RESUMO
Cancers from sun-exposed skin accumulate "driver" mutations, causally implicated in oncogenesis. Because errors incorporated during translesion synthesis (TLS) opposite UV lesions would generate these mutations, TLS mechanisms are presumed to underlie cancer development. To address the role of TLS in skin cancer formation, we determined which DNA polymerase is responsible for generating UV mutations, analyzed the relative contributions of error-free TLS by Polη and error-prone TLS by Polθ to the replication of UV-damaged DNA and to genome stability, and examined the incidence of UV-induced skin cancers in Polθ-/-, Polη-/-, and Polθ-/- Polη-/- mice. Our findings that the incidence of skin cancers rises in Polθ-/- mice and is further exacerbated in Polθ-/- Polη-/- mice compared with Polη-/- mice support the conclusion that error-prone TLS by Polθ provides a safeguard against tumorigenesis and suggest that cancer formation can ensue in the absence of somatic point mutations.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Neoplasias Cutâneas/metabolismo , Animais , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/fisiologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Instabilidade Genômica/genética , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Pele/citologia , Pele/metabolismo , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , DNA Polimerase tetaRESUMO
Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/enzimologia , Genes Precoces , Proteínas Imediatamente Precoces/biossíntese , Transdução de Sinais , Transcrição Gênica , Proteínas ras/metabolismo , Animais , Simulação por Computador , Ativação Enzimática , Retroalimentação Fisiológica , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Luz , Camundongos , Modelos Genéticos , Células NIH 3T3 , Optogenética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de Célula Única , Fatores de Tempo , Transcriptoma , Transfecção , Regulação para CimaRESUMO
Low-dose 5-aminolevulinic acid photodynamic therapy (ALA-PDT) has been used to cope with skin photoaging, and is thought to involve DNA damage repair responses. However, it is still unknown how low-dose ALA-PDT regulates DNA damage repair to curb skin photoaging. We established a photoaging model using human dermal fibroblasts (HDFs) and rat skin. RNA-sequencing (RNA-seq) analysis was conducted to identify differentially expressed genes (DEGs) in HDFs before and after low-dose ALA-PDT treatment, followed by bioinformatics analysis. Senescence-associated ß-galactosidase (SA-ß-gal) staining was employed to assess skin aging-related manifestations and Western blotting to evaluate the expression of associated proteins. A comet assay was used to detect cellular DNA damage, while immunofluorescence to examine the expression of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in cells and skin tissues. In both in vivo and in vitro models, low-dose ALA-PDT alleviated the manifestations of ultraviolet B (UVB)-induced skin photoaging. Low-dose ALA-PDT significantly reduced DNA damage in photoaged HDFs. Furthermore, low-dose ALA-PDT accelerated the clearance of the photoproduct 8-oxo-dG in photoaged HDFs and superficial dermis of photoaged rat skin. RNA-seq analysis suggested that low-dose ALA-PDT upregulated the expression of key genes in the base excision repair (BER) pathway. Further functional validation showed that inhibition on BER expression by using UPF1069 significantly suppressed SA-ß-gal activity, G2/M phase ratio, expression of aging-associated proteins P16, P21, P53, and MUTYH proteins, as well as clearance of the photoproduct 8-oxo-dG in photoaged HDFs. Low-dose ALA-PDT exerts anti-photoaging effects by activating the BER signalling pathway.
Assuntos
Ácido Aminolevulínico , Dano ao DNA , Reparo do DNA , Fibroblastos , Fotoquimioterapia , Transdução de Sinais , Envelhecimento da Pele , Raios Ultravioleta , Ácido Aminolevulínico/farmacologia , Reparo do DNA/efeitos dos fármacos , Animais , Raios Ultravioleta/efeitos adversos , Humanos , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Fotoquimioterapia/métodos , Ratos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Pele/patologia , Masculino , Fármacos Fotossensibilizantes/farmacologia , 8-Hidroxi-2'-Desoxiguanosina/metabolismoRESUMO
Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.
