RESUMO
The interaction of radiation with matter takes place through energy transfer and is accomplished especially by ionized atoms or molecules. The effect of radiation on biological systems involves multiple physical, chemical and biological steps. Direct effects result in a large number of reactive oxygen species (ROS) within and outside and inside of the cells as well, which are responsible for oxidative stress. Indirect effects are defined as alteration of normal biological processes and cellular components (DNA, protein, lipids, etc.) caused by the reactive oxygen species directly induced by radiation. In this work, a classical design of an electrochemical (EC) three-electrodes system was employed for analyzing the effects of proton beam radiation on melanoma B16 cell line. In order to investigate the effect of proton radiation on the B16 cells, the cells were grown on the EC surface and irradiated. After optimization of the experimental set-up and dosimetry, the radiobiological experiments were performed at doses ranging between 0 and 2 Gy and the effect of proton beam irradiation on the cells was evaluated by the means of cyclic voltammetry and measuring the open circuit potential between working and reference electrodes.
Assuntos
Linhagem Celular/efeitos da radiação , Técnicas Eletroquímicas , Melanoma Experimental , Estresse Oxidativo , Terapia com Prótons , Animais , CamundongosRESUMO
Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.
Assuntos
Neoplasias Esofágicas , Imunidade , Fator Regulador 1 de Interferon , Fator de Transcrição STAT1 , Linhagem Celular/efeitos da radiação , Neoplasias Esofágicas/genética , Humanos , Imunidade/efeitos da radiação , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/efeitos da radiação , Interferon Tipo I , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/efeitos da radiação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/efeitos da radiaçãoRESUMO
In this paper I provide a personal perspective on future prospects for cell and gene therapy for osteoarthritis (OA) and how mammalian protein production platforms, virally transfected and irradiated protein packaging cell lines may be used as "cellular factories" for over-production of therapeutic proteins and growth factors, particularly in the context of intra-articular regenerative therapies. I will also speculate on future opportunities and challenges in this area of research and how new innovations in biotechnology will impact on the field of cell and gene therapy for OA, related osteoarticular disorders and the broader discipline of regenerative medicine for musculoskeletal disorders. Mammalian protein production platforms are likely to have a significant impact on synovial joint diseases that are amenable to cell and gene therapy using therapeutic proteins and growth factors. Future cell and gene therapy for OA will need to re-consider the current strategies that employ primary, aged and senescent cells with feeble regenerative properties and seriously consider the use of mammalian protein production platforms.
Assuntos
Linhagem Celular/efeitos da radiação , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Osteoartrite/terapia , Transfecção , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Articulações/patologia , Osteoartrite/genética , Osteoartrite/patologiaRESUMO
Far-infrared radiation (FIR) exerts numerous beneficial effects on health and cell physiology. Recent studies revealed that the biological effects of FIR are independent of thermal effects. There is no proper method for measuring the parameters of the non-thermal biological effects of FIR, which limits its biomedical application. In this study, we established a cell detection platform using epithelial cell migration to measure the limits of the biological effects of FIR. FIR promoted the migration of rat renal tubular epithelial cells as revealed by our standardized detection method. We defined the ratio of the FIR-promoted migration area to the migration area of the control group as the FIR biological index (FBI). An increase of the FBI was highly associated with FIR-promoted mitochondrial function. Through FBI detection, we revealed the limits of the biological effects of FIR, including effective irradiation time, wavelengths, and temperature. FBI detection can be used to clarify important parameters of the biological effects of FIR in biomedical studies and health industry applications.
