scholarly journals BAP1 Status Determines the Sensitivity of Malignant Mesothelioma Cells to Gemcitabine Treatment

2019 ◽  
Vol 20 (2) ◽  
pp. 429 ◽  
Author(s):  
Alice Guazzelli ◽  
Parisa Meysami ◽  
Emyr Bakker ◽  
Constantinos Demonacos ◽  
Antonio Giordano ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Malignant Mesothelioma ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Late Apoptosis ◽  
Mutant Cells ◽  
Gemcitabine Treatment

Malignant mesothelioma (MMe) is a cancer with poor prognosis and resistance to standard treatments. Recent reports have highlighted the role of the BRCA1 associated protein 1 gene (BAP1) in the development of MMe. In this study, the chemosensitivity of human mesothelioma cell lines carrying BAP1 wild-type (WT), mutant and silenced was analysed. The BAP1 mutant cells were significantly less sensitive than BAP1 WT cell lines to the clinically relevant drug gemcitabine. Silencing of BAP1 significantly increased resistance of MMe cells to gemcitabine. Cell cycle analysis suggested that gemcitabine induced Sub-G1 phase accumulation of the BAP1 WT cells and increased in the S-phase in both BAP1 WT and mutant cells. Analysis of the role of BAP1 in apoptosis suggested that gemcitabine induced early apoptosis in both BAP1 WT and BAP1 mutant cells but with a much higher degree in the WT cells. Effects on the population of cells in late apoptosis, which can mark necrosis and necroptosis, could not be seen in the mutant cells, highlighting the possibility that BAP1 plays a role in several types of cell death. Significantly decreased DNA damage in the form of double-strand breaks was observed in gemcitabine-treated BAP1 mutant cells, compared to BAP1 WT cells under the same conditions. After BAP1 silencing, a significant decrease in DNA damage in the form of double-strand breaks was observed compared to cells transfected with scramble siRNA. Taken together, the results presented in this manuscript shed light on the role of BAP1 in the response of MMe cells to gemcitabine treatment and in particular in the control of the DNA damage response, therefore providing a potential route for more efficient MMe therapy.

scholarly journals SET8 Is a Potential Therapeutic Target in MM

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4435-4435
Author(s):  
Herviou Laurie ◽  
Fanny Izard ◽  
Elke De Bruyne ◽  
Eva Desmedt ◽  
Anqi Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Damage Response

Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of the Cell-Intrinsic DNA Damage Response by the Innate Immune Machinery

2021 ◽  
Vol 22 (23) ◽  
pp. 12761
Author(s):  
Thomas J. Hayman ◽  
Peter M. Glazer
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Innate Immune ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Immune Mediated ◽  
Damage Response

Maintenance of genomic integrity is crucial for cell survival. As such, elegant DNA damage response (DDR) systems have evolved to ensure proper repair of DNA double-strand breaks (DSBs) and other lesions that threaten genomic integrity. Towards this end, most therapeutic studies have focused on understanding of the canonical DNA DSB repair pathways to enhance the efficacy of DNA-damaging therapies. While these approaches have been fruitful, there has been relatively limited success to date and potential for significant normal tissue toxicity. With the advent of novel immunotherapies, there has been interest in understanding the interactions of radiation therapy with the innate and adaptive immune responses, with the ultimate goal of enhancing treatment efficacy. While a substantial body of work has demonstrated control of the immune-mediated (extrinsic) responses to DNA-damaging therapies by several innate immune pathways (e.g., cGAS–STING and RIG-I), emerging work demonstrates an underappreciated role of the innate immune machinery in directly regulating tumor cell-intrinsic/cell-autonomous responses to DNA damage.

Bestimmung der DNA-Schädigung in vitro

2010 ◽  
Vol 49 (S 01) ◽  
pp. S64-S68
Author(s):  
E. Dikomey
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Chromosome Aberrations ◽  
Genetic Factors ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cell Inactivation ◽  
Repair Mechanisms ◽  
Break Repair

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

scholarly journals RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

2021 ◽  
Author(s):  
Sang-Min Jang ◽  
Christophe E. Redon ◽  
Haiqing Fu ◽  
Fred E. Indig ◽  
Mirit I. Aladjem
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Cell Sorting ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ubiquitinated Proteins ◽  
Damage Response ◽  
Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

scholarly journals Differential Roles of ATM- and Chk2-Mediated Phosphorylations of Hdmx in Response to DNA Damage

2006 ◽  
Vol 26 (18) ◽  
pp. 6819-6831 ◽  
Author(s):  
Yaron Pereg ◽  
Suzanne Lam ◽  
Amina Teunisse ◽  
Sharon Biton ◽  
Erik Meulmeester ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Genomic Stability ◽  
Double Strand Breaks ◽  
Nuclear Accumulation ◽  
Strand Breaks ◽  
Damage Response ◽  
Subsequent Degradation ◽  
Atm Protein

ABSTRACT The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

scholarly journals Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

2018 ◽  
Vol 115 (51) ◽  
pp. E11961-E11969 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Michael T. Kimble ◽  
Lorraine S. Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Inhibitory Effects ◽  
Checkpoint Signaling ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Damage Response ◽  
End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

scholarly journals Live-cell chromosome dynamics in Arabidopsis thaliana reveals increased chromatin mobility in response to DNA damage

