RESUMO
Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fitocromo B/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Modelos Genéticos , Fitocromo A/genética , Fitocromo A/metabolismo , Fitocromo B/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica/efeitos da radiação , Homologia de Sequência de Aminoácidos , Ativação Transcricional/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Recently, therapeutic effects of extremely low-frequency electromagnetic field (ELF-EMF) as complementary and alternative medicine, used in the oncology field to control disease symptoms. Micro RNAs (miRs) are responsible for the post-transcriptional regulation of gene expression in the cell. This study aimed to evaluate the expression changes of miR-144 and miR-375 in the human gastric adenocarcinoma cell line (AGS) under the exposure of ELF-EMF. MATERIALS AND METHODS: AGS cells were exposed to magnetic flux densities of 0.2 and 2 mT for 18 h, continuously and discontinuously (1.5 h on/1.5 h off). Cell viability was evaluated by MTT assay. Changes of miR-144 expression levels in AGS cells immediately after exposure and 18 and 36 h after the exposure cut-off was calculated by QRT-PCR. RESULTS: The cell viability of AGS cells was decreased under the exposure of 0.2 and 2 mT EMFs when compared to the control. Up-regulation of miR-144 and miR-375 were observed in AGS cells under the exposure of magnetic fields. CONCLUSIONS: The results indicated that the miR levels were significantly decreased 18 and 36 h after finishing the exposure, but not reached the normal range. The results of this investigation indicated that weak and moderate intermittent 50 Hz ELF-EMFs can induce changes in miRNA expression.
Assuntos
Campos Eletromagnéticos , MicroRNAs/genética , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Ativação Transcricional/efeitos da radiaçãoRESUMO
Quercetin 3-O-ß-D-glucuronide (Q-3-G), the glucuronide conjugate of quercetin, has been reported as having anti-inflammatory properties in the lipopolysaccharide-stimulated macrophages, as well as anticancer and antioxidant properties. Unlike quercetin, which has been extensively described to possess a wide range of pharmacological activities including skin protective effects, the pharmacological benefits and mechanisms Q-3-G in the skin remained to be elucidated. This study focused on characterizing the skin protective properties, including anti-inflammatory and antioxidant properties, of Q-3-G against UVB-induced or H2O2-induced oxidative stress, the hydration effects, and antimelanogenesis activities using human keratinocytes (HaCaT) and melanoma (B16F10) cells. Q-3-G down-regulated the expression of the pro-inflammatory gene and cytokine such as cyclooxygenase-2 (COX-2) and tumor necrosis factor (TNF)-α in H2O2 or UVB-irradiated HaCaT cells. We also showed that Q-3-G exhibits an antioxidant effect using free radical scavenging assays, flow cytometry, and an increased expression of nuclear factor erythroid 2- related factor 2 (Nrf2). Q-3-G reduced melanin production in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. The hydration effects and mechanisms of Q-3-G were examined by evaluating the moisturizing factor-related genes, such as transglutaminase-1 (TGM-1), filaggrin (FLG), and hyaluronic acid synthase (HAS)-1. In addition, Q-3-G increased the phosphorylation of c-Jun, Jun N-terminal kinase (JNK), Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4), and TAK1, involved in the MAPKs/AP-1 pathway, and the phosphorylation of IκBα, IκB kinase (IKK)-α, Akt, and Src, involved in the NF-κB pathway. Taken together, we have demonstrated that Q-3-G exerts anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis properties in human keratinocytes and melanoma cells through NF-κB and AP-1 pathways.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Queratinócitos/patologia , Melaninas/metabolismo , Melanoma Experimental/patologia , NF-kappa B/metabolismo , Quercetina/análogos & derivados , Fator de Transcrição AP-1/metabolismo , Células HEK293 , Células HaCaT , Humanos , Peróxido de Hidrogênio/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Modelos Biológicos , Quercetina/química , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiação , Raios UltravioletaRESUMO
Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
Assuntos