Assuntos
Células Epiteliais/efeitos da radiação , Raios Infravermelhos , Animais , Linhagem Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Mitocôndrias/efeitos da radiação , RatosRESUMO
Ulcerative colitis2 (UC) is an inflammatory bowel disease3 (IBD) that causes long-lasting inflammation and ulcers in the human digestive tract. The repair function of TLR4 in the intestinal epithelium is still unknown. Here, wild-type4 (WT) mice, TLR4-knockout mice5 (KO; TLR4-/-) and commensal-depleted mice were used as dextran sulfate sodium6 (DSS)-induced or radiation-induced colitis and injury models to explore the role of TLR4 signaling in intestinal injury. Exogenous lipopolysaccharide7 (LPS) promoted DSS-induced inflammatory cytokines and aggravated intestinal damage. TLR4 deficiency and commensal bacterial depletion inhibited the toxic effects of LPS, but these mice were more susceptible to DSS-induced and radiation-induced intestinal damage. Compared with WT mice, neither DSS nor radiation promoted production of more inflammatory cytokines in the guts of TLR4-KO and commensal-depleted mice. Introducing the cytokine repair factors, PGE2 and GM-CSF, increased the cytokine levels in the guts of DSS-induced colitis mice. We hypothesized that TLR4 and its ligands repaired the epithelium after DSS-induced and radiation-induced intestinal damage by upregulating PGE2 and GM-CSF. Transwell migration assays suggested that LPS, IL6, TNF, PGE2 and GM-CSF promoted intestinal cell migration, and cell viability analysis suggested that these factors protected against radiation-induced intestinal damage. Our data underscore the importance of the balancing role of TLR4 in intestinal injury and repair.
Assuntos
Linhagem Celular/efeitos da radiação , Colite/induzido quimicamente , Colite/fisiopatologia , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Receptor 4 Toll-Like/efeitos da radiação , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
BACKGROUND Exposure to ionizing radiation (IR) induces severe damage in multiple human tissues. The testes are extremely sensitive to IR, and testes irradiation can result in infertility and abnormality. A novel and safe radioprotector for testes injury from IR is needed. Polydatin (PD) has been proved to have anti-oxidant and anti-inflammatory effects, indicating its potential application in radiation protection. MATERIAL AND METHODS Male wild-type C57BL/6 mice (8 weeks old) were exposed to ionizing radiation. At different times after irradiation, testes were isolated and subjected to hematoxylin-eosin (HE) staining and TUNEL staining, as well as related quantification. ELISA assay was used to measure the level of inflammatory cytokines, and apoptosis proteins were detected by Western blot assay. Intracellular ROS was measured by DCFH-DA flow cytometry method. RESULTS In the present study, we demonstrated that polydatin effectively alleviated testes injury and retained sperm viability. PD pretreatment also inhibited cell apoptosis caused by irradiation. Radiation-induced decrease of FSH and testosterone was also inhibited by PD treatment. Finally, we showed that PD obviously reduced the ROS level, using DCFH-DA method. We also found that PD reduced the concentration of the oxidative products MDA and 8-OHdG. PD also inhibited apoptosis-related proteins such as Bax and caspase 3. CONCLUSIONS Our data proved that polydatin effectively alleviated testes injury after irradiation, mainly through reducing ROS and oxidative stress. Our findings suggest polydatin as a potential radioprotector for testes radiation damage.
Assuntos
Glucosídeos/farmacologia , Estilbenos/farmacologia , Testículo/efeitos dos fármacos , Testículo/efeitos da radiação , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Glucosídeos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Lesões Experimentais por Radiação/tratamento farmacológico , Protetores contra Radiação/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estilbenos/uso terapêuticoRESUMO
High-dose ionizing radiation is sufficient for breaking DNA strands, leading to cell death and mutations. By contrast, the effects of fractionated ionizing radiation on human-derived cells remain unclear. To better understand the genotoxic effects of fractionated ionizing radiation, as well as the cellular recovery rate, we investigated the frequency of micronucleus (MN) formation in various types of human cells. We irradiated cells with fractionated X-ray doses of 2 Gy at a rate of 0.0635 Gy/min, separated into two to eight smaller doses. After irradiation, we investigated the frequency of MN formation. In addition, we investigated the rate of decrease in MN frequency after irradiation with 1 or 2 Gy X-rays at various recovery periods. Fractionated irradiation decreased MN frequency in a dose-dependent manner. When the total dose of X-rays was the same, the MN frequencies were lower after fractionated X-ray irradiation than acute irradiation in every cell type examined. The rate of MN decrease was faster in KMST-6 cells, which were derived from a human embryo, than in the other cells. The rate of MN decrease was higher in cells exposed to fractionated X-rays than in those exposed to acute irradiation. Recovery rates were very similar among cell lines, except in KMST-6 cells, which recovered more rapidly than other cell types.
