RESUMO
Towards improving the efficacy of radiotherapy, one approach is to target the molecules and processes mediating cellular radioresponse. Along these lines, translational control of gene expression has been established as a fundamental component of cellular radioresponse, which suggests that the molecules participating in this process (i.e., the translational machinery) can serve as determinants of radiosensitivity. Moreover, the proteins comprising the translational machinery are often overexpressed in tumor cells suggesting the potential for tumor specific radiosensitization. Studies to date have shown that inhibiting proteins involved in translation initiation, the rate-limiting step in translation, specifically the three members of the eIF4F cap binding complex eIF4E, eIF4G, and eIF4A as well as the cap binding regulatory kinases mTOR and Mnk1/2, results in the radiosensitization of tumor cells. Because ribosomes are required for translation initiation, inhibiting ribosome biogenesis also appears to be a strategy for radiosensitization. In general, the radiosensitization induced by targeting the translation initiation machinery involves inhibition of DNA repair, which appears to be the consequence of a reduced expression of proteins critical to radioresponse. The availability of clinically relevant inhibitors of this component of the translational machinery suggests opportunities to extend this approach to radiosensitization to patient care.
Assuntos
Biomarcadores Tumorais , Neoplasias/genética , Iniciação Traducional da Cadeia Peptídica/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Tolerância a Radiação/genética , Animais , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo , Neoplasias/radioterapia , Processamento de Proteína Pós-Traducional , Radioterapia , Ribossomos/metabolismo , Transdução de SinaisRESUMO
DNA damage causes cancer, impairs development and accelerates aging. Transcription-blocking lesions and transcription-coupled repair defects lead to developmental failure and premature aging in humans. Following DNA repair, homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that, in Caenorhabditis elegans, removal of the WRAD complex of the MLL/COMPASS H3K4 methyltransferase exacerbates developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylases SPR-5 and AMX-1 promotes developmental growth and extends lifespan amid ultraviolet-induced damage. We demonstrate that DNA-damage-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis and protein homeostasis and regulates the recovery of protein biosynthesis that ensures survival following genotoxic stress. Our study uncovers a role for H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis required for developmental growth and longevity after DNA damage.
Assuntos
Caenorhabditis elegans/genética , Reparo do DNA , DNA de Helmintos/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Tamanho da Ninhada/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , DNA de Helmintos/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Homeostase/efeitos da radiação , Longevidade/efeitos da radiação , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Biossíntese de Proteínas/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Raios UltravioletaRESUMO
Non-melanoma skin cancer (NMSC) is the most common form of cancer. Ultraviolet-B (UVB) radiation has been shown to be a complete carcinogen in the development of NMSC. The mammalian target of rapamycin complex 1 (mTORC1) is upregulated by UVB. Ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, is also upregulated in response to UVB. However, the interplay between these two pathways after UVB exposure remains unclear. The studies described here compare mRNA stability between normal human keratinocytes (HaCaT cells) and HaCaT cells with low levels of raptor to investigate whether the induction of ODC by UVB is dependent on mTORC1. We show that the knockdown of mTORC1 activity led to decreased levels of ODC protein both before and after exposure to 20 mJ/cm2 UVB. ODC mRNA was less stable in cells with decreased mTORC1 activity. Polysome profiles revealed that the initiation of ODC mRNA translation did not change in UVB-treated cells. We have shown that the ODC transcript is stabilized by the RNA-binding protein human antigen R (HuR). To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability in human keratinocytes exposed to UVB. We show an increased cytoplasmic localization of HuR after UVB exposure in wild-type cells. The ablation of HuR via CRISPR/Cas9 did not alter the stability of the ODC message, suggesting the involvement of other trans-acting factors. These data suggest that in human keratinocytes, ODC mRNA stability is regulated, in part, by an mTORC1-dependent mechanism after UVB exposure.