Luz , Anticorpos de Cadeia Única/metabolismo , Animais , Calorimetria , Deinococcus/metabolismo , Dimerização , Células HEK293 , Humanos , Interferometria , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Fitocromo/química , Plasmídeos/genética , Plasmídeos/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Ativação Transcricional/efeitos da radiaçãoRESUMO
BACKGROUND: Radiation-induced skin injury is one of the main adverse effects and a dose-limiting factor of radiotherapy without feasible treatment. The underlying mechanism of this disease is still limited. OBJECTIVE: To investigate the potential molecular pathways and mechanisms of radiation-induced skin injury. METHODS: mRNA expression profiles were determined by Affymetrix Human HTA2.0 microarray.IFI6 overexpression and knockdown were mediated by lentivirus. The functional changes of skin cells were measured by flow cytometry, ROS probe and Edu probe. Protein distribution was detected by immunofluorescence experiment, and IFI6-interacting proteins were detected by immunoprecipitation (IP) combined with mass spectrometry. The global gene changes in IFI6-overexpressed skin cells after irradiation were detected by RNA-seq. RESULTS: mRNA expression profiling showed 50 upregulated and 13 down regulated genes and interferon alpha inducible protein 6 (IFI6) was top upregulated. Overexpression of IFI6 promoted cell proliferation and reduced cell apoptosis as well as ROS production following radiation, and conversely, increased the radiosensitivity of HaCaT and human skin fibroblast (WS1). IFI6 was translocated into nucleus in irradiated skin cells and the interacting relationship with mitochondrial single-stranded DNA-binding protein 1 (SSBP1), which could enhance the transcriptional activity of heat shock transcription factor 1 (HSF1).IFI6 augmented HSF1 activity following radiation in HaCaT and WS1 cells. RNA-seq analysis showed IFI6 modulated virus infection and cellular response to stress pathways, which may help to further explore how IFI6 regulate the transcriptional activity of HSF1. CONCLUSION: This study reveals that IFI6 is induced by ionizing radiation and confers radioprotection in skin cells.
Assuntos
Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas Mitocondriais/metabolismo , Tolerância a Radiação/genética , Radiodermite/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistência à Doença/genética , Técnicas de Silenciamento de Genes , Células HaCaT , Humanos , Proteínas Mitocondriais/genética , RNA-Seq , Radiodermite/patologia , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Pele/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
Embryonic development is particularly vulnerable to stress and DNA damage, as mutations can accumulate through cell proliferation in a wide number of cells and organs. However, the biological effects of chronic exposure to ionising radiation (IR) at low and moderate dose rates (< 6 mGy/h) remain largely controversial, raising concerns for environmental protection. The present study focuses on the molecular effects of IR (0.005 to 50 mGy/h) on zebrafish embryos at the gastrula stage (6 hpf), at both the transcriptomics and epigenetics levels. Our results show that exposure to IR modifies the expression of genes involved in mitochondrial activity from 0.5 to 50 mGy/h. In addition, important developmental pathways, namely, the Notch, retinoic acid, BMP and Wnt signalling pathways, were altered at 5 and 50 mGy/h. Transcriptional changes of genes involved in the morphogenesis of the ectoderm and mesoderm were detected at all dose rates, but were prominent from 0.5 to 50 mGy/h. At the epigenetic level, exposure to IR induced a hypomethylation of DNA in the promoter of genes that colocalised with both H3K27me3 and H3Kme4 histone marks and correlated with changes in transcriptional activity. Finally, pathway enrichment analysis demonstrated that the DNA methylation changes occurred in the promoter of important developmental genes, including morphogenesis of the ectoderm and mesoderm. Together, these results show that the transcriptional program regulating morphogenesis in gastrulating embryos was modified at dose rates greater than or equal to 0.5 mGy/h, which might predict potential neurogenesis and somitogenesis defects observed at similar dose rates later in development.