Assuntos
Ciclina D1 , Inibidor de Quinase Dependente de Ciclina p21 , Reparo do DNA , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Humanos , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos da radiação , Células HeLa , Proteínas Tirosina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Fase S , Fase G1 , Melanoma/genética , Melanoma/patologia , Células Cultivadas , Raios Ultravioleta/efeitos adversos , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/efeitos da radiação , Quinases DyrkRESUMO
Skin photoaging is mostly caused by ultraviolet A (UVA), although active medications to effectively counteract UVA-induced photoaging have not yet been created. Resveratrol, a naturally occurring polyphenol found in the skin of grapes, has been shown to have various biological functions such as anti-inflammatory and antioxidant characteristics. However, the role of resveratrol in UVA-induced photoaging has not been clarified. We investigated the mechanism of action of resveratrol by UVA irradiation of human skin fibroblasts (HSF) and innovatively modified a mouse model of photoaging. The results demonstrated that resveratrol promoted AMP-activated protein kinase (AMPK) phosphorylation to activate autophagy, reduce reactive oxygen species (ROS) production, inhibit apoptosis, and restore normal cell cycle to alleviate UVA-induced photoaging. In addition, subcutaneous injection of resveratrol not only improved the symptoms of roughness, erythema, and increased wrinkles in the skin of UVA photodamaged mice, but also alleviated epidermal hyperkeratosis and hyperpigmentation, reduced inflammatory responses, and inhibited collagen fiber degradation. In conclusion, our studies proved that resveratrol can treat UVA-induced photoaging and elucidated the possible molecular mechanisms involved, providing a new therapeutic strategy for future anti-aging.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Fibroblastos , Resveratrol , Envelhecimento da Pele , Pele , Raios Ultravioleta , Resveratrol/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Animais , Raios Ultravioleta/efeitos adversos , Humanos , Masculino , Proteínas Quinases Ativadas por AMP/metabolismo , Camundongos , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Pele/patologia , Pele/metabolismo , Estilbenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiaçãoRESUMO
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.
Assuntos
Proteínas de Caenorhabditis elegans/química , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Schizosaccharomyces pombe/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Cisplatino/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Formaldeído/química , Células HeLa , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Seabuckthorn pulp oil (SBO) is used in beauty products because of its rich lipophilic substances with high nutraceutical and cosmeceutical potential. However, the mechanism through which SBO enhances skin elasticity remains unclear. Therefore, in this study, we examined the anti-photoaging activity of SBO in normal human dermal fibroblasts (NHDF) under ultraviolet (UV) irradiation. Pretreatment with SBO significantly suppressed UV-B-induced cell toxicity and collagen degradation, suggesting that SBO contains anti-photoaging substances. Further, palmitoleic acid, the main component of SBO, maintained cell viability and collagen levels in UV-B-irradiated NHDF by suppressing the expression of matrix metalloproteinase 1 and acted on the inhibition of p38 and JNK phosphorylation and nuclear translocation of nuclear factor-kappa B. These findings suggest the utility of SBO as an anti-photoaging agent.
Assuntos
Sobrevivência Celular , Fibroblastos , Hippophae , Metaloproteinase 1 da Matriz , Óleos de Plantas , Raios Ultravioleta , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Raios Ultravioleta/efeitos adversos , Hippophae/química , Metaloproteinase 1 da Matriz/metabolismo , Óleos de Plantas/farmacologia , Óleos de Plantas/química , Sobrevivência Celular/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , NF-kappa B/metabolismo , Colágeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiaçãoRESUMO
Numerous studies have demonstrated the efficacy of extremely low frequency-pulsed electromagnetic fields (ELF-PEMF) in accelerating the wound healing process in vitro and in vivo. Our study focuses specifically on ELF-PEMF applied with the Magnomega® device and aims to assess their effect during the main stages of the proliferative phase of dermal wound closure, in vitro. Thus, after the characterization of the EMFs delivered by the Magnomega® unit, primary culture of human dermal fibroblasts (HDFs) were exposed, or not for the control culture, to 10-12 and 100 Hz ELF-PEMF. These parameters are used in clinical practice by physiotherapists in order to enhance healing of dermal lesions in patients. HDFs proliferation was first assessed and revealed an increase in the expression of one of the two genetic markers of cell proliferation tested (PCNA and MKI67), after initial exposure of the cells to 10-12 Hz PEMF. Next, migration of HDFs was investigated by performing scratch assays on HDF layers. The observed wound closure kinetics corroborate the early organization of actin stress fibers that was revealed in the cytoplasm of HDFs exposed to 100 Hz ELF-PEMF. Also, maturation of HDFs into myofibroblasts was significantly increased in cells exposed to 10-12 or to 100 Hz PEMF. The present study is the first to demonstrate, in vitro, an early stimulation of HDFs, after their exposure to ELF-PEMF delivered by the Magnomega® device, which could contribute to an acceleration of the wound healing process.