Assuntos
Queratinócitos/enzimologia , Queratinócitos/efeitos da radiação , Ornitina Descarboxilase/metabolismo , Proteína Regulatória Associada a mTOR/genética , Linhagem Celular , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ornitina Descarboxilase/genética , Biossíntese de Proteínas/efeitos da radiação , Estabilidade de RNA/efeitos da radiação , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Raios UltravioletaRESUMO
Intracellular processes can be localized for efficiency or regulation. For example, localized mRNA translation by chloroplastic ribosomes occurs in the biogenesis of PSII, one of the two photosystems of the photosynthetic electron transport chain in the chloroplasts of plants and algae. The biogenesis of PSI and PSII requires the synthesis and assembly of their constituent polypeptide subunits, pigments, and cofactors. Although these biosynthetic pathways are well characterized, less is known about when and where they occur in developing chloroplasts. Here, we used fluorescence microscopy in the unicellular alga Chlamydomonas reinhardtii to reveal spatiotemporal organization in photosystem biogenesis. We focused on translation by chloroplastic ribosomes and chlorophyll biosynthesis in two developmental contexts of active photosystem biogenesis: (1) growth of the mature chloroplast and (2) greening of a nonphotosynthetic chloroplast. The results reveal that a translation zone is the primary location of the biogenesis of PSI and PSII. This discretely localized region within the chloroplast contrasts with the distributions of photosystems throughout this organelle and, therefore, is likely a hub where anabolic pathways converge for photosystem biogenesis.plantcell;31/12/3057/FX1F1fx1.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Chlamydomonas/metabolismo , Cloroplastos/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Chlamydomonas/genética , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/genética , Clorofila/biossíntese , Cloroplastos/efeitos da radiação , Mitose/genética , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/efeitos da radiação , RNA Mensageiro/genética , Tilacoides/metabolismoRESUMO
Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.
Assuntos
Acetilcarnitina/urina , Síndrome Aguda da Radiação/diagnóstico , Carnitina/urina , Hipoxantina/sangue , Metabolômica/métodos , Taurina/sangue , Irradiação Corporal Total/métodos , Acetilcarnitina/sangue , Síndrome Aguda da Radiação/sangue , Síndrome Aguda da Radiação/patologia , Síndrome Aguda da Radiação/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Carnitina/sangue , Cromatografia Líquida , Citrulina/sangue , Citrulina/urina , Metabolismo Energético/genética , Metabolismo Energético/efeitos da radiação , Ácidos Graxos/sangue , Ácidos Graxos/urina , Feminino , Hipoxantina/urina , Macaca mulatta , Masculino , Espectrometria de Massas , Metaboloma/genética , Metaboloma/efeitos da radiação , Prolina/sangue , Prolina/urina , Biossíntese de Proteínas/efeitos da radiação , Curva ROC , Taurina/urinaRESUMO
Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.
Assuntos
Células Artificiais/metabolismo , Fotossíntese , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , Células Artificiais/efeitos dos fármacos , Metabolismo Energético/efeitos da radiação , Luz , Organelas/metabolismo , Organelas/efeitos da radiação , Fotossíntese/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Lipossomas Unilamelares/metabolismoRESUMO
The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.
Assuntos
Relógios Circadianos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Sistemas CRISPR-Cas , Relógios Circadianos/genética , Relógios Circadianos/efeitos da radiação , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos da radiação , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos da radiação , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Elementos de Resposta/genética , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiaçãoRESUMO
CD40L/interleukin-4 (IL-4) stimulation occurs in vivo in the tumor microenvironment and induces global translation to varying degrees in individuals with chronic lymphocytic leukemia (CLL) in vitro. However, the implications of CD40L/IL-4 for the translation of specific genes is not known. To determine the most highly translationally regulated genes in response to CD40L/IL-4, we carried out ribosome profiling, a next-generation sequencing method. Significant differences in the translational efficiency of DNA damage response genes, specifically ataxia-telangiectasia-mutated kinase (ATM) and the MRE11/RAD50/NBN (MRN) complex, were observed between patients, suggesting different patterns of translational regulation. We confirmed associations between CD40L/IL-4 response and baseline ATM levels, induction of ATM, and phosphorylation of the ATM targets, p53 and H2AX. X-irradiation was used to demonstrate that CD40L/IL-4 stimulation tended to improve DNA damage repair. Baseline ATM levels, independent of the presence of 11q deletion, correlated with overall survival (OS). Overall, we suggest that there are individual differences in translation of specific genes, including ATM, in response to CD40L/IL-4 and that these interpatient differences might be clinically important.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Ligante de CD40/imunologia , Dano ao DNA , Interleucina-4/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Biossíntese de Proteínas/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ligante de CD40/genética , Feminino , Raios gama , Histonas/genética , Histonas/imunologia , Humanos , Interleucina-4/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/efeitos da radiação , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