Assuntos
Linhagem Celular/efeitos da radiação , Fracionamento da Dose de Radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Testes para Micronúcleos , Pré-Escolar , Dano ao DNA , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Células HeLa , Humanos , Lactente , Cinética , Doses de Radiação , Radiação Ionizante , Radiografia , Reprodutibilidade dos Testes , Raios XRESUMO
PURPOSE: To scrutinize the apoptotic and genotoxic effects of low-intensity ultrasound and an ultrasound contrast agent (SonoVue; Bracco Diagnostics Inc., EU) on human peripheral mononuclear blood cells (PMBCs). METHODS: PMBCs were subjected to a low-intensity ultrasound field (1-MHz frequency; spatial peak temporal average intensity 0.18 W/cm2) followed by analysis for apoptosis and DNA damage (single-strand breaks + double-strand breaks). The comet assay was then repeated after 2 h to examine the ability of cells to repair DNA breaks. RESULTS: The results demonstrated that low-intensity ultrasound was capable of selectively inducing apoptosis in leukemic PMBCs, but not in healthy cells. The introduction of ultrasound contrast agent SonoVue resulted in an increase in apoptosis in both groups. DNA analysis after ultrasound exposure indicated that ultrasound triggered DNA damage in leukemic PMBCs (66.05 ± 13.36%), while the damage was minimal (7.01 ± 0.89%) in control PMBCs. However, both cell lines demonstrated an ability to repair DNA single- and double-strand breaks 2 h after sonication. CONCLUSIONS: The study demonstrated that low-intensity ultrasound selectively induced apoptosis in cancer PMBCs. Ultrasound-induced DNA damage was observed primarily in leukemic PMBCs. Nevertheless, both cell lines were able to repair ultrasound-mediated DNA strand breaks.
Assuntos
Apoptose/efeitos da radiação , Dano ao DNA/efeitos da radiação , Leucemia/patologia , Leucócitos Mononucleares/efeitos da radiação , Ondas Ultrassônicas , Adulto , Linhagem Celular/efeitos da radiação , Linhagem Celular Tumoral/efeitos da radiação , Ensaio Cometa , Meios de Contraste/administração & dosagem , Voluntários Saudáveis , Humanos , Masculino , Fosfolipídeos/administração & dosagem , Hexafluoreto de Enxofre/administração & dosagemRESUMO
BACKGROUND AND OBJECTIVE: Low-level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low-level ultrahigh-frequency (UHF) and ultrashort-pulse (USP) blue laser irradiation on osteoblasts. STUDY DESIGN/ MATERIALS AND METHODS: The MC3T3-E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF-USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), and osteopontin (Opn) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI-induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. RESULTS: LLLI with the 405 nm UHF-USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non-irradiated control and LLLI using continuous-wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose-dependent manner up to 9.4 J/cm2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post-irradiation revealed no differences between the LLLI and control groups; however, promotion of cell proliferation and ALP activity by LLLI was significantly inhibited by CPZ. CONCLUSION: LLLI with a 405 nm UHF-USP blue laser enhances extracellular calcification of osteoblasts by upregulating proliferation and differentiation via TRPV1. Lasers Surg. Med. 50:340-352, 2018. © 2017 Wiley Periodicals, Inc.
Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Osteoblastos/fisiologia , Osteoblastos/efeitos da radiação , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/efeitos da radiação , Fosfatase Alcalina/metabolismo , Animais , Calcinose/fisiopatologia , Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Lasers de Corante/uso terapêutico , Camundongos , Osteopontina/metabolismo , Osteopontina/efeitos da radiação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Regulação para CimaRESUMO
Clitocybin A, an isoindolinone from Clitocybe aurantiaca, was investigated to assess its anti-wrinkle properties, through reactive oxygen species (ROS)-scavenging and elastase inhibitory activities, procollagen synthesis, and matrix metalloproteinase-1 (MMP-1) expression, in human primary dermal fibroblast-neonatal (HDF-N) cells. Clitocybin A exhibited no significant cytotoxicity up to 10 ppm in HDF-N cells, with cell viability and cell proliferation activity greater than 94.6% and 91.9%, respectively. Strong and concentration-dependent ROS radical scavenging activities of clitocybin A were observed following irradiation with UVB at 30 mJ/cm2. Furthermore, clitocybin A treatment of cells at 0.1, 1, and 10 ppm exhibited decreased elastase activity, in a concentration-dependent manner, by 1.97%, 6.6%, and 8.31%, respectively, versus the control group. The effects of clitocybin A on procollagen synthesis and MMP-1 expression were investigated. Clitocybin A treatment of cells at 1, 5, and 10 ppm increased procollagen synthesis, by 67.9%, 74.4%, and 112.9%, respectively, versus the control group. At these concentrations, MMP-1 expression decreased significantly following UV irradiation. Together, these findings suggest that clitocybin A may be an effective ingredient for use in anti-wrinkle cosmetic products.
Assuntos
Agaricales/química , Sequestradores de Radicais Livres/farmacologia , Isoindóis/antagonistas & inibidores , Micélio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Isoindóis/administração & dosagem , Isoindóis/química , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 1 da Matriz/efeitos da radiação , Elastase Pancreática/efeitos dos fármacos , Elastase Pancreática/metabolismo , Pró-Colágeno/antagonistas & inibidores , Pró-Colágeno/biossíntese , Pró-Colágeno/efeitos da radiação , Espécies Reativas de Oxigênio/efeitos da radiação , Espalhamento de Radiação , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Raios UltravioletaRESUMO
OBJECTIVE: To investigate the changes of proliferative activity and reactive oxygen species level of human epidermal cell line HaCaT after being irradiated with low-energy 633 nm red light. METHODS: Irradiation distance was determined through preliminary experiment. HaCaT cells were conventionally sub-cultured with RPMI 1640 culture medium containing 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells of the third passage were used in the following experiments. (1) Cells were divided into blank control group and 0.082, 0.164, 0.245, 0.491, 1.472, 2.453, 4.910, and 9.810 J/cm(2) irradiation groups according to the random number table, with 3 wells in each group. Cells in blank control group were not irradiated, while cells in the latter 8 irradiation groups were irradiated with 633 nm red light for 10, 20, 30, 60, 180, 300, 600, and 1 200 s in turn. Cells were reirradiated once every 8 hours. After being irradiated for 48 hours (6 times) in irradiation groups, the proliferative activity of cells in 9 groups was determined with cell counting kit 8 and microplate reader (denoted as absorbance value). (2) Another batch of cells were grouped and irradiated as in experiment (1). After being irradiated for once in irradiation groups, cells in 9 groups were conventionally cultured for 60 min with detection reagent of reactive oxygen species. At post culture minute (PCM) 0 (immediately), 30, 60, and 120, reactive oxygen species level of cells was determined with microplate reader (denoted as absorbance value). (3) Another batch of cells were divided into blank control group, 0.082, 0.491, 2.453, and 9.810 J/cm(2) irradiation groups, and positive control group. Cells in blank control group and positive control group were not irradiated (positive control reagent of reactive oxygen species was added to cells in positive control group), and cells in irradiation groups were irradiated as in experiment (1) for once. The expression of reactive oxygen species in cells of each group was observed by confocal laser scanning microscope. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, and t test. RESULTS: (1) Irradiation distance was 10 cm. Proliferative activity of cells in blank control group and 0.082, 0.164, 0.245, 0.491, 1.472, 2.453, 4.910, and 9.810 J/cm(2) irradiation groups was 1.000, 1.116±0.031, 1.146±0.016, 1.162±0.041, 1.179±0.016, 1.207±0.016, 1.247±0.040, 1.097±0.059, and 0.951±0.118, respectively. Compared with that in blank control group, proliferative activity of cells in 0.082-2.453 J/cm(2) irradiation groups was significantly higher (with t values from -22.803 to -6.779, P values below 0.05). Proliferative activity of cells in 4.910 and 9.810 J/cm(2) irradiation groups was similar to that in blank control group (with t values respectively -2.854 and 0.711, P values above 0.05). (2) Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 0 and 30 in 0.164-2.453 J/cm(2) irradiation groups (with t values from -12.453 to -4.684, P<0.05 or P<0.01), while that showed no significant change in 0.082, 4.910, and 9.810 J/cm(2) irradiation groups (with t values from -3.925 to -0.672, P values above 0.05). Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 60 in 0.082-2.453 J/cm(2) irradiation groups (with t values from -11.387 to -4.717, P<0.05 or P<0.01). Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 120 in 0.491-2.453 J/cm(2) irradiation groups (with t values from -10.657 to -6.644, P<0.05 or P<0.01). (3) Compared with that in blank control group, the expression of reactive oxygen species of cells was increased in 0.082, 0.491, and 2.453 J/cm(2) irradiation groups and positive control group. The expression of reactive oxygen species of cells in 9.810 J/cm(2) irradiation group was attenuated when compared with the expressions in the other irradiation groups. Reactive oxygen species expressed in mitochondria of cells in each group. CONCLUSIONS: Low-energy 633 nm red light can enhance the proliferation of human epidermal cell line HaCaT, and the effect is closely related to the increase of reactive oxygen species produced by mitochondria after being stimulated by red light irradiation.
Assuntos
Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Epiderme/efeitos da radiação , Raios Infravermelhos , Espécies Reativas de Oxigênio , Proliferação de Células/fisiologia , Meios de Cultura , Humanos , Distribuição AleatóriaRESUMO
Human corneal epithelial (HCE-T) and human lens epithelial (SRA01/04) cells derived from the human eye were exposed to 60 gigahertz (GHz) millimeter-wavelength radiation for 24 h. There was no statistically significant increase in the micronucleus (MN) frequency in cells exposed to 60 GHz millimeter-wavelength radiation at 1 mW/cm² compared with sham-exposed controls and incubator controls. The MN frequency of cells treated with bleomycin for 1 h provided positive controls. The comet assay, used to detect DNA strand breaks, and heat shock protein (Hsp) expression also showed no statistically significant effects of exposure. These results indicate that exposure to millimeter-wavelength radiation has no effect on genotoxicity in human eye cells.
Assuntos
Linhagem Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Olho , Proteínas de Choque Térmico/metabolismo , Exposição à Radiação/efeitos adversos , Ondas de Rádio/efeitos adversos , Bleomicina , Ensaio Cometa , Humanos , Cristalino , Micro-OndasRESUMO
DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm-6-MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild-type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non-catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non-catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Benzo(a)pireno/toxicidade , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA/genética , Teste de Complementação Genética , Humanos , Peróxido de Hidrogênio/toxicidade , Metanossulfonato de Metila/toxicidade , Mitomicina/toxicidade , Mutagênicos/química , Mutagênicos/toxicidade , Raios Ultravioleta , Vitamina K 3/toxicidadeRESUMO
Ginsenosides, also known as ginseng saponins, are the principal bioactive ingredients of ginseng, which are responsible for its diverse pharmacological activities. The present work aimed to assess skin anti-photoaging properties of ginsenoside Rb2 (Rb2), one of the predominant protopanaxadiol-type ginsenosides, in human epidermal keratinocyte HaCaT cells under UV-B irradiation. When the cultured keratinocytes were subjected to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced reactive oxygen species elevation and matrix metalloproteinase-2 expression and secretion. However, Rb2 at the used concentrations was unable to modulate cellular survivals in the UV-B-irradiated keratinocytes. In brief, Rb2 possesses a protective role against the photoaging of human keratinocyte cells under UV-B irradiation.