Assuntos
Proliferação de Células , Campos Eletromagnéticos , Fibroblastos , Medicina Regenerativa , Pele , Cicatrização , Humanos , Cicatrização/efeitos da radiação , Pele/citologia , Pele/lesões , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Medicina Regenerativa/métodos , Movimento Celular , Células CultivadasRESUMO
OBJECTIVE: This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment. Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used. J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.
Assuntos
Fibroblastos , Proteínas Filagrinas , Metaloproteinase 1 da Matriz , Fator 2 Relacionado a NF-E2 , Fator de Necrose Tumoral alfa , Raios Ultravioleta , Humanos , Raios Ultravioleta/efeitos adversos , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Pele/efeitos da radiação , Pele/efeitos dos fármacos , Pele/metabolismo , Protetores Solares/administração & dosagem , Protetores Solares/química , Protetores Solares/farmacologia , Aminoácidos/administração & dosagem , Aminoácidos/farmacologia , Aminoácidos/química , Interleucina-1alfa/metabolismo , Histamina/sangue , Creme para a Pele/administração & dosagem , Biomarcadores/metabolismo , Colágeno Tipo I , Proteínas de Filamentos Intermediários/metabolismo , Antígeno Ki-67/metabolismo , Dímeros de Pirimidina , Células CultivadasRESUMO
The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias/patologia , Síndromes de Tricotiodistrofia/enzimologia , Animais , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Epoprostenol , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Camundongos , Pele/patologia , Transcrição Gênica , Síndromes de Tricotiodistrofia/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genéticaRESUMO
Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.
Assuntos
Dano ao DNA/efeitos da radiação , Metilação de DNA/genética , Replicação do DNA/genética , Pele/efeitos da radiação , Metilação de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Fibroblastos/efeitos da radiação , Genoma Humano/genética , Genoma Humano/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Genômica/métodos , Humanos , Mutação INDEL/efeitos da radiação , Melanócitos/efeitos da radiação , Mutagênese/genética , Mutagênese/efeitos da radiação , Pele/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
The purpose of this research was to investigate the effect of toluidine blue (TB) mediated photodynamic therapy (PDT) on the inhibition of lipopolysaccharide (LPS)-induced inflammation in rat gingival fibroblasts through in vitro experiments. Rat gingival fibroblasts were divided into five groups: (1) control, (2) LPS treatment, (3) laser treatment, (4) TB treatment (1.0 µg/mL), and (5) PDT treatment (TB plus laser irradiation at 320 mW/cm2 for 240 s). After 24 h, cell growth activity was measured using MTT assay. The levels of receptor activator for nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in the cell culture supernatant were measured using enzyme-linked immunosorbent assay (ELISA). Nuclear proteins were extracted and the phosphorylation levels of phosphorylated nuclear factor-κB/p65 (p-p65) and phosphorylated inhibitor of nuclear factor-κB (p-IκBα) were determined using Western Blot. MTT results showed no significant difference in cell viability between the groups (P > 0.05). After LPS induction, OPG expression decreased, RANKL expression increased, and the OPG/RANKL ratio decreased, which was different from the control group (P < 0.05). After PDT treatment, OPG expression increased, RANKL expression decreased (P < 0.05), and the OPG/RANKL ratio increased (P < 0.05). Compared to the control group, there was no significant difference in OPG and RANKL expression or the OPG/RANKL ratio (P > 0.05). The activation of NF-κB was closely related to the phosphorylation levels of p-p65 and p-IκBα. LPS significantly up-regulated p-p65 and p-IκBα expression (P < 0.05), while PDT treatment decreased their phosphorylation levels (P < 0.05). TB-PDT treatment can inhibit NF-κB signaling pathway activation, decrease RANKL and OPG expression, and reduce the OPG/RANKL ratio, thereby reducing inflammation and playing a role in periodontitis treatment.
