on the frequencies of chromosome aberrations in the peripheral blood lymphocytes in 17 777 persons from HBRA and 10 386 from the control area (CA).Cochrane's Q and I2 statistics were used to evaluate heterogeneity among studies and pooling odds ratio (OR) with 95％ confidence intervals (95％ CI) were calculated using random-effect models.Publication bias was also calculated by funnel plot,Egger linear regression test and Begg rank correlation test.Results The pooling OR of translocation [OR(95％ CI) =1.57(1.10-2.24)] and unstable chromosome aberration [OR(95％ CI) =2.04(1.32-3.14)] of long-term exposed inhabitants living in HBRA were higher than control.The subgroup analysis showed that,fortranslocation,the OR (95％ CI) were 1.24 (1.09-1.42,I2 =0.00) for male,1.37 (1.17-1.60,I2 =0.00) for female,1.17 (1.05-1.30,I2 =69.50％) for adults,and 1.38 (1.25-1.51,I2 =0.00) for Chinese.For unstable chromosome aberration,the OR (95％ CI) were 3.78 (2.40-5.97,I2 =0.00) for female,2.60 (2.25-3.00,I2 =69.60％) for adults,1.03(0.85-1.24,I2 =0.00) for children,3.19 (2.46-4.13,I2 =21.60％) for Iranian,and 1.64 (1.33-2.02,I2 =0.00) for Chinese.HBRA,age and sex differences were the reasons of above heterogeneity.Conclusions To the inhabitants living in HBRA,the frequencies of translocation and unstable chromosome aberration of peripheral blood lymphocytes are higher than those in control area.More comprehensive analysis should be performed to assess the health risk in HBRA inhabitants,which may arise new stragety in radiation protection.
ABSTRACT
Objective To investigate the effect of salt-induced kinase 2 (SIK2) in the G2/M checkpoint in response to ionizing radiation and the possible mechanism.Methods HeLa cells were irradiated with 60Co γ-rays.The cell model of knockdown SIK2 expression was constrcuted by transfecting HeLa cells with a pSicoR-based lentivirus vector of expressing SIK2 shRNA by lipofectamin 2000.Western blot and flow cytometry were performed to measure the changes of SIK2 protein level and cell cycle distribution.The phosphorylated histone protein H3 on Ser 10 was used as a molecular marker of mitotic cells for detecting the function of G2/M checkpoint.Results The expression level of SIK2 protein increased in HeLa cells after 60Co γ-ray irradiation.A cell model of knockdown SIK2 expression was successfully generated by transfecting the specific shRNA against SIK2.Depression of SIK2 significantly increased the cellular sensitivity at 1,2,4,6 Gy post-irradiation (t =-3.445,-2.581,-3.251,-2.553,P ＜0.05),and led cells to release earlier from the G2/M boundary arrest compared to control cells at 5,6 h post-irradiation (t =4.341,6.500,P ＜ 0.05).Western blot analysis indicated that the irradiation-induced phosphorylated CHK2/T68 in SIK2 knock-down cells was earlier than that in control cells.Conclusions salt-induced kinase 2 (SIK2) participates in the regulation of G2/M checkpoint induced by ionizing radiation and affects cellular radiosensitivity.
ABSTRACT
Objective To comprehensively analyze the effect of low-dose radiation on the lens of the eye of radiation workers.Methods The papers dealing with the relationship between occupational radiation exposure and the lens of the eye were collected by retrieving documents of the domestic and foreign medical information databases with references to other papers.There were 28 papers finalized with 17 608 workers included in the Meta-analysis.Stata12.0 was used for Meta-analysis,Q-test and I2 statistic for heterogeneity test,and funnel regression method,Begg's rank method and Egger's regression method for publication bias.Results The pooling odds ratio (OR) opacity in radiation workers were 2.51 (2.01,3.13),4.03 (2.77,5.85),respectively.Conclusions Low-dose radiation may lead to negative impact on ocular lens under the current occupational protection conditions.The proportion of posterior subcapsular opacity in radiation-related cataract is higher than that in age-related cataract.It is important to strengthen radiation protection of ocular lens.