Assuntos
Metilação de DNA/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Morfogênese/genética , Organogênese/genética , Regiões Promotoras Genéticas , Radiação Ionizante , Ativação Transcricional/efeitos da radiação , Peixe-Zebra/genética , Animais , Biologia Computacional/métodos , Ectoderma/embriologia , Ectoderma/metabolismo , Ectoderma/efeitos da radiação , Perfilação da Expressão Gênica , Mesoderma/embriologia , Mesoderma/metabolismo , Mesoderma/efeitos da radiação , Transcriptoma , Peixe-Zebra/embriologiaRESUMO
Nanosecond pulsed electric field (nsPEF) has emerged as a promising tool for hepatocellular carcinoma ablation recently. However, little is known about how nsPEF affects liver regeneration while being applied to eliminate liver lesions. Besides, the impact of nsPEF ablation on liver function should also be taken into consideration in the process. In this paper, we study the impact of nsPEF ablation on liver function by the measurement of serum levels of AST and ALT as well as liver regeneration and relevant molecular mechanisms in vivo. We found that mouse liver function exhibited a temporary injury without weight loss after ablation. In addition, local hepatic nsPEF ablation promoted significant proliferation of hepatocytes of the whole liver with an increase in HGF level. Moreover, the proliferation of hepatocytes was dramatically inhibited by the inhibitor of c-Met. Of interest, the periablational area is characterized by high level of PDGF and a large amount of activated hepatic stellate cells. Furthermore, neutralizing PDGF was able to significantly inhibit liver regeneration, the increased HGF level, and the accumulation of activated HSCs. Our findings demonstrated that nsPEF not only was a safe ablation approach but also could stimulate the regeneration of the whole liver through the activation of the HGF/c-Met pathway by upregulation of PDGF within the periablational zone.
Assuntos
Fator de Crescimento de Hepatócito/genética , Regeneração Hepática/genética , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-met/genética , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Radiação Eletromagnética , Regulação da Expressão Gênica/efeitos da radiação , Hepatócitos/efeitos da radiação , Humanos , Fígado/crescimento & desenvolvimento , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Regeneração Hepática/efeitos da radiação , Camundongos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Ativação Transcricional/efeitos da radiaçãoRESUMO
High energy laser, particularly 532 nm, is widely used in defense and medical applications and there is need to address its occupational safety. Thermal and non-thermal effects of 532 nm high energy laser on skin are cause of concern. This study indicates impact of 532 nm laser on rat skin and first of its kind of attempt to understand transcriptional activation of genes as an early response following laser exposure. Skin of experimental rats were exposed to 532 nm radiance at 0.1, 0.25 and 0.50 W/cm2 for 10 sec. Thermographic changes of skin exposed to 532 nm laser exhibited increased Tmax temperature in radiance dependent manner. After thermal imaging, skin of experimental rats was collected 1 h post laser exposure for studying differential gene expression. The skin exposed to lower power density (0.1 W/cm2) did not show significant changes in expression of gene pathways studied. At moderate radiance (0.25 W/cm2), predominantly canonical wnt/B-catenin pathway genes notch1, axin2, ccdn1, wnt5a and redox homeostasis genes; txn1, nqo1 and txnrd1 were expressed. At higher radiance (0.5 W/cm2), significant repression of genes related to wound healing process particularly notch/wnt pathway viz. hes5, wnt1, wn3b with higher expression of dab2 was recorded. The data obtained from these studies would help in drawing safety limits for skin exposure to 532 nm laser. Further, genes expressed at moderate and high level of radiance exposure to skin were distinct and differential and provide new avenue to configure pathway to counteract laser induced delay in tissue injury and hair follicular damage.