Assuntos
Fibroblastos , Gengiva , Lipopolissacarídeos , Osteoprotegerina , Fotoquimioterapia , Ligante RANK , Cloreto de Tolônio , Animais , Fotoquimioterapia/métodos , Ratos , Gengiva/efeitos dos fármacos , Gengiva/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Ligante RANK/metabolismo , Osteoprotegerina/metabolismo , Células Cultivadas , Inflamação , NF-kappa B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , FosforilaçãoRESUMO
The aim of this study was to evaluate the antiproliferative properties of low-level laser therapy (LLLT) on gingival fibroblasts obtained from calcium channel blocker-induced gingival overgrowth (GO). Gingival fibroblasts of patients with GO were compared to healthy gingival fibroblasts (H). Both cells were exposed to LLLT (685 nm wavelength, 25mW power, diode laser) and compared to those not treated with LLLT. Cell proliferation and viability were measured with MTT assay at baseline and after 24 and 72 h. TGF-ß1, CTGF, and collagen Type 1 levels were evaluated with Enzyme-Linked Immunosorbent Assay (ELISA). LLLT significantly decreased the proliferation of GO fibroblasts (p < 0.05) while leading to a significantly higher proliferation in H fibroblasts compared to the untreated cells (p < 0.05). GO cells showed significantly higher CTGF, TGF-ß, and collagen Type 1 expression than the H cells (p < 0.05). LLLT significantly reduced CTGF levels in GO cells compared to the control group (p < 0.05). In H cells, CTGF and TGF-ß levels were also significantly decreased in response to LLLT compared to the control group (p < 0.05). While LLLT significantly reduced collagen expression in the H group (p < 0.05), it did not significantly impact the GO cells. LLLT significantly reduced the synthesis of the growth factors and collagen in both groups with an antiproliferative effect on the gingival fibroblasts from calcium channel blocker-induced GO, suggesting that it can offer a therapeutic approach in the clinical management of drug-induced GO, reversing the fibrotic changes.
Assuntos
Bloqueadores dos Canais de Cálcio , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo , Fibroblastos , Gengiva , Crescimento Excessivo da Gengiva , Terapia com Luz de Baixa Intensidade , Humanos , Fibroblastos/efeitos da radiação , Fibroblastos/efeitos dos fármacos , Terapia com Luz de Baixa Intensidade/métodos , Crescimento Excessivo da Gengiva/induzido quimicamente , Crescimento Excessivo da Gengiva/radioterapia , Crescimento Excessivo da Gengiva/terapia , Bloqueadores dos Canais de Cálcio/farmacologia , Proliferação de Células/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Gengiva/efeitos da radiação , Gengiva/citologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Sobrevivência Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Lasers Semicondutores/uso terapêutico , Masculino , Adulto , FemininoRESUMO
The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.
Assuntos
Autofagia , Proliferação de Células , Senescência Celular , Reparo do DNA , Fibroblastos , Humanos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Autofagia/efeitos da radiação , Senescência Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Raios X/efeitos adversos , Proliferação de Células/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Histonas/metabolismo , Relação Dose-Resposta à Radiação , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Células Cultivadas , Dano ao DNA/efeitos da radiaçãoRESUMO
Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number.
Assuntos
Dano ao DNA , Fibroblastos , Histonas , Tomografia Computadorizada por Raios X , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Tomografia Computadorizada por Raios X/métodos , Histonas/metabolismo , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Relação Dose-Resposta à Radiação , Epitélio Pigmentado da Retina/efeitos da radiação , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Reparo do DNA , Transferência Linear de EnergiaRESUMO
Allomyrina dichotoma larvae (ADL) is an insect type that is used ethnopharmacologically to treat various diseases; however, its use as an antiaging treatment has not been widely studied. Previously, we found that an ethyl acetate (EA) fraction derived from an ADL extract (ADLE) has a high polyphenol content and antioxidant properties. In this study, we identified the underlying molecular mechanism for the protective effect of the EA fraction against UVB-induced photodamage in vitro and ex vivo. UVB treatment increased intracellular reactive oxygen species levels and DNA damage; the latter of which was significantly decreased following cotreatment with the EA fraction. Biological markers of aging, such as p16INK4a, p21WAF1, and senescence-associated ß-gal levels, were induced by UVB treatment but significantly suppressed following EA-fraction treatment. UVB-induced upregulation of matrix metalloproteinase (MMP)-1 and downregulation of COL1A1 were also reversed by EA-fraction treatment in both cells and a 3D skin model, which resulted in increased keratin and collagen deposition. Moreover, EA-fraction treatment inhibited the phosphorylation of MAPKs (p38, ERK, and JNK) and nuclear factor (NF-)-kB and decreased the levels of inflammatory cytokines in UVB-treated cells. The results indicate that an EA fraction from ADLE ameliorates UVB-induced degradation of COL1A1 by inhibiting MMP expression and inactivating the MAPK/NF-κB p65/AP-1 signaling pathway involved in this process.
Assuntos
Acetatos , Fibroblastos , Larva , Envelhecimento da Pele , Raios Ultravioleta , Humanos , Raios Ultravioleta/efeitos adversos , Animais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Acetatos/farmacologia , Acetatos/química , Larva/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/genética , NF-kappa B/metabolismoRESUMO
DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.
Assuntos
Histonas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Adenoviridae/genética , Animais , Células Cultivadas , Quinase 1 do Ponto de Checagem , Meios de Cultura Livres de Soro , Dano ao DNA , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Células HCT116 , Histonas/genética , Humanos , Camundongos , Modelos Genéticos , Fosforilação/efeitos da radiação , Proteínas Quinases/análise , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Raios UltravioletaRESUMO
Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i.e., of GAPDH or PKM2). Through ultra-short (13)C labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.
Assuntos
Proteínas de Transporte/metabolismo , Peróxido de Hidrogênio/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Proteínas de Membrana/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/efeitos da radiação , Hormônios Tireóideos/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolômica/métodos , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.
Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Ferroquelatase/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase I/genética , RNA Ribossômico/genética , Fatores de Transcrição/genética , Linhagem Celular Transformada , Sobrevivência Celular , Imunoprecipitação da Cromatina , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Ferroquelatase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Raios UltravioletaRESUMO
An in vitro study was designed to evaluate the effects of photobiomodulation (PBM) with 915-nm diode laser on human gingival fibroblast (HGF) cells under hyperglycemic condition. The HGF cells were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 30 mM glucose concentration for 48 h to mimic the hyperglycemic condition. Subsequently, the cells received three sessions of PBM (915 nm, continuous emission mode, 200 mW, energy density values of 3.2, 6, and 9.2 J/cm2). Twenty-four hours post-irradiation, cell proliferation, expression of interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) were assessed with MTT and real-time polymerase chain reaction (PCR) tests, respectively. Also, reactive oxygen species (ROS) production was measured using CM-H2DCFDA fluorimetry. No changes were detected in the cell proliferation rate between the high glucose control group and laser-treated cells, while VEGF and IL-6 gene expression levels increased significantly after PBM in the high glucose-treated cells group. ROS level was significantly decreased in the irradiated cells in high-glucose medium compared with the high glucose control group. Our study revealed the inductive role of 915-nm-mediated PBM on VEGF and the inflammatory response while concurrently reducing reactive oxygen species production in HGF cells in hyperglycemic conditions.