The effects of the high-dose ionizing radiation used in radiotherapy have been thoroughly demonstrated in vitro and in vivo. However, the effects of low-dose ionizing radiation (LDIR) such as computed tomography-guided biopsies and X-ray fluoroscopy on skin cells remain controversial. This study investigated the molecular effects of LDIR on the human primary keratinocytes (HPKs) and U937 cells, monocytes-like cell lines. These cells were exposed to 0.1 Gray (Gy) X-ray as LDIR. The modulation of transcription was assessed using a cDNA array, and the protein expression after LDIR exposure was investigated using isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis at 24 hours. These effects were confirmed by immunoblotting analysis. The direct effects of LDIR on the U937 cells and HPKs and the bystander effects of irradiated HPKs on U937 cells were also investigated. LDIR downregulated c-Myc in both U937 cells and HPKs, and upregulated the p21WAF1/CIP1 protein expression in U937 cells along with the activation of TGFß and protein phosphatase 2A (PP2A). In HPKs, LDIR downregulated the mTOR signaling with repression of S6 and 4EBP1 activation. Similar changes were observed as bystander effects of LDIR. Our findings suggest that LDIR inhibits protein synthesis and induces the cytokines activation associated with inflammation via direct and bystander effects, which might recapitulate the effects of LDIR in inflammated skin structures.
Assuntos
Ciclo Celular/efeitos da radiação , Queratinócitos/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Células U937/efeitos da radiação , Raios X/efeitos adversos , Expressão Gênica/efeitos da radiação , Humanos , Immunoblotting , Queratinócitos/metabolismo , Espectrometria de Massas , Redes e Vias Metabólicas/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica , Células U937/metabolismoRESUMO
In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.
Assuntos
Síndrome Aguda da Radiação/genética , Regulação da Expressão Gênica/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Síndrome Aguda da Radiação/fisiopatologia , Adulto , Animais , Relação Dose-Resposta à Radiação , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Papio/genética , Biossíntese de Proteínas/genética , Triagem , Irradiação Corporal TotalRESUMO
Objectives: 4E-BP1 is a family member of eIF4E binding proteins (4E-BPs) which act as the suppressors of cap-dependent translation of RNA via competitively associating with cap-bound eIF4E. RNA translation regulation is an important manner to control the cellular responses to a series of stress conditions such as ionizing radiation (IR)-induced DNA damage response and cell cycle controlling. This study aimed to determine the mechanism of 4E-BP1 stabilization and its potential downstream target(s) in the response to IR. Methods: PI3Ks kinase inhibitors were used to determine the signaling control of 4E-BP1 phosphorylation and protein stability. shRNA strategy was employed to silence the expression of 4E-BP1 in HeLa and HepG2 cells, and determine its effect on the irradiation-induced CHK2 phosphorylation. The protein degradation/stability was investigated by western blotting on the condition of blocking novel protein synthesis by cycloheximide (CHX). Results: The phosphorylation of 4E-BP1 at Thr37/46 was significantly increased in both HepG2 and HeLa cells by ionizing radiation. Depression of 4E-BP1 by shRNA strategy resulted in an incomplete G2 arrest at the early stage of 2 hours post-irradiation, as well as a higher accumulation of mitotic cells at 10 and 12 hours post-irradiation as compared to the control cells. Consistently, the CHK2 phosphorylation at Thr68 induced by IR was also attenuated by silencing 4E-BP1 expression. Both PI3K and DNA-PKcs kinase inhibitors significantly decreased the protein level of 4E-BP1, which was associated with the accelerated degradation mediated by ubiquitination-proteasome pathway. Conclusion: PI3K kinase activity is necessary for maintaining 4E-BP1 stability. Our results also suggest 4E-BP1 a novel biological role of regulating cell cycle G2 checkpoint in responding to IR stress in association with controlling CHK2 phosphorylation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Quinase do Ponto de Checagem 2/genética , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/genética , Biossíntese de Proteínas/genética , Proteínas de Ciclo Celular , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Células HeLa , Células Hep G2 , Humanos , Fosforilação/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Radiação Ionizante , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiaçãoRESUMO
Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns and are indispensable for antibacterial and antiviral immunity. Our previous report showed that ionizing radiation increases the cell surface expressions of TLR2 and TLR4 and enhances their responses to agonists in human monocytic THP1 cells. The present study investigated how ionizing radiation increases the cell surface expressions of TLR2 and TLR4 in THP1 cells. The THP1 cells treated or not treated with pharmaceutical agents such as cycloheximide and N-acetyl-L-cysteine (NAC) were exposed to X-ray irradiation, following which the expressions of TLRs and mitogen-activated protein kinase were analyzed. X-ray irradiation increased the mRNA expressions of TLR2 and TLR4, and treatment with a protein synthesis inhibitor cycloheximide abolished the radiation-induced upregulation of their cell surface expressions. These results indicate that radiation increased those receptors through de novo protein synthesis. Furthermore, treatment with an antioxidant NAC suppressed not only the radiation-induced upregulation of cell surface expressions of TLR2 and TLR4, but also the radiation-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Since it has been shown that the inhibitor for JNK can suppress the radiation-induced upregulation of TLR expression, the present results suggest that ionizing radiation increased the cell surface expressions of TLR2 and TLR4 through reactive oxygen species-mediated JNK activation.