Assuntos
Lasers/efeitos adversos , Pele/efeitos da radiação , Fatores de Transcrição/genética , Transcrição Gênica/efeitos da radiação , Animais , Expressão Gênica/efeitos da radiação , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Via de Sinalização Wnt/efeitos da radiação , beta Catenina/genéticaRESUMO
The circadian clock provides a time-keeping mechanism that synchronizes various biological activities with the surrounding environment. Arabidopsis (Arabidopsis thaliana) CIRCADIAN CLOCK ASSOCIATED1 (CCA1), encoding a MYB-related transcription factor, is a key component of the core oscillator of the circadian clock, with peak expression in the morning. The molecular mechanisms regulating the light induction and rhythmic expression of CCA1 remain elusive. In this study, we show that two phytochrome signaling proteins, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its paralog FAR-RED IMPAIRED RESPONSE1 (FAR1), are essential for the light-induced expression of CCA1 FHY3 and FAR1 directly bind to the CCA1 promoter and activate its expression, whereas PHYTOCHROME INTERACTING FACTOR5 (PIF5) directly binds to its promoter and represses its expression. Furthermore, PIF5 and TIMING OF CAB EXPRESSION1 physically interact with FHY3 and FAR1 to repress their transcriptional activation activity on CCA1 expression. These findings demonstrate that the photosensory-signaling pathway integrates with circadian oscillators to orchestrate clock gene expression. This mechanism might form the molecular basis of the regulation of the clock system by light in response to daily changes in the light environment, thus increasing plant fitness.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Luz , Proteínas Nucleares/metabolismo , Fitocromo/metabolismo , Fatores de Transcrição/genética , Sequência de Bases , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Regiões Promotoras Genéticas , Ligação Proteica/efeitos da radiação , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiaçãoRESUMO
T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.
Assuntos
Imunoterapia Adotiva , Luz , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos da radiação , Animais , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Criptocromos/genética , Criptocromos/metabolismo , Citotoxicidade Imunológica/imunologia , Citotoxicidade Imunológica/efeitos da radiação , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Ativação Linfocitária/efeitos da radiação , Camundongos , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos Quiméricos/genética , Ativação Transcricional/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The endothelium contributes to the pathophysiology of adverse effects caused by conventional (genotoxic) anticancer therapeutics (cAT). The relevance of structurally different types of cAT-induced DNA lesions for eliciting selected endothelial stress responses is largely unknown. Here, we analyzed the cAT-induced formation of DNA double-strand breaks (DSB), transcription blockage and DNA damage response (DDR) in time kinetic analyses employing a monolayer of primary human endothelial cells (HUVEC). We observed that the degree of cAT-induced transcription blockage, the number of DSB and activation of DDR-related factors diverge. For instance, ionizing radiation caused the formation of numerous DSB and triggerd a substantial activation of ATM/Chk2 signaling, which however were not accompanied by a significant transcription inhibition. By contrast, the DNA cross-linking cAT cisplatin triggered a rapid and substantial blockage of transcription, which yet was not reflected by an appreciable number of DSB or increased levels of pATM/pChk2. In general, cAT-stimulated ATM-dependent phosphorylation of Kap1 (Ser824) and p53 (Ser15) reflected best cAT-induced transcription blockage. In conclusion, cAT-induced formation of DSB and profound activation of prototypical DDR factors is independent of the inhibition of RNA polymerase II-regulated transcription in an endothelial monolayer. We suggest that DSB formed directly or indirectly following cAT-treatment do not act as comprehensive triggers of superior signaling pathways shutting-down transcription while, at the same time, causing an appreciable stimulation of the DDR. Rather, it appears that distinct cAT-induced DNA lesions elicit diverging signaling pathways, which separately control transcription vs. DDR activity in the endothelium.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Cisplatino/farmacologia , Proteína 28 com Motivo Tripartido/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Cultura Primária de Células , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiaçãoRESUMO
Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.
