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Anticancer Research Mar 2024Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown...
BACKGROUND/AIM
Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells.
MATERIALS AND METHODS
We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis.
RESULTS
Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells.
CONCLUSION
Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.
Topics: Humans; Temozolomide; Glioblastoma; Senotherapeutics; Flavonols; Antineoplastic Agents; Flavonoids; Apoptosis; DNA Damage; Cell Line, Tumor; DNA
PubMed: 38423634
DOI: 10.21873/anticanres.16884 -
BioRxiv : the Preprint Server For... Feb 2024When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the...
When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage in . We report that translesion synthesis, rather than template switching, is the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.
PubMed: 38405884
DOI: 10.1101/2024.02.13.580051 -
Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.Applied Microbiology and Biotechnology Feb 2024Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated...
Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.
Topics: Saccharomyces cerevisiae; CRISPR-Cas Systems; DNA Damage; Mitomycin; Luciferases; DNA
PubMed: 38300351
DOI: 10.1007/s00253-024-13020-w -
G3 (Bethesda, Md.) Mar 2024We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA...
We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.
Topics: Histones; Methyl Methanesulfonate; Molecular Chaperones; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; DNA Repair; DNA Damage; Chromatin; Cell Cycle Proteins; Histone Acetyltransferases; Acetylation; Sordariales
PubMed: 38261383
DOI: 10.1093/g3journal/jkae019 -
Molecular Microbiology Feb 2024
Correction to "SUMOylation of yeast Pso2 enhances its translocation and accumulation in the mitochondria and suppresses methyl methanesulfonate-induced mitochondrial DNA damage".
PubMed: 38219261
DOI: 10.1111/mmi.15226 -
Scientific Data Jan 2024A wealth of proteogenomic data has been generated using cancer samples to deepen our understanding of the mechanisms of cancer and how biological networks are altered in...
A wealth of proteogenomic data has been generated using cancer samples to deepen our understanding of the mechanisms of cancer and how biological networks are altered in association with somatic mutation of tumor suppressor genes, such as TP53 and PTEN. To generate functional signatures of TP53 or PTEN loss, we profiled the RNA and phosphoproteomes of the MCF10A epithelial cell line, along with its congenic TP53- or PTEN-knockout derivatives, upon perturbation with the monofunctional DNA alkylating agent methyl methanesulfonate (MMS) vs. mock treatment. To enable quantitative and reproducible mass spectrometry data generation, the cell lines were SILAC-labeled (stable isotope labeling with amino acids in cell culture), and the experimental design included label swapping and biological replicates. All data are publicly available and may be used to advance our understanding of the TP53 and PTEN tumor suppressor genes and to provide functional signatures for bioinformatic analyses of proteogenomic datasets.
Topics: Humans; DNA Damage; Epithelial Cells; Mutation; Neoplasms; PTEN Phosphohydrolase; RNA; Tumor Suppressor Protein p53
PubMed: 38177134
DOI: 10.1038/s41597-023-02829-1 -
International Journal of Molecular... Dec 2023DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6)...
DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6 localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6 only localized in MICs and newly developing MACs. knockout led to aberrant nuclear division during vegetative growth. The mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6 interacted with Msh2 and MMR-independent factors. Downregulation of expression affected the stability of Msh6. In addition, knockout led to the upregulated expression of several homologs at different developmental stages. Msh6 is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in .
Topics: DNA Mismatch Repair; Tetrahymena thermophila; MutS Homolog 2 Protein; Escherichia coli; DNA-Binding Proteins; Meiosis; Gametogenesis
PubMed: 38139447
DOI: 10.3390/ijms242417619 -
Biochemistry. Biokhimiia Nov 2023Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage...
Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.
Topics: Humans; DNA-Directed DNA Polymerase; A549 Cells; Cisplatin; Hydrogen Peroxide; DNA Replication; DNA Damage; Adenocarcinoma of Lung; DNA Primase; Multifunctional Enzymes
PubMed: 38105210
DOI: 10.1134/S0006297923110214 -
Molecular & Cellular Proteomics : MCP Jan 2024In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR...
In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.
Topics: Saccharomyces cerevisiae; Proteome; Chromatography, Liquid; Tandem Mass Spectrometry; Ubiquitination; Saccharomyces cerevisiae Proteins; DNA Damage; DNA Repair
PubMed: 38101750
DOI: 10.1016/j.mcpro.2023.100695 -
The Plant Journal : For Cell and... Mar 2024Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast,...
Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the K /K value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.
Topics: Sorghum; Reverse Genetics; Plant Breeding; Mutation; Phenotype; Edible Grain; Ethyl Methanesulfonate; Genome, Plant
PubMed: 38100514
DOI: 10.1111/tpj.16582