Assuntos
Ginsenosídeos/farmacologia , Queratinócitos/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ginsenosídeos/química , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Raios UltravioletaRESUMO
Low-dose hyper-radiosensitivity (HRS) has been reported in normal human lymphoblastoid cell lines for exposures at ≤ 20 cGy, but the cytogenetic effects of oxygen (O2 ) levels in tissue culture medium on HRS have not been evaluated. We asked whether HRS was lost in G2-irradiated cells grown in atmospheres of 2.5% or 5% O2 , compared to responses by cells cultured in ambient O2 (21%). The results indicate a loss of HRS when cells are cultured and irradiated either in 2.5% or 5% O2 . We then evaluated whether low O2 levels either before or after exposure were responsible for the loss of HRS. For cells irradiated in 5% O2 , subsequent immediate re-oxygenation to ambient O2 levels restored the HRS effect, while cells cultured and irradiated at ambient O2 levels and then transferred to 5% O2 exhibited little or no HRS, indicating that ambient O2 levels after, but not before, radiation substantially affect the amounts of cytogenetic damage. HRS was not observed when cells were irradiated in G1. At doses of 40-400 cGy there was significantly less cytogenetic damage when cells were recovering from radiation at low O2 levels than at ambient O2 levels. Here we provide the first cytogenetic evidence for the loss of HRS at low O2 levels in G2-irradiated cells; these results suggest that at low O2 levels for all doses evaluated there is either less damage to DNA, perhaps because of lower amounts of reactive oxygen species, or that DNA damage repair pathways are activated more efficiently.
Assuntos
Relação Dose-Resposta à Radiação , Fase G2/efeitos da radiação , Testes para Micronúcleos/métodos , Linhagem Celular/efeitos da radiação , Meios de Cultivo Condicionados/química , Fase G1/genética , Fase G1/efeitos da radiação , Fase G2/efeitos dos fármacos , Fase G2/genética , Raios gama , Humanos , Modelos Lineares , Oxigênio/metabolismo , Oxigênio/farmacologiaRESUMO
Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800 MHz, strength of 30 V/m on oxidation-reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60 min, specific absorption rate was calculated to be 1.6 W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2',7'-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p < 0.05) increased after 10 min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation-reduction equilibrium within the growing cells.
Assuntos
Linhagem Celular/efeitos da radiação , Oxirredução/efeitos da radiação , Ondas de Rádio/efeitos adversos , Animais , Sobrevivência Celular , Colorimetria , Cricetinae , Radiação Eletromagnética , Ensaio de Imunoadsorção Enzimática , Proteínas/química , Espécies Reativas de Oxigênio/químicaRESUMO
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.
Assuntos
Reparo do DNA , Proteína de Replicação A/metabolismo , Ciclo Celular , Linhagem Celular/efeitos da radiação , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA/efeitos da radiação , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Raios UltravioletaRESUMO
RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Galinhas , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Hidroxiureia/farmacologia , Dados de Sequência Molecular , Proteínas de Ligação a RNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Radiação Ionizante , Vertebrados/genéticaRESUMO
This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Técnicas In Vitro , Diferenciação Celular/efeitos da radiação , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Células-Tronco Embrionárias/efeitos da radiação , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Peróxido de Hidrogênio/toxicidade , Mitomicina/toxicidade , Testes de MutagenicidadeRESUMO
Relative Biological Effectiveness (RBE) values are used to characterise the biological efficiency of different radiation qualities relative to photon irradiations. The RBE-high linear energy transfer (LET) relation for ion irradiations presents general features that the authors propose to look at using a nanometric description of the energy deposition of these ion irradiations (protons and alphas of different energies). In this work, the simulation of the energy transfer points in the tracks was made by Monte Carlo method using the Geant4-DNA processes and a nanometric description of the target of interest for studying biological effects, the DNA molecule. Results were obtained concerning the sensitive volume to be considered for direct DNA clustered damages that could be related to late biological effects.