Assuntos
Luz , Microscopia Confocal , Microscopia de Fluorescência , Optogenética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Ionizing radiation (IR) can promote migration and invasion of cancer cells, but the basis for this phenomenon has not been fully elucidated. IR increases expression of glucose-regulated protein 78kDa (GRP78) on the surface of cancer cells (CS-GRP78), and this up-regulation is associated with more aggressive behavior, radioresistance, and recurrence of cancer. Here, using various biochemical and immunological methods, including flow cytometry, cell proliferation and migration assays, Rho activation and quantitative RT-PCR assays, we investigated the mechanism by which CS-GRP78 contributes to radioresistance in pancreatic ductal adenocarcinoma (PDAC) cells. We found that activated α2-Macroglobulin (α2M*) a ligand of the CS-GRP78 receptor, induces formation of the AKT kinase (AKT)/DLC1 Rho-GTPase-activating protein (DLC1) complex and thereby increases Rho activation. Further, CS-GRP78 activated the transcriptional coactivators Yes-associated protein (YAP) and tafazzin (TAZ) in a Rho-dependent manner, promoting motility and invasiveness of PDAC cells. We observed that radiation-induced CS-GRP78 stimulates the nuclear accumulation of YAP/TAZ and increases YAP/TAZ target gene expressions. Remarkably, targeting CS-GRP78 with C38 monoclonal antibody (Mab) enhanced radiosensitivity and increased the efficacy of radiation therapy by curtailing PDAC cell motility and invasion. These findings reveal that CS-GRP78 acts upstream of YAP/TAZ signaling and promote migration and radiation-resistance in PDAC cells. We therefore conclude that, C38 Mab is a promising candidate for use in combination with radiation therapy to manage PDAC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/radioterapia , Proteínas de Choque Térmico/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Fatores de Transcrição/metabolismo , Aciltransferases , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pancreáticas/patologia , Tolerância a Radiação , Ativação Transcricional/efeitos da radiação , Proteínas de Sinalização YAPRESUMO
Chemotherapy and radiation are more effective in wild-type (WT) p53 tumors due to p53 activation. This is one rationale for developing drugs that reactivate mutant p53 to synergize with chemotherapy and radiation. Zinc metallochaperones (ZMC) are a new class of mutant p53 reactivators that restore WT structure and function to zinc-deficient p53 mutants. We hypothesized that the thiosemicarbazone, ZMC1, would synergize with chemotherapy and radiation. Surprisingly, this was not found. We explored the mechanism of this and found the reactive oxygen species (ROS) activity of ZMC1 negates the signal on p53 that is generated with chemotherapy and radiation. We hypothesized that a zinc scaffold generating less ROS would synergize with chemotherapy and radiation. The ROS effect of ZMC1 is generated by its chelation of redox active copper. ZMC1 copper binding (K Cu) studies reveal its affinity for copper is approximately 108 greater than Zn2+ We identified an alternative zinc scaffold (nitrilotriacetic acid) and synthesized derivatives to improve cell permeability. These compounds bind zinc in the same range as ZMC1 but bound copper much less avidly (106- to 107-fold lower) and induced less ROS. These compounds were synergistic with chemotherapy and radiation by inducing p53 signaling events on mutant p53. We explored other combinations with ZMC1 based on its mechanism of action and demonstrate that ZMC1 is synergistic with MDM2 antagonists, BCL2 antagonists, and molecules that deplete cellular reducing agents. We have identified an optimal Cu2+:Zn2+ binding ratio to facilitate development of ZMCs as chemotherapy and radiation sensitizers. Although ZMC1 is not synergistic with chemotherapy and radiation, it is synergistic with a number of other targeted agents.
Assuntos
Cobre/metabolismo , Metalochaperonas/metabolismo , Mutação , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Terapia Combinada , Humanos , Metalochaperonas/genética , Camundongos , Ligação Proteica , Piridinas/farmacologia , Radiação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
BACKGROUND/AIM: Epigallocatechin-3-gallate (EGCG) is a major polyphenolic component of green tea. EGCG plays a potential role in radio-sensitizing cancer cells. The combined effect of EGCG and radiation was investigated in a colorectal cancer cell line, focusing on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) autophagy signalling. MATERIALS AND METHODS: HCT-116 cells were treated with 12.5 µM EGCG for different periods of time, 2 Gy radiation, or both. Cell viability was determined with the WST-8 assay. The number of colonies was determined with the colony formation assay. mRNA expression of LC3 and caspase-9 was analyzed with quantitative real-time polymerase chain reaction. RESULTS: Combination treatment with EGCG and radiation significantly decreased the growth of HCT-116 cells. The number of colonies was reduced to 34.2% compared to the control group. Immunofluorescence microscopy images showed that nuclear translocation of Nrf2 was significantly increased when cells were treated with the combination of EGCG and radiation compared to the control and single-treatment groups. Combined treatment with EGCG and radiation significantly induced LC3 and caspase-9 mRNA expression. CONCLUSION: EGCG increased the sensitivity of colorectal cancer cells to radiation by inhibiting cell proliferation and inducing Nrf2 nuclear translocation and autophagy.