2021 ◽  
Author(s):  
Anis Meschichi ◽  
Adrien Sicard ◽  
Frédéric Pontvianne ◽  
Svenja Reeck ◽  
Stefanie Rosa
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Genome Instability ◽  
High Mobility ◽  
Double Strand Breaks ◽  
Nuclear Space ◽  
Strand Breaks ◽  
Homologous Sequences ◽  
Damage Response ◽  
Repair Mechanisms

Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage potentially leading to translocations and genome instability. Homologous recombination (HR) is a conservative repair pathway in which intact homologous sequences are used as a template for repair. How damaged DNA molecules search for homologous sequences in the crowded space of the cell nucleus is, however, still poorly understood, especially in plants. Here, we measured global chromosome and DSB site mobility, in Arabidopsis thaliana, by tracking the motion of specific loci using the lacO/LacI tagging system and two GFP-tagged HR regulators, RAD51 and RAD54. We observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Interestingly, we observed that DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease of DSB mobility is observed, which seems to be associated to the relocation of DSBs to the nucleus periphery. Altogether, our data suggest that changes in chromatin mobility are triggered in response to DNA damage, and that this may act as a mechanism to enhance the physical search within the nuclear space to locate a homologous template during homology-directed DNA repair.

scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

scholarly journals CDK and Mec1/Tel1-catalyzed phosphorylation of Sae2 regulate different responses to DNA damage

2019 ◽  
Vol 47 (21) ◽  
pp. 11238-11249 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Valerie E Garcia ◽  
Lorraine S Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Phosphorylation Site ◽  
Physiological Role ◽  
Checkpoint Signaling ◽  
Strand Breaks ◽  
Checkpoint Kinases ◽  
Damage Response ◽  
End Resection

Abstract Sae2 functions in the DNA damage response by controlling Mre11-Rad50-Xrs2 (MRX)-catalyzed end resection, an essential step for homology-dependent repair of double-strand breaks (DSBs), and by attenuating DNA damage checkpoint signaling. Phosphorylation of Sae2 by cyclin-dependent kinase (CDK1/Cdc28) activates the Mre11 endonuclease, while the physiological role of Sae2 phosphorylation by Mec1 and Tel1 checkpoint kinases is not fully understood. Here, we compare the phenotype of sae2 mutants lacking the main CDK (sae2-S267A) or Mec1 and Tel1 phosphorylation sites (sae2-5A) with sae2Δ and Mre11 nuclease defective (mre11-nd) mutants. The phosphorylation-site mutations confer DNA damage sensitivity, but not to the same extent as sae2Δ. The sae2-S267A mutation is epistatic to mre11-nd for camptothecin (CPT) sensitivity and synergizes with sgs1Δ, whereas sae2-5A synergizes with mre11-nd and exhibits epistasis with sgs1Δ. We find that attenuation of checkpoint signaling by Sae2 is mostly independent of Mre11 endonuclease activation but requires Mec1 and Tel1-dependent phosphorylation of Sae2. These results support a model whereby CDK-catalyzed phosphorylation of Sae2 activates resection via Mre11 endonuclease, whereas Sae2 phosphorylation by Mec1 and Tel1 promotes resection by the Dna2-Sgs1 and Exo1 pathways indirectly by dampening the DNA damage response.

scholarly journals Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin

Tumor Biology ◽  
2017 ◽  
Vol 39 (3) ◽  
pp. 101042831769430 ◽  
Author(s):  
Juan Lv ◽  
Ying Qian ◽  
Xiaoyan Ni ◽  
Xiuping Xu ◽  
Xuejun Dong
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Feedback Regulation ◽  
Double Strand Breaks ◽  
Gene Clone ◽  
Methyl Methanesulfonate ◽  
Strand Breaks ◽  
Damage Response ◽  
Dna Replication And Repair ◽  
Mcf 7

The methyl methanesulfonate and ultraviolet-sensitive gene clone 81 protein is a structure-specific nuclease that plays important roles in DNA replication and repair. Knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 has been found to sensitize cancer cells to chemotherapy. However, the underlying molecular mechanism is not well understood. We found that methyl methanesulfonate and ultraviolet-sensitive gene clone 81 was upregulated and the ATM/Chk2 pathway was activated at the same time when MCF-7 cells were treated with cisplatin. By using lentivirus targeting methyl methanesulfonate and ultraviolet-sensitive gene clone 81 gene, we showed that knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 enhanced cell apoptosis and inhibited cell proliferation in MCF-7 cells under cisplatin treatment. Abrogation of ATM/Chk2 pathway inhibited cell viability in MCF-7 cells in response to cisplatin. Importantly, we revealed that ATM/Chk2 was required for the upregulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 resulted in inactivation of ATM/Chk2 pathway in response to cisplatin. Meanwhile, knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 activated the p53/Bcl-2 pathway in response to cisplatin. These data suggest that the ATM/Chk2 may promote the repair of DNA damage caused by cisplatin by sustaining methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and the double-strand breaks generated by methyl methanesulfonate and ultraviolet-sensitive gene clone 81 may activate the ATM/Chk2 pathway in turn, which provide a novel mechanism of how methyl methanesulfonate and ultraviolet-sensitive gene clone 81 modulates DNA damage response and repair.

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