ABSTRACT
Objective To establish an experimental model for the study of α-particle-induced bystander effect of DNA damage and investigate the characteristics of bystander DNA double-strand break (DSB).Methods The red fluorescence fusion protein of HsBrkl-RFP was used to mark the cytoplasm of one cell line to distinguish the irradiated target cells (HFS-RFP) and the non-irradiated bystander cells (HFS) in the co-culture cellular model.After α-particle irradiation,cellular DSB and its repair kinetics were analyzed by the immunofluorescence staining of γH2AX and laser confocal microscope observation.Results A bystander studying model was established by co-culturing human HFS-RFP cells with its partner HSF cells.After 0.1 Gy or 0.2 Gy α-particle irradiation,the similar kinetics of γH2AX foci production and abatement were observed in both irradiated HFS-RFP cells and non-irradiated bystander HFS cells,in which the highest level of γH2AX foci was detected at 1 h post-irradiation.The second peak of γH2AX foci formation appeared at 8 h post-irradiation,which possibly indicates the occurrence of secondary DSB.However,the production of secondary DSB in the bystander cells was weaker than that in the irradiated cells.Conclusions The cell co-culture model can be used for bystander effect investigation.Bystander DSB can be effectively induce by irradiation and the secondary breakage of DNA DSB in the bystander cells may relative to the consequential biochemical processing of clustered DNA damage.
ABSTRACT
Objective To clarify the mechanism of immediate early response gene 5 (ler5)transcription induced by radiation. Methods Deletant construction, site-specific mutagenesis,electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to forecast the promoter region,binding sites and transcription factors of Ier5 gene in HeLa cells.Results The promoter region of Ier5 gene might be in the region of Ier5 -8 deletant ( -408 - -238 bp).The Ier5 gene had two transcription factors of GCF and NFI,and GCF had two binding sites located in the region of - 388 - - 382 bp and - 274 - - 270 bp of Ier5 promoter.The binding site of NFI was located in - 362 --357 bp of Ier5 promoter. GCF could inhibit the expression of Ier5 gene and this inhibition was diminished when the radiation dose increased. In contrast, NFI increased the expression of Ier5.Conclusions The most possible region of Ier5 promoter is from -408 to - 238 bp which has two binding sites for the radiosensitivity transcription factors of GCF and NFI that could negatively and positively regulate the expression of Ier5 respectively.
ABSTRACT
Objective To evaluate the radioprotective effect of vanillin derivative VND3207 on DNA damage induced by different LET ionizing radiation.Methods The plasmid DNA in liquid was irradiated by 60Co γ-rays, proton or 7Li heavy ion with or without VND3207.The conformation changes of plasmid DNA were assessed by agarose gel electrophoresis and the quantification was done using gel imaging system.Results The DNA damage induced by proton and 7Li heavy ion was much more serious as compared with that by 60Co γ-rays, and the vanillin derivative VND3207 could efficiently decrease the DNA damage induced by all three types of irradiation sources, which was expressed as a significantly reduced ratio of open circular form (OC) of plasmid DNA.The radioprotective effect of VND3207 increased with the increasing of drug concentration.The protective efficiencies of 200 μmol/L VND3207 were 85.3% (t =3.70,P =0.033), 73.3% (t = 10.58, P =0.017)and 80.4% (t =8.57,P =0.008)on DNA damage induction by 50 Gy of γ-rays, proton and 7Li heavy ion, respectively.It seemed that the radioprotection of VND3207 was more effective on DNA damage induced by high LET heavy ion than that by proton.Conclusions VND3207 has a protective effect against the genotoxicity of different LET ionizing radiation, especially for γ-rays and 7 Li heavy ion.