Assuntos
Catequina/análogos & derivados , Neoplasias Colorretais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Radiossensibilizantes/farmacologia , Caspase 9/genética , Catequina/farmacologia , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HCT116 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fator 2 Relacionado a NF-E2/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiaçãoRESUMO
Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch ('Blue-OFF'), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.
Assuntos
Optogenética/métodos , Proteínas Repressoras/genética , Ativação Transcricional/efeitos da radiação , Animais , Células CHO , Cricetulus , Regulação da Expressão Gênica/efeitos da radiação , Luz , Modelos Teóricos , Estimulação Luminosa , Estabilidade Proteica/efeitos da radiação , Proteólise/efeitos da radiaçãoRESUMO
This study aims to assess whether a 1950 MHz radiofrequency (RF) electromagnetic field could protect human neuroblastoma SH-SY5Y cells against a subsequent treatment with menadione, a chemical agent inducing DNA damage via reactive oxygen species formation. Cells were pre-exposed for 20 h to specific absorption rate of either 0.3 or 1.25 W/kg, and 3 h after the end of the exposure, they were treated with 10 µM menadione (MD) for 1 h. No differences were observed between sham- and RF-exposed samples. A statistically significant reduction in menadione-induced DNA damage was detected in cells pre-exposed to either 0.3 or 1.25 W/kg (P < 0.05). Moreover, our analyses of gene expression revealed that the pre-exposure to RF almost inhibited the dramatic loss of glutathione peroxidase-based antioxidant scavenging efficiency that was induced by MD, and in parallel strongly enhanced the gene expression of catalase-based antioxidant protection. In addition, RF abolished the MD-dependent down-regulation of oxoguanine DNA glycosylase, which is a critical DNA repairing enzyme. Overall, our findings suggested that RF pre-exposure reduced menadione-dependent DNA oxidative damage, most probably by enhancing antioxidant scavenging efficiency and restoring DNA repair capability. Our results provided some insights into the molecular mechanisms underlying the RF-induced adaptive response in human neuroblastoma cells challenged with menadione.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Terapia por Radiofrequência , Vitamina K 3/efeitos adversos , Linhagem Celular Tumoral , Campos Eletromagnéticos , Humanos , Neuroblastoma/etiologia , Neuroblastoma/genética , Neuroblastoma/terapia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fatores de Proteção , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiaçãoRESUMO
On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.
Assuntos
Optogenética/métodos , Transcrição Gênica/efeitos da radiação , Animais , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Luz , Engenharia de Proteínas/métodos , Proteínas/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Ativação Transcricional/efeitos da radiaçãoRESUMO
Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Proteínas/metabolismo , Saccharomyces/enzimologia , Saccharomyces/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Clonagem Molecular , Perfilação da Expressão Gênica , Microbiologia Industrial/métodos , Regiões Promotoras Genéticas , Proteínas/genética , Saccharomyces/genética , Saccharomyces/crescimento & desenvolvimento , TemperaturaRESUMO
Considerable data has been generated to elucidate the transcriptional response of cells to ultraviolet radiation (UVR) exposure providing a mechanistic understanding of UVR-induced cellular responses. However, using these data to support standards development has been challenging. In this study, we apply benchmark dose (BMD) modeling of transcriptional data to derive thresholds of gene responsiveness following exposure to solar-simulated UVR. Human epidermal keratinocytes were exposed to three doses (10, 20, 150 kJ/m2 ) of solar simulated UVR and assessed for gene expression changes 6 and 24 hr postexposure. The dose-response curves for genes with p-fit values (≥ 0.1) were used to derive BMD values for genes and pathways. Gene BMDs were bi-modally distributed, with a peak at â¼16 kJ/m2 and â¼108 kJ/m2 UVR exposure. Genes/pathways within Mode 1 were involved in cell signaling and DNA damage response, while genes/pathways in the higher Mode 2 were associated with immune response and cancer development. The median value of each Mode coincides with the current human exposure limits for UVR and for the minimal erythemal dose, respectively. Such concordance implies that the use of transcriptional BMD data may represent a promising new approach for deriving thresholds of actinic effects. Environ. Mol. Mutagen. 59:502-515, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.