RESUMO
To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.
Assuntos
Células-Tronco Embrionárias Murinas/efeitos da radiação , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Transcriptoma , Raios XRESUMO
The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological ß-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Transdução de Sinal Luminoso , Miócitos Cardíacos/metabolismo , Optogenética/métodos , Animais , Cálcio/metabolismo , Cátions Bivalentes , Diferenciação Celular , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Átrios do Coração/citologia , Átrios do Coração/efeitos da radiação , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos da radiação , Luz , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos da radiaçãoRESUMO
Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2Ct, fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Genoma , Proteína de Replicação A/genética , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , DNA/metabolismo , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Osteoblastos , Fosforilação/efeitos da radiação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteólise/efeitos da radiação , Proteína de Replicação A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
BACKGROUND: Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis. METHODS: Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs. RESULTS: In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice. CONCLUSIONS: The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.
Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades/genética , Instabilidade Genômica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Radiação IonizanteRESUMO
Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxazóis/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/patologia , Células-Tronco Embrionárias Murinas/efeitos da radiação , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/efeitos da radiação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Células-Tronco/patologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
DNA double-strand breaks (DSB) elicit a ubiquitylation cascade that controls DNA repair pathway choice. This cascade involves the ubiquitylation of histone H2A by the RNF168 ligase and the subsequent recruitment of RIF1, which suppresses homologous recombination (HR) in G1 cells. The RIF1-dependent suppression is relieved in S/G2 cells, allowing PALB2-driven HR to occur. With the inhibitory impact of RIF1 relieved, it remains unclear how RNF168-induced ubiquitylation influences HR. Here, we uncover that RNF168 links the HR machinery to H2A ubiquitylation in S/G2 cells. We show that PALB2 indirectly recognizes histone ubiquitylation by physically associating with ubiquitin-bound RNF168. This direct interaction is mediated by the newly identified PALB2-interacting domain (PID) in RNF168 and the WD40 domain in PALB2, and drives DNA repair by facilitating the assembly of PALB2-containing HR complexes at DSBs. Our findings demonstrate that RNF168 couples PALB2-dependent HR to H2A ubiquitylation to promote DNA repair and preserve genome integrity.
Assuntos
DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Histonas/metabolismo , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Células HEK293 , Histonas/genética , Humanos , Lasers de Excimer , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Raios XRESUMO
The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA , Proteína Forkhead Box O3/genética , Linfócitos/metabolismo , Lisina Acetiltransferase 5/genética , Receptor Notch1/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ligação Competitiva , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteína Forkhead Box O3/metabolismo , Raios gama , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Linfócitos/efeitos da radiação , Lisina Acetiltransferase 5/metabolismo , Metformina/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Ligação Proteica , Receptor Notch1/metabolismo , Transdução de SinaisRESUMO
The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis.
Assuntos
Células-Tronco Adultas/efeitos da radiação , Compartimento Celular/efeitos da radiação , Células-Tronco Embrionárias Murinas/efeitos da radiação , Células-Tronco Neurais/efeitos da radiação , Radiação Ionizante , Células-Tronco Adultas/citologia , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Ligase Dependente de ATP/deficiência , DNA Ligase Dependente de ATP/metabolismo , Relação Dose-Resposta à Radiação , Humanos , Camundongos , Camundongos Mutantes , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , SíndromeRESUMO
Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.