Assuntos
Membrana Celular/metabolismo , Monócitos/metabolismo , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/efeitos da radiação , Acetilcisteína/farmacologia , Linhagem Celular , Membrana Celular/efeitos da radiação , Ceramidas/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Monócitos/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiaçãoRESUMO
The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure.
Assuntos
Morte Celular/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Luz/efeitos adversos , Células Fotorreceptoras/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Morte Celular/efeitos da radiação , Linhagem Celular Transformada , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/efeitos da radiação , Citosol/efeitos dos fármacos , Citosol/efeitos da radiação , Células Ependimogliais/citologia , Células Ependimogliais/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Técnicas In Vitro , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição NF-E2/genética , Fator de Transcrição NF-E2/metabolismo , Células Fotorreceptoras/efeitos da radiação , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Retina/citologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/prevenção & controle , Fatores de Tempo , Triterpenos/químicaRESUMO
The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Biossíntese de Proteínas/efeitos da radiação , Proteoma/efeitos da radiação , Proteômica , Animais , Linhagem Celular , Telefone Celular , Humanos , Camundongos , Transcriptoma/efeitos da radiação , Tecnologia sem FioRESUMO
BACKGROUND: Skin hyperpigmentary disorders including postinflammatory hyperpigmentation, melasma, solar lentigines, and conditions like freckles are common. The light-emitting diodes (LEDs) are the latest category of nonthermal and noninvasive phototherapy to be considered in skin pigmentation disorder treatment. PURPOSE: The purpose of this study was to investigate the effects of 660-nm LED on inhibition of melanogenesis. We investigated whether a 660-nm LED affected melanin synthesis in in vitro and in vivo models, and we explored the mechanisms involved. METHODS: The inhibitory effect of 660-nm LED on melanin synthesis was evaluated in B16F10 cells and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of 660-nm LED. RESULTS: Interestingly, 660-nm LED inhibited alpha-melanocyte-stimulating hormone-induced tyrosinase activity in B16F10 cells. We also found that 660-nm LED decreased MITF and tyrosinase expression and induced the activation of ERK. These findings suggest that the depigmenting effects of 660-nm LED result from downregulation of MITF and tyrosinase expression due to increased ERK activity. The 660-nm LED reduced UVB-induced melanogenesis in the skin of HRM-2 via downregulation of tyrosinase and MITF. CONCLUSION: These findings suggest 660-nm LED is a potentially depigmentation strategy.
Assuntos
Luz , Melaninas/biossíntese , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Biossíntese de Proteínas/efeitos da radiação , Pele/efeitos da radiação , Animais , Linhagem Celular Tumoral , Oxirredutases Intramoleculares/metabolismo , Antígeno MART-1/análise , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Pelados , Fator de Transcrição Associado à Microftalmia/análise , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Monofenol Mono-Oxigenase/análise , Oxirredutases/metabolismo , Proteínas S100/análise , Pele/química , alfa-MSH/metabolismoRESUMO
BACKGROUND: Skin fibrosis is a debilitating condition that significantly impacts patient quality of life. Ultraviolet phototherapy is currently used to treat several diseases featuring skin fibrosis. High-fluence light-emitting diode-generated red light (HF-LED-RL) does not cause DNA damage associated with skin cancer, and it is generally regarded as safe, portable, and cost-effective. Early clinical observations suggest that LED-generated light may possess antifibrotic effects, although these findings are largely unexplored. Previously published research demonstrated that HF-LED-RL decreases fibroblast proliferation and collagen in vitro. OBJECTIVE: The goal of this study was to compare the combination effects of HF-LED-RL alone with HF-LED-RL in combination with resveratrol. MATERIALS AND METHODS: It is hypothesized that resveratrol, an active ingredient in red wine, a potent antioxidant scavenger of reactive oxygen species, and an inhibitor of collagen production, may synergistically decrease fibroblast proliferation and collagen production when combined with HF-LED-RL. RESULTS: In this study, evidence is provided that resveratrol combined with HF-LED-RL acts synergistically to decrease fibroblast proliferation and procollagen 1A1 production, and this represents a new potential therapeutic modality that is termed the "photobotanical" effect due to the combined light and botanical properties observed. CONCLUSION: The study, discovery, and use of photobotanical combinations may usher in new therapeutics or phototherapy adjuvants for the treatment of dermatologic diseases.
Assuntos
Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Colágeno Tipo I/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Estilbenos/farmacologia , Linhagem Celular , Cadeia alfa 1 do Colágeno Tipo I , Cor , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , ResveratrolRESUMO
The health benefits of natural sunlight have been noted since the rise of civilization, even without the knowledge of its mechanisms of action. Currently, phototherapy remains an effective and widely used treatment for a variety of skin diseases. Ultraviolet radiation, from either the sun or artificial light sources, has a profound immunomodulatory effect that is responsible for its beneficial clinical outcomes. Ultraviolet radiation mostly induces the innate while suppressing the adaptive immune system, leading to both local and systemic effects. It is antigen specific, acts on both effector and regulatory T cells, alters antigen-presenting cell function, and induces the secretion of cytokines and soluble mediators. This review provides an overview of the immunologic mechanisms by which ultraviolet radiation is responsible for the therapeutic effects of phototherapy.
Assuntos
Imunidade Adaptativa/efeitos da radiação , Sistema Imunitário/efeitos da radiação , Imunidade Inata/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Pele/efeitos da radiação , Raios Ultravioleta , Animais , Citocinas/biossíntese , Dano ao DNA/efeitos da radiação , Fenômenos Eletromagnéticos , Humanos , Tolerância Imunológica/efeitos da radiação , Receptores Toll-Like/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Terapia UltravioletaRESUMO
Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078-87. ©2016 AACR.
Assuntos
Glioblastoma/patologia , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/patologia , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos da radiação , Imunofluorescência , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/radioterapia , Complexo de Golgi/genética , Complexo de Golgi/efeitos da radiação , Humanos , Mitocôndrias/genética , Mitocôndrias/efeitos da radiação , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Polirribossomos/genética , Polirribossomos/efeitos da radiação , RNA Mensageiro/genética , Radiação Ionizante , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation.
Assuntos
Fibroblastos/efeitos da radiação , Proteoma/genética , Radiação Ionizante , Células-Tronco/efeitos da radiação , Linhagem Celular , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Humanos , Transferência Linear de Energia , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/efeitos da radiação , Proteoma/efeitos da radiação , Proteômica , Tolerância a Radiação/genética , Células-Tronco/metabolismoRESUMO
Plant NB-LRR proteins confer resistance to multiple pathogens, including viruses. Although the recognition of viruses by NB-LRR proteins is highly specific, previous studies have suggested that NB-LRR activation results in a response that targets all viruses in the infected cell. Using an inducible system to activate NB-LRR defenses, we find that NB-LRR signaling does not result in the degradation of viral transcripts, but rather prevents them from associating with ribosomes and translating their genetic material. This indicates that defense against viruses involves the repression of viral RNA translation. This repression is specific to viral transcripts and does not involve a global shutdown of host cell translation. As a consequence of the repression of viral RNA translation, NB-LRR responses induce a dramatic increase in the biogenesis of RNA processing bodies (PBs). We demonstrate that other pathways that induce translational repression, such as UV irradiation and RNAi, also induce PBs. However, by investigating the phosphorylation status of eIF2α and by using suppressors of RNAi we show that the mechanisms leading to PB induction by NB-LRR signaling are different from these stimuli, thus defining a distinct type of translational control and anti-viral mechanism in plants.