RESUMO
Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.
RESUMO
WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n = 11), intellectual disability (n = 9), epilepsy (n = 7), and autism spectrum disorder (n = 4). Additional phenotypic features included abnormal growth parameters (n = 7), heart anomalies (n = 2), and hearing loss (n = 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.
Assuntos
Transtorno do Espectro Autista , Proteínas de Drosophila , Transtornos do Desenvolvimento da Linguagem , Transtornos do Neurodesenvolvimento , Animais , Humanos , Transtorno do Espectro Autista/genética , Drosophila melanogaster/genética , Transtornos do Neurodesenvolvimento/genética , Análise por Conglomerados , Cromatina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas de Drosophila/genéticaRESUMO
The 53BP1-RIF1-shieldin pathway maintains genome stability by suppressing nucleolytic degradation of DNA ends at double-strand breaks (DSBs). Although RIF1 interacts with damaged chromatin via phospho-53BP1 and facilitates recruitment of the shieldin complex to DSBs, it is unclear whether other regulatory cues contribute to this response. Here, we implicate methylation of histone H3 at lysine 4 by SETD1A-BOD1L in the recruitment of RIF1 to DSBs. Compromising SETD1A or BOD1L expression or deregulating H3K4 methylation allows uncontrolled resection of DNA ends, impairs end-joining of dysfunctional telomeres, and abrogates class switch recombination. Moreover, defects in RIF1 localization to DSBs are evident in patient cells bearing loss-of-function mutations in SETD1A. Loss of SETD1A-dependent RIF1 recruitment in BRCA1-deficient cells restores homologous recombination and leads to resistance to poly(ADP-ribose)polymerase inhibition, reinforcing the clinical relevance of these observations. Mechanistically, RIF1 binds directly to methylated H3K4, facilitating its recruitment to, or stabilization at, DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a Telômeros , Proteína BRCA1/genética , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
We recently identified that methylation of lysine 4 of histone H3 (H3K4) by SETD1A (SET domain containing 1A) maintains genome stability by protecting newly-replicated DNA from degradation. Mechanistically, SETD1A-dependent histone methylation regulates nucleosome mobilisation by FANCD2 (FA complementation group D2), a crucial step in maintaining genome integrity with important implications in chemo-sensitivity.
RESUMO
Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.
Assuntos
DNA/biossíntese , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , Células A549 , DNA/genética , Replicação do DNA/fisiologia , Epigênese Genética/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Metilação , Chaperonas Moleculares/genética , Nucleossomos/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells in the matrix of corn starch gel. This biocatalyst was used for repeated batch fermentations of glucose and grape must at various sugar concentrations (110-280 g/L) and low-temperature winemaking (5 degrees C). The biocatalyst retained its operational stability for a long period, and it was proved to be capable of producing dry and semisweet wines. The produced wines were analyzed for volatile byproducts by GC and GC-MS, and the results showed an increase in the number and amount of esters by immobilized cells. In addition, an increase in the percentages of esters and a decrease in those of alcohols with the drop of fermentation temperature were reported. The activation energy (E(a)) was lower (approximately 36%) and the reaction rate constant (k) was higher (approximately 78% at 30 degrees C and approximately 265% at 15 degrees C) in the case of immobilized cells compared to free cells, especially at low temperatures. These results show that corn starch gel may act as a promoter for the enzymes that are involved in the process or as a catalyst of the alcoholic fermentation and can explain the capability of immobilized cells for extremely low-temperature winemaking. Therefore, these results open a new way for research to find new catalysts in biotechnological processes.
Assuntos
Células Imobilizadas/metabolismo , Fermentação , Saccharomyces cerevisiae/metabolismo , Vinho/análise , Catálise , Células Imobilizadas/química , Ésteres/análise , Etanol/metabolismo , Cinética , Saccharomyces cerevisiae/química , Amido/química , Volatilização , Zea mays/químicaRESUMO
BACKGROUND: A study was conducted at the University Hospital of Patras between January 2002 and December 2003 to investigate antibiotic resistance patterns and clonality of Salmonella enterica in southwestern Greece. METHODS: Ninety-five isolates recovered from different outpatients were characterized by specific antisera and were tested for their susceptibility to various antimicrobial agents. Clones were identified by pulsed-field gel electrophoresis (PFGE) of XbaI chromosomal DNA digests. RESULTS: Five serotypes were characterized among 95 isolates, recovered mainly from children, with the predominance of Salmonella enteritidis followed by Salmonella typhimurium. The strains were further distinguished by PFGE to 16 clones. The majority of S.enteritidis (61 strains) belonged to clone A, already characterized in Greece, while S.typhimurium (18 isolates) belonged to 3 clones, exhibiting multiresistant phenotypes. CONCLUSIONS: One clone S.enteritidis, type A, circulates in southwestern Greece, mainly during summer, while clonality was also observed among S.